Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth

PubMed Central

Kiselinova, Maja; De Spiegelaere, Ward; Buzon, Maria Jose; Malatinkova, Eva; Lichterfeld, Mathias; Vandekerckhove, Linos

2016-01-01

The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4–2.6) between time point 1 and 2; and median of 31 days (IQR: 28–36) between time point 2 and 3. Patients were median of 6 years (IQR: 3–12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2–8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA) was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85), us HIV-1 RNA (p = 0.029, R² = 0.40), and VOA (p = 0.041, R2 = 0.44). Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54). The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1). Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the replication-competent virus in ART suppressed patients. PMID:26938995

Herpes simplex virus type 2 (HSV-2) genital shedding in HSV-2-/HIV-1-co-infected women receiving effective combination antiretroviral therapy.

PubMed

Péré, Héléne; Rascanu, Aida; LeGoff, Jérome; Matta, Mathieu; Bois, Frédéric; Lortholary, Olivier; Leroy, Valériane; Launay, Odile; Bélec, Laurent

2016-03-01

The dynamics of genital shedding of HSV-2 DNA was assessed in HIV-1-infected women taking combination antiretroviral therapy (cART). HIV-1 RNA, HIV-1 DNA and HSV DNA loads were measured during 12-18 months using frozen plasma, PBMC and cervicovaginal lavage samples from 22 HIV-1-infected women, including 17 women naive for antiretroviral therapy initiating cART and 5 women with virological failure switching to a new regimen. Nineteen (86%) women were HSV-2-seropositive. Among HSV-2-/HIV-1-co-infected women, HIV-1 RNA loads showed a rapid fall from baseline after one month of cART, in parallel in paired plasma and cervicovaginal secretions. In contrast, HIV-1 DNA loads did not show significant variations from baseline up to 18 months of treatment in both systemic and genital compartments. HSV DNA was detected at least once in 12 (63%) of 19 women during follow up: HSV-2 shedding in the genital compartment was observed in 11% of cervicovaginal samples at baseline and in 16% after initiating or switching cART. Cervicovaginal HIV-1 RNA loads were strongly associated with plasma HIV-1 RNA loads over time, but not with cervicovaginal HSV DNA loads. Reactivation of genital HSV-2 replication frequently occurred despite effective cART in HSV-2-/HIV-1-co-infected women. Genital HSV-2 replication under cART does not influence cervicovaginal HIV-1 RNA or DNA shedding. © The Author(s) 2015.

Genital shedding of human immunodeficiency virus type 1 DNA during pregnancy: association with immunosuppression, abnormal cervical or vaginal discharge, and severe vitamin A deficiency.

PubMed

John, G C; Nduati, R W; Mbori-Ngacha, D; Overbaugh, J; Welch, M; Richardson, B A; Ndinya-Achola, J; Bwayo, J; Krieger, J; Onyango, F; Kreiss, J K

1997-01-01

The presence of human immunodeficiency virus type 1 (HIV-1) in genital secretions may be a determinant of vertical HIV-1 transmission. Cervical and vaginal secretions from HIV-1-seropositive pregnant women were evaluated to determine prevalence and correlates of HIV-1-infected cells in the genital tract. HIV-1 DNA was detected by polymerase chain reaction in 32% of 212 cervical and 10% of 215 vaginal specimens. Presence of HIV-1 DNA in the cervix was associated with cervical mucopus and a significantly lower absolute CD4 cell count (354 vs. 469, P < .001). An absolute CD4 cell count <200 was associated with a 9.6-fold increased odds of cervical HIV-1 DNA detection compared with a count > or = 500 (95% confidence interval, 2.8-34.2). Detection of vaginal HIV- 1 DNA was associated with abnormal vaginal discharge, lower absolute CD4 cell count, and severe vitamin A deficiency. Presence of HIV-1-infected cells in genital secretions was associated with immunosuppression and abnormal cervical or vaginal discharge.

Random integration of SV40 in SV40-transformed, immortalized human fibroblasts.

PubMed

Hara, H; Kaji, H

1987-02-01

We have studied the relationship between immortalization of SV40-transformed human embryonic fibroblasts and their SV40 integration sites. From several independently transformed cell pools, we have isolated clones which do not harbor unintegrated SV40 DNA. We have analysed whole-cell DNA from these clones, using the Southern blot method. Our results suggest that no specific integration sites in the cellular genome exist which are a prerequisite for the immortalization process. Although some integration sites were found to be predominant in pre-crisis clones, they could not be detected in the post-crisis clones. This suggests that none of these predominating sites is selected for during the crisis period.

HIV-1 DNA predicts disease progression and post-treatment virological control

PubMed Central

Williams, James P; Hurst, Jacob; Stöhr, Wolfgang; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Cooper, David; Schechter, Mauro; Tambussi, Giuseppe; Fidler, Sarah; Carrington, Mary; Babiker, Abdel; Weber, Jonathan

2014-01-01

In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials. Clinical trial registration: ISRCTN76742797 and EudraCT2004-000446-20 DOI: http://dx.doi.org/10.7554/eLife.03821.001 PMID:25217531

Impact of the Central Polypurine Tract on the Kinetics of Human Immunodeficiency Virus Type 1 Vector Transduction

PubMed Central

Van Maele, Bénédicte; De Rijck, Jan; De Clercq, Erik; Debyser, Zeger

2003-01-01

Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) show great promise as gene carriers for future gene therapy. Insertion of a fragment containing the central polypurine tract (cPPT) in HIV-1 vector constructs is known to enhance transduction efficiency drastically, reportedly by facilitating the nuclear import of HIV-1 cDNA through a central DNA flap. We have studied the impact of the cPPT on the kinetics of HIV-1 vector transduction by real-time PCR. The kinetics of total HIV-1 DNA, two-long-terminal-repeat (2-LTR) circles, and, by an Alu-PCR, integrated proviral DNA were monitored. About 6 to 12 h after transduction, the total HIV-1 DNA reached a maximum level, followed by a steep decrease. The 2-LTR circles peaked after 24 to 48 h and were diluted upon cell division. Integration of HIV-1 DNA was first detected at 12 h postinfection. When HIV-1 vectors that contained the cPPT were used, DNA synthesis was similar but a threefold higher amount of 2-LTR circles was detected, confirming the impact on nuclear import. Moreover, a 10-fold increase in the amount of integrated DNA was observed in the presence of the cPPT. Only in the absence of the cPPT was a saturation in 2-LTR circle formation seen at a high multiplicity of infection, suggesting a role for the cPPT in overcoming a barrier to the nuclear import of HIV-1 DNA. A major effect of the central DNA flap on the juxtaposition of both LTRs is unlikely, since transduction with HIV-1 vectors containing ectopic cPPT fragments resulted in increased amounts of 2-LTR circles as well as integrated DNA. Inhibitors of transduction by cPPT-containing HIV vectors were also studied by real-time PCR. The reverse transcriptase inhibitor azidothymidine (AZT) and the nonnucleoside reverse transcriptase inhibitor α-APA clearly inhibited viral DNA synthesis, whereas integrase inhibitors such as the diketo acid L-708,906 and the pyranodipyrimidine V-165 specifically inhibited integration. PMID:12663775

Variability of human immunodeficiency virus-1 in the female genital reservoir during genital reactivation of herpes simplex virus type 2.

PubMed

LeGoff, J; Roques, P; Jenabian, M-A; Charpentier, C; Brochier, C; Bouhlal, H; Gresenguet, G; Frost, E; Pepin, J; Mayaud, P; Belec, L

2015-09-01

Clinical and subclinical genital herpes simplex virus type 2 (HSV-2) reactivations have been associated with increases in human immunodeficiency virus (HIV)-1 genital shedding. Whether HSV-2 shedding contributes to the selection of specific genital HIV-1 variants remains unknown. We evaluated the genetic diversity of genital and blood HIV-1 RNA and DNA in 14 HIV-1/HSV-2-co-infected women, including seven with HSV-2 genital reactivation, and seven without as controls. HIV-1 DNA and HIV-1 RNA env V1-V3 sequences in paired blood and genital samples were compared. The HSV-2 selection pressure on HIV was estimated according to the number of synonymous substitutions (dS), the number of non-synonymous substitutions (dN) and the dS/dN ratio within HIV quasi-species. HIV-1 RNA levels in cervicovaginal secretions were higher in women with HSV-2 replication than in controls (p0.02). Plasma HIV-1 RNA and genital HIV-1 RNA and DNA were genetically compartmentalized. No differences in dS, dN and the dS/dN ratio were observed between the study groups for either genital HIV-1 RNA or plasma HIV-1 RNA. In contrast, dS and dN in genital HIV-1 DNA were significantly higher in patients with HSV-2 genital reactivation (p <0.01 and p <0.05, respectively). The mean of the dS/dN ratio in genital HIV-1 DNA was slightly higher in patients with HSV-2 genital replication, indicating a trend for purifying selection (p 0.056). HSV-2 increased the genetic diversity of genital HIV-1 DNA. These observations confirm molecular interactions between HSV-2 and HIV-1 at the genital tract level. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

CRISPR/Cas9 Inhibits Multiple Steps of HIV-1 Infection.

PubMed

Yin, Lijuan; Hu, Siqi; Mei, Shan; Sun, Hong; Xu, Fengwen; Li, Jian; Zhu, Weijun; Liu, Xiaoman; Zhao, Fei; Zhang, Di; Cen, Shan; Liang, Chen; Guo, Fei

2018-05-09

CRISPR/Cas9 is an adaptive immune system where bacteria and archaea have evolved to resist the invading viruses and plasmid DNA by creating site-specific double-strand breaks in DNA. This study tested this gene editing system in inhibiting human immunodeficiency virus type 1 (HIV-1) infection by targeting the viral long terminal repeat and the gene coding sequences. Strong inhibition of HIV-1 infection by Cas9/gRNA was observed, which resulted not only from insertions and deletions (indels) that were introduced into viral DNA due to Cas9 cleavage, but also from the marked decrease in the levels of the late viral DNA products and the integrated viral DNA. This latter defect might have reflected the degradation of viral DNA that has not been immediately repaired after Cas9 cleavage. It was further observed that Cas9, when solely located in the cytoplasm, inhibits HIV-1 as strongly as the nuclear Cas9, except that the cytoplasmic Cas9 does not act on the integrated HIV-1 DNA and thus cannot be used to excise the latent provirus. Together, the results suggest that Cas9/gRNA is able to target and edit HIV-1 DNA both in the cytoplasm and in the nucleus. The inhibitory effect of Cas9 on HIV-1 is attributed to both the indels in viral DNA and the reduction in the levels of viral DNA.

Cellular HIV-1 DNA Levels in Drug Sensitive Strains Are Equivalent to Those in Drug Resistant Strains in Newly-Diagnosed Patients in Europe

PubMed Central

Demetriou, Victoria L.; van de Vijver, David A. M. C.; Kousiappa, Ioanna; Balotta, Claudia; Clotet, Bonaventura; Grossman, Zehava; Jørgensen, Louise B.; Lepej, Snjezana Z.; Levy, Itzchak; Nielsen, Claus; Paraskevis, Dimitrios; Poljak, Mario; Roman, Francois; Ruiz, Lidia; Schmidt, Jean-Claude; Vandamme, Anne-Mieke; Van Laethem, Kristel; Vercauteren, Jurgen; Kostrikis, Leondios G.

2010-01-01

Background HIV-1 genotypic drug resistance is an important threat to the success of antiretroviral therapy and transmitted resistance has reached 9% prevalence in Europe. Studies have demonstrated that HIV-1 DNA load in peripheral blood mononuclear cells (PBMC) have a predictive value for disease progression, independently of CD4 counts and plasma viral load. Methodology/Principal Findings Molecular-beacon-based real-time PCR was used to measure HIV-1 second template switch (STS) DNA in PBMC in newly-diagnosed HIV-1 patients across Europe. These patients were representative for the HIV-1 epidemic in the participating countries and were carrying either drug-resistant or sensitive viral strains. The assay design was improved from a previous version to specifically detect M-group HIV-1 and human CCR5 alleles. The findings resulted in a median of 3.32 log10 HIV-1 copies/106 PBMC and demonstrated for the first time no correlation between cellular HIV-1 DNA load and transmitted drug-resistance. A weak association between cellular HIV-1 DNA levels with plasma viral RNA load and CD4+ T-cell counts was also reconfirmed. Co-receptor tropism for 91% of samples, whether or not they conferred resistance, was CCR5. A comparison of pol sequences derived from RNA and DNA, resulted in a high similarity between the two. Conclusions/Significance An improved molecular-beacon-based real-time PCR assay is reported for the measurement of HIV-1 DNA in PBMC and has investigated the association between cellular HIV-1 DNA levels and transmitted resistance to antiretroviral therapy in newly-diagnosed patients from across Europe. The findings show no correlation between these two parameters, suggesting that transmitted resistance does not impact disease progression in HIV-1 infected individuals. The CCR5 co-receptor tropism predominance implies that both resistant and non-resistant strains behave similarly in early infection. Furthermore, a correlation found between RNA- and DNA-derived sequences in the pol region suggests that genotypic drug-resistance testing could be carried out on either template. PMID:20544014

Genital Shedding of Human Immunodeficiency Virus Type 1 DNA during Pregnancy: Association with Immunosuppression, Abnormal Cervical or Vaginal Discharge, and Severe Vitamin A Deficiency

PubMed Central

John, Grace C.; Nduati, Ruth W.; Mbori-Ngacha, Dorothy; Overbaugh, Julie; Welch, Mary; Richardson, Barbra A.; Ndinya-Achola, Jeckoniah; Bwayo, Job; Krieger, John; Onyango, Francis; Kreiss, Joan K.

2012-01-01

The presence of human immunodeficiency virus type 1 (HIV-1) in genital secretions may be a determinant of vertical HIV-1 transmission. Cervical and vaginal secretions from HIV-1–seropositive pregnant women were evaluated to determine prevalence and correlates of HIV-1–infected cells in the genital tract. HIV-1 DNA was detected by polymerase chain reaction in 32% of 212 cervical and 10% of 215 vaginal specimens. Presence of HIV-1 DNA in the cervix was associated with cervical mucopus and a significantly lower absolute CD4 cell count (354 vs. 469, P < .001). An absolute CD4 cell count <200 was associated with a 9.6-fold increased odds of cervical HIV-1 DNA detection compared with a count ≥500 (95% confidence interval, 2.8–34.2). Detection of vaginal HIV-1 DNA was associated with abnormal vaginal discharge, lower absolute CD4 cell count, and severe vitamin A deficiency. Presence of HIV-1–infected cells in genital secretions was associated with immunosuppression and abnormal cervical or vaginal discharge. PMID:8985196

USF-related transcription factor, HIV-TF1, stimulates transcription of human immunodeficiency virus-1.

PubMed

Maekawa, T; Sudo, T; Kurimoto, M; Ishii, S

1991-09-11

The transcription factor HIV-TF1, which binds to a region about 60 bp upstream from the enhancer of the human immunodeficiency virus-1 (HIV-1), was purified from human B cells. HIV-TF1 had a molecular weight of 39,000. Binding of HIV-TF1 to the HIV long terminal repeat (LTR) activated transcription from the HIV promoter in vitro. The HIV-TF1-binding site in HIV LTR was similar to the site recognized by upstream stimulatory factor (USF) in the adenovirus major late promoter. DNA-binding properties of HIV-TF1 suggested that HIV-TF1 might be identical or related to USF. Interestingly, treatment of purified HIV-TF1 by phosphatase greatly reduced its DNA-binding activity, suggesting that phosphorylation of HIV-TF1 was essential for DNA binding. The disruption of HIV-TF1-binding site induced a 60% decrease in the level of transcription from the HIV promoter in vivo. These results suggest that HIV-TF1 is involved in transcriptional regulation of HIV-1.

Cross-subtype Detection of HIV-1 Using Reverse Transcription and Recombinase Polymerase Amplification

PubMed Central

Lillis, Lorraine; Lehman, Dara A.; Siverson, Joshua B.; Weis, Julie; Cantera, Jason; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie; Boyle, David S.

2016-01-01

A low complexity diagnostic test that rapidly and reliably detects HIV infection in infants at the point of care could facilitate early treatment, improving outcomes. However, many infant HIV diagnostics can only be performed in laboratory settings. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that can rapidly amplify proviral DNA from multiple subtypes of HIV-1 in under twenty minutes without complex equipment. In this study we added reverse transcription (RT) to RPA to allow detection of both HIV-1 RNA and DNA. We show that this RT-RPA HIV-1 assay has a limit of detection of 10 to 30 copies of an exact sequence matched DNA or RNA, respectively. In addition, at 100 copies of RNA or DNA, the assay detected 171 of 175 (97.7 %) sequence variants that represent all the major subtypes and recombinant forms of HIV-1 Groups M and O. This data suggests that the application of RT-RPA for the combined detection of HIV-1 viral RNA and proviral DNA may prove a highly sensitive tool for rapid and accurate diagnosis of infant HIV. PMID:26821087

Dynamics of cellular HIV-1 DNA levels over 144 weeks of darunavir/ritonavir monotherapy versus triple therapy in the MONET trial.

PubMed

Geretti, Anna Maria; Arribas, Jose R; Lathouwers, Erkki; Foster, Geraldine M; Yakoob, Rabia; Kinloch, Sabine; Hill, Andrew; van Delft, Yvon; Moecklinghoff, Christiane

2013-01-01

In patients receiving combination antiretroviral therapy (ART), switching to monotherapy with ritonavir-boosted darunavir (DRV/r) can maintain plasma HIV-1 RNA suppression with no treatment-emergent drug resistance; effects on cellular HIV-1 DNA burden are less well characterized. In MONET, patients on stable combination ART for at least 6 months with plasma HIV-1 RNA <50 copies/mL and no history of virologic failure switched to DRV/r 800/100 mg once daily, either alone (n = 127) or with 2 nucleos(t)ide reverse transcriptase inhibitors (NRTIs) (n = 129). In a representative subset of 146 patients, total HIV-1 DNA load in peripheral blood mononuclear cells (PBMC) was tested retrospectively at baseline, week 48, week 96, and week 144. Mean HIV-1 DNA levels at baseline vs week 144 were 2.50 vs 2.49 log10 copies/106 PBMC in the monotherapy arm and 2.59 vs 2.61 log10 copies/106 PBMC in the triple therapy arm, with mean (median) changes of -0.05 (-0.03) and +0.03 (+0.01) log10 copies/106 PBMC in the 2 arms, respectively. Overall baseline HIV-1 DNA levels were higher in patients with nadir CD4 counts <200 cell/µL (P<.05) and in patients who over 144 weeks experienced at least 1 HIV-1 RNA measurement >50 copies/mL (P < .05). In this substudy of the MONET trial, HIV-1 DNA levels remained stable during 144 weeks of either DRV/r monotherapy or triple therapy with DRV/r + 2 NRTIs. In both treatment arms, baseline HIV-1 DNA levels were predicted by the nadir CD4 cell count and predictive of plasma HIV-1 RNA detection during follow-up.

Clinical comparison of branched DNA and reverse transcriptase-PCR and nucleic acid sequence-based amplification assay for the quantitation of circulating recombinant form_BC HIV-1 RNA in plasma.

PubMed

Pan, Pinliang; Tao, Xiaoxia; Zhang, Qi; Xing, Wenge; Sun, Xianguang; Pei, Lijian; Jiang, Yan

2007-12-01

To investigate the correlation between three viral load assays for circulating recombinant form (CRF)_BC. Recent studies in HIV-1 molecular epidemiology, reveals that CRF_BC is the dominant subtype of HIV-1 virus in mainland China, representing over 45% of the HIV-1 infected population. The performances of nucleic acid sequence-based amplification (NASBA), branched DNA (bDNA) and reverse transcriptase polymerase chain reaction (RT-PCR) were compared for the HIV-1 viral load detection and quantitation of CRF_BC in China. Sixteen HIV-1 positive and three HIV-1 negative samples were collected. Sequencing of the positive samples in the gp41 region was conducted. The HIV-1 viral load values were determined using bDNA, RT-PCR and NASBA assays. Deming regression analysis with SPSS 12.0 (SPS Inc., Chicago, Illinois, USA) was performed for data analysis. Sequencing and phylogenetic analysis of env gene (gp41) region of the 16 HIV-1 positive clinical specimens from Guizhou Province in southwest China revealed the dominance of the subtype CRF_BC in that region. A good correlation of their viral load values was observed among three assays. Pearson's correlation between RT-PCR and bDNA is 0.969, Lg(VL)RT-PCR = 0.969 * Lg(VL)bDNA + 0.55; Pearson's correlation between RT-PCR and NASBA is 0.968, Lg(VL)RT-PCR = 0.968 * Lg(VL)NASBA + 0.937; Pearson's correlation between NASBA and bDNA is 0.980, Lg(VL)NASBA = 0.980 * Lg(VL)bDNA - 0.318. When testing with 3 different assays, RT-PCR, bDNA and NASBA, the group of 16 HIV-1 positive samples showed the viral load value was highest for RT-PCR, followed by bDNA then NASBA, which is consistent with the former results in subtype B. The three viral load assays are highly correlative for CRF_BC in China.

HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial.

PubMed

Nilsson, Charlotta; Hejdeman, Bo; Godoy-Ramirez, Karina; Tecleab, Teghesti; Scarlatti, Gabriella; Bråve, Andreas; Earl, Patricia L; Stout, Richard R; Robb, Merlin L; Shattock, Robin J; Biberfeld, Gunnel; Sandström, Eric; Wahren, Britta

2015-01-01

We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers. HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA. The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients. Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use. International Standard Randomised Controlled Trial Number (ISRCTN) 60284968.

HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial

PubMed Central

Nilsson, Charlotta; Hejdeman, Bo; Godoy-Ramirez, Karina; Tecleab, Teghesti; Scarlatti, Gabriella; Bråve, Andreas; Earl, Patricia L.; Stout, Richard R.; Robb, Merlin L.; Shattock, Robin J.; Biberfeld, Gunnel; Sandström, Eric; Wahren, Britta

2015-01-01

Background We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers. Methods HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA. Results The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients. Conclusion Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use. Trial Registration International Standard Randomised Controlled Trial Number (ISRCTN) 60284968 PMID:26121679

Highly active antiretroviral therapy started during pregnancy or postpartum suppresses HIV-1 RNA, but not DNA, in breast milk.

PubMed

Shapiro, Roger L; Ndung'u, Thumbi; Lockman, Shahin; Smeaton, Laura M; Thior, Ibou; Wester, Carolyn; Stevens, Lisa; Sebetso, Gaseene; Gaseitsiwe, Simani; Peter, Trevor; Essex, Max

2005-09-01

The ability of highly active antiretroviral therapy (HAART) to reduce human immunodeficiency virus type 1 (HIV-1) RNA and DNA in breast milk has not been described. We compared breast-milk HIV-1 RNA and DNA loads of women in Botswana who received HAART (nevirapine, lamivudine, and zidovudine) and women who did not receive HAART. Women in the HAART group received treatment for a median of 98 days (range, 67-222 days) at the time of breast-milk sampling; 23 (88%) of 26 had whole breast-milk HIV-1 RNA loads <50 copies/mL, compared with 9 (36%) of 25 women who did not receive HAART (P=.0001). This finding remained significant in a multivariate logistic-regression model (P = .0006). The whole-milk HIV-1 DNA load was unaffected by HAART. Of women who received HAART, 13 (50%) of 26 had HIV-1 DNA loads <10 copies/10(6) cells, compared with 15 (65%) of 23 who did not receive HAART (P = .39). HAART suppressed cell-free HIV-1 RNA in breast milk and may therefore reduce mother-to-child transmission (MTCT) of HIV-1 via breast-feeding. However, HAART initiated during pregnancy or early after delivery had no apparent effect on cell-associated HIV-1 DNA loads in breast milk. Clinical trials to determine MTCT among breast-feeding women receiving HAART are needed.

Comparative evaluation of the QUANTIPLEX HIV-1 RNA 2.0 and 3.0 (bDNA) assays and the AMPLICOR HIV-1 MONITOR v1.5 test for the quantitation of human immunodeficiency virus type 1 RNA in plasma.

PubMed

Anastassopoulou, C G; Touloumi, G; Katsoulidou, A; Hatzitheodorou, H; Pappa, M; Paraskevis, D; Lazanas, M; Gargalianos, P; Hatzakis, A

2001-01-01

HIV-1 RNA measurements from 84 plasma specimens obtained with the QUANTIPLEX HIV-1 RNA 2.0 and 3.0 (bDNA) assays (Chiron Diagnostics, Emeryville, CA) and with the AMPLICOR HIV-1 MONITOR Test, version 1.5 with ultra-sensitive specimen preparation (Roche Diagnostic Systems, Inc., Branchburg, NJ) were compared. The absolute RNA values of tested specimens differed significantly between bDNA 2.0 and bDNA 3.0 or Monitor v1.5 measurements (Wilcoxon signed-rank test P<0.001). Results generated with bDNA 3.0 or with Monitor v1.5 were approximately twofold greater than those generated with bDNA 2.0, with smaller differences at higher HIV-1 RNA levels and greater differences at RNA levels below 1000 copies per ml. Although highly correlated (r=0.92 and 0.86, respectively), viral load data generated with bDNA 2.0 and either bDNA 3.0 or Monitor v1.5 were in poor agreement. Concordant results (difference in log(10) copies per ml <0.5) were found at frequencies of 80% for bDNA 2.0 and bDNA 3.0 and only at 58.5% for bDNA 2.0 and Monitor v1.5. In contrast, bDNA 3.0 and Monitor v1.5 measurements were highly correlated (r=0.96) and in good agreement (92.7%).

Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA.

PubMed

Herzner, Anna-Maria; Hagmann, Cristina Amparo; Goldeck, Marion; Wolter, Steven; Kübler, Kirsten; Wittmann, Sabine; Gramberg, Thomas; Andreeva, Liudmila; Hopfner, Karl-Peter; Mertens, Christina; Zillinger, Thomas; Jin, Tengchuan; Xiao, Tsan Sam; Bartok, Eva; Coch, Christoph; Ackermann, Damian; Hornung, Veit; Ludwig, Janos; Barchet, Winfried; Hartmann, Gunther; Schlee, Martin

2015-10-01

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

Cross-subtype detection of HIV-1 using reverse transcription and recombinase polymerase amplification.

PubMed

Lillis, Lorraine; Lehman, Dara A; Siverson, Joshua B; Weis, Julie; Cantera, Jason; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie; Boyle, David S

2016-04-01

A low complexity diagnostic test that rapidly and reliably detects HIV infection in infants at the point of care could facilitate early treatment, improving outcomes. However, many infant HIV diagnostics can only be performed in laboratory settings. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that can rapidly amplify proviral DNA from multiple subtypes of HIV-1 in under twenty minutes without complex equipment. In this study we added reverse transcription (RT) to RPA to allow detection of both HIV-1 RNA and DNA. We show that this RT-RPA HIV-1 assay has a limit of detection of 10-30 copies of an exact sequence matched DNA or RNA, respectively. In addition, at 100 copies of RNA or DNA, the assay detected 171 of 175 (97.7%) sequence variants that represent all the major subtypes and recombinant forms of HIV-1 Groups M and O. This data suggests that the application of RT-RPA for the combined detection of HIV-1 viral RNA and proviral DNA may prove a highly sensitive tool for rapid and accurate diagnosis of infant HIV. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of blood collection filter papers for HIV-1 DNA PCR.

PubMed

Masciotra, Silvina; Khamadi, Samoel; Bilé, Ebi; Puren, Adrian; Fonjungo, Peter; Nguyen, Shon; Girma, Mulu; Downing, Robert; Ramos, Artur; Subbarao, Shambavi; Ellenberger, Dennis

2012-10-01

The collection of dried blood spots (DBS) on Whatman 903 cards has facilitated for years the detection of HIV-1 in infants by DNA PCR as early as 4-6 weeks after birth in resource-limited settings (RLS), but alternate blood collection devices are proving to be necessary. The qualitative detection of HIV-1 DNA by PCR from DBS prepared on three commercially available blood collection cards was evaluated at the Centers for Disease Control and Prevention (CDC) and in four laboratories in Africa. DBS were prepared on Ahlstrom grade 226, Munktell TFN and Whatman 903, and stored under a variety of conditions. DBS were stored at ambient temperature (RT), 37°C with high humidity, and -20°C for varying lengths of time. The presence of HIV-1 DNA was tested using Roche Amplicor HIV-1 DNA (v 1.5) weekly for 4 weeks and at weeks 8 and 12 (RT and 37°C), at weeks 4, 8, and 18 (-20°C) of storage. DBS specimens were also tested after international shipment at RT. In addition, after nearly 3 years storage at -20°C, DBS were also evaluated independently using the COBAS Ampliprep/TaqMan HIV-1 Qual and Abbott RealTime HIV-1 Qualitative tests. HIV-1 DNA was detected equally well on the three blood collection cards regardless of storage conditions and PCR assay. Ahlstrom 226 and Munktell TFN papers were comparable to Whatman 903 for HIV-1 DNA detection and may be considered as optional blood collection devices in resource-limited countries. Published by Elsevier B.V.

Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

PubMed

Zhang, Shi-Meng; Zhang, He; Yang, Tian-Yi; Ying, Tian-Yi; Yang, Pei-Xiang; Liu, Xiao-Dan; Tang, Sheng-Jian; Zhou, Ping-Kun

2014-01-01

HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

Priming with a simplified intradermal HIV-1 DNA vaccine regimen followed by boosting with recombinant HIV-1 MVA vaccine is safe and immunogenic: a phase IIa randomized clinical trial.

PubMed

Munseri, Patricia J; Kroidl, Arne; Nilsson, Charlotta; Joachim, Agricola; Geldmacher, Christof; Mann, Philipp; Moshiro, Candida; Aboud, Said; Lyamuya, Eligius; Maboko, Leonard; Missanga, Marco; Kaluwa, Bahati; Mfinanga, Sayoki; Podola, Lilly; Bauer, Asli; Godoy-Ramirez, Karina; Marovich, Mary; Moss, Bernard; Hoelscher, Michael; Gotch, Frances; Stöhr, Wolfgang; Stout, Richard; McCormack, Sheena; Wahren, Britta; Mhalu, Fred; Robb, Merlin L; Biberfeld, Gunnel; Sandström, Eric; Bakari, Muhammad

2015-01-01

Intradermal priming with HIV-1 DNA plasmids followed by HIV-1MVA boosting induces strong and broad cellular and humoral immune responses. In our previous HIVIS-03 trial, we used 5 injections with 2 pools of HIV-DNA at separate sites for each priming immunization. The present study explores whether HIV-DNA priming can be simplified by reducing the number of DNA injections and administration of combined versus separated plasmid pools. In this phase IIa, randomized trial, priming was performed using 5 injections of HIV-DNA, 1000 μg total dose, (3 Env and 2 Gag encoding plasmids) compared to two "simplified" regimens of 2 injections of HIV-DNA, 600 μg total dose, of Env- and Gag-encoding plasmid pools with each pool either administered separately or combined. HIV-DNA immunizations were given intradermally at weeks 0, 4, and 12. Boosting was performed intramuscularly with 108 pfu HIV-MVA at weeks 30 and 46. 129 healthy Tanzanian participants were enrolled. There were no differences in adverse events between the groups. The proportion of IFN-γ ELISpot responders to Gag and/or Env peptides after the second HIV-MVA boost did not differ significantly between the groups primed with 2 injections of combined HIV-DNA pools, 2 injections with separated pools, and 5 injections with separated pools (90%, 97% and 97%). There were no significant differences in the magnitude of Gag and/or Env IFN-γ ELISpot responses, in CD4+ and CD8+ T cell responses measured as IFN-γ/IL-2 production by intracellular cytokine staining (ICS) or in response rates and median titers for binding antibodies to Env gp160 between study groups. A simplified intradermal vaccination regimen with 2 injections of a total of 600 μg with combined HIV-DNA plasmids primed cellular responses as efficiently as the standard regimen of 5 injections of a total of 1000 μg with separated plasmid pools after boosting twice with HIV-MVA. World Health Organization International Clinical Trials Registry Platform PACTR2010050002122368.

A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load.

PubMed

Malnati, Mauro S; Scarlatti, Gabriella; Gatto, Francesca; Salvatori, Francesca; Cassina, Giulia; Rutigliano, Teresa; Volpi, Rosy; Lusso, Paolo

2008-01-01

Quantification of human immunodeficiency virus type-1 (HIV-1) proviral DNA is increasingly used to measure the HIV-1 cellular reservoirs, a helpful marker to evaluate the efficacy of antiretroviral therapeutic regimens in HIV-1-infected individuals. Furthermore, the proviral DNA load represents a specific marker for the early diagnosis of perinatal HIV-1 infection and might be predictive of HIV-1 disease progression independently of plasma HIV-1 RNA levels and CD4(+) T-cell counts. The high degree of genetic variability of HIV-1 poses a serious challenge for the design of a universal quantitative assay capable of detecting all the genetic subtypes within the main (M) HIV-1 group with similar efficiency. Here, we describe a highly sensitive real-time PCR protocol that allows for the correct quantification of virtually all group-M HIV-1 strains with a higher degree of accuracy compared with other methods. The protocol involves three stages, namely DNA extraction/lysis, cellular DNA quantification and HIV-1 proviral load assessment. Owing to the robustness of the PCR design, this assay can be performed on crude cellular extracts, and therefore it may be suitable for the routine analysis of clinical samples even in developing countries. An accurate quantification of the HIV-1 proviral load can be achieved within 1 d from blood withdrawal.

Simultaneous Runs of the Bayer VERSANT HIV-1 Version 3.0 and HCV bDNA Version 3.0 Quantitative Assays on the System 340 Platform Provide Reliable Quantitation and Improved Work Flow

PubMed Central

Elbeik, Tarek; Markowitz, Norman; Nassos, Patricia; Kumar, Uday; Beringer, Scott; Haller, Barbara; Ng, Valerie

2004-01-01

Branched DNA (bDNA) assays to quantify human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of three distinct steps, including sample processing, hybridization, and detection, and utilize the System 340 platform for plate incubation and washing. Sample processing differs: HIV-1 from 1 ml of plasma is concentrated by high-speed centrifugation, whereas HCV plasma or serum samples are used without concentration. The first step of hybridization involves viral lysis at 63°C: HIV-1 is performed in a heat block, whereas HCV is performed in System 340. The remaining hybridization and detection steps are similar for HIV-1 and HCV and executed on System 340. In the present study, the HIV-1 bDNA assay was adapted for viral lysis in the System 340 platform. The adaptation, test method 2, includes a 20-s vortex of concentrated viral pellet and lysis working solution, transfer of viral lysate to the 96-well capture plate, and transfer to System 340 programmed for HCV assay specifications. With test method 2, specificity and quantification were within assay specifications. HCV bDNA methodology remains unchanged. Hence, an HIV-1 and an HCV bDNA can be run simultaneously on System 340. With simultaneous testing, laboratories can run full plates, as well as combinations of full and partial plates. Also, simultaneous HIV-1 and HCV bDNA permits labor consolidation and improved workflow while maintaining multitasking and rapid patient result turnaround. PMID:15243070

Simultaneous runs of the Bayer VERSANT HIV-1 version 3.0 and HCV bDNA version 3.0 quantitative assays on the system 340 platform provide reliable quantitation and improved work flow.

PubMed

Elbeik, Tarek; Markowitz, Norman; Nassos, Patricia; Kumar, Uday; Beringer, Scott; Haller, Barbara; Ng, Valerie

2004-07-01

Branched DNA (bDNA) assays to quantify human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of three distinct steps, including sample processing, hybridization, and detection, and utilize the System 340 platform for plate incubation and washing. Sample processing differs: HIV-1 from 1 ml of plasma is concentrated by high-speed centrifugation, whereas HCV plasma or serum samples are used without concentration. The first step of hybridization involves viral lysis at 63 degrees C: HIV-1 is performed in a heat block, whereas HCV is performed in System 340. The remaining hybridization and detection steps are similar for HIV-1 and HCV and executed on System 340. In the present study, the HIV-1 bDNA assay was adapted for viral lysis in the System 340 platform. The adaptation, test method 2, includes a 20-s vortex of concentrated viral pellet and lysis working solution, transfer of viral lysate to the 96-well capture plate, and transfer to System 340 programmed for HCV assay specifications. With test method 2, specificity and quantification were within assay specifications. HCV bDNA methodology remains unchanged. Hence, an HIV-1 and an HCV bDNA can be run simultaneously on System 340. With simultaneous testing, laboratories can run full plates, as well as combinations of full and partial plates. Also, simultaneous HIV-1 and HCV bDNA permits labor consolidation and improved workflow while maintaining multitasking and rapid patient result turnaround.

Broad and potent immune responses to a low dose intradermal HIV-1 DNA boosted with HIV-1 recombinant MVA among healthy adults in Tanzania☆,☆☆

PubMed Central

Bakari, Muhammad; Aboud, Said; Nilsson, Charlotta; Francis, Joel; Buma, Deus; Moshiro, Candida; Aris, Eric A.; Lyamuya, Eligius F.; Janabi, Mohamed; Godoy-Ramirez, Karina; Joachim, Agricola; Polonis, Victoria R.; Bråve, Andreas; Earl, Patricia; Robb, Merlin; Marovich, Mary; Wahren, Britta; Pallangyo, Kisali; Biberfeld, Gunnel; Mhalu, Fred; Sandström, Eric

2016-01-01

Background We conducted a phase I/II randomized placebo-controlled trial with the aim of exploring whether priming with a low intradermal dose of a multiclade, multigene HIV-1 DNA vaccine could improve the immunogenicity of the same vaccine given intramuscularly prior to boosting with a heterologous HIV-1 MVA among healthy adults in Dar es Salaam, Tanzania. Methods Sixty HIV-uninfected volunteers were randomized to receive DNA plasmid vaccine 1 mg intradermally (id), n = 20, or 3.8 mg intramuscularly (im), n = 20, or placebo, n = 20, using a needle-free injection device. DNA plasmids encoding HIV-1 genes gp160 subtype A, B, C; rev B; p17/p24 gag A, B and Rtmut B were given at weeks 0, 4 and 12. Recombinant MVA (108 pfu) expressing HIV-1 Env, Gag, Pol of CRF01_AE or placebo was administered im at month 9 and 21. Results The vaccines were well tolerated. Two weeks after the third HIV-DNA injection, 22/38 (58%) vaccinees had IFN-γ ELISpot responses to Gag. Two weeks after the first HIV-MVA boost all 35 (100%) vaccinees responded to Gag and 31 (89%) to Env. Two to four weeks after the second HIV-MVA boost, 28/29 (97%) vaccinees had IFN-γ ELISpot responses, 27 (93%) to Gag and 23 (79%) to Env. The id-primed recipients had significantly higher responses to Env than im recipients. Intracellular cytokine staining for Gag-specific IFN-γ/IL-2 production showed both CD8+ and CD4+ T cell responses. All vaccinees had HIV-specific lymphoproliferative responses. All vaccinees reacted in diagnostic HIV serological tests and 26/29 (90%) had antibodies against gp160 after the second HIV-MVA boost. Furthermore, while all of 29 vaccinee sera were negative for neutralizing antibodies against clade B, C and CRF01 AE pseudoviruses in the TZM-bl neutralization assay, in a PBMC assay, the response rate ranged from 31% to 83% positives, depending upon the clade B or CRF01_AE virus tested. This vaccine approach is safe and highly immunogenic. Low dose, id HIV-DNA priming elicited higher and broader cell-mediated immune responses to Env after HIV-MVA boost compared to a higher HIV-DNA priming dose given im. Three HIV-DNA priming immunizations followed by two HIV-MVA boosts efficiently induced Env-antibody responses. PMID:21864626

Integrated HIV DNA accumulates prior to treatment while episomal HIV DNA records ongoing transmission afterwards.

PubMed

Murray, John M; McBride, Kristin; Boesecke, Christoph; Bailey, Michelle; Amin, Janaki; Suzuki, Kazuo; Baker, David; Zaunders, John J; Emery, Sean; Cooper, David A; Koelsch, Kersten K; Kelleher, Anthony D

2012-03-13

We investigated the dynamics of HIV RNA and HIV DNA levels after the commencement of raltegravir-based antiretroviral therapy (ART) in primary (PHI) and chronically HIV-infected (CHI) individuals (the PINT study). We recruited 8 PHI and 8 CHI ART-naive individuals who commenced a 1-year combination regimen of Truvada and the integrase inhibitor raltegravir. Nonlinear mixed effects modelling was used to determine multiphasic decay of plasma HIV RNA levels (pVL), as well as dynamics of total, episomal [2-long terminal repeats (LTR)] and integrated HIV DNA in CD4 T cells from peripheral blood. Although pVL decreased faster through first and second phase for PHI individuals there was no difference in the final level reaching a mean of 9 copies/ml by week 16 that was maintained thereafter. Total HIV DNA and integrated HIV DNA levels from CHI patients were significantly higher than from PHI patients. However, at no time did 2-LTR levels differ between groups. Of note, 2-LTR circles exhibited an initial increase peaking at week 3 followed by biphasic decay with a half-life of 29 days. Second phase integrated HIV DNA levels were significantly correlated with duration of infection and consistent with this form of infection occurring at approximately 100 000 integration events per day in the absence of ART, achieving its 50% level 2 years after infection. Integrated HIV DNA levels accumulate with duration of untreated HIV infection. The relatively short half-life and high levels of 2-LTR circles after 1 year support continued HIV transmission during ART.

Comparative DNA Methylation Profiling Reveals an Immunoepigenetic Signature of HIV-related Cognitive Impairment

PubMed Central

Corley, Michael J.; Dye, Christian; D’Antoni, Michelle L.; Byron, Mary Margaret; Yo, Kaahukane Leite-Ah; Lum-Jones, Annette; Nakamoto, Beau; Valcour, Victor; SahBandar, Ivo; Shikuma, Cecilia M.; Ndhlovu, Lishomwa C.; Maunakea, Alika K.

2016-01-01

Monocytes/macrophages contribute to the neuropathogenesis of HIV-related cognitive impairment (CI); however, considerable gaps in our understanding of the precise mechanisms driving this relationship remain. Furthermore, whether a distinct biological profile associated with HIV-related CI resides in immune cell populations remains unknown. Here, we profiled DNA methylomes and transcriptomes of monocytes derived from HIV-infected individuals with and without CI using genome-wide DNA methylation and gene expression profiling. We identified 1,032 CI-associated differentially methylated loci in monocytes. These loci related to gene networks linked to the central nervous system (CNS) and interactions with HIV. Most (70.6%) of these loci exhibited higher DNA methylation states in the CI group and were preferentially distributed over gene bodies and intergenic regions of the genome. CI-associated DNA methylation states at 12 CpG sites associated with neuropsychological testing performance scores. CI-associated DNA methylation also associated with gene expression differences including CNS genes CSRNP1 (P = 0.017), DISC1 (P = 0.012), and NR4A2 (P = 0.005); and a gene known to relate to HIV viremia, THBS1 (P = 0.003). This discovery cohort data unveils cell type-specific DNA methylation patterns related to HIV-associated CI and provide an immunoepigenetic DNA methylation “signature” potentially useful for corroborating clinical assessments, informing pathogenic mechanisms, and revealing new therapeutic targets against CI. PMID:27629381

A Multiplex PCR Approach for Detecting Dual Infections and Recombinants Involving Major HIV Variants

PubMed Central

Cappy, Pierre; De Oliveira, Fabienne; Gueudin, Marie; Alessandri-Gradt, Elodie

2016-01-01

The cocirculation of different HIV types and groups can lead to dual infections and recombinants, which hinder diagnosis and therapeutic management. We designed two multiplex PCRs (mPCRs) coupled with capillary electrophoresis to facilitate the detection of such infections. The first, MMO2, targets three variants (HIV-1/M, HIV-1/O, and HIV-2), and the second, MMO, targets HIV-1/M and HIV-1/O. These mPCRs were validated on DNA and RNA extracts from 19 HIV-1/M, 12 HIV-1/O, and 13 HIV-2 cultures and from mixtures simulating dual infections. They were then assessed with DNA and RNA extracts from samples of 47 clinical monoinfections and HIV-1/M+O dual infections or infections with HIV-1/MO recombinants. Both mPCRs had excellent specificity. Sensitivities ranged from 80 to 100% for in vitro samples and from 58 to 100% for clinical samples, with the results obtained depending on the material used and the region of the genome concerned. Sensitivity was generally lower for DNA than for RNA and for amplifications of the integrase and matrix regions. In terms of global detection (at least one target gene for each strain), both mPCRs yielded a detection rate of 100% for in vitro samples. MMO2 detected 100% of the clinical strains from DNA and 97% from RNA, whereas MMO detected 100% of the strains from both materials. Thus, for in vitro and clinical samples, MMO2 was a useful tool for detecting dual infections with HIV-1 and HIV-2 (referred to as HIV-1+HIV-2) and HIV-1/M+O, and MMO was useful for detecting both MO dual infections and MO mosaic patterns. PMID:26912747

Comparison of the Gen-Probe Aptima HIV-1 and Abbott HIV-1 qualitative assays with the Roche Amplicor HIV-1 DNA assay for early infant diagnosis using dried blood spots.

PubMed

Nelson, Julie A E; Hawkins, J Tyler; Schanz, Maria; Mollan, Katie; Miller, Melissa B; Schmitz, John L; Fiscus, Susan A

2014-08-01

The current gold standard for infant diagnosis of HIV-1 is the Roche Amplicor Qualitative DNA assay, but it is being phased out. Compare the Abbott qualitative assay and the Gen-Probe Aptima assay to the gold standard Roche DNA assay using dried blood spots (DBS). The Gen-Probe Aptima and Abbott qualitative HIV-1 assays were compared to the Roche DNA assay for early infant diagnosis. Specificity and sensitivity were determined for the three assays using DBS from 50 HIV-exposed uninfected infants and 269 HIV-1 infected adults from North Carolina, respectively. All of the negative and 151 of the positive DBS had valid results on the 3 different assays, and an additional 118 positive DBS had valid results on the Roche DNA and Aptima assays. All three assays were very specific. The Roche DNA assay was the most sensitive (96.7%) over a wide range of HIV PVL, including samples with PVL<400 copies/ml. Restricted to samples with PVL>400 copies/ml, the Gen-Probe Aptima assay had sensitivity (96.5%) comparable to the Roche DNA assay (98.8%). The Abbott Qualitative assay was the least sensitive and only had sensitivity above 95% among samples with PVL over 1000 copies/ml. The Abbott HIV-1 Qualitative assay was not as sensitive as the comparator assays, so it would not be a useful replacement assay, especially for infants taking antiretroviral prophylaxis. The Gen-Probe Aptima assay is an adequate replacement option for infant diagnosis using DBS. Copyright © 2014 Elsevier B.V. All rights reserved.

HIV DNA in CD14+ reservoirs is associated with regional brain atrophy in patients naive to combination antiretroviral therapy.

PubMed

Kallianpur, Kalpana J; Valcour, Victor G; Lerdlum, Sukalaya; Busovaca, Edgar; Agsalda, Melissa; Sithinamsuwan, Pasiri; Chalermchai, Thep; Fletcher, James L K; Tipsuk, Somporn; Shikuma, Cecilia M; Shiramizu, Bruce T; Ananworanich, Jintanat

2014-07-17

To examine associations between regional brain volumes and HIV DNA in peripheral CD14 cells (monocytes) among HIV-infected individuals naive to combination antiretroviral therapy (cART). A prospective study of HIV-infected Thai individuals who met Thai national criteria for cART initiation. Enrolment was stratified by HIV DNA in a blinded fashion. CD14 cells were isolated from peripheral mononuclear cells to high purity (median 91.4% monocytes by flow cytometry), and HIV DNA was quantified by multiplex real-time PCR. Baseline regional brain volumes obtained by T1-weighted 1.5-Tesla MRI were compared between HIV DNA groups using analysis of covariance (ANCOVA). We studied 60 individuals with mean (SD) age of 34.7 (7.0) years, CD4 T-lymphocyte count of 232 (137) cells/μl and log10 plasma HIV RNA of 4.8 (0.73). Median (interquartile range, IQR) HIV DNA copy number per 10 CD14 cells was 54 (102). Using our previously determined optimal cut-point of 45 copies/10 cells for this cohort, a threshold value above which CD14 HIV DNA identified HIV-associated neurocognitive disorders (HANDs), we found that CD14 HIV DNA  ≥ 45 copies/10 cells was associated with reduced volumes of the nucleus accumbens (P=0.021), brainstem (P=0.033) and total gray matter (P=0.045) independently of age, CD4 cell count and intracranial volume. HIV DNA burden in CD14 monocytes is directly linked to brain volumetric loss. Our findings implicate peripheral viral reservoirs in HIV-associated brain atrophy and support their involvement in the neuropathogenesis of HAND, underscoring the need for therapies that target these cells.

Epstein-Barr virus DNA loads in adult human immunodeficiency virus type 1-infected patients receiving highly active antiretroviral therapy

NASA Technical Reports Server (NTRS)

Ling, Paul D.; Vilchez, Regis A.; Keitel, Wendy A.; Poston, David G.; Peng, Rong Sheng; White, Zoe S.; Visnegarwala, Fehmida; Lewis, Dorothy E.; Butel, Janet S.

2003-01-01

Patients with human immunodeficiency virus type 1 (HIV-1) infection are at high risk of developing Epstein-Barr virus (EBV)-associated lymphoma. However, little is known of the EBV DNA loads in patients receiving highly active antiretroviral therapy (HAART). Using a real-time quantitative polymerase chain reaction assay, we demonstrated that significantly more HIV-1-infected patients receiving HAART than HIV-1-uninfected volunteers had detectable EBV DNA in blood (57 [81%] of 70 vs. 11 [16%] of 68 patients; P=.001) and saliva (55 [79%] of 68 vs. 37 [54%] of 68 patients; P=.002). The mean EBV loads in blood and saliva samples were also higher in HIV-1-infected patients than in HIV-1-uninfected volunteers (P=.001). The frequency of EBV detection in blood was associated with lower CD4+ cell counts (P=.03) among HIV-1-infected individuals, although no differences were observed in the EBV DNA loads in blood or saliva samples in the HIV-1-infected group. Additional studies are needed to determine whether EBV-specific CD4+ and CD8+ cells play a role in the pathogenesis of EBV in HIV-1-infected patients receiving HAART.

Impact of HIV type 1 subtype variation on viral RNA quantitation.

PubMed

Parekh, B; Phillips, S; Granade, T C; Baggs, J; Hu, D J; Respess, R

1999-01-20

We evaluated the performance of three HIV-1 RNA quantitation methods (Amplicor HIV-1 MONITOR-1.0, NASBA, and Quantiplex HIV RNA 2.0 [branched DNA (bDNA)]) using plasma specimens (N = 60) from individuals from Asia and Africa infected with one of three HIV-1 subtypes (A, Thai B [B'] or E; N = 20 each). Our results demonstrate that of the 20 subtype A specimens, 19 were quantifiable by the bDNA assay compared with 15 by the MONITOR-1.0 and 13 by NASBA. Of those quantifiable, the mean log10 difference was 0.93 between bDNA and MONITOR-1.0 and 0.46 between bDNA and NASBA. For subtype B' specimens, the correlation among methods was better with only 2 specimens missed by NASBA and 3 by the bDNA assay. However the missed specimens had viral burden near the lower limit (1000 copies/ml) for these assays. For the 20 subtype E specimens, MONITOR-1.0 and NASBA quantified RNA in 17 and 14 specimens, respectively, as compared with 19 specimens quantified by the bDNA assay. The correlation among different assays, especially between bDNA/NASBA and MONITOR-1.0/NASBA, was poor, although the mean log10 difference for subtype E specimens was 0.4 between bDNA and MONITOR-1.0 and only 0.08 between bDNA and NASBA. The addition of a new primer set, designed for non-B HIV-1 subtypes, to the existing MONITOR assay (MONITOR-1.0+) resulted in RNA detection in all 60 specimens and significantly improved the efficiency of quantitation for subtypes A and E. Our data indicate that HIV-1 subtype variation can have a major influence on viral load quantitation by different methods. Periodic evaluation and modification of these quantitative methods may be necessary to ensure reliable quantification of divergent viruses.

Relationship of HIV Reservoir Characteristics with Immune Status and Viral Rebound Kinetics in an HIV Therapeutic Vaccine Study

PubMed Central

Li, Jonathan Z.; Heisey, Andrea; Ahmed, Hayat; Wang, Hongying; Zheng, Lu; Carrington, Mary; Wrin, Terri; Schooley, Robert T.; Lederman, Michael M.; Kuritzkes, Daniel R.

2014-01-01

Objectives To evaluate the impact of therapeutic HIV vaccination on the HIV reservoir, and assess the relationship of the viral reservoir with HIV-specific immune status and viral rebound kinetics. Design Retrospective analysis of ACTG A5197, a randomized, placebo-controlled trial of a therapeutic rAd5 HIV-1 gag vaccine. Methods Participants received vaccine/placebo at weeks 0, 4, and 26 prior to a 16-week analytic treatment interruption (ATI) at week 38. Cell-associated HIV-1 RNA and DNA (CA-RNA and CA-DNA) and HIV-1 residual viremia (RV) were quantified at weeks 0, 8, and 38. HIV-specific CD4+/CD8+ activity were assessed by an intracellular cytokine staining assay. Results At study entry, CA-RNA and CA-DNA levels were correlated inversely with the numbers of HIV-specific CD4+ interferon-γ-producing cells (CA-RNA: r = −0.23, P=0.03 and CA-DNA: r = −0.28, P<0.01, N=93). Therapeutic HIV vaccination induced HIV-specific CD4+ activity, but did not significantly affect levels of CA-RNA or CA-DNA. Vaccine recipients with undetectable RV at week 8 had higher frequencies of HIV-specific CD4+ and CD8+ interferon-γ-producing cells (undetectable versus detectable RV: 277 versus 161 CD4+ cells/106 lymphocytes, P=0.03 and 1326 versus 669 CD8+ cells/106 lymphocytes, P=0.04). Pre-ATI CA-RNA and CA-DNA were associated with post-ATI plasma HIV set point (CA-RNA: r = 0.51, P<0.01 and CA-DNA: r = 0.47, P<0.01). Conclusions Vaccine-induced T-cell responses were associated with a modest transient effect on RV, but more potent immune responses and/or combination treatment with latency-reversing agents are needed to reduce the HIV reservoir. HIV reservoir measures may act as biomarkers of post-ATI viral rebound kinetics. PMID:25254301

Protocol for the use of a rapid real-time PCR method for the detection of HIV-1 proviral DNA using double-stranded primer.

PubMed

Pau, Chou-Pong; Wells, Susan K; Granade, Timothy C

2012-01-01

This chapter describes a real-time PCR method for the detection of HIV-1 proviral DNA in whole blood samples using a novel double-stranded primer system. The assay utilizes a simple commercially available DNA extraction method and a rapid and easy-to-perform real-time PCR protocol to consistently detect a minimum of four copies of HIV-1 group M proviral DNA in as little as 90 min after sample (whole blood) collection. Co-amplification of the human RNase P gene serves as an internal control to monitor the efficiency of both the DNA extraction and amplification. Once the assay is validated properly, it may be suitable as an alternative confirmation test for HIV-1 infections in a variety of HIV testing venues including the mother-to-child transmission testing sites, clinics, and diagnostic testing centers.

Mitochondrial DNA Haplogroup A Decreases the Risk of Drug Addiction but Conversely Increases the Risk of HIV-1 Infection in Chinese Addicts.

PubMed

Zhang, A-Mei; Hu, Qiu-Xiang; Liu, Feng-Liang; Bi, Rui; Yang, Bi-Qing; Zhang, Wen; Guo, Hao; Logan, Ian; Zheng, Yong-Tang; Yao, Yong-Gang

2016-08-01

Drug addiction is one of the most serious social problems in the world today and addicts are always at a high risk of acquiring HIV infection. Mitochondrial impairment has been reported in both drug addicts and in HIV patients undergoing treatment. In this study, we aimed to investigate whether mitochondrial DNA (mtDNA) haplogroup could affect the risk of drug addiction and HIV-1 infection in Chinese. We analyzed mtDNA sequence variations of 577 Chinese intravenous drug addicts (289 with HIV-1 infection and 288 without) and compared with 2 control populations (n = 362 and n = 850). We quantified the viral load in HIV-1-infected patients with and without haplogroup A status and investigated the potential effect of haplogroup A defining variants m.4824A > G and m.8794C > T on the cellular reactive oxygen species (ROS) levels by using an allotopic expression assay. mtDNA haplogroup A had a protective effect against drug addiction but appeared to confer an increased risk of HIV infection in addicts. HIV-1-infected addicts with haplogroup A had a trend for a higher viral load, although the mean viral load was similar between carriers of haplogroup A and those with other haplogroup. Hela cells overexpressing allele m.8794 T showed significantly decreased ROS levels as compared to cells with the allele m.8794C (P = 0.03). Our results suggested that mtDNA haplogroup A might protect against drug addiction but increase the risk of HIV-1 infection. The contradictory role of haplogroup A might be caused by an alteration in mitochondrial function due to a particular mtDNA ancestral variant.

APOBEC3G inhibits HIV-1 RNA elongation by inactivating the viral trans-activation response element.

PubMed

Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

2014-07-29

Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription. Copyright © 2014 Elsevier Ltd. All rights reserved.

Scaffold attachment factor B suppresses HIV-1 infection of CD4+ cells by preventing binding of RNA polymerase II to HIV-1's long terminal repeat.

PubMed

Ma, Li; Sun, Li; Jin, Xia; Xiong, Si-Dong; Wang, Jian-Hua

2018-06-10

The 5' end of HIV-1 long terminal repeat (LTR) promoter plays an essential role in driving viral transcription and productive infection. Multiple host and viral factors regulate LTR activity and modulate HIV-1 latency. Manipulation of the HIV-1 LTR provides a potential therapeutic strategy for combating HIV-1 persistence. In this study, we identified an RNA-/DNA-binding protein, Scaffold Attachment Factor B (SAFB1) as a host-cell factor that represses HIV-1 transcription. We found that SAFB1 bound to HIV-1 5`-LTR and significantly repressed 5`-LTR-driven-viral transcription and HIV-1 infection of CD4 + T cells. Mechanistically, SAFB1-mediated repression of HIV-1 transcription and infection was independent of its RNA- and DNA-binding capacities, instead, by binding to phosphorylated RNA polymerase II (RNA pol II), SAFB1 blocked its recruitment to the HIV-1 LTR. Of note, the SAFB1-mediated repression of HIV-1 transcription from proviral DNA maintained HIV-1 latency in CD4 + T cells. In summary, our findings reveal that SAFB1 binds to HIV-1-LTR and physically interacts with phosphorylated RNA pol II, repressing HIV-1 transcription initiation and elongation. Our findings improve the understanding of host modulation of HIV-1 transcription and latency and provide a new host-cell target for improved anti-HIV-1 therapies. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Performance of NucliSens HIV-1 EasyQ Version 2.0 compared with six commercially available quantitative nucleic acid assays for detection of HIV-1 in China.

PubMed

Xu, Sihong; Song, Aijing; Nie, Jianhui; Li, Xiuhua; Wang, Youchun

2010-10-01

Six HIV-1 viral load assays have been widely used in China. These include the Cobas Amplicor HIV-1 Monitor Version 1.5 ('Amplicor'), Cobas AmpliPrep/Cobas TaqMan HIV-1 test Version 1.0 ('CAP/CTM'), Versant HIV-1 RNA Version 3.0 (branched DNA [bDNA]-based assay; 'Versant bDNA'), Abbott RealTime HIV-1 assay ('Abbott RealTime'), NucliSens HIV-1 QT (nucleic acid sequence-based amplification assay; 'NucliSens NASBA'), and NucliSens EasyQ HIV-1 Version 1.1 ('EasyQ V1.1'). Recently, an updated version of EasyQ V1.1, NucliSens EasyQ HIV-1 Version 2.0 ('EasyQ V2.0') was introduced into China. It is important to evaluate the impact of HIV-1 genotypes on the updated assay compared with the other commercial available assays in China. A total of 175 plasma samples with different HIV-1 clades prevalent in China were collected from treatment-naïve patients. The viral loads of those samples were determined with the seven HIV-1 viral load assays, and the quantitative differences between them were evaluated. Overall, EasyQ V2.0 exhibited a significant correlation (R = 0.769-0.850, p ≤ 0.001) and high agreement (94.77-97.13%, using the Bland-Altman model) with the other six assays. Although no significant differences between EasyQ V2.0 and the other six assays were observed when quantifying clade B' samples, there were statistically significant differences between EasyQ V2.0 and the Amplicor, Versant bDNA, and Abbott RealTime assays when quantifying clade BC samples, and between EasyQ V2.0 and the Versant bDNA and Abbott RealTime assays when quantifying clade AE samples. For clade BC samples, the quantitative differences between EasyQ V2.0 and the Amplicor, Versant bDNA, and Abbott RealTime assays exceeded 0.5 log(10) IU/mL in approximately 50% of samples and exceeded 1 log(10) IU/mL in approximately 15% of samples. For clade AE samples, the quantitative differences between EasyQ V2.0 and the CAP/CTM, Versant bDNA, and Abbott RealTime assays exceeded 0.5 log(10) IU/mL in approximately 50% of samples, and the differences between EasyQ V2.0 and CAP/CTM exceeded 1 log(10) IU/mL in approximately 15% of samples. Genotypes may affect the quantification of HIV-1 RNA, especially in clade BC samples with respect to EasyQ V2.0 and the Amplicor, Versant bDNA, or Abbott RealTime assays, and in clade AE samples with respect to EasyQ V2.0 and the Versant bDNA or Abbott RealTime assays. It is therefore strongly suggested that, where possible, the HIV-1 viral load in infected patients be quantified at follow-up by the same version of the same assay that was used initially.

Peripheral Blood Mononuclear Cells HIV DNA Levels Impact Intermittently on Neurocognition

PubMed Central

Cysique, Lucette A.; Hey-Cunningham, William J.; Dermody, Nadene; Chan, Phillip; Brew, Bruce J.; Koelsch, Kersten K.

2015-01-01

Objectives To determine the contribution of peripheral blood mononuclear cells’ (PBMCs) HIV DNA levels to HIV-associated dementia (HAD) and non-demented HIV-associated neurocognitive disorders (HAND) in chronically HIV-infected adults with long-term viral suppression on combined antiretroviral treatment (cART). Methods Eighty adults with chronic HIV infection on cART (>97% with plasma and CSF HIV RNA <50 copies/mL) were enrolled into a prospective observational cohort and underwent assessments of neurocognition and pre-morbid cognitive ability at two visits 18 months apart. HIV DNA in PBMCs was measured by real-time PCR at the same time-points. Results At baseline, 46% had non-demented HAND; 7.5% had HAD. Neurocognitive decline occurred in 14% and was more likely in those with HAD (p<.03). Low pre-morbid cognitive ability was uniquely associated with HAD (p<.05). Log10 HIV DNA copies were stable between study visits (2.26 vs. 2.22 per 106 PBMC). Baseline HIV DNA levels were higher in those with lower pre-morbid cognitive ability (p<.04), and higher in those with no ART treatment during HIV infection 1st year (p = .03). Baseline HIV DNA was not associated with overall neurocognition. However, % ln HIV DNA change was associated with decline in semantic fluency in unadjusted and adjusted analyses (p = .01-.03), and motor-coordination (p = .02-.12) to a lesser extent. Conclusions PBMC HIV DNA plays a role in HAD pathogenesis, and this is moderated by pre-morbid cognitive ability in the context of long-term viral suppression. While the HIV DNA levels in PBMC are not associated with current non-demented HAND, increasing HIV DNA levels were associated with a decline in neurocognitive functions associated with HAND progression. PMID:25853424

APOBEC3G Inhibits HIV-1 RNA Elongation by Inactivating the Viral Trans-Activation Response Element

PubMed Central

Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

2014-01-01

Deamination of cytidine residues in viral DNA (vDNA) is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient HIV-1 replication. dC to dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here we demonstrate that A3G provides an additional layer of defence against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. Finally, we show that free ssDNA termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3′+5′ ends is sufficient for A3G deamination, identifying A3G as an efficient mutator, and that deamination of (−)SSDNA results in an early block of HIV-1 transcription. PMID:24859335

Evaluation of performance across the dynamic range of the Abbott RealTime HIV-1 assay as compared to VERSANT HIV-1 RNA 3.0 and AMPLICOR HIV-1 MONITOR v1.5 using serial dilutions of 39 group M and O viruses.

PubMed

Swanson, Priscilla; Huang, Shihai; Abravaya, Klara; de Mendoza, Carmen; Soriano, Vincent; Devare, Sushil G; Hackett, John

2007-04-01

Performance of the Abbott m2000 instrument system and the Abbott RealTime HIV-1 assay was evaluated using a panel of 37 group M (subtypes A-D, F, G, CRF01_AE, CRF02_AG and unique recombinant forms) and 2 group O virus isolates. Testing was performed on 273 sample dilutions and compared to VERSANT HIV-1 RNA 3.0 (bDNA) and AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5) test results. RealTime HIV-1, bDNA, and Monitor v1.5 tests quantified 87%, 78%, and 81% of samples, respectively. RealTime HIV-1 detected an additional 31 samples at < 40 copies/mL. For group M, RealTime HIV-1 dilution profiles and viral loads were highly correlated with bDNA and Monitor v1.5 values; 87% and 89% of values were within 0.5 log(10) copies/mL. In contrast, the group O viruses were not detected by Monitor v1.5 and were substantially underquantified by approximately 2 log(10) copies/mL in bDNA relative to the RealTime HIV-1 assay. Sequence analysis revealed that RealTime HIV-1 primer/probe binding sites are highly conserved and exhibit fewer nucleotide mismatches relative to Monitor v1.5. The automated m2000 system and RealTime HIV-1 assay offer the advantages of efficient sample processing and throughput with reduced "hands-on" time while providing improved sensitivity, expanded dynamic range and reliable quantification of genetically diverse HIV-1 strains.

Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification.

PubMed

Boyle, David S; Lehman, Dara A; Lillis, Lorraine; Peterson, Dylan; Singhal, Mitra; Armes, Niall; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie

2013-04-02

Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes.

Inhibition of recombinase polymerase amplification by background DNA: a lateral flow-based method for enriching target DNA.

PubMed

Rohrman, Brittany; Richards-Kortum, Rebecca

2015-02-03

Recombinase polymerase amplification (RPA) may be used to detect a variety of pathogens, often after minimal sample preparation. However, previous work has shown that whole blood inhibits RPA. In this paper, we show that the concentrations of background DNA found in whole blood prevent the amplification of target DNA by RPA. First, using an HIV-1 RPA assay with known concentrations of nonspecific background DNA, we show that RPA tolerates more background DNA when higher HIV-1 target concentrations are present. Then, using three additional assays, we demonstrate that the maximum amount of background DNA that may be tolerated in RPA reactions depends on the DNA sequences used in the assay. We also show that changing the RPA reaction conditions, such as incubation time and primer concentration, has little effect on the ability of RPA to function when high concentrations of background DNA are present. Finally, we develop and characterize a lateral flow-based method for enriching the target DNA concentration relative to the background DNA concentration. This sample processing method enables RPA of 10(4) copies of HIV-1 DNA in a background of 0-14 μg of background DNA. Without lateral flow sample enrichment, the maximum amount of background DNA tolerated is 2 μg when 10(6) copies of HIV-1 DNA are present. This method requires no heating or other external equipment, may be integrated with upstream DNA extraction and purification processes, is compatible with the components of lysed blood, and has the potential to detect HIV-1 DNA in infant whole blood with high proviral loads.

HIV shedding in cervico-vaginal secretions in pregnant women.

PubMed

Gardella, Barbara; Roccio, Marianna; Maccabruni, Anna; Mariani, Bianca; Panzeri, Lucia; Zara, Francesca; Spinillo, Arsenio

2011-07-01

The purpose of this study was to evaluate the presence of HIV-1 in cervico-vaginal secretions of pregnant as compared to non-pregnant HIV-seropositive women. We compared 43 known HIV seropositive pregnant patients versus 241 age-matched (± 2 years) control non-pregnant HIV-seropositive subjects. In pregnant patients blood and cervico-vaginal samples were obtained during each trimester of pregnancy. In control subjects the same samples were obtained at enrolment. HIV-1 RNA was measured in plasma; proviral HIV-1 DNA, cell-associated and cell-free HIV-1 RNA in cervico-vaginal secretion by competitive polymerase chain reaction (cRT-PCR) and reverse transcriptase PCR. The genital shedding of HIV-DNA (22/43 as compared to 79/241, p = 0.02), and cell-free HIV-RNA detection (26/43 as compared to 72/241, p < .001) was more common in first-trimester pregnant than in non pregnant women. Pregnancy correlated with a significant positive trend in the cervico-vaginal load of HIV-DNA (Spearman Rho= 0.149, p= 0.012), and cell-free HIV-RNA (Spearman Rho= 0.253, p < .001), but not of HIV-RNA transcripts (Spearman Rho = 0.06, p= 0.31). After correction for potential confounders, first trimester pregnant women had increased rates of genital HIV- DNA (odds ratio = 1.94, 95% confidence interval = 1.01 3.78) and cell-free HIV-RNA (odds ratio = 4.07, 95% confidence interval = 1.97 8.41) detection compared to nonpregnant controls. The shedding of genital HIV was increased in pregnant compared to non pregnant subjects, even in patients with undetectable viremia. In this low-risk HIV-positive population the risks of vertical or horizontal transmissions should not be underestimated.

HIV-1 Tat protein induces DNA damage in human peripheral blood B-lymphocytes via mitochondrial ROS production.

PubMed

El-Amine, Rawan; Germini, Diego; Zakharova, Vlada V; Tsfasman, Tatyana; Sheval, Eugene V; Louzada, Ruy A N; Dupuy, Corinne; Bilhou-Nabera, Chrystèle; Hamade, Aline; Najjar, Fadia; Oksenhendler, Eric; Lipinski, Marс; Chernyak, Boris V; Vassetzky, Yegor S

2018-05-01

Human immunodeficiency virus (HIV) infection is associated with B-cell malignancies in patients though HIV-1 is not able to infect B-cells. The rate of B-cell lymphomas in HIV-infected individuals remains high even under the combined antiretroviral therapy (cART) that reconstitutes the immune function. Thus, the contribution of HIV-1 to B-cell oncogenesis remains enigmatic. HIV-1 induces oxidative stress and DNA damage in infected cells via multiple mechanisms, including viral Tat protein. We have detected elevated levels of reactive oxygen species (ROS) and DNA damage in B-cells of HIV-infected individuals. As Tat is present in blood of infected individuals and is able to transduce cells, we hypothesized that it could induce oxidative DNA damage in B-cells promoting genetic instability and malignant transformation. Indeed, incubation of B-cells isolated from healthy donors with purified Tat protein led to oxidative stress, a decrease in the glutathione (GSH) levels, DNA damage and appearance of chromosomal aberrations. The effects of Tat relied on its transcriptional activity and were mediated by NF-κB activation. Tat stimulated oxidative stress in B-cells mostly via mitochondrial ROS production which depended on the reverse electron flow in Complex I of respiratory chain. We propose that Tat-induced oxidative stress, DNA damage and chromosomal aberrations are novel oncogenic factors favoring B-cell lymphomas in HIV-1 infected individuals. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Intradermal HIV-1 DNA Immunization Using Needle-Free Zetajet Injection Followed by HIV-Modified Vaccinia Virus Ankara Vaccination Is Safe and Immunogenic in Mozambican Young Adults: A Phase I Randomized Controlled Trial.

PubMed

Viegas, Edna Omar; Tembe, Nelson; Nilsson, Charlotta; Meggi, Bindiya; Maueia, Cremildo; Augusto, Orvalho; Stout, Richard; Scarlatti, Gabriella; Ferrari, Guido; Earl, Patricia L; Wahren, Britta; Andersson, Sören; Robb, Merlin L; Osman, Nafissa; Biberfeld, Gunnel; Jani, Ilesh; Sandström, Eric

2017-11-27

We assessed the safety and immunogenicity of HIV-DNA priming using Zetajet™, a needle-free device intradermally followed by intramuscular HIV-MVA boosts, in 24 healthy Mozambicans. Volunteers were randomized to receive three immunizations of 600 μg (n = 10; 2 × 0.1 ml) or 1,200 μg (n = 10; 2 × 0.2 ml) of HIV-DNA (3 mg/ml), followed by two boosts of 10 8 pfu HIV-MVA. Four subjects received placebo saline injections. Vaccines and injections were safe and well tolerated with no difference between the two priming groups. After three HIV-DNA immunizations, IFN-γ ELISpot responses to Gag were detected in 9/17 (53%) vaccinees, while none responded to Envelope (Env). After the first HIV-MVA, the overall response rate to Gag and/or Env increased to 14/15 (93%); 14/15 (93%) to Gag and 13/15 (87%) to Env. There were no significant differences between the immunization groups in frequency of response to Gag and Env or magnitude of Gag responses. Env responses were significantly higher in the higher dose group (median 420 vs. 157.5 SFC/million peripheral blood mononuclear cell, p = .014). HIV-specific antibodies to subtype C gp140 and subtype B gp160 were elicited in all vaccinees after the second HIV-MVA, without differences in titers between the groups. Neutralizing antibody responses were not detected. Two (13%) of 16 vaccinees, one in each of the priming groups, exhibited antibodies mediating antibody-dependent cellular cytotoxicity to CRF01_AE. In conclusion, HIV-DNA vaccine delivered intradermally in volumes of 0.1-0.2 ml using Zetajet was safe and well tolerated. Priming with the 1,200 μg dose of HIV-DNA generated higher magnitudes of ELISpot responses to Env.

Susceptibility Testing by Polymerase Chain Reaction DNA Quantitation: A Method to Measure Drug Resistance of Human Immunodeficiency Virus Type 1 Isolates

NASA Astrophysics Data System (ADS)

Eron, Joseph J.; Gorczyca, Paul; Kaplan, Joan C.; D'Aquila, Richard T.

1992-04-01

Polymerase chain reaction (PCR) DNA quantitation (PDQ) susceptibility testing rapidly and directly measures nucleoside sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates. PCR is used to quantitate the amount of HIV-1 DNA synthesized after in vitro infection of peripheral blood mononuclear cells. The relative amounts of HIV-1 DNA in cell lysates from cultures maintained at different drug concentrations reflect drug inhibition of virus replication. The results of PDQ susceptibility testing of 2- or 3-day cultures are supported by assays measuring HIV-1 p24 antigen production in supernatants of 7- or 10-day cultures. DNA sequence analyses to identify mutations in the reverse transcriptase gene that cause resistance to 3'-azido-3'-deoxythymidine also support the PDQ results. With the PDQ method, both infectivity titration and susceptibility testing can be performed on supernatants from primary cultures of peripheral blood mononuclear cells. PDQ susceptibility testing should facilitate epidemiologic studies of the clinical significance of drug-resistant HIV-1 isolates.

Comparison of the Quantiplex Version 3.0 Assay and a Sensitized Amplicor Monitor Assay for Measurement of Human Immunodeficiency Virus Type 1 RNA Levels in Plasma Samples

PubMed Central

Highbarger, Helene C.; Alvord, W. Gregory; Jiang, Min Kang; Shah, Akram S.; Metcalf, Julia A.; Lane, H. Clifford; Dewar, Robin L.

1999-01-01

This study evaluated correlation and agreement between version 3 of the Quantiplex human immunodeficiency virus type 1 (HIV-1) RNA assay (v3 branched DNA [bDNA]) and a sensitized Amplicor HIV-1 Monitor assay (reverse transcription [RT]-PCR) for the measurement of HIV RNA. Three hundred eighteen samples from 59 randomly selected, HIV-1-seropositive persons on various drug protocols from the National Institute of Allergy and Infectious Diseases HIV outpatient clinic were studied. The results indicate that v3 bDNA and RT-PCR are highly correlated (r = 0.98) and are in good agreement (mean difference in log10 copies/ml ± 2 standard deviations = 0.072 ± 0.371). The relationship between values obtained by both assays is given by the following equation: log10v3 bDNA = −0.0915 + 1.0052 · log10RT-PCR. This represents a 1.026-fold difference between log10RT-PCR values and log10v3 bDNA values. PMID:10523562

High viral load in lymph nodes and latent human immunodeficiency virus (HIV) in peripheral blood cells of HIV-1-infected chimpanzees.

PubMed Central

Saksela, K; Muchmore, E; Girard, M; Fultz, P; Baltimore, D

1993-01-01

We have examined human immunodeficiency virus type 1 (HIV-1) infection in chimpanzees by analyzing HIV-1 DNA and RNA in lymph nodes and peripheral mononuclear cells (PBMCs). Like certain asymptomatic HIV-infected persons, these chimpanzees had no detectable viral replication in their PBMCs. However, viral replication and a high viral load were observed in the lymphatic tissue. Despite the absence of viral replication in PBMCs, 1/1,000 to 1/10,000 of the PBMCs contained HIV-1 proviral DNA, and HIV transcription could be rapidly induced in these cells in vitro. These results provide direct evidence of cellular latency of HIV in vivo and suggest that HIV infection in chimpanzees may be a useful model for clinical latency of HIV infection in humans. Images PMID:8230463

Comparison of the COBAS TAQMAN HIV-1 HPS with VERSANT HIV-1 RNA 3.0 assay (bDNA) for plasma RNA quantitation in different HIV-1 subtypes.

PubMed

Gomes, Perpétua; Palma, Ana Carolina; Cabanas, Joaquim; Abecasis, Ana; Carvalho, Ana Patrícia; Ziermann, Rainer; Diogo, Isabel; Gonçalves, Fátima; Lobo, Céu Sousa; Camacho, Ricardo

2006-08-01

Quantitation of HIV-1 RNA levels in plasma has an undisputed prognostic value and is extremely important for evaluating response to antiretroviral therapy. The purpose of this study was to evaluate the performance of the real-time PCR COBAS TaqMan 48 analyser, comparing it to the existing VERSANT 3.0 (bDNA) for HIV-1 RNA quantitation in plasma of individuals infected with different HIV-1 subtypes (104 blood samples). A positive linear correlation between the two tests (r2 = 0.88) was found. Quantitation by the COBAS TaqMan assay was approximately 0.32log10 higher than by bDNA. The relationship between the two assays was similar within all subtypes with a Deming regression of <1 and <0 for the Bland-Altman plots. Overall, no significant differences were found in plasma viral load quantitation in different HIV-1 subtypes between both assays; therefore these assays are suitable for viral load quantitation of highly genetically diverse HIV-1 plasma samples.

[Immune response induced by HIV DNA vaccine combined with recombinant adeno-associated virus].

PubMed

Liu, Yan-zheng; Zhou, Ling; Wang, Qi; Ye, Shu-qing; Li, Hong-xia; Zeng, Yi

2004-09-01

HIV-1 DNA vaccine and recombinant adeno-associated virus (rAAV) expressing gagV3 gene of HIV-1 subtype B were constructed and BALB/c mice were immunized by vaccination regimen consisting of consecutive priming with DNA vaccine and boosting with rAAV vaccine; the CTL and antibody response were detected and compared with those induced by DNA vaccine or rAAV vaccine separately. HIV-1 subtype B gagV3 gene was inserted into the polyclonal site of plasmid pCI-neo, DNA vaccine pCI-gagV3 was thereby constructed; pCI-gagV3 was transfected into p815 cells, G-418-resistant cells were obtained through screening transfected cells with G418, the expression of HIV-1 antigen in G-418-resistant cells was detected by EIA; BALB/c mice were immunized with pCI-gagV3 and the immune response was tested; BALB/c mouse immunized with pCI-gagV3 and combined with rAAV expressing the same gagV3 genes were tested for antibody level in sera by EIA method and cytotoxicity response by LDH method. pCI-gagV3 could express HIV-1 gene in p815 cells; pCI-gagV3 could induce HIV-1 specific humoral and cell-mediated immune response in BALB/c mice. The HIV-1 specific antibody level was 1/20; when the ratio of effector cells: target cells was 50:1, the average specific cytotoxicity was 41.7%; there was no evident increase in the antibody level induced by pCI-gagV3 combined with rAAV, but there was increase in CTL response, the average specific cytotoxicity was 61.3% when effector cells: target cells ratio was 50:1. HIV-1 specific cytotoxicity in BALB/c mice can be increased by immunization of BALB/c mice with DNA vaccine combined with rAAV vaccine.

HIV-1 activation of innate immunity depends strongly on the intracellular level of TREX1 and sensing of incomplete reverse transcription products.

PubMed

Kumar, Swati; Morrison, James H; Dingli, David; Poeschla, Eric

2018-05-16

TREX1 has been reported to degrade cytosolic immune-stimulatory DNA, including viral DNA generated during HIV-1 infection, but the dynamic range of its capacity to suppress innate immune stimulation is unknown and its full role in the viral life cycle remains unclear. A main purpose of our study was to determine how the intracellular level of TREX1 affects HIV-1 activation and avoidance of innate immunity. Using stable over-expression and CRISPR-mediated gene disruption, we engineered a range of TREX1 levels in human THP-1 monocytes. Increasing the level of TREX1 dramatically suppressed HIV-1 induction of interferon-stimulated genes (ISGs). Productive infection and integrated proviruses were equal to increased. Knocking out TREX1 impaired viral infectivity, increased early viral cDNA and caused ten-fold or greater increases in HIV-1 ISG induction. Knockout of cyclic GMP-AMP synthase (cGAS) abrogated all ISG induction. Moreover, cGAS knockout produced no increase in single cycle infection, establishing that HIV-1 DNA-triggered signaling is not rapid enough to impair the initial ISG-triggering infection cycle. Disruption of the HIV-1 capsid by PF74 also induced ISGs and this was TREX1 level-dependent, required reverse transcriptase catalysis, and was eliminated by cGAS gene knockout. Thus, the intracellular level of TREX1 pivotally modulates innate immune induction by HIV-1. Partial HIV-1 genomes are the TREX1 target and are sensed by cGAS. The nearly complete lack of innate immune induction despite equal to increased viral integration observed when the TREX1 protein level is experimentally elevated indicates that integration-competent genomes are shielded from cytosolic sensor-effectors during uncoating and transit to the nucleus. IMPORTANCE Much remains unknown about how TREX1 influences HIV-1 replication, whether it targets full-length viral DNA versus partial intermediates, how intracellular TREX1 protein levels correlate with ISG induction, and whether TREX1 digestion of cytoplasmic DNA and subsequent cGAS pathway activation affects both initial and subsequent cycles of infection. To answer these questions, we experimentally varied the intracellular level of TREX1 and show that this strongly determines the innate immunogenicity of HIV-1. In addition, several lines of evidence including time of addition experiments with drugs that impair reverse transcription or capsid integrity showed that the pathogen-associated molecular patterns sensed after viral entry contain DNA, are TREX1 and cGAS substrates, and are derived from incomplete RT products. In contrast, the experiments demonstrate that full-length integration competent viral DNA is immune to TREX1. Treatment approaches that reduce TREX1 levels or facilitate release of DNA intermediates may advantageously combine enhanced innate immunity with antiviral effects. Copyright © 2018 American Society for Microbiology.

Negative Feedback Regulation of HIV-1 by Gene Editing Strategy.

PubMed

Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-Bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

2016-08-16

The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells.

Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

NASA Astrophysics Data System (ADS)

Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

2016-01-01

APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

Effects of anticoagulant, processing delay, and assay method (branched DNA versus reverse transcriptase PCR) on measurement of human immunodeficiency virus type 1 RNA levels in plasma.

PubMed

Kirstein, L M; Mellors, J W; Rinaldo, C R; Margolick, J B; Giorgi, J V; Phair, J P; Dietz, E; Gupta, P; Sherlock, C H; Hogg, R; Montaner, J S; Muñoz, A

1999-08-01

We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (P < 0.05) but small after 6 h (bDNA assay, -0.12 log(10) copies/ml; RT-PCR, -0.05 log(10) copies/ml) and after 18 h (bDNA assay, -0.27 log(10) copies/ml; RT-PCR, -0.15 log(10) copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, -0.002 log(10) copies/ml; RT-PCR, -0.02 log(10) copies/ml), but it was after 18 h (bDNA assay, -0.09 log(10) copies/ml; RT-PCR, -0.09 log(10) copies/ml). Only 4% of samples processed after 6 h lost more than 50% (>/=0.3 log(10) copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4(+)-cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (-0.05 to -0.12 log(10) copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4(+)-cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP.

The effect of treatment of vaginal infections on shedding of human immunodeficiency virus type 1.

PubMed

Wang, C C; McClelland, R S; Reilly, M; Overbaugh, J; Emery, S R; Mandaliya, K; Chohan, B; Ndinya-Achola, J; Bwayo, J; Kreiss, J K

2001-04-01

To assess the effect of treatment of vaginal infections on vaginal shedding of cell-free human immunodeficiency virus type 1 (HIV-1) and HIV-1-infected cells, HIV-1-seropositive women were examined before and after treatment of Candida vulvovaginitis, Trichomonas vaginitis, and bacterial vaginosis. For Candida (n=98), vaginal HIV-1 RNA decreased from 3.36 to 2.86 log(10) copies/swab (P<.001), as did the prevalence of HIV-1 DNA (36% to 17%; odds ratio [OR], 2.8; 95% confidence interval [CI], 1.3-6.5). For Trichomonas vaginitis (n=55), HIV-1 RNA decreased from 3.67 to 3.05 log(10) copies/swab (P<.001), but the prevalence of HIV-1 DNA remained unchanged (22%-25%; OR, 0.8; 95% CI, 0.3-2.2). For bacterial vaginosis (n=73), neither the shedding of HIV-1 RNA (from 3.11 to 2.90 log(10) copies/swab; P=.14) nor the prevalence of DNA (from 21% to 23%; OR, 0.8; 95% CI, 0.3-2.0) changed. Vaginal HIV-1 decreased 3.2- and 4.2-fold after treating Candida and Trichomonas, respectively. These data suggest that HIV-1 transmission intervention strategies that incorporate diagnosis and treatment of these prevalent infections warrant evaluation.

Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing

PubMed Central

Kaminski, Rafal; Chen, Yilan; Fischer, Tracy; Tedaldi, Ellen; Napoli, Alessandro; Zhang, Yonggang; Karn, Jonathan; Hu, Wenhui; Khalili, Kamel

2016-01-01

We employed an RNA-guided CRISPR/Cas9 DNA editing system to precisely remove the entire HIV-1 genome spanning between 5′ and 3′ LTRs of integrated HIV-1 proviral DNA copies from latently infected human CD4+ T-cells. Comprehensive assessment of whole-genome sequencing of HIV-1 eradicated cells ruled out any off-target effects by our CRISPR/Cas9 technology that might compromise the integrity of the host genome and further showed no effect on several cell health indices including viability, cell cycle and apoptosis. Persistent co-expression of Cas9 and the specific targeting guide RNAs in HIV-1-eradicated T-cells protected them against new infection by HIV-1. Lentivirus-delivered CRISPR/Cas9 significantly diminished HIV-1 replication in infected primary CD4+ T-cell cultures and drastically reduced viral load in ex vivo culture of CD4+ T-cells obtained from HIV-1 infected patients. Thus, gene editing using CRISPR/Cas9 may provide a new therapeutic path for eliminating HIV-1 DNA from CD4+ T-cells and potentially serve as a novel and effective platform toward curing AIDS. PMID:26939770

DNA Vaccine Molecular Adjuvants SP-D-BAFF and SP-D-APRIL Enhance Anti-gp120 Immune Response and Increase HIV-1 Neutralizing Antibody Titers

PubMed Central

Gupta, Sachin; Clark, Emily S.; Termini, James M.; Boucher, Justin; Kanagavelu, Saravana; LeBranche, Celia C.; Abraham, Sakhi; Montefiori, David C.

2015-01-01

ABSTRACT Broadly neutralizing antibodies (bNAbs) specific for conserved epitopes on the HIV-1 envelope (Env) are believed to be essential for protection against multiple HIV-1 clades. However, vaccines capable of stimulating the production of bNAbs remain a major challenge. Given that polyreactivity and autoreactivity are considered important characteristics of anti-HIV bNAbs, we designed an HIV vaccine incorporating the molecular adjuvants BAFF (B cell activating factor) and APRIL (a proliferation-inducing ligand) with the potential to facilitate the maturation of polyreactive and autoreactive B cells as well as to enhance the affinity and/or avidity of Env-specific antibodies. We designed recombinant DNA plasmids encoding soluble multitrimers of BAFF and APRIL using surfactant protein D as a scaffold, and we vaccinated mice with these molecular adjuvants using DNA and DNA-protein vaccination strategies. We found that immunization of mice with a DNA vaccine encoding BAFF or APRIL multitrimers, together with interleukin 12 (IL-12) and membrane-bound HIV-1 Env gp140, induced neutralizing antibodies against tier 1 and tier 2 (vaccine strain) viruses. The APRIL-containing vaccine was particularly effective at generating tier 2 neutralizing antibodies following a protein boost. These BAFF and APRIL effects coincided with an enhanced germinal center (GC) reaction, increased anti-gp120 antibody-secreting cells, and increased anti-gp120 functional avidity. Notably, BAFF and APRIL did not cause indiscriminate B cell expansion or an increase in total IgG. We propose that BAFF and APRIL multitrimers are promising molecular adjuvants for vaccines designed to induce bNAbs against HIV-1. IMPORTANCE Recent identification of antibodies that neutralize most HIV-1 strains has revived hopes and efforts to create novel vaccines that can effectively stimulate HIV-1 neutralizing antibodies. However, the multiple immune evasion properties of HIV have hampered these efforts. These include the instability of the gp120 trimer, the inaccessibility of the conserved sequences, highly variable protein sequences, and the loss of HIV-1-specific antibody-producing cells during development. We have shown previously that tumor necrosis factor (TNF) superfamily ligands, including BAFF and APRIL, can be multitrimerized using the lung protein SP-D (surfactant protein D), enhancing immune responses. Here we show that DNA or DNA-protein vaccines encoding BAFF or APRIL multitrimers, IL-12p70, and membrane-bound HIV-1 Env gp140 induced tier 1 and tier 2 neutralizing antibodies in a mouse model. BAFF and APRIL enhanced the immune reaction, improved antibody binding, and increased the numbers of anti-HIV-1 antibody-secreting cells. Adaptation of this vaccine design may prove useful in designing preventive HIV-1 vaccines for humans. PMID:25631080

Global analysis of host-pathogen interactions that regulate early stage HIV-1 replication

PubMed Central

König, Renate; Zhou, Yingyao; Elleder, Daniel; Diamond, Tracy L.; Bonamy, Ghislain M.C.; Irelan, Jeffrey T.; Chiang, Chih-yuan; Tu, Buu P.; De Jesus, Paul D.; Lilley, Caroline E.; Seidel, Shannon; Opaluch, Amanda M.; Caldwell, Jeremy S.; Weitzman, Matthew D.; Kuhen, Kelli L.; Bandyopadhyay, Sourav; Ideker, Trey; Orth, Anthony P.; Miraglia, Loren J.; Bushman, Frederic D.; Young, John A.; Chanda, Sumit K.

2008-01-01

Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA damage response and RNA splicing were identified as important modulators of early stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of post-translational modification, and nucleic acid binding proteins. Finally, fifteen proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multi-scale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate early steps of HIV-1 infection. PMID:18854154

Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination.

PubMed

Boyer, J D; Ugen, K E; Wang, B; Agadjanyan, M; Gilbert, L; Bagarazzi, M L; Chattergoon, M; Frost, P; Javadian, A; Williams, W V; Refaeli, Y; Ciccarelli, R B; McCallus, D; Coney, L; Weiner, D B

1997-05-01

Novel approaches for the generation of more effective vaccines for HIV-1 are of significant importance. In this report we analyze the immunogenicity and efficacy of an HIV-1 DNA vaccine encoding env, rev and gag/pol in a chimpanzee model system. The immunized animals developed specific cellular and humoral immune responses. Animals were challenged with a heterologous chimpanzee titered stock of HIV-1 SF2 virus and followed for 48 weeks after challenge. Polymerase chain reaction coupled with reverse transcription (RT-PCR) results indicated infection in the control animal, whereas those animals vaccinated with the DNA constructs were protected from the establishment of infection. These studies serve as an important benchmark for the use of DNA vaccine technology for the production of protective immune responses.

Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells.

PubMed

Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R; Hosmane, Nina N; Capoferri, Adam A; Bruner, Katherine M; Pollack, Ross A; Zhang, Hao; Drummond, Michael Bradley; Siliciano, Janet M; Siliciano, Robert; Stivers, James T

2016-09-20

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection.

Monophosphoryl lipid A enhances both humoral and cell-mediated immune responses to DNA vaccination against human immunodeficiency virus type 1.

PubMed Central

Sasaki, S; Tsuji, T; Hamajima, K; Fukushima, J; Ishii, N; Kaneko, T; Xin, K Q; Mohri, H; Aoki, I; Okubo, T; Nishioka, K; Okuda, K

1997-01-01

To enhance immunity induced by DNA vaccination against human immunodeficiency virus type 1 (HIV-1), we evaluated the efficacy of monophosphoryl lipid A (MPL), an adjuvant of bacterial origin. BALB/c mice were intramuscularly injected with immunogenic DNA, encoding the env and rev genes of the HIV-1(IIIB) strain, formulated with MPL dissolved in different vehicles (MPL in stable emulsion and MPL in aqueous formulation). The sera from mice immunized with the two preparations of MPL revealed 2(6) to 2(9) times higher HIV-1-specific immunoglobulin G (IgG) titers than the sera from mice immunized without MPL. In virus neutralization tests for HIV-1(IIIB), by p24 assay and antifusion assay of infected MOLT-4 cells, MPL tends to elicit antibody more protective than antibody elicited without adjuvant. MPL also elicited stronger delayed-type hypersensitivity and cytotoxic-T-lymphocyte activity against HIV-1(IIIB) compared to DNA alone. HIV-1-specific IgG subclass analysis showed that MPL tends to facilitate IgG2a production, suggesting enhancement of a predominant T-helper-type-1 response, and this enhancement may help to facilitate protective-antibody induction. Furthermore, a chloramphenicol acetyltransferase (CAT) assay was employed to determine whether MPL affected the gene expression process. Interestingly, both MPL preparations reduced CAT activity in the muscle injected with CAT expression vector but increased anti-CAT antibody production. These results indicate that MPL acts as an effective adjuvant for immunogenic DNA injection despite reduced expression of encoding protein in muscle. We conclude that MPL has a strong adjuvant effect on DNA vaccination against HIV-1. PMID:9284115

Armored Long RNA Controls or Standards for Branched DNA Assay for Detection of Human Immunodeficiency Virus Type 1▿

PubMed Central

Zhan, Sien; Li, Jinming; Xu, Ruihuan; Wang, Lunan; Zhang, Kuo; Zhang, Rui

2009-01-01

The branched DNA (bDNA) assay is a reliable method for quantifying the RNA of human immunodeficiency virus type 1 (HIV-1). The positive controls and standards for this assay for the detection of HIV-1 consist of naked RNA, which is susceptible to degradation by RNase. Armored RNA is a good candidate for an RNase-resistant positive control or standard. However, its use has been limited by the maximal length of the exogenous RNA packaged into virus-like particles by routine armored RNA technology. In the present study, we produced armored long RNA (armored L-RNA) controls or standards (AR-HIV-pol-3034b) for a bDNA assay of HIV-1 by increasing the amount and affinity of the pac sites (the pac site is a specific 19-nucleotide stem-loop region located at the 5′ terminus of the MS2 bacteriophage replicase gene) by a one-plasmid double-expression system. AR-HIV-pol-3034b was completely resistant to DNase and RNase, was stable in normal human EDTA-preserved plasma at 4°C for at least 6 months, and produced reproducible, linear results in the Versant HIV-1 RNA 3.0 assay. In conclusion, AR-HIV-pol-3034b could act as a positive control or standard in a bDNA assay for the detection of HIV-1. In addition, the one-plasmid double-expression system can be used as a better platform than the one-plasmid expression system and the two-plasmid coexpression system for expressing armored L-RNA. PMID:19494069

Armored long RNA controls or standards for branched DNA assay for detection of human immunodeficiency virus type 1.

PubMed

Zhan, Sien; Li, Jinming; Xu, Ruihuan; Wang, Lunan; Zhang, Kuo; Zhang, Rui

2009-08-01

The branched DNA (bDNA) assay is a reliable method for quantifying the RNA of human immunodeficiency virus type 1 (HIV-1). The positive controls and standards for this assay for the detection of HIV-1 consist of naked RNA, which is susceptible to degradation by RNase. Armored RNA is a good candidate for an RNase-resistant positive control or standard. However, its use has been limited by the maximal length of the exogenous RNA packaged into virus-like particles by routine armored RNA technology. In the present study, we produced armored long RNA (armored L-RNA) controls or standards (AR-HIV-pol-3034b) for a bDNA assay of HIV-1 by increasing the amount and affinity of the pac sites (the pac site is a specific 19-nucleotide stem-loop region located at the 5' terminus of the MS2 bacteriophage replicase gene) by a one-plasmid double-expression system. AR-HIV-pol-3034b was completely resistant to DNase and RNase, was stable in normal human EDTA-preserved plasma at 4 degrees C for at least 6 months, and produced reproducible, linear results in the Versant HIV-1 RNA 3.0 assay. In conclusion, AR-HIV-pol-3034b could act as a positive control or standard in a bDNA assay for the detection of HIV-1. In addition, the one-plasmid double-expression system can be used as a better platform than the one-plasmid expression system and the two-plasmid coexpression system for expressing armored L-RNA.

Quantitation of human immunodeficiency virus type 1 in breast milk.

PubMed

Ghosh, M K; Kuhn, L; West, J; Semrau, K; Decker, D; Thea, D M; Aldrovandi, G M

2003-06-01

The distribution and stability of human immunodeficiency virus type 1 (HIV-1) in breast milk (BM) components remain largely unknown. Inhibitory effects, if any, of BM on HIV RNA and DNA PCR amplification are poorly understood. We have addressed these issues by using virus-spiked BM samples from HIV-negative women. BM samples from HIV-negative women were spiked with HIV-1 virions or cells containing a single integrated copy of HIV DNA (8E5/LAV). After incubation under different experimental conditions, viral RNA was detected by the Roche Amplicor UltraSensitive assay in whole-milk, skim milk, and lipid fractions. We found excellent correlation between HIV-1 input copy and recovery in whole milk (r = 0.965, P < 0.0001), skim milk (r = 0.972, P < 0.0001), and the lipid fraction (r = 0.905, P < 0.001). PCR inhibition was observed in less than 10% of the spiked samples. Similar levels of inhibition were noted in BM samples collected from HIV-infected women. HIV proviral DNA was detected in BM samples using real-time PCR (linear correlation between the threshold cycle versus log DNA copy number, >0.982). The effects of incubation duration and temperature and repeated freeze-thaw cycles on HIV RNA recovery were analyzed. HIV RNA levels were remarkably stable in whole milk after three freeze-thaw cycles and for up to 30 h at room temperature. Our findings improve the understanding of the dynamics of HIV detection in BM and the conditions for BM sample collection, storage, and processing.

Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex.

PubMed

Grigorov, Boyan; Bocquin, Anne; Gabus, Caroline; Avilov, Sergey; Mély, Yves; Agopian, Audrey; Divita, Gilles; Gottikh, Marina; Witvrouw, Myriam; Darlix, Jean-Luc

2011-07-01

Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1.

Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex

PubMed Central

Grigorov, Boyan; Bocquin, Anne; Gabus, Caroline; Avilov, Sergey; Mély, Yves; Agopian, Audrey; Divita, Gilles; Gottikh, Marina; Witvrouw, Myriam; Darlix, Jean-Luc

2011-01-01

Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1. PMID:21447560

Clinical Comparison of an Enhanced-Sensitivity Branched-DNA Assay and Reverse Transcription-PCR for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma

PubMed Central

Nolte, Frederick S.; Boysza, Jodi; Thurmond, Cathy; Clark, W. Scott; Lennox, Jeffrey L.

1998-01-01

The performance characteristics of an enhanced-sensitivity branched-DNA assay (bDNA) (Quantiplex HIV-1 version 2.0; Chiron Corp., Emeryville, Calif.) and a reverse transcription (RT)-PCR assay (AMPLICOR HIV-1 Monitor; Roche Diagnostic Systems, Inc., Branchburg, N.J.) were compared in a molecular diagnostic laboratory. Samples used in this evaluation included linearity and reproducibility panels made by dilution of a human immunodeficiency virus type 1 (HIV-1) stock culture of known virus particle count in HIV-1-negative plasma, a subtype panel consisting of HIV-1 subtypes A through F at a standardized level, and 64 baseline plasma specimens from HIV-1-infected individuals. Plots of log10 HIV RNA copies per milliliter versus log10 nominal virus particles per milliliter demonstrated that both assays were linear over the stated dynamic ranges (bDNA, r = 0.98; RT-PCR, r = 0.99), but comparison of the slopes of the regression lines (bDNA, m = 0.96; RT-PCR, m = 0.83) suggested that RT-PCR had greater proportional systematic error. The between-run coefficients of variation for bDNA and RT-PCR were 24.3 and 34.3%, respectively, for a sample containing 1,650 nominal virus particles/ml and 44.0 and 42.7%, respectively, for a sample containing 165 nominal virus particles/ml. Subtypes B, C, and D were quantitated with similar efficiencies by bDNA and RT-PCR; however, RT-PCR was less efficient in quantitating subtypes A, E, and F. One non-B subtype was recognized in our clinical specimens based on the ratio of values obtained with the two methods. HIV-1 RNA was quantitated in 53 (83%) baseline plasma specimens by bDNA and in 55 (86%) specimens by RT-PCR. RT-PCR values were consistently greater than bDNA values, with population means of 142,419 and 67,580 copies/ml, respectively (P < 0.01). The results were highly correlated (r = 0.91), but the agreement was poor (mean difference in log10 copies per milliliter ± 2 standard deviations, 0.45 ± 0.61) for the 50 clinical specimens that gave discrete values with both methods. PMID:9508301

Multicenter Comparison of Roche COBAS AMPLICOR MONITOR Version 1.5, Organon Teknika NucliSens QT with Extractor, and Bayer Quantiplex Version 3.0 for Quantification of Human Immunodeficiency Virus Type 1 RNA in Plasma

PubMed Central

Murphy, Donald G.; Côté, Louise; Fauvel, Micheline; René, Pierre; Vincelette, Jean

2000-01-01

The performance and characteristics of Roche COBAS AMPLICOR HIV-1 MONITOR version 1.5 (CA MONITOR 1.5) UltraSensitive (usCA MONITOR 1.5) and Standard (stCA MONITOR 1.5) procedures, Organon Teknika NucliSens HIV-1 RNA QT with Extractor (NucliSens), and Bayer Quantiplex HIV RNA version 3.0 (bDNA 3.0) were compared in a multicenter trial. Samples used in this study included 460 plasma specimens from human immunodeficiency virus (HIV) type 1 (HIV-1)-infected persons, 100 plasma specimens from HIV antibody (anti-HIV)-negative persons, and culture supernatants of HIV-1 subtype A to E isolates diluted in anti-HIV-negative plasma. Overall, bDNA 3.0 showed the least variation in RNA measures upon repeat testing. For the Roche assays, usCA MONITOR 1.5 displayed less variation in RNA measures than stCA MONITOR 1.5. NucliSens, at an input volume of 2 ml, showed the best sensitivity. Deming regression analysis indicated that the results of all three assays were significantly correlated (P < 0.0001). However, the mean difference in values between CA MONITOR 1.5 and bDNA 3.0 (0.274 log10 RNA copies/ml; 95% confidence interval, 0.192 to 0.356) was significantly different from 0, indicating that CA MONITOR 1.5 values were regularly higher than bDNA 3.0 values. Upon testing of 100 anti-HIV-negative plasma specimens, usCA MONITOR 1.5 and NucliSens displayed 100% specificity, while bDNA 3.0 showed 98% specificity. NucliSens quantified 2 of 10 non-subtype B viral isolates at 1 log10 lower than both CA MONITOR 1.5 and bDNA 3.0. For NucliSens, testing of specimens with greater than 1,000 RNA copies/ml at input volumes of 0.1, 0.2, and 2.0 ml did not affect the quality of results. Additional factors differing between assays included specimen throughput and volume requirements, limit of detection, ease of execution, instrument work space, and costs of disposal. These characteristics, along with assay performance, should be considered when one is selecting a viral load assay. PMID:11060065

Using RT-PCR and bDNA assays to measure non-clade B HIV-1 subtype RNA.

PubMed

Pasquier, C; Sandres, K; Salama, G; Puel, J; Izopet, J

1999-08-01

The performance of the new version of RT-PCR assay (Amplicor HIV-1 Monitor v1.5) was assessed. The quantification of non-B subtype HIV-1 plasma RNA (30A, 1C, 1D, 3E, 2F, 3G) obtained using Monitor v1.5 was compared to the former version of this assay (Monitor v1.0) and to the Quantiplex v2.0 bDNA assay. The new primers used in Monitor v1.5 were similar to the former version in both specificity and sensitivity. The new primers corrected the detection and quantification defect observed previously for HIV-1 non-B subtypes and gave slightly higher RNA concentrations than those measured using the bDNA assay (+0.39 log copies/ml).

Structural Insights into the HIV-1 Minus-strand Strong-stop DNA*

PubMed Central

Chen, Yingying; Maskri, Ouerdia; Chaminade, Françoise; René, Brigitte; Benkaroun, Jessica; Godet, Julien; Mély, Yves; Mauffret, Olivier; Fossé, Philippe

2016-01-01

An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first strand transfer that requires base pairing of the R region at the 3′-end of the genomic RNA with the complementary r region at the 3′-end of minus-strand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing. For this purpose, we investigated ssDNA using structural probes (nucleases and potassium permanganate). This study is the first to determine the secondary structure of the full-length HIV-1 ssDNA in the absence or presence of NC. The probing data and phylogenetic analysis support the folding of ssDNA into three stem-loop structures and the presence of four high-affinity binding sites for NC. Our results support a model for the NC-mediated annealing process in which the preferential binding of NC to four sites triggers unfolding of the three-dimensional structure of ssDNA, thus facilitating interaction of the r sequence of ssDNA with the R sequence of the genomic RNA. In addition, using gel retardation assays and ssDNA mutants, we show that the NC-mediated annealing process does not rely on a single pathway (zipper intermediate or kissing complex). PMID:26668324

Effects of Anticoagulant, Processing Delay, and Assay Method (Branched DNA versus Reverse Transcriptase PCR) on Measurement of Human Immunodeficiency Virus Type 1 RNA Levels in Plasma

PubMed Central

Kirstein, Lynn M.; Mellors, John W.; Rinaldo, Charles R.; Margolick, Joseph B.; Giorgi, Janis V.; Phair, John P.; Dietz, Edith; Gupta, Phalguni; Sherlock, Christopher H.; Hogg, Robert; Montaner, J. S. G.; Muñoz, Alvaro

1999-01-01

We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (P < 0.05) but small after 6 h (bDNA assay, −0.12 log10 copies/ml; RT-PCR, −0.05 log10 copies/ml) and after 18 h (bDNA assay, −0.27 log10 copies/ml; RT-PCR, −0.15 log10 copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, −0.002 log10 copies/ml; RT-PCR, −0.02 log10 copies/ml), but it was after 18 h (bDNA assay, −0.09 log10 copies/ml; RT-PCR, −0.09 log10 copies/ml). Only 4% of samples processed after 6 h lost more than 50% (≥0.3 log10 copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4+-cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (−0.05 to −0.12 log10 copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4+-cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP. PMID:10405379

Cost assessment of the automated VERSANT 440 Molecular System versus the semi-automated System 340 bDNA Analyzer platforms.

PubMed

Elbeik, Tarek; Loftus, Richard A; Beringer, Scott

2007-11-01

Labor, supply and waste were evaluated for HIV-1 and HCV bDNA on the semi-automated System 340 bDNA Analyzer and the automated VERSANT 440 Molecular System (V440). HIV-1 sample processing was evaluated using a 24- and 48-position centrifuge rotor. Vigilance time (hands-on manipulations plus incubation time except initial target hybridization) and disposables were approximately 37 and 12% lower for HIV-1, and 64 and 31% lower for HCV bDNA, respectively, with V440. Biohazardous solid waste was approximately twofold lower for both assays and other waste types were the same for either assay on both platforms. HIV-1 sample processing vigilance time for the 48-position rotor was reduced by 2 h. V440 provides cost savings and improved workflow.

An Enhanced Synthetic Multiclade DNA Prime Induces Improved Cross-Clade-Reactive Functional Antibodies when Combined with an Adjuvanted Protein Boost in Nonhuman Primates

PubMed Central

Wise, Megan C.; Hutnick, Natalie A.; Pollara, Justin; Myles, Devin J. F.; Williams, Constance; Yan, Jian; LaBranche, Celia C.; Khan, Amir S.; Sardesai, Niranjan Y.; Montefiori, David; Barnett, Susan W.; Zolla-Pazner, Susan; Ferrari, Guido

2015-01-01

ABSTRACT The search for an efficacious human immunodeficiency virus type 1 (HIV-1) vaccine remains a pressing need. The moderate success of the RV144 Thai clinical vaccine trial suggested that vaccine-induced HIV-1-specific antibodies can reduce the risk of HIV-1 infection. We have made several improvements to the DNA platform and have previously shown that improved DNA vaccines alone are capable of inducing both binding and neutralizing antibodies in small-animal models. In this study, we explored how an improved DNA prime and recombinant protein boost would impact HIV-specific vaccine immunogenicity in rhesus macaques (RhM). After DNA immunization with either a single HIV Env consensus sequence or multiple constructs expressing HIV subtype-specific Env consensus sequences, we detected both CD4+ and CD8+ T-cell responses to all vaccine immunogens. These T-cell responses were further increased after protein boosting to levels exceeding those of DNA-only or protein-only immunization. In addition, we observed antibodies that exhibited robust cross-clade binding and neutralizing and antibody-dependent cellular cytotoxicity (ADCC) activity after immunization with the DNA prime-protein boost regimen, with the multiple-Env formulation inducing a more robust and broader response than the single-Env formulation. The magnitude and functionality of these responses emphasize the strong priming effect improved DNA immunogens can induce, which are further expanded upon protein boost. These results support further study of an improved synthetic DNA prime together with a protein boost for enhancing anti-HIV immune responses. IMPORTANCE Even with effective antiretroviral drugs, HIV remains an enormous global health burden. Vaccine development has been problematic in part due to the high degree of diversity and poor immunogenicity of the HIV Env protein. Studies suggest that a relevant HIV vaccine will likely need to induce broad cellular and humoral responses from a simple vaccine regimen due to the resource-limited setting in which the HIV pandemic is most rampant. DNA vaccination lends itself well to increasing the amount of diversity included in a vaccine due to the ease of manufacturing multiple plasmids and formulating them as a single immunization. By increasing the number of Envs within a formulation, we were able to show an increased breadth of responses as well as improved functionality induced in a nonhuman primate model. This increased breadth could be built upon, leading to better coverage against circulating strains with broader vaccine-induced protection. PMID:26085155

Levels of HIV-1 persistence on antiretroviral therapy are not associated with markers of inflammation or activation

PubMed Central

Bosch, Ronald J.; Macatangay, Bernard J.; Rinaldo, Charles R.; Riddler, Sharon A.; Mellors, John W.

2017-01-01

Antiretroviral therapy (ART) reduces levels of HIV-1 and immune activation but both can persist despite clinically effective ART. The relationships among pre-ART and on-ART levels of HIV-1 and activation are incompletely understood, in part because prior studies have been small or cross-sectional. To address these limitations, we evaluated measures of HIV-1 persistence, inflammation, T cell activation and T cell cycling in a longitudinal cohort of 101 participants who initiated ART and had well-documented sustained suppression of plasma viremia for a median of 7 years. During the first 4 years following ART initiation, HIV-1 DNA declined by 15-fold (93%) whereas cell-associated HIV-1 RNA (CA-RNA) fell 525-fold (>99%). Thereafter, HIV-1 DNA levels continued to decline slowly (5% per year) with a half-life of 13 years. Participants who had higher HIV-1 DNA and CA-RNA before starting treatment had higher levels while on ART, despite suppression of plasma viremia for many years. Markers of inflammation and T cell activation were associated with plasma HIV-1 RNA levels before ART was initiated but there were no consistent associations between these markers and HIV-1 DNA or CA-RNA during long-term ART, suggesting that HIV-1 persistence is not driving or driven by inflammation or activation. Higher levels of inflammation, T cell activation and cycling before ART were associated with higher levels during ART, indicating that immunologic events that occurred well before ART initiation had long-lasting effects despite sustained virologic suppression. These findings should stimulate studies of viral and host factors that affect virologic, inflammatory and immunologic set points prior to ART initiation and should inform the design of strategies to reduce HIV-1 reservoirs and dampen immune activation that persists despite ART. PMID:28426825

The Majority of HIV Type 1 DNA in Circulating CD4+ T Lymphocytes Is Present in Non-Gut-Homing Resting Memory CD4+ T Cells

PubMed Central

Xu, Yin; Bailey, Michelle; Seddiki, Nabila; Suzuki, Kazuo; Murray, John M.; Gao, Yuan; Yan, Celine; Cooper, David A.; Kelleher, Anthony D.; Koelsch, Kersten K.; Zaunders, John

2013-01-01

Abstract Memory CD4+ T lymphocytes in peripheral blood that express integrins α4ß7 preferentially recirculate through gut-associated lymphoid tissue (GALT), a proposed site of significant HIV-1 replication. Tregs and activated CD4+ T cells in GALT could also be particularly susceptible to infection. We therefore hypothesized that infection of these subsets of memory CD4+ T cells may contribute disproportionately to the HIV-1 reservoir. A cross-sectional study of CD4+ T cell subsets of memory CD45RO+ cells in peripheral blood mononuclear cells (PBMCs) was conducted using leukapheresis from eight subjects with untreated chronic HIV-1 infection. Real-time polymerase chain reaction (PCR) was used to quantify total and integrated HIV-1 DNA levels from memory CD4+ T cells sorted into integrin β7+ vs. β7−, CD25+CD127low Treg vs. CD127high, and activated CD38+ vs. CD38−. More than 80% of total HIV-1 DNA was found to reside in the integrin β7-negative non-gut-homing subset of CD45RO+ memory CD4+ T cells. Less than 10% was found in highly purified Tregs or CD38+ activated memory cells. Similarly, integrated HIV-1 DNA copies were found to be more abundant in resting non-gut-homing memory CD4+ T cells (76%) than in their activated counterparts (23%). Our investigations showed that the majority of both total and integrated HIV-1 DNA was found within non-gut-homing resting CD4+ T cells. PMID:23971972

Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products

PubMed Central

Ludwig, Linda B; Ambrus, Julian L; Krawczyk, Kristie A; Sharma, Sanjay; Brooks, Stephen; Hsiao, Chiu-Bin; Schwartz, Stanley A

2006-01-01

Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ) protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). Results Inspection of published sequences revealed a potential transcription initiator element (INR) situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR) suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s) could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s) were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK) sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP) sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The novel HAPs are encoded in a region of the LTR that has already been shown to be deleted in some HIV-infected long-term survivors and represent new potential targets for vaccine development. PMID:17090330

Cocaine modulates HIV-1 integration in primary CD4+ T cells: implications in HIV-1 pathogenesis in drug-abusing patients

PubMed Central

Addai, Amma B.; Pandhare, Jui; Paromov, Victor; Mantri, Chinmay K.; Pratap, Siddharth; Dash, Chandravanu

2015-01-01

Epidemiologic studies suggest that cocaine abuse worsens HIV-1 disease progression. Increased viral load has been suggested to play a key role for the accelerated HIV disease among cocaine-abusing patients. The goal of this study was to investigate whether cocaine enhances proviral DNA integration as a mechanism to increase viral load. We infected CD4+ T cells that are the primary targets of HIV-1 in vivo and treated the cells with physiologically relevant concentrations of cocaine (1 µM–100 µM). Proviral DNA integration in the host genome was measured by nested qPCR. Our results illustrated that cocaine from 1 µM through 50 µM increased HIV-1 integration in CD4+ T cells in a dose-dependent manner. As integration can be modulated by several early postentry steps of HIV-1 infection, we examined the direct effects of cocaine on viral integration by in vitro integration assays by use of HIV-1 PICs. Our data illustrated that cocaine directly increases viral DNA integration. Furthermore, our MS analysis showed that cocaine is able to enter CD4+ T cells and localize to the nucleus-. In summary, our data provide strong evidence that cocaine can increase HIV-1 integration in CD4+ T cells. Therefore, we hypothesize that increased HIV-1 integration is a novel mechanism by which cocaine enhances viral load and worsens disease progression in drug-abusing HIV-1 patients. PMID:25691383

HIV-1 concentrations in human breast milk before and after weaning

PubMed Central

Kuhn, Louise; Kim, Hae-Young; Walter, Jan; Thea, Donald M.; Sinkala, Moses; Mwiya, Mwiya; Kankasa, Chipepo; Decker, Don; Aldrovandi, Grace M.

2015-01-01

Concentrations of HIV-1 RNA and DNA in mucosal compartments influence the risk of sexual transmission and mother-to-child transmission of HIV-1. Breast milk production is physiologically regulated such that supply is a function of infant demand but whether demand also influences HIV-1 dynamics in breast milk is unknown. We tested whether minor and major changes in feeding frequency influence breast milk viral concentrations in 958 HIV-1-infected women, who were followed with their infants for 24 months as part of a trial in Lusaka, Zambia. Women were randomized to wean abruptly at 4 months or to continue breastfeeding for a duration of their own choosing. Two weeks after breastfeeding cessation i.e. weaning (4.5 months) HIV-1 concentrations in breast milk were substantially higher (median RNA 2,708 copies/ml and DNA 14 copies/ml) than if breastfeeding continued (median RNA <50 copies/ml and DNA <1 copy/ml, p<0.0001). Among those continuing breastfeeding, HIV-1 concentrations in milk were higher if breastfeeding was non-exclusive (median RNA 293 copies/ml and DNA 2 copies/ml, p=0.0006). Elevated milk viral concentrations after stopping breastfeeding explained higher than expected rates of late postnatal HIV transmission in those who weaned early. Changes in the frequency of breastfeeding peri-weaning and with non-exclusive breastfeeding influenced milk viral concentrations. This may explain the reduced risk of HIV-1 transmission associated with exclusive breastfeeding and may explain why early weaning does not achieve the magnitude of HIV prevention predicted by models. Our results support continuation of maternal antiretroviral drug interventions over the full duration of time when any breast milk exposures are likely to occur after planned weaning. PMID:23596203

Interlaboratory quality control of total HIV-1 DNA load measurement for multicenter reservoir studies.

PubMed

Gantner, Pierre; Mélard, Adeline; Damond, Florence; Delaugerre, Constance; Dina, Julia; Gueudin, Marie; Maillard, Anne; Sauné, Karine; Rodallec, Audrey; Tuaillon, Edouard; Plantier, Jean-Christophe; Rouzioux, Christine; Avettand-Fenoel, Véronique

2017-11-01

Viral reservoirs represent an important barrier to HIV cure. Accurate markers of HIV reservoirs are needed to develop multicenter studies. The aim of this multicenter quality control (QC) was to evaluate the inter-laboratory reproducibility of total HIV-1-DNA quantification. Ten laboratories of the ANRS-AC11 working group participated by quantifying HIV-DNA with a real-time qPCR assay (Biocentric) in four samples (QCMD). Good reproducibility was found between laboratories (standard deviation ≤ 0.2 log 10 copies/10 6 PBMC) for the three positive QC that were correctly classified by each laboratory (QC1

Elevated cytokine and chemokine levels in the placenta are associated with in-utero HIV-1 mother-to-child transmission.

PubMed

Kumar, Surender B; Rice, Cara E; Milner, Danny A; Ramirez, Nilsa C; Ackerman, William E; Mwapasa, Victor; Turner, Abigail Norris; Kwiek, Jesse J

2012-03-27

To determine whether there is an association between cytokine and chemokine levels in plasma isolated from the placenta and HIV-1 mother-to-child transmission (MTCT). We designed a case-control study of HIV-infected, pregnant women enrolled in the Malaria and HIV in Pregnancy cohort. Participants were recruited in Blantyre, Malawi, from 2000 to 2004. Patients were women whose children were HIV-1 DNA-positive at birth (in-utero MTCT) or HIV-1 DNA-negative at birth and HIV-1 DNA-positive at 6 weeks postpartum (intrapartum MTCT); controls were women whose children were HIV-1 DNA-negative both at birth and 6 weeks postpartum. After delivery, blood was isolated from an incision on the basal plate of the placenta. We used a Bio-Plex human cytokine assay (Bio-Rad, Hercules, California USA) to simultaneously quantify 27 cytokines, chemokines and growth factors in placental plasma. HIV-1 RNA copies were quantified with the Roche Amplicor kit. Levels of interleukin (IL) 4, IL-5, IL-6, IL-7, IL-9, eotaxin, IL-1Ra and interferon gamma-induced protein 10 (IP-10) were significantly elevated in placental plasma isolated from cases of in-utero HIV-1 MTCT. In contrast, only granulocyte colony-stimulating factor was elevated in placental plasma isolated from cases of intrapartum MTCT. After adjusting for maternal age, gestational age and peripheral CD4(+) T-cell count, every log(10) increase in placental IP-10 was associated with a three-fold increase in the prevalence of in-utero HIV-1 MTCT. Elevated cytokine and chemokine levels in placental plasma were associated with in-utero and not intrapartum MTCT. IP-10, which is both a T-cell chemokine and potentiator of HIV-replication, was robustly and independently associated with prevalent, in-utero MTCT.

Performance Characteristics of the QUANTIPLEX HIV-1 RNA 3.0 Assay for Detection and Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma

PubMed Central

Erice, Alejo; Brambilla, Donald; Bremer, James; Jackson, J. Brooks; Kokka, Robert; Yen-Lieberman, Belinda; Coombs, Robert W.

2000-01-01

The QUANTIPLEX HIV-1 RNA assay, version 3.0 (a branched DNA, version 3.0, assay [bDNA 3.0 assay]), was evaluated by analyzing spiked and clinical plasma samples and was compared with the AMPLICOR HIV-1 MONITOR Ultrasensitive (ultrasensitive reverse transcription-PCR [US-RT-PCR]) method. A panel of spiked plasma samples that contained 0 to 750,000 copies of human immunodeficiency virus type 1 (HIV-1) RNA per ml was tested four times in each of four laboratories (1,344 assays). Negative results (<50 copies/ml) were obtained in 30 of 32 (94%) assays with seronegative samples, 66 of 128 (52%) assays with HIV-1 RNA at 50 copies/ml, and 5 of 128 (4%) assays with HIV-1 RNA at 100 copies/ml. The assay was linear from 100 to 500,000 copies/ml. The within-run standard deviation (SD) of the log10 estimated HIV-1 RNA concentration was 0.08 at 1,000 to 500,000 copies/ml, increased below 1,000 copies/ml, and was 0.17 at 100 copies/ml. Between-run reproducibility at 100 to 500 copies/ml was <0.10 log10 in most comparisons. Interlaboratory differences across runs were ≤0.10 log10 at all concentrations examined. A subset of the panel (25 to 500 copies/ml) was also analyzed by the US-RT-PCR assay. The within-run SD varied inversely with the log10 HIV-1 RNA concentration but was higher than the SD for the bDNA 3.0 assay at all concentrations. Log-log regression analysis indicated that the two methods produced very similar estimates at 100 to 500 copies/ml. In parallel testing of clinical specimens with low HIV-1 RNA levels, 80 plasma samples with <50 copies/ml by the US-RT-PCR assay had <50 copies/ml when they were retested by the bDNA 3.0 assay. In contrast, 11 of 78 (14%) plasma samples with <50 copies/ml by the bDNA 3.0 assay had ≥50 copies/ml when they were retested by the US-RT-PCR assay (median, 86 copies/ml; range, 50 to 217 copies/ml). Estimation of bDNA 3.0 values of <50 copies/ml by extending the standard curve of the assay showed that these samples with discrepant results had higher HIV-1 RNA levels than the samples with concordant results (median, 34 versus 17 copies/ml; P = 0.0051 by the Wilcoxon two-sample test). The excellent reproducibility, broad linear range, and good sensitivity of the bDNA 3.0 assay make it a very attractive method for quantitation of HIV-1 RNA levels in plasma. PMID:10921936

Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells

PubMed Central

Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R; Hosmane, Nina N; Capoferri, Adam A; Bruner, Katherine M; Pollack, Ross A; Zhang, Hao; Drummond, Michael Bradley; Siliciano, Janet M; Siliciano, Robert; Stivers, James T

2016-01-01

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection. DOI: http://dx.doi.org/10.7554/eLife.18447.001 PMID:27644592

Impact of monotherapy on HIV-1 reservoir, immune activation, and co-infection with Epstein-Barr virus

PubMed Central

Petrara, Maria Raffaella; Cattelan, Anna Maria; Sasset, Lolita; Freguja, Riccardo; Carmona, Francesco; Sanavia, Silvia; Zanchetta, Marisa; Del Bianco, Paola

2017-01-01

Objectives Although monotherapy (mART) effectiveness in maintaining viral suppression and CD4 cell count has been extensively examined in HIV-1-infected patients, its impact on HIV-1 reservoir, immune activation, microbial translocation and co-infection with Epstein-Barr Virus (EBV) is unclear. Methods This retrospective study involved 32 patients who switched to mART; patients were studied at baseline, 48 and 96 weeks after mART initiation. Thirty-two patients who continued combined antiretroviral therapy (cART) over the same period of time were included in the study. Markers of HIV-1 reservoir (HIV-1 DNA and intracellular HIV-1 RNA) were quantified by real-time PCR. Markers of T-(CD3+CD8+CD38+) and B-(CD19+CD80/86+ and CD19+CD10-CD21lowCD27+) cell activation were evaluated by flow cytometry. Plasma levels of microbial translocation markers were quantified by real-time PCR (16S ribosomal DNA and mitochondrial [mt]DNA) or by ELISA (LPS and sCD14). EBV was typed and quantified by multiplex real-time PCR. Results At baseline, no differences were found between mART and cART groups. Three (10%) mART-treated patients had a virological failure vs none in the cART group. Levels of HIV-1 DNA, intracellular HIV-1 RNA and EBV-DNA remained stable in the mART group, while decreased significantly in the cART group. Percentages of T- and B-activated cells significantly increased in the mART-treated patients, while remained at low levels in the cART-treated ones (p = 0.014 and p<0.001, respectively). Notably, levels of mtDNA remained stable in the cART group, but significantly rose in the mART one (p<0.001). Conclusions Long-term mART is associated with higher levels of T- and B-cell activation and, conversely to cART, does not reduce the size of HIV-1 reservoir and EBV co-infection. PMID:28926641

Comparative evaluation of the Cobas Amplicor HIV-1 Monitor Ultrasensitive Test, the new Cobas AmpliPrep/Cobas Amplicor HIV-1 Monitor Ultrasensitive Test and the Versant HIV RNA 3.0 assays for quantitation of HIV-1 RNA in plasma samples.

PubMed

Berger, Annemarie; Scherzed, Lina; Stürmer, Martin; Preiser, Wolfgang; Doerr, Hans Wilhelm; Rabenau, Holger Felix

2005-05-01

There are several commercially available assays for the quantitation of HIV RNA. A new automated specimen preparation system, the Cobas AmpliPrep, was developed to automate this last part of the PCR. We compared the results obtained by the Roche Cobas Amplicor HIV-1 Monitor Ultrasensitive Test (MCA, manual sample preparation) with those by the Versant HIV-1 RNA 3.0 assay (bDNA). Secondly we compared the MCA with the new Cobas AmpliPrep/Cobas Amplicor HIV Monitor Ultrasensitive Test (CAP/CA, automated specimen preparation) by investigating clinical patient samples and a panel of HIV-1 non-B subtypes. Furthermore, we assessed the assay throughput and workflow (especially hands-on time) for all three assays. Seventy-two percent of the 140 investigated patient samples gave concordant results in the bDNA and MCA assays. The MCA values were regularly higher than the bDNA values. One sample was detected only by the MCA within the linear range of quantification. In contrast, 38 samples with results <50 copies/ml in the MCA showed in the bDNA results between 51 and 1644 copies/ml (mean value 74 copies/ml); 21 of these specimens were shown to have detectable HIV RNA < 50 copies/ml in the MCA assay. The overall agreement between the MCA and the CAP/CA was 94.3% (551/584). The quantification results showed significant correlation, although the CAP/CA generated values slightly lower than those generated by the manual procedure. We found that the CAP/CA produced comparable results with the MCA test in a panel of HIV-1 non-B subtypes. All three assays showed comparable results. The bDNA provides a high sample throughput without the need of full automation. The new CAP/CA provides reliable test results with no HIV-subtype specific influence and releases time for other works in the laboratory; thus it is suitable for routine diagnostic PCR.

TaqMan RT-PCR and VERSANT HIV-1 RNA 3.0 (bDNA) assay Quantification of HIV-1 RNA viral load in breast milk.

PubMed

Israel-Ballard, Kiersten; Ziermann, Rainer; Leutenegger, Christian; Di Canzio, James; Leung, Kimmy; Strom, Lynn; Abrams, Barbara; Chantry, Caroline

2005-12-01

Transmission of HIV via breast milk is a primary cause of pediatric HIV infection in developing countries. Reliable methods to detect breast milk viral load are important. To correlate the ability of the VERSANT HIV 3.0 (bDNA) assay to real-time (RT) TaqMan PCR in quantifying breast milk HIV-1 RNA. Forty-six breast milk samples that had been spiked with cell-free HIV-1 and eight samples spiked with cell-associated HIV-1 were assayed for HIV-1 RNA by both VERSANT HIV 3.0 and TaqMan RNA assays. Only assays on the cell-free samples were statistically compared. Both a Deming regression slope and a Bland-Altman slope indicated a linear relationship between the two assays. TaqMan quantitations were on average 2.6 times higher than those of HIV 3.0. A linear relationship was observed between serial dilutions of spiked cell-free HIV-1 and both the VERSANT HIV 3.0 and the TaqMan RNA assays. The two methods correlated well although the VERSANT HIV 3.0 research protocol quantified HIV-1 RNA slightly lower than TaqMan.

Conserved Elements Vaccine for HIV | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the National Cancer Institute (NCI) developed a DNA vaccine using conserved elements of HIV-1 Gag, administered in a prime-boost vaccination protocol. Two of the HIV Gag CE DNA vectors have been tested in a rhesus macaque model. Priming with the Gag CE vaccine and boosting with full length Gag DNA showed increased immune responses when compared to vaccination with Gag alone. Researchers seek licensing and/or co-development research collaborations for development this DNA vaccine.

Suppression of APOBEC3-mediated restriction of HIV-1 by Vif

PubMed Central

Feng, Yuqing; Baig, Tayyba T.; Love, Robin P.; Chelico, Linda

2014-01-01

The APOBEC3 restriction factors are a family of deoxycytidine deaminases that are able to suppress replication of viruses with a single-stranded DNA intermediate by inducing mutagenesis and functional inactivation of the virus. Of the seven human APOBEC3 enzymes, only APOBEC3-D, -F, -G, and -H appear relevant to restriction of HIV-1 in CD4+ T cells and will be the focus of this review. The restriction of HIV-1 occurs most potently in the absence of HIV-1 Vif that induces polyubiquitination and degradation of APOBEC3 enzymes through the proteasome pathway. To restrict HIV-1, APOBEC3 enzymes must be encapsidated into budding virions. Upon infection of the target cell during reverse transcription of the HIV-1 RNA into (-)DNA, APOBEC3 enzymes deaminate cytosines to form uracils in single-stranded (-)DNA regions. Upon replication of the (-)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite to the uracils thereby inducing C/G to T/A mutations that can functionally inactivate HIV-1. APOBEC3G is the most studied APOBEC3 enzyme and it is known that Vif attempts to thwart APOBEC3 function not only by inducing its proteasomal degradation but also by several degradation-independent mechanisms, such as inhibiting APOBEC3G virion encapsidation, mRNA translation, and for those APOBEC3G molecules that still become virion encapsidated, Vif can inhibit APOBEC3G mutagenic activity. Although most Vif variants can induce efficient degradation of APOBEC3-D, -F, and -G, there appears to be differential sensitivity to Vif-mediated degradation for APOBEC3H. This review examines APOBEC3-mediated HIV restriction mechanisms, how Vif acts as a substrate receptor for a Cullin5 ubiquitin ligase complex to induce degradation of APOBEC3s, and the determinants and functional consequences of the APOBEC3 and Vif interaction from a biological and biochemical perspective. PMID:25206352

Influenza vaccination of HIV-1-positive and HIV-1-negative former intravenous drug users.

PubMed

Amendola, A; Boschini, A; Colzani, D; Anselmi, G; Oltolina, A; Zucconi, R; Begnini, M; Besana, S; Tanzi, E; Zanetti, A R

2001-12-01

The immunogenicity of an anti-influenza vaccine was assessed in 409 former intravenous drug user volunteers and its effect on the levels of HIV-1 RNA, proviral DNA and on CD4+ lymphocyte counts in a subset HIV-1-positive subjects was measured. HIV-1-positive individuals (n = 72) were divided into three groups on the basis of their CD4+ lymphocyte counts, while the 337 HIV-1-negative participants were allocated into group four. Haemagglutination inhibiting (HI) responses varied from 45.8 to 70% in the HIV-1-positive subjects and were significantly higher in group four (80.7% responses to the H1N1 strain, 81.6% to the H3N2 strain, and 83% to the B strain). The percentage of subjects with HI protective antibody titres (> or = 1:40) increased significantly after vaccination, especially in HIV-1 uninfected subjects. Immunization caused no significant changes in CD4+ counts and in neither plasma HIV-1 RNA nor proviral DNA levels. Therefore, vaccination against influenza may benefit persons infected by HIV-1. Copyright 2001 Wiley-Liss, Inc.

Keeping your armour intact: how HIV-1 evades detection by the innate immune system: HIV-1 capsid controls detection of reverse transcription products by the cytosolic DNA sensor cGAS.

PubMed

Maelfait, Jonathan; Seiradake, Elena; Rehwinkel, Jan

2014-07-01

HIV-1 infects dendritic cells (DCs) without triggering an effective innate antiviral immune response. As a consequence, the induction of adaptive immune responses controlling virus spread is limited. In a recent issue of Immunity, Lahaye and colleagues show that intricate interactions of HIV capsid with the cellular cofactor cyclophilin A (CypA) control infection and innate immune activation in DCs. Manipulation of HIV-1 capsid to increase its affinity for CypA results in reduced virus infectivity and facilitates access of the cytosolic DNA sensor cGAS to reverse transcribed DNA. This in turn induces a strong host response. Here, we discuss these findings in the context of recent developments in innate immunity and consider the implications for disease control and vaccine design. © 2014 The Authors. Bioessays published by WILEY Periodicals, Inc.

Evaluation of the clinical sensitivity for the quantification of human immunodeficiency virus type 1 RNA in plasma: Comparison of the new COBAS TaqMan HIV-1 with three current HIV-RNA assays--LCx HIV RNA quantitative, VERSANT HIV-1 RNA 3.0 (bDNA) and COBAS AMPLICOR HIV-1 Monitor v1.5.

PubMed

Katsoulidou, Antigoni; Petrodaskalaki, Maria; Sypsa, Vana; Papachristou, Eleni; Anastassopoulou, Cleo G; Gargalianos, Panagiotis; Karafoulidou, Anastasia; Lazanas, Marios; Kordossis, Theodoros; Andoniadou, Anastasia; Hatzakis, Angelos

2006-02-01

The COBAS TaqMan HIV-1 test (Roche Diagnostics) was compared with the LCx HIV RNA quantitative assay (Abbott Laboratories), the Versant HIV-1 RNA 3.0 (bDNA) assay (Bayer) and the COBAS Amplicor HIV-1 Monitor v1.5 test (Roche Diagnostics), using plasma samples of various viral load levels from HIV-1-infected individuals. In the comparison of TaqMan with LCx, TaqMan identified as positive 77.5% of the 240 samples versus 72.1% identified by LCx assay, while their overall agreement was 94.6% and the quantitative results of samples that were positive by both methods were strongly correlated (r=0.91). Similarly, in the comparison of TaqMan with bDNA 3.0, both methods identified 76.3% of the 177 samples as positive, while their overall agreement was 95.5% and the quantitative results of samples that were positive by both methods were strongly correlated (r=0.95). Finally, in the comparison of TaqMan with Monitor v1.5, TaqMan identified 79.5% of the 156 samples as positive versus 80.1% identified by Monitor v1.5, while their overall agreement was 95.5% and the quantitative results of samples that were positive by both methods were strongly correlated (r=0.96). In conclusion, the new COBAS TaqMan HIV-1 test showed excellent agreement with other widely used commercially available tests for the quantitation of HIV-1 viral load.

Quantitative and Cost Comparison of Ultrasensitive Human Immunodeficiency Virus Type 1 RNA Viral Load Assays: Bayer bDNA Quantiplex Versions 3.0 and 2.0 and Roche PCR Amplicor Monitor Version 1.5

PubMed Central

Elbeik, Tarek; Charlebois, Edwin; Nassos, Patricia; Kahn, James; Hecht, Frederick M.; Yajko, David; Ng, Valerie; Hadley, Keith

2000-01-01

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA as a measure of viral load has greatly improved the monitoring of therapies for infected individuals. With the significant reductions in viral load now observed in individuals treated with highly active anti-retroviral therapy (HAART), viral load assays have been adapted to achieve greater sensitivity. Two commercially available ultrasensitive assays, the Bayer Quantiplex HIV-1 bDNA version 3.0 (bDNA 3.0) assay and the Roche Amplicor HIV-1 Monitor Ultrasensitive version 1.5 (Amplicor 1.5) assay, are now being used to monitor HIV-1-infected individuals. Both of these ultrasensitive assays have a reported lower limit of 50 HIV-1 RNA copies/ml and were developed from corresponding older generation assays with lower limits of 400 to 500 copies/ml. However, the comparability of viral load data generated by these ultrasensitive assays and the relative costs of labor, disposables, and biohazardous wastes were not determined in most cases. In this study, we used matched clinical plasma samples to compare the quantification of the newer bDNA 3.0 assay with that of the older bDNA 2.0 assay and to compare the quantification and costs of the bDNA 3.0 assay and the Amplicor 1.5 assay. We found that quantification by the bDNA 3.0 assay was approximately twofold higher than that by the bDNA 2.0 assay and was highly correlated to that by the Amplicor 1.5 assay. Moreover, cost analysis based on labor, disposables, and biohazardous wastes showed significant savings with the bDNA 3.0 assay as compared to the costs of the Amplicor 1.5 assay. PMID:10699005

Quantitative and cost comparison of ultrasensitive human immunodeficiency virus type 1 RNA viral load assays: Bayer bDNA quantiplex versions 3.0 and 2.0 and Roche PCR Amplicor monitor version 1.5.

PubMed

Elbeik, T; Charlebois, E; Nassos, P; Kahn, J; Hecht, F M; Yajko, D; Ng, V; Hadley, K

2000-03-01

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA as a measure of viral load has greatly improved the monitoring of therapies for infected individuals. With the significant reductions in viral load now observed in individuals treated with highly active anti-retroviral therapy (HAART), viral load assays have been adapted to achieve greater sensitivity. Two commercially available ultrasensitive assays, the Bayer Quantiplex HIV-1 bDNA version 3.0 (bDNA 3.0) assay and the Roche Amplicor HIV-1 Monitor Ultrasensitive version 1.5 (Amplicor 1.5) assay, are now being used to monitor HIV-1-infected individuals. Both of these ultrasensitive assays have a reported lower limit of 50 HIV-1 RNA copies/ml and were developed from corresponding older generation assays with lower limits of 400 to 500 copies/ml. However, the comparability of viral load data generated by these ultrasensitive assays and the relative costs of labor, disposables, and biohazardous wastes were not determined in most cases. In this study, we used matched clinical plasma samples to compare the quantification of the newer bDNA 3.0 assay with that of the older bDNA 2.0 assay and to compare the quantification and costs of the bDNA 3.0 assay and the Amplicor 1.5 assay. We found that quantification by the bDNA 3.0 assay was approximately twofold higher than that by the bDNA 2.0 assay and was highly correlated to that by the Amplicor 1.5 assay. Moreover, cost analysis based on labor, disposables, and biohazardous wastes showed significant savings with the bDNA 3.0 assay as compared to the costs of the Amplicor 1.5 assay.

On the use of the KMR unintegrated parton distribution functions

NASA Astrophysics Data System (ADS)

Golec-Biernat, Krzysztof; Staśto, Anna M.

2018-06-01

We discuss the unintegrated parton distribution functions (UPDFs) introduced by Kimber, Martin and Ryskin (KMR), which are frequently used in phenomenological analyses of hard processes with transverse momenta of partons taken into account. We demonstrate numerically that the commonly used differential definition of the UPDFs leads to erroneous results for large transverse momenta. We identify the reason for that, being the use of the ordinary PDFs instead of the cutoff dependent distribution functions. We show that in phenomenological applications, the integral definition of the UPDFs with the ordinary PDFs can be used.

HIV dynamics linked to memory CD4+ T cell homeostasis.

PubMed

Murray, John M; Zaunders, John; Emery, Sean; Cooper, David A; Hey-Nguyen, William J; Koelsch, Kersten K; Kelleher, Anthony D

2017-01-01

The dynamics of latent HIV is linked to infection and clearance of resting memory CD4+ T cells. Infection also resides within activated, non-dividing memory cells and can be impacted by antigen-driven and homeostatic proliferation despite suppressive antiretroviral therapy (ART). We investigated whether plasma viral level (pVL) and HIV DNA dynamics could be explained by HIV's impact on memory CD4+ T cell homeostasis. Median total, 2-LTR and integrated HIV DNA levels per μL of peripheral blood, for 8 primary (PHI) and 8 chronic HIV infected (CHI) individuals enrolled on a raltegravir (RAL) based regimen, exhibited greatest changes over the 1st year of ART. Dynamics slowed over the following 2 years so that total HIV DNA levels were equivalent to reported values for individuals after 10 years of ART. The mathematical model reproduced the multiphasic dynamics of pVL, and levels of total, 2-LTR and integrated HIV DNA in both PHI and CHI over 3 years of ART. Under these simulations, residual viremia originated from reactivated latently infected cells where most of these cells arose from clonal expansion within the resting phenotype. Since virion production from clonally expanded cells will not be affected by antiretroviral drugs, simulations of ART intensification had little impact on pVL. HIV DNA decay over the first year of ART followed the loss of activated memory cells (120 day half-life) while the 5.9 year half-life of total HIV DNA after this point mirrored the slower decay of resting memory cells. Simulations had difficulty reproducing the fast early HIV DNA dynamics, including 2-LTR levels peaking at week 12, and the later slow loss of total and 2-LTR HIV DNA, suggesting some ongoing infection. In summary, our modelling indicates that much of the dynamical behavior of HIV can be explained by its impact on memory CD4+ T cell homeostasis.

Subcutaneous administration CpG-ODNs acts as a potent adjuvant for an HIV-1-tat-based vaccine candidate to elicit cellular immunity in BALB/c mice.

PubMed

Panahi, Zeinab; Abdoli, Asghar; Mosayebi, Ghasem; Mahdavi, Mehdi; Bahrami, Fariborz

2018-03-01

To evaluate the combined effects of CpG oligodeoxynucleotides (CpG-ODNs) adjuvant and subcutaneous injection route on efficacy of a HIV-1-tat DNA vaccine candidate using BALB/c mice as an animal model. Evaluation of cellular and humoral immunity of mice injected subcutaneously with HIV-1-tat gene cloned into a pcDNA3.1 vector indicated that significant levels of IFN-γ cytokine secretion (900 pg/ml), lymphocyte proliferation (2.5 stimulation index) and IgG 2a (1.45 absorbance 450 nm) production could be achieved. These indicators of stimulated cellular immunity were elicited 2 weeks after the last injection (P < 0.05). Formulation of HIV-1-tat DNA vaccine candidate with CpG-ODNs as an adjuvant while administrated subcutaneously are a promising approach to induce effective cellular immunity responses against HIV-1 infection.

New Highly Sensitive Real-Time PCR Assay for HIV-2 Group A and Group B DNA Quantification.

PubMed

Bertine, Mélanie; Gueudin, Marie; Mélard, Adeline; Damond, Florence; Descamps, Diane; Matheron, Sophie; Collin, Fidéline; Rouzioux, Christine; Plantier, Jean-Christophe; Avettand-Fenoel, Véronique

2017-09-01

HIV-2 infection is characterized by a very low replication rate in most cases and low progression. This necessitates an approach to patient monitoring that differs from that for HIV-1 infection. Here, a new highly specific and sensitive method for HIV-2 DNA quantification was developed. The new test is based on quantitative real-time PCR targeting the long terminal repeat (LTR) and gag regions and using an internal control. Analytical performance was determined in three laboratories, and clinical performance was determined on blood samples from 63 patients infected with HIV-2 group A ( n = 35) or group B ( n = 28). The specificity was 100%. The 95% limit of detection was three copies/PCR and the limit of quantification was six copies/PCR. The within-run coefficients of variation were between 1.03% at 3.78 log 10 copies/PCR and 27.02% at 0.78 log 10 copies/PCR. The between-run coefficient of variation was 5.10%. Both manual and automated nucleic acid extraction methods were validated. HIV-2 DNA loads were detectable in blood cells from all 63 patients. When HIV-2 DNA was quantifiable, median loads were significantly higher in antiretroviral-treated than in naive patients and were similar for groups A and B. HIV-2 DNA load was correlated with HIV-2 RNA load ( r = 0.68; 95% confidence interval [CI], 0.4 to 0.8; P < 0.0001). Our data show that this new assay is highly sensitive and quantifies the two main HIV-2 groups, making it useful for the diagnosis of HIV-2 infection and for pathogenesis studies on HIV-2 reservoirs. Copyright © 2017 American Society for Microbiology.

The Frequency of Cytidine Editing of Viral DNA Is Differentially Influenced by Vpx and Nucleosides during HIV-1 or SIVMAC Infection of Dendritic Cells

PubMed Central

Nguyen, Xuan-Nhi; Barateau, Véronique; Wu, Nannan; Berger, Gregory; Cimarelli, Andrea

2015-01-01

Two cellular factors are currently known to modulate lentiviral infection specifically in myeloid cells: SAMHD1 and APOBEC3A (A3A). SAMHD1 is a deoxynucleoside triphosphohydrolase that interferes with viral infection mostly by limiting the intracellular concentrations of dNTPs, while A3A is a cytidine deaminase that has been described to edit incoming vDNA. The restrictive phenotype of myeloid cells can be alleviated through the direct degradation of SAMHD1 by the HIV-2/SIVSM Vpx protein or else, at least in the case of HIV-1, by the exogenous supplementation of nucleosides that artificially overcome the catabolic activity of SAMHD1 on dNTPs. Here, we have used Vpx and dNs to explore the relationship existing between vDNA cytidine deamination and SAMHD1 during HIV-1 or SIVMAC infection of primary dendritic cells. Our results reveal an interesting inverse correlation between conditions that promote efficient infection of DCs and the extent of vDNA editing that may reflect the different susceptibility of vDNA to cytoplasmic effectors during the infection of myeloid cells. PMID:26496699

Hepatitis B Virus Vaccination in HIV-1-Infected Young Adults: A Tool to Reduce the Size of HIV-1 Reservoirs?

PubMed

Bekele, Yonas; Graham, Rebecka Lantto; Soeria-Atmadja, Sandra; Nasi, Aikaterini; Zazzi, Maurizio; Vicenti, Ilaria; Naver, Lars; Nilsson, Anna; Chiodi, Francesca

2017-01-01

During anti-retroviral therapy (ART) HIV-1 persists in cellular reservoirs, mostly represented by CD4+ memory T cells. Several approaches are currently being undertaken to develop a cure for HIV-1 infection through elimination (or reduction) of these reservoirs. Few studies have so far been conducted to assess the possibility of reducing the size of HIV-1 reservoirs through vaccination in virologically controlled HIV-1-infected children. We recently conducted a vaccination study with a combined hepatitis A virus (HAV) and hepatitis B virus (HBV) vaccine in 22 HIV-1-infected children. We assessed the size of the virus reservoir, measured as total HIV-1 DNA copies in blood cells, pre- and postvaccination. In addition, we investigated by immunostaining whether the frequencies of CD4+ and CD8+ T cells and parameters of immune activation and proliferation on these cells were modulated by vaccination. At 1 month from the last vaccination dose, we found that 20 out of 22 children mounted a serological response to HBV; a majority of children had antibodies against HAV at baseline. The number of HIV-1 DNA copies in blood at 1 month postvaccination was reduced in comparison to baseline although this reduction was not statistically significant. A significant reduction of HIV-1 DNA copies in blood following vaccination was found in 12 children. The frequencies of CD4+ (naïve, effector memory) and CD8+ (central memory) T-cell subpopulations changed following vaccinations and a reduction in the activation and proliferation pattern of these cells was also noticed. Multivariate linear regression analysis revealed that the frequency of CD8+ effector memory T cells prior to vaccination was strongly predictive of the reduction of HIV-1 DNA copies in blood following vaccination of the 22 HIV-1-infected children. The results of this study suggest a beneficial effect of vaccination to reduce the size of virus reservoir in HIV-1-infected children receiving ART. A reduced frequency of activated CD4+ cells and an increase in central memory CD8+ T cells were associated with this finding. Further studies should assess whether vaccination is a possible tool to reduce HIV-1 reservoirs.

Hepatitis B Virus Vaccination in HIV-1-Infected Young Adults: A Tool to Reduce the Size of HIV-1 Reservoirs?

PubMed Central

Bekele, Yonas; Graham, Rebecka Lantto; Soeria-Atmadja, Sandra; Nasi, Aikaterini; Zazzi, Maurizio; Vicenti, Ilaria; Naver, Lars; Nilsson, Anna; Chiodi, Francesca

2018-01-01

During anti-retroviral therapy (ART) HIV-1 persists in cellular reservoirs, mostly represented by CD4+ memory T cells. Several approaches are currently being undertaken to develop a cure for HIV-1 infection through elimination (or reduction) of these reservoirs. Few studies have so far been conducted to assess the possibility of reducing the size of HIV-1 reservoirs through vaccination in virologically controlled HIV-1-infected children. We recently conducted a vaccination study with a combined hepatitis A virus (HAV) and hepatitis B virus (HBV) vaccine in 22 HIV-1-infected children. We assessed the size of the virus reservoir, measured as total HIV-1 DNA copies in blood cells, pre- and postvaccination. In addition, we investigated by immunostaining whether the frequencies of CD4+ and CD8+ T cells and parameters of immune activation and proliferation on these cells were modulated by vaccination. At 1 month from the last vaccination dose, we found that 20 out of 22 children mounted a serological response to HBV; a majority of children had antibodies against HAV at baseline. The number of HIV-1 DNA copies in blood at 1 month postvaccination was reduced in comparison to baseline although this reduction was not statistically significant. A significant reduction of HIV-1 DNA copies in blood following vaccination was found in 12 children. The frequencies of CD4+ (naïve, effector memory) and CD8+ (central memory) T-cell subpopulations changed following vaccinations and a reduction in the activation and proliferation pattern of these cells was also noticed. Multivariate linear regression analysis revealed that the frequency of CD8+ effector memory T cells prior to vaccination was strongly predictive of the reduction of HIV-1 DNA copies in blood following vaccination of the 22 HIV-1-infected children. The results of this study suggest a beneficial effect of vaccination to reduce the size of virus reservoir in HIV-1-infected children receiving ART. A reduced frequency of activated CD4+ cells and an increase in central memory CD8+ T cells were associated with this finding. Further studies should assess whether vaccination is a possible tool to reduce HIV-1 reservoirs. PMID:29375579

Safety and Immunogenicity of a Recombinant Adenovirus Serotype 35-Vectored HIV-1 Vaccine in Adenovirus Serotype 5 Seronegative and Seropositive Individuals.

PubMed

Fuchs, Jonathan D; Bart, Pierre-Alexandre; Frahm, Nicole; Morgan, Cecilia; Gilbert, Peter B; Kochar, Nidhi; DeRosa, Stephen C; Tomaras, Georgia D; Wagner, Theresa M; Baden, Lindsey R; Koblin, Beryl A; Rouphael, Nadine G; Kalams, Spyros A; Keefer, Michael C; Goepfert, Paul A; Sobieszczyk, Magdalena E; Mayer, Kenneth H; Swann, Edith; Liao, Hua-Xin; Haynes, Barton F; Graham, Barney S; McElrath, M Juliana

2015-05-01

Recombinant adenovirus serotype 5 (rAd5)-vectored HIV-1 vaccines have not prevented HIV-1 infection or disease and pre-existing Ad5 neutralizing antibodies may limit the clinical utility of Ad5 vectors globally. Using a rare Ad serotype vector, such as Ad35, may circumvent these issues, but there are few data on the safety and immunogenicity of rAd35 directly compared to rAd5 following human vaccination. HVTN 077 randomized 192 healthy, HIV-uninfected participants into one of four HIV-1 vaccine/placebo groups: rAd35/rAd5, DNA/rAd5, and DNA/rAd35 in Ad5-seronegative persons; and DNA/rAd35 in Ad5-seropositive persons. All vaccines encoded the HIV-1 EnvA antigen. Antibody and T-cell responses were measured 4 weeks post boost immunization. All vaccines were generally well tolerated and similarly immunogenic. As compared to rAd5, rAd35 was equally potent in boosting HIV-1-specific humoral and cellular immunity and responses were not significantly attenuated in those with baseline Ad5 seropositivity. Like DNA, rAd35 efficiently primed rAd5 boosting. All vaccine regimens tested elicited cross-clade antibody responses, including Env V1/V2-specific IgG responses. Vaccine antigen delivery by rAd35 is well-tolerated and immunogenic as a prime to rAd5 immunization and as a boost to prior DNA immunization with the homologous insert. Further development of rAd35-vectored prime-boost vaccine regimens is warranted.

Nucleocapsid Protein Annealing of a Primer-Template Enhances (+)-Strand DNA Synthesis and Fidelity by HIV-1 Reverse Transcriptase†

PubMed Central

Kim, Jiae; Roberts, Anne; Yuan, Hua; Xiong, Yong; Anderson, Karen S.

2012-01-01

Human immunodeficiency virus type-1 (HIV-1) requires reverse transcriptase (RT) and HIV-1 nucleocapsid protein (NCp7) for proper viral replication. HIV-1 NCp7 has been shown to enhance various steps in reverse transcription including tRNA initiation and strand transfer which may be mediated through interactions with RT as well as RNA and DNA oligonucleotides. With the use of DNA oligonucleotides, we have examined the interaction of NCp7 with RT and the kinetics of reverse transcription during (+)-strand synthesis with an NCp7-facilitated annealed primer-template. Using a pre-steady state kinetics approach, the NCp7-annealed primer-template has a substantial increase (3-7 fold) in the rate of incorporation (kpol) by RT as compared to heat annealed primer-template with single nucleotide incorporation. There was also a 2-fold increase in the binding affinity constant (Kd) of the nucleotide. These differences in kpol and Kd were not through direct interactions between HIV-1 RT and NCp7. When examining extension by RT, the data suggests that the NCp7-annealed primer-template facilitates the formation of a longer product more quickly compared to the heat annealed primer-template. This enhancement in rate is mediated through interactions with NCp7’s zinc fingers and N-terminal domain and nucleic acids. The NCp7-annealed primer-template also enhances the fidelity of RT (3-fold) by slowing the rate of incorporation of an incorrect nucleotide. Taken together, this study elucidates a new role of NCp7 by facilitating DNA-directed DNA synthesis during reverse transcription by HIV-1 RT that may translate into enhanced viral fitness and offers an avenue to exploit for targeted therapeutic intervention against HIV. PMID:22210155

Effect of therapeutic intensification followed by HIV DNA prime and rAd5 boost vaccination on HIV-specific immunity and HIV reservoir (EraMune 02): a multicentre randomised clinical trial.

PubMed

Achenbach, Chad J; Assoumou, Lambert; Deeks, Steven G; Wilkin, Timothy J; Berzins, Baiba; Casazza, Joseph P; Lambert-Niclot, Sidonie; Koup, Richard A; Costagliola, Dominique; Calvez, Vincent; Katlama, Christine; Autran, Brigitte; Murphy, Robert L

2015-03-01

Achievement of a cure for HIV infection might need reactivation of latent virus and improvement of HIV-specific immunity. As an initial step, in this trial we assessed the effect of antiretroviral therapy intensification and immune modulation with a DNA prime and recombinant adenovirus 5 (rAd5) boost vaccine. In this multicentre, randomised, open-label, non-comparative, phase 2 clinical trial, we enrolled eligible adults 18-70 years of age with chronic HIV-1 infection on suppressive antiretroviral therapy with current CD4 count of at least 350 cells per μL and HIV DNA between 10 and 1000 copies per 10(6) peripheral blood mononuclear cells. After an 8 week lead-in of antiretroviral intensification therapy (standard dose raltegravir and dose-adjusted maraviroc based on baseline antiretroviral therapy), patients were randomly assigned (1:1) to receive antiretroviral therapy intensification alone or intensification plus injections of HIV DNA prime vaccine (4 mg VRC-HIVDNA016-00-VP) at weeks 8, 12, and 16, followed by HIV rAd5 boost vaccine (10(10) particle units of VRC-HIVADV014-00-VP) at week 32. Randomisation was computer generated in permuted blocks of six and was stratified by study site. The primary endpoint was a 0·5 log10 or greater decrease in HIV DNA in peripheral blood mononuclear cells at week 56. This study is registered with ClinicalTrials.gov, number NCT00976404. Between Nov 29, 2010, and Oct 28, 2011, we enrolled 28 eligible patients from three academic HIV clinics in the USA. After the 8 week lead-in of antiretroviral intensification therapy, 14 patients were randomly assigned to continue antiretroviral therapy intensification alone and 14 to intensification plus vaccine. Enrolled participants had median CD4 count of 636 cells per μL, median HIV DNA 170 copies per 10(6) peripheral blood mononuclear cells, and duration of antiretroviral therapy of 13 years. The median amount of HIV DNA did not change significantly between baseline and week 56 in the antiretroviral therapy intensification plus vaccine group. One participant in the antiretroviral therapy intensification alone group reached the primary endpoint, with 0·55 log10 decrease in HIV DNA in peripheral blood mononuclear cells. Both treatments were well tolerated. No severe or systemic reactions to vaccination occurred, and five serious adverse events were recorded during the study, most of which resolved spontaneously or were judged unrelated to study treatments. Antiretroviral therapy intensification followed by DNA prime and rAd5 boost vaccine did not significantly increase HIV expression or reduce the latent HIV reservoir. A multifaceted approach that includes stronger activators of HIV expression and novel immune modulators will probably be needed to reduce the latent HIV reservoir and allow for long-term control in patients off antiretroviral therapy. Objectif Recherche Vaccin SIDA (ORVACS). Copyright © 2015 Elsevier Ltd. All rights reserved.

Subtype C gp140 Vaccine Boosts Immune Responses Primed by the South African AIDS Vaccine Initiative DNA-C2 and MVA-C HIV Vaccines after More than a 2-Year Gap

PubMed Central

Mayer, Kenneth H.; Elizaga, Marnie L.; Bekker, Linda-Gail; Allen, Mary; Morris, Lynn; Montefiori, David; De Rosa, Stephen C.; Sato, Alicia; Gu, Niya; Tomaras, Georgia D.; Tucker, Timothy; Barnett, Susan W.; Mkhize, Nonhlanhla N.; Shen, Xiaoying; Downing, Katrina; Williamson, Carolyn; Pensiero, Michael; Corey, Lawrence; Williamson, Anna-Lise

2016-01-01

A phase I safety and immunogenicity study investigated South African AIDS Vaccine Initiative (SAAVI) HIV-1 subtype C (HIV-1C) DNA vaccine encoding Gag-RT-Tat-Nef and gp150, boosted with modified vaccinia Ankara (MVA) expressing matched antigens. Following the finding of partial protective efficacy in the RV144 HIV vaccine efficacy trial, a protein boost with HIV-1 subtype C V2-deleted gp140 with MF59 was added to the regimen. A total of 48 participants (12 U.S. participants and 36 Republic of South Africa [RSA] participants) were randomized to receive 3 intramuscular (i.m.) doses of SAAVI DNA-C2 of 4 mg (months 0, 1, and 2) and 2 i.m. doses of SAAVI MVA-C of 1.45 × 109 PFU (months 4 and 5) (n = 40) or of a placebo (n = 8). Approximately 2 years after vaccination, 27 participants were rerandomized to receive gp140/MF59 at 100 μg or placebo, as 2 i.m. injections, 3 months apart. The vaccine regimen was safe and well tolerated. After the DNA-MVA regimen, CD4+ T-cell and CD8+ T-cell responses occurred in 74% and 32% of the participants, respectively. The protein boost increased CD4+ T-cell responses to 87% of the subjects. All participants developed tier 1 HIV-1C neutralizing antibody responses as well as durable Env binding antibodies that recognized linear V3 and C5 peptides. The HIV-1 subtype C DNA-MVA vaccine regimen showed promising cellular immunogenicity. Boosting with gp140/MF59 enhanced levels of binding and neutralizing antibodies as well as CD4+ T-cell responses to HIV-1 envelope. (This study has been registered at ClinicalTrials.gov under registration no. NCT00574600 and NCT01423825.) PMID:27098021

Template-independent DNA synthesis activity associated with the reverse transcriptase of the long terminal repeat retrotransposon Tf1.

PubMed

Oz-Gleenberg, Iris; Herzig, Eytan; Hizi, Amnon

2012-01-01

Reverse transcriptases (RTs) possess a non-templated addition (NTA) activity while synthesizing DNA with blunt-ended DNA primer/templates. Interestingly, the RT of the long terminal repeat retrotransposon Tf1 has an NTA activity that is substantially higher than that of HIV-1 or murine leukemia virus RTs. By performing steady state kinetics, we found that the differences between the NTA activities of Tf1 and HIV-1 RTs can be explained by the substantially lower K(M) value for the incoming dNTP of Tf1 RT (while the differences between the apparent k(cat) values of these two RTs are relatively small). Furthermore, the K(M) values, calculated for both RTs with the same dNTP, are much lower for the template-dependent synthesis (TDS) than those of NTA. However, TDS of HIV-1 RT is higher than that of Tf1 RT. The overall relative order of the apparent k(cat)/K(M) values for dATP is: HIV-1 RT (TDS) > Tf1 RT (TDS) > Tf1 RT (NTA) > HIV-1 RT (NTA). Under the employed conditions, Tf1 RT can add up to seven nucleotides to the blunt-ended substrate, while the other RTs add mostly a single nucleotide. The NTA activity of Tf1 RT is restricted to DNA primers. Furthermore, the NTA activity of Tf1 and HIV-1 RTs is suppressed by ATP, as it competes with the incoming dATP (although ATP is not incorporated by the NTA activity of the RTs). The unusually high NTA activity of Tf1 RT can explain why, after completing cDNA synthesis, the in vivo generated Tf1 cDNA has relatively long extra sequences beyond the highly conserved CA at its 3'-ends. © 2011 The Authors Journal compilation © 2011 FEBS.

Meticulous plasma isolation is essential to avoid false low-level viraemia in Roche Cobas HIV-1 viral load assays.

PubMed

Mortier, Virginie; Vancoillie, Leen; Dauwe, Kenny; Staelens, Delfien; Demecheleer, Els; Schauvliege, Marlies; Dinakis, Sylvie; Van Maerken, Tom; Dessilly, Géraldine; Ruelle, Jean; Verhofstede, Chris

2017-10-24

Pre-analytical sample processing is often overlooked as a potential cause of inaccurate assay results. Here we demonstrate how plasma, extracted from standard EDTA-containing blood collection tubes, may contain traces of blood cells consequently resulting in a false low-level HIV-1 viral load when using Roche Cobas HIV-1 assays. The presence of human DNA in Roche Cobas 4800 RNA extracts and in RNA extracts from the Abbott HIV-1 RealTime assay was assessed by quantifying the human albumin gene by means of quantitative PCR. RNA was extracted from plasma samples before and after an additional centrifugation and tested for viral load and DNA contamination. The relation between total DNA content and viral load was defined. Elevated concentrations of genomic DNA were detected in 28 out of 100 Cobas 4800 extracts and were significantly more frequent in samples processed outside of the AIDS Reference Laboratory. An association between genomic DNA presence and spurious low-level viraemia results was demonstrated. Supplementary centrifugation of plasma before RNA extraction eliminated the contamination and the false viraemia. Plasma isolated from standard EDTA-containing blood collection tubes may contain traces of HIV DNA leading to false viral load results above the clinical cutoff. Supplementary centrifugation of plasma before viral load analysis may eliminate the occurrence of this spurious low-level viraemia.

Evidence of Subclinical mtDNA Alterations in HIV-Infected Pregnant Women Receiving Combination Antiretroviral Therapy Compared to HIV-Negative Pregnant Women

PubMed Central

Money, Deborah M.; Wagner, Emily C.; Maan, Evelyn J.; Chaworth-Musters, Tessa; Gadawski, Izabelle; van Schalkwyk, Julie E.; Forbes, John C.; Burdge, David R.; Albert, Arianne Y. K.; Lohn, Zoe; Côté, Hélène C. F.

2015-01-01

Introduction Combination antiretroviral therapy (cART) can effectively prevent vertical transmission of HIV but there is potential risk of adverse maternal, foetal or infant effects. Specifically, the effect of cART use during pregnancy on mitochondrial DNA (mtDNA) content in HIV-positive (HIV+) women is unclear. We sought to characterize subclinical alterations in peripheral blood mtDNA levels in cART-treated HIV+ women during pregnancy and the postpartum period. Methods This prospective longitudinal observational cohort study enrolled both HIV+ and HIV-negative (HIV-) pregnant women. Clinical data and blood samples were collected at three time points in pregnancy (13-<23 weeks, 23-<30 weeks, 30–40 weeks), and at delivery and six weeks post-partum in HIV+ women. Peripheral blood mtDNA to nuclear DNA (nDNA) ratio was measured by qPCR. Results Over a four year period, 63 HIV+ and 42 HIV- women were enrolled. HIV+ women showed significantly lower mtDNA/nDNA ratios compared to HIV- women during pregnancy (p = 0.003), after controlling for platelet count and repeated measurements using a multivariable mixed-effects model. Ethnicity, gestational age (GA) and substance use were also significantly associated with mtDNA/nDNA ratio (p≤0.02). Among HIV+ women, higher CD4 nadir was associated with higher mtDNA/nDNA ratios (p<0.0001), and these ratio were significantly lower during pregnancy compared to the postpartum period (p<0.0001). Conclusions In the context of this study, it was not possible to distinguish between mtDNA effects related to HIV infection versus cART therapy. Nevertheless, while mtDNA levels were relatively stable over time in both groups during pregnancy, they were significantly lower in HIV+ women compared to HIV- women. Although no immediate clinical impact was observed on maternal or infant health, lower maternal mtDNA levels may exert long-term effects on women and children and remain a concern. Improved knowledge of such subclinical alterations is another step toward optimizing the safety and efficacy of cART regimens during pregnancy. PMID:26247211

Evidence of Subclinical mtDNA Alterations in HIV-Infected Pregnant Women Receiving Combination Antiretroviral Therapy Compared to HIV-Negative Pregnant Women.

PubMed

Money, Deborah M; Wagner, Emily C; Maan, Evelyn J; Chaworth-Musters, Tessa; Gadawski, Izabelle; van Schalkwyk, Julie E; Forbes, John C; Burdge, David R; Albert, Arianne Y K; Lohn, Zoe; Côté, Hélène C F

2015-01-01

Combination antiretroviral therapy (cART) can effectively prevent vertical transmission of HIV but there is potential risk of adverse maternal, foetal or infant effects. Specifically, the effect of cART use during pregnancy on mitochondrial DNA (mtDNA) content in HIV-positive (HIV+) women is unclear. We sought to characterize subclinical alterations in peripheral blood mtDNA levels in cART-treated HIV+ women during pregnancy and the postpartum period. This prospective longitudinal observational cohort study enrolled both HIV+ and HIV-negative (HIV-) pregnant women. Clinical data and blood samples were collected at three time points in pregnancy (13-<23 weeks, 23-<30 weeks, 30-40 weeks), and at delivery and six weeks post-partum in HIV+ women. Peripheral blood mtDNA to nuclear DNA (nDNA) ratio was measured by qPCR. Over a four year period, 63 HIV+ and 42 HIV- women were enrolled. HIV+ women showed significantly lower mtDNA/nDNA ratios compared to HIV- women during pregnancy (p = 0.003), after controlling for platelet count and repeated measurements using a multivariable mixed-effects model. Ethnicity, gestational age (GA) and substance use were also significantly associated with mtDNA/nDNA ratio (p≤0.02). Among HIV+ women, higher CD4 nadir was associated with higher mtDNA/nDNA ratios (p<0.0001), and these ratio were significantly lower during pregnancy compared to the postpartum period (p<0.0001). In the context of this study, it was not possible to distinguish between mtDNA effects related to HIV infection versus cART therapy. Nevertheless, while mtDNA levels were relatively stable over time in both groups during pregnancy, they were significantly lower in HIV+ women compared to HIV- women. Although no immediate clinical impact was observed on maternal or infant health, lower maternal mtDNA levels may exert long-term effects on women and children and remain a concern. Improved knowledge of such subclinical alterations is another step toward optimizing the safety and efficacy of cART regimens during pregnancy.

Longitudinal studies on maternal HIV-1 variants by biological phenotyping, sequence analysis and viral load.

PubMed

Renta, J Y; Cadilla, C L; Vega, M E; Hillyer, G V; Estrada, C; Jiménez, E; Abreu, E; Méndez, I; Gandía, J; Meléndez-Guerrero, L M

1997-11-01

In this study, the HIV-1 variant viruses from ten pregnant women and their infants were isolated and characterized longitudinally in order to determine the role that viral envelope (gp120-V3 loop) gene variation and viral tropism play in vertical transmission. Biological phenotyping of each HIV variant was accomplished by growth in MT-2, and macrophages from healthy and non-HIV-infected donors. Genetic characterization of the variants was accomplished by DNA sequence analysis. All the women enrolled in this study received ZDV therapy. Virus was cultured from eight out of ten env V3-PCR positive mothers. HIV-1 isolates were all non-syncitium inducing variants. None of the mothers were found to transmit HIV, as determined by DNA PCR and quantitative co-cultures on their infants which were seronegative for HIV-1 through one year after birth. Viral cultures from infant blood samples were negative and infants were all healthy. However, nested env V3-PCR detected proviral DNA in five out of ten infants. In contrast, conventional gag-PCR was negative in the same five infants. Sequences of the five maternal-infant pairs were different, suggesting unique infant HIV-1 variants. The three highest maternal viral load values corresponded to infants that were env V3-PCR positive. These results suggest that HIV-1 particles are transmitted from ZDV-treated mothers to infants. Infant follow up is recommended to determine if HIV-1 has been inhibited by the immune system of the infants.

Decreased mitochondrial DNA content in subcutaneous fat from HIV-infected women taking antiretroviral therapy as measured at delivery.

PubMed

Nasi, Milena; Pinti, Marcello; Chiesa, Elisabetta; Fiore, Simona; Manzini, Serena; Del Giovane, Cinzia; D'Amico, Roberto; Palai, Nicoletta; Campatelli, Carlo; Sabbatini, Francesca; Roccio, Marianna; Tibaldi, Cecilia; Masuelli, Giulia; Mussini, Cristina; Ferrazzi, Enrico; d'Arminio Monforte, Antonella; Cossarizza, Andrea

2011-01-01

Increasing numbers of pregnant HIV-positive women are receiving combination antiretroviral regimens for preventing mother-to-child virus transmission or for treating the infection itself. Several studies have demonstrated that nucleoside reverse transcriptase inhibitors (NRTIs) induce mitochondrial toxicity by several mechanisms, including depletion of mitochondrial DNA (mtDNA). By the quantification of mtDNA levels, we studied mitochondrial toxicity in HIV-positive women at delivery and the possible correlations with antiretroviral regimens, viroimmunological and metabolic parameters. We analysed 68 HIV-positive women enrolled in the Italian Prospective Cohort Study on Efficacy and Toxicity of Antiretroviral in Pregnancy (TARGET Study); all were taking ≥1 NRTI. We quantified mtDNA copies per cell in subcutaneous fat samples collected during delivery. At the 3rd, 6th and 9th month of pregnancy, we collected data concerning CD4(+) T-cell count, plasma HIV RNA, total and high-density lipoprotein (HDL) cholesterol, fasting plasma glucose and triglycerides. As a control, we analysed mtDNA levels in abdominal subcutaneous fat samples from 23 HIV-seronegative women at delivery. mtDNA content was significantly lower in HIV-infected women when compared with HIV-negative controls. mtDNA content varied independently from viroimmunological, lipid and glucose parameters at the different months, with the exceptions of triglycerides at the 9th month and of HDL at the 6th month of pregnancy. In subcutaneous tissue from women taking NRTI-based antiretroviral regimens, we observed a significant decrease of mtDNA content, compared with uninfected women not on antiviral treatment. Moreover, a significant correlation was noted between mtDNA content and HDL cholesterol and triglycerides.

Comparative evaluation of the COBAS AmpliPrep/COBAS TaqMan HIV type 1 test (CAP/CTM) and VERSANT HIV type 1 RNA 3.0 assay (bDNA) for quantifying HIV type 1 viral loads in China.

PubMed

Xu, Sihong; Song, Aijing; Nie, Jianhui; Li, Xiuhua; Li, Jingyun; Bao, Zuoyi; Wang, Youchun

2008-11-01

In the present study, 277 clinical samples from untreated and treated HIV-1-infected patients with different clades were used to assess the agreement between the COBAS AmpliPrep/COBAS TaqMan HIV-1 test (CAP/CTM) and VERSANT HIV-1 RNA 3.0 Assay (bDNA). A qualitative comparison of the results of the two assays showed concordance for 255 positive and 15 negative samples (94.95%, kappa = 0.798). However, seven samples with viral loads close to the lower limit of detection for CAP/CTM were negative by bDNA. A significant correlation (r = 0.881, p < 0.001) was observed for 253 samples with viral loads within the dynamic ranges of the two assays, and Bland-Altman analysis showed good agreement (96.05%) between the two assays for these 253 samples [mean (+/-2 SD), 0.389(-0.385, 1.163)]. Furthermore, ART drugs had no impact on the performances of the two assays. For samples with different clades predominant in China, the fitted regression line differed significantly from the line of equality, although significant correlations (r = 0.850-0.891, p < 0.001) and good agreements (92.86-97.25%) were found for the two assays. The mean differences for clade B' and BC samples were significant (p < 0.01). Good precision for clade B' samples was achieved for the CAP/CTM (CV: 20.73%) and bDNA (CV: 12.19%) assays. Furthermore, for clades B', BC, and AE, both assays exhibited good linearities (r = 0.9773-0.9998). Thus, the CAP/CTM and bDNA assays could be useful for quantifying HIV-1 RNA in routine clinical samples and monitoring viral loads in treated and untreated HIV-infected patients in China.

Direct and quantitative detection of HIV-1 RNA in human plasma with a branched DNA signal amplification assay.

PubMed

Urdea, M S; Wilber, J C; Yeghiazarian, T; Todd, J A; Kern, D G; Fong, S J; Besemer, D; Hoo, B; Sheridan, P J; Kokka, R

1993-11-01

To determine the relative effect of sample matrix on the quantitation of HIV RNA in plasma. Two HIV-positive specimens were diluted into five and 10 different HIV-negative plasma samples, respectively. Branched DNA signal amplification technology and reverse-transcriptase polymerase chain reaction were used to measure the viral load. In one sample the viral load by polymerase chain reaction ranged from undetectable to 1.9 x 10(5) copies/ml, and the branched DNA results ranged from 2.6 x 10(4) to 4.2 x 10(4) HIV RNA equivalent/ml. In the other sample the corresponding figures were 6.3 x 10(4) to 5.5 x 10(5) copies/ml and 5.7 x 10(4) to 7.5 x 10(4) HIV RNA equivalents/ml. In contrast to reverse-transcriptase polymerase chain reaction the branched DNA signal amplification assay does not require a separate extraction step or enzymatic amplification of the target. Therefore this measurement is less affected by the sample matrix and the signal generated is directly proportional to the viral load.

Low-level viremia and proviral DNA impede immune reconstitution in HIV-1-infected patients receiving highly active antiretroviral therapy.

PubMed

Ostrowski, Sisse R; Katzenstein, Terese L; Thim, Per T; Pedersen, Bente K; Gerstoft, Jan; Ullum, Henrik

2005-02-01

Immunological and virological consequences of low-level viremia in human immunodeficiency virus (HIV) type 1-infected patients receiving highly active antiretroviral therapy (HAART) remain to be determined. For 24 months, 101 HAART-treated, HIV-1-infected patients with HIV RNA levels 20 copies/mL at >/=1 visit (dVL patients) (median increase, 81 copies/mL [interquartile range, 37-480 copies/mL]). dVL patients had higher concentrations of CD8 cells, activated and memory T cells, and proviral DNA, compared with uVL patients (P<.05). A higher HIV RNA level was independently associated with reduced CD4 gain (P<.001). A higher HIV RNA level also was associated with increases in activated CD8(+)CD38(+) and CD8(+)HLA-DR(+) cells (P<.05), and a higher level of activated CD8(+)CD38(+) cells was independently associated with reduced CD4 gain (P<.05). A higher proviral DNA level was associated with increases in CD4(+)CD45RA(-)CD28(-) effector cells and reductions in naive CD4(+)CD45RA(+)CD62L(+) and CD8(+)CD45RA(+)CD62L(+) cells (P<.05). Higher levels of activated CD4(+)HLA-DR(+) and early differentiated CD4(+)CD45RA(-)CD28(+) cells predicted increased risk of subsequent detectable viremia in patients with undetectable HIV RNA (P<.05). These findings indicate that low-level viremia and proviral DNA are intimately associated with the immunological and virological equilibrium in patients receiving HAART.

Persistence of human immunodeficiency virus type 1 subtype B DNA in dried-blood samples on FTA filter paper.

PubMed

Li, Chung-Chen; Beck, Ingrid A; Seidel, Kristy D; Frenkel, Lisa M

2004-08-01

The stability of human immunodeficiency virus type 1 (HIV-1) DNA in whole blood collected on filter paper (FTA Card) was evaluated. After >4 years of storage at room temperature in the dark our qualitative assay detected virus at a rate similar to that of our initial test (58 of 60, 97%; P = 0.16), suggesting long-term HIV-1 DNA stability.

Persistence of Human Immunodeficiency Virus Type 1 Subtype B DNA in Dried-Blood Samples on FTA Filter Paper

PubMed Central

Li, Chung-Chen; Beck, Ingrid A.; Seidel, Kristy D.; Frenkel, Lisa M.

2004-01-01

The stability of human immunodeficiency virus type 1 (HIV-1) DNA in whole blood collected on filter paper (FTA Card) was evaluated. After >4 years of storage at room temperature in the dark our qualitative assay detected virus at a rate similar to that of our initial test (58 of 60, 97%; P = 0.16), suggesting long-term HIV-1 DNA stability. PMID:15297546

HIV-1 DNA levels in peripheral blood mononuclear cells and cannabis use are associated with intermittent HIV shedding in semen of men who have sex with men on successful antiretroviral regimens.

PubMed

Ghosn, Jade; Leruez-Ville, Marianne; Blanche, Jérôme; Delobelle, Aurore; Beaudoux, Céline; Mascard, Laurence; Lecuyer, Hervé; Canestri, Ana; Landman, Roland; Zucman, David; Ponscarme, Diane; Rami, Agathe; Viard, Jean-Paul; Spire, Bruno; Rouzioux, Christine; Costagliola, Dominique; Suzan-Monti, Marie

2014-06-01

Few data exist on the efficacy of combined antiretroviral therapy (cART) in semen of human immunodeficiency virus type 1 (HIV-1) infected men who have sex with men (MSM) with sustained control of HIV replication in blood. HIV-1 infected MSM on successful cART for >6 months were enrolled. HIV-RNA was quantified in seminal plasma (spVL) and in blood plasma (bpVL) from 2 paired samples collected 4 weeks apart. Relationship between spVL and bpVL (measured by an ultrasensitive assay, LOQ 10 copies/mL), total peripheral blood mononuclear cells (PBMC)-associated HIV-DNA, sexually transmitted infections (STIs), and self-reported socio-behavioral characteristics was assessed using GEE logistic regression. In total, 157 patients were included. Median time with bpVL <50 copies/mL was 3.3 years. spVL was detectable in 23/304 samples (prevalence 7.6%). Median spVL was 145 cp/mL (100-1475). spVL was detectable on the first, on the second, and on both samples in 5, 14, and 2 men, respectively. In sum, 33 individuals (21%) had STIs (asymptomatic in 24/33). Residual bpVL was undetectable by ultrasensitive assay in 225/300 samples (75%). After multivariable adjustments, PBMC-associated HIV-DNA (OR 2.6[1.2; 6.0], for HIV-DNA > 2.5 log10 cp/10(6) PBMC, P = .02), and cannabis use during sexual intercourse (OR 2.8[1.2; 6.7], P = .02) were the only factors associated significantly with spVL. We show that HIV-RNA can be detected intermittently in semen of HIV-1 infected MSM despite successful cART. The size of blood HIV-1 reservoir predicted spVL detection. Our results indicated also that the possible effect of cannabis should be taken into account when developing prevention interventions targeted toward HIV-infected MSM on successful cART. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes.

PubMed

Balasubramaniam, Muthukumar; Davids, Benem; Addai, Amma B; Pandhare, Jui; Dash, Chandravanu

2017-02-22

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3' ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Quantification of HIV-1 DNA using real-time recombinase polymerase amplification.

PubMed

Crannell, Zachary Austin; Rohrman, Brittany; Richards-Kortum, Rebecca

2014-06-17

Although recombinase polymerase amplification (RPA) has many advantages for the detection of pathogenic nucleic acids in point-of-care applications, RPA has not yet been implemented to quantify sample concentration using a standard curve. Here, we describe a real-time RPA assay with an internal positive control and an algorithm that analyzes real-time fluorescence data to quantify HIV-1 DNA. We show that DNA concentration and the onset of detectable amplification are correlated by an exponential standard curve. In a set of experiments in which the standard curve and algorithm were used to analyze and quantify additional DNA samples, the algorithm predicted an average concentration within 1 order of magnitude of the correct concentration for all HIV-1 DNA concentrations tested. These results suggest that quantitative RPA (qRPA) may serve as a powerful tool for quantifying nucleic acids and may be adapted for use in single-sample point-of-care diagnostic systems.

Replication of Human Herpesviruses Is Associated with Higher HIV DNA Levels during Antiretroviral Therapy Started at Early Phases of HIV Infection

PubMed Central

Anderson, Christy M.; Var, Susanna R.; Oliveira, Michelli F.; Lada, Steven M.; Vargas, Milenka V.; Little, Susan J.; Richman, Douglas D.; Strain, Matthew C.; Pérez-Santiago, Josué; Smith, Davey M.

2016-01-01

ABSTRACT Asymptomatic replication of human herpesviruses (HHV) is frequent in HIV-infected men and is associated with increased T-cell activation and HIV disease progression. We hypothesized that the presence of replication of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) (the most frequently detected HHV) might influence HIV DNA decay during antiretroviral therapy (ART). We investigated 607 peripheral blood mononuclear cell (PBMC) samples from 107 CMV-seropositive, HIV-infected men who have sex with men, who started ART within a median of 3 months from their estimated date of infection (EDI) and were monitored for a median of 19 months thereafter. Levels of HIV, CMV, and EBV DNA and cellular HIV RNA were measured by droplet digital PCR (ddPCR) for each time point. Using a general linear mixed-effect regression model, we evaluated associations between the presence of detectable CMV DNA and EBV DNA levels and HIV DNA decay and cellular HIV RNA levels, while adjusting for peak HIV RNA, nadir CD4+ count, CD4/CD8 ratio, CMV IgG levels, time from EDI to ART initiation, time from ART initiation to virologic suppression, detectable CMV DNA pre-ART, and age. The presence of intermittent CMV DNA in PBMC during ART was significantly associated with slower decay of HIV DNA (P = 0.011) but not with increased cellular HIV RNA transcription or more detectable 2-long terminal repeat circles. Higher levels of EBV DNA were also associated with higher levels of HIV DNA (P < 0.001) and increased unspliced cellular HIV RNA transcription (P = 0.010). These observations suggest that replication of HHV may help maintain a larger HIV DNA reservoir, but the underlying mechanisms remain unclear. IMPORTANCE Over three-fourths of HIV-infected men have at least one actively replicating human herpesvirus (HHV) in their mucosal secretions at any one time. Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are the most common, and although it is often asymptomatic, such CMV and EBV replication is associated with higher levels of immune activation and HIV disease progression. We hypothesized that HHV-associated activation of HIV-infected CD4+ T cells might lead to increased HIV DNA. This study found that detectable CMV in blood cells of HIV-infected men was associated with slower decay of HIV DNA even during antiretroviral therapy (ART) that was started during early HIV infection. Similarly, levels of EBV DNA were associated with higher levels of HIV DNA during ART. If this observation points to a causal pathway, interventions that control CMV and EBV replication may be able to reduce the HIV reservoir, which might be relevant to current HIV cure efforts. PMID:26842469

Comparison of the RealTime HIV-1, COBAS TaqMan 48 v1.0, Easy Q v1.2, and Versant v3.0 assays for determination of HIV-1 viral loads in a cohort of Canadian patients with diverse HIV subtype infections.

PubMed

Church, Deirdre; Gregson, Daniel; Lloyd, Tracie; Klein, Marina; Beckthold, Brenda; Laupland, Kevin; Gill, M John

2011-01-01

HIV clinics in Canada provide care to an increasing number of patients born outside of Canada with HIV-1 non-B subtype infections. Because the Easy Q HIV-1 v1.2 assay (EQ; bioMérieux) failed to detect some non-B subtype infections, a multiassay HIV-1 viral load (VL) study was conducted with patients with diverse HIV subtype infections. Patients were enrolled from the Southern Alberta HIV Clinic (SAC), Calgary, Alberta, Canada (n = 349) and the McGill HIV Clinic (MHC), Montreal, Quebec, Canada (n = 20) and had four or five tubes of blood drawn for testing by EQ and three other commercial HIV VL assays: (i) the Versant 3.0 HIV-1 test, with the Versant 440 instrument (branched DNA [bDNA]; Siemens), (ii) the RealTime HIV-1 test, with the m2000rt instrument (m2000rt; Abbott Molecular Diagnostics), and (iii) the COBAS AmpliPrep TaqMan HIV-1 48 test (CAP-CTM; Roche Molecular Diagnostics). Blood was processed according to the individual manufacturer's requirements and stored frozen at -86°C. The HIV subtype was known for patients who had undergone HIV genotypic resistance testing (Virco, Belgium). Data analyses were done using standard statistical methods within Stata 9.0 (StataCorp, College Station, TX). A total of 371 samples were tested on 369 patients, of whom 291 (81%) had a Virco genotype result of B (195; 53%) or non-B (96; 26%) subtypes A to D and F to K, as well as circulating recombinant forms (CRFs) (i.e., CRF01_AE and CRF02_AG). Most (58/78; 74%) patients of unknown subtype were recent African emigrants who likely have non-subtype B infection. Overall bias was small in pairwise Bland-Altman plots, but the limits of agreement between assays were wide. Discordant viral load results occurred for 98 samples and were due to missing values, false negatives, and significant underquantification that varied by HIV subtype. Results were obtained for all 371 samples with m2000rt, but for only 357 (97%) with CAP-CTM, 338 (92%) with EQ, and 276 (75%) with bDNA due to errors/equipment failures. False-negative results (nondetection of viral RNA versus other assay results) occurred for all platforms, as follows: for m2000rt, 8 (2%) [B(4) and non-B(4) subtypes], CAP-CTM, 9 (2.5%) [B(6) and non-B(3) subtypes]; EQ, 20 (6%) [B(7) and non-B(13) subtypes]; bDNA, 5 (2%) [B(1) and C(4)]. EQ and bDNA had the highest rates of underquantification by ≥ 1.0 log(10) copies/ml, mainly for HIV non-B subtypes. Performance significantly varied between HIV VL platforms according to subtype. HIV viral diversity in the population being tested must be considered in selection of the viral load platform.

SUN2 Modulates HIV-1 Infection and Latency through Association with Lamin A/C To Maintain the Repressive Chromatin.

PubMed

Sun, Wei-Wei; Jiao, Shi; Sun, Li; Zhou, Zhaocai; Jin, Xia; Wang, Jian-Hua

2018-05-01

The postintegrational latency of HIV-1 is characterized by reversible silencing of long terminal repeat (LTR)-driven transcription of the HIV genome. It is known that the formation of repressive chromatin at the 5'-LTR of HIV-1 proviral DNA impedes viral transcription by blocking the recruitment of positive transcription factors. How the repressive chromatin is formed and modulated during HIV-1 infection remains elusive. Elucidation of which chromatin reassembly factor mediates the reorganization of chromatin is likely to facilitate the understanding of the host's modulation of HIV-1 transcription and latency. Here we revealed that "Sad1 and UNC84 domain containing 2" (SUN2), an inner nuclear membrane protein, maintained the repressive chromatin and inhibited HIV LTR-driven transcription of proviral DNA through an association with lamin A/C. Specifically, lamin A/C tethered SUN2 to the nucleosomes 1 and 2 of the HIV-1 5'-LTR to block the initiation and elongation of HIV-1 transcription. SUN2 knockdown converted chromatin to an active form and thus enhanced the phosphorylation of RNA polymerase II and its recruitment to the 5'-LTR HIV-1 proviral DNA, leading to reactivation of HIV-1 from latency. Conversely, the exogenous factors such as tumor necrosis factor alpha (TNF-α) induced reactivation, and the replication of HIV-1 led to the disassociation between SUN2 and lamin A/C, suggesting that disruption of the association between SUN2 and lamin A/C to convert the repressive chromatin to the active form might be a prerequisite for the initiation of HIV-1 transcription and replication. Together, our findings indicate that SUN2 is a novel chromatin reassembly factor that helps to maintain chromatin in a repressive state and consequently inhibits HIV-1 transcription. IMPORTANCE Despite the successful use of scores of antiretroviral drugs, HIV latency poses a major impediment to virus eradication. Elucidation of the mechanism of latency facilitates the discovery of new therapeutic strategies. It has been known that the formation of repressive chromatin at the 5'-LTR of HIV-1 proviral DNA impedes viral transcription and maintains viral latency, but how the repressive chromatin is formed and modulated during HIV-1 infection remains elusive. In this study, we performed in-depth virological and cell biological studies and discovered that an inner nuclear membrane protein, SUN2, is a novel chromatin reassembly factor that maintains repressive chromatin and thus modulates HIV-1 transcription and latency: therefore, targeting SUN2 may lead to new strategies for HIV cure. Copyright © 2018 Sun et al.

Torque Teno Virus in HIV-infected transgender in Surakarta, Indonesia

NASA Astrophysics Data System (ADS)

Hartono; Agung Prasetyo, Afiono; Fanani, Mohammad

2018-05-01

Torque Teno Virus (TTV) is a circular single-stranded DNA virus that may co-infected with human immunodeficiency virus (HIV), especially in the high-risk community e.g. the transgender performing high-riskbehavior. TTV shows an increased viremia in HIV patients and maybe influence the HIV clinical progression. Blood samples collected from transgender performing high-riskbehavior in Surakarta were tested by serological and molecular assays to detect the presence of HIV infection. The blood samples with HIV positive status were then tested by a nested polymerase chain reaction (PCR) to detect the presentation of TTV DNA. The amplified PCR products were molecularly cloned and subjected to sequence analysis. TTV DNA was detected in 40.0% HIV-positive samples. The molecular characterization revealed that the most prevalent was genogroup 3, followed by genogroup 2 and 1, respectively. TTV was detected in HIV-infected transgender performing high-riskbehavior in Surakarta with high infection rate.

Short communication: identification of a novel HIV type 1 subtype H/J recombinant in Canada with discordant HIV viral load (RNA) values in three different commercial assays.

PubMed

Kim, John E; Beckthold, Brenda; Chen, Zhaoxia; Mihowich, Jennifer; Malloch, Laurie; Gill, Michael John

2007-11-01

The presence of HIV-1 non-B subtypes is increasing worldwide. This poses challenges to commercial diagnostic and viral load (RNA) monitoring tests that are predominantly based on HIV-1 subtype B strains. Based on phylogenetic analysis of the gag, pol, and env gene regions, we describe the first HIV-1 H/J recombinant in Canada that presented divergent viral load values. DNA sequence analysis of the gag gene region further revealed that genetic diversity between this H/J recombinant and the primers and probes used in the bio-Merieux Nuclisens HIV-1 QT (Nuclisens) and Roche Amplicor Monitor HIV-1, v1.5 (Monitor) viral RNA assays can erroneously lead to undetectable viral load values. This observation appears to be more problematic in the Nuclisens assay. In light of increasing genetic diversity in HIV worldwide we recommend that DNA sequencing of HIV, especially in the gag gene region targeted by primers and probes used in molecular diagnostic and viral load tests, be incorporated into clinical monitoring practices.

Virological and immunological responses to raltegravir and dolutegravir in the gut-associated lymphoid tissue of HIV-infected men and women.

PubMed

Weber, Michael D; Andrews, Elizabeth; Prince, Heather A; Sykes, Craig; Rosen, Elias P; Bay, Camden; Shaheen, Nicholas J; Madanick, Ryan D; Dellon, Evan S; De Paris, Kristina; Nelson, Julie Ae; Gay, Cynthia L; Kashuba, Angela Dm

2018-05-01

Raltegravir (RTG) and dolutegravir (DTG) have different pharmacokinetic patterns in the gastrointestinal tract. To determine if this results in pharmacodynamic differences, we compared HIV RNA, HIV DNA, and immunological markers in gut-associated lymphoid tissue (GALT) of HIV-infected participants receiving RTG or DTG with tenofovir+emtricitabine (TDF/FTC). GALT specimens from the terminal ileum, splenic flexure, and rectum were obtained by colonoscopy at a single time point in 20 adults treated with RTG (n=10) or DTG (n=10) with HIV RNA <50 copies/mL. Flow cytometry, drug concentrations, and HIV RNA and DNA were analyzed in tissue. CD4/8 + T cells were tested for γδ TCR, and markers of T cell activation and exhaustion. Data are reported as median (Q1,Q3). 15 men and 5 women were enrolled. There was no difference in time since HIV diagnosis for those on RTG [9.5 (4-22) yr] and DTG [17 (1-24) yr] (p = 0.6), although time on RTG [5.4 (2.3-6.7) yr] was greater than DTG [1.0 (0.1-1.5) yr] (P < 0.001). Concentrations of RTG and DTG in rectal tissue (RT) were similar to previous reports: median tissue:plasma ratio was 11.25 for RTG and 0.44 for DTG. RNA:DNA ratios were [1.14 (0.18-5.10)] for the RTG group and [0.90 (0.30-18.87)] for the DTG group (p = 0.95). No differences (p ≥ 0.1) between CD4 + and CD8 + T cell markers were found. RTG produced higher tissue exposures than DTG, but no significant differences in GALT HIV RNA, DNA, or most immunologic markers were observed.

Genotypes of JC virus, DNA of cytomegalovirus, and proviral DNA of human immunodeficiency virus in eyes of acquired immunodeficiency syndrome patients.

PubMed

Eberwein, Philipp; Hansen, Lutz L; Agostini, Hansjürgen T

2005-02-01

JC virus (JCV) is a human polyomavirus that exists in at least eight different genotypes as a result of coevolution with different human populations all over the world. Well adapted to its host, it usually persists in the kidneys and possibly the brain. If the host becomes immunodeficient, JCV can cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). There is increasing evidence that JCV is transactivated by cytomegalovirus (CMV) and the human immunodeficiency virus (HIV). Both CMV and HIV can infect the retina of acquired immunodeficiency syndrome (AIDS) patients, causing severe necrosis in the case of CMV retinitis or a mild HIV-associated vasculopathy, with bleeding and cotton wool spots. The authors therefore investigated by polymerase chain reaction (PCR) whether DNA of these three viruses was detectable in paraffin-embedded eyes of AIDS patients with a clinical history of CMV retinitis. From a total of 65 eyes, JCV was detected in 21 (32%). Thirty-six (55%) were positive for CMV and 6 (9%) for proviral DNA of HIV. JCV and CMV were found in 13 eyes, JCV and HIV in 3 eyes, CMV and HIV in 1 eye, and DNA from all three viruses in 1 eye. The JCV genotypes were types 1A, 2A, 2E, 3, and 4. In 21 eyes of patients without AIDS, only one sample was JCV positive. In conclusion, JCV DNA can be detected in ocular tissue of AIDS patients at a significantly higher level than in eyes of nonimmunosuppressed patients. Further investigations will help to decide if JCV contributes to the retinopathy caused by CMV and HIV.

Vaccination with Combination DNA and Virus-Like Particles Enhances Humoral and Cellular Immune Responses upon Boost with Recombinant Modified Vaccinia Virus Ankara Expressing Human Immunodeficiency Virus Envelope Proteins.

PubMed

Gangadhara, Sailaja; Kwon, Young-Man; Jeeva, Subbiah; Quan, Fu-Shi; Wang, Baozhong; Moss, Bernard; Compans, Richard W; Amara, Rama Rao; Jabbar, M Abdul; Kang, Sang-Moo

2017-12-19

Heterologous prime boost with DNA and recombinant modified vaccinia virus Ankara (rMVA) vaccines is considered as a promising vaccination approach against human immunodeficiency virus (HIV-1). To further enhance the efficacy of DNA-rMVA vaccination, we investigated humoral and cellular immune responses in mice after three sequential immunizations with DNA, a combination of DNA and virus-like particles (VLP), and rMVA expressing HIV-1 89.6 gp120 envelope proteins (Env). DNA prime and boost with a combination of VLP and DNA vaccines followed by an rMVA boost induced over a 100-fold increase in Env-specific IgG antibody titers compared to three sequential immunizations with DNA and rMVA. Cellular immune responses were induced by VLP-DNA and rMVA vaccinations at high levels in CD8 T cells, CD4 T cells, and peripheral blood mononuclear cells secreting interferon (IFN)-γ, and spleen cells producing interleukin (IL)-2, 4, 5 cytokines. This study suggests that a DNA and VLP combination vaccine with MVA is a promising strategy in enhancing the efficacy of DNA-rMVA vaccination against HIV-1.

Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA.

PubMed

Darlix, J L; Vincent, A; Gabus, C; de Rocquigny, H; Roques, B

1993-08-01

Two DNA strand transfer reactions take place during reverse transcription of the retroviral genome. The first transfer, that of the minus-strand strong stop DNA from the 5' end of the viral RNA to the 3' end, has been studied in vitro with two RNAs mimicking the 5' and 3' regions of the HIV1 genome and with nucleocapsid protein, NCp7, and reverse transcriptase. The results show that NCp7 strongly activates the 5' to 3' DNA strand transfer during reverse transcription while a basic peptide resembling NCp7 is inactive. Activation of the first transfer by several NCp7 derived peptides and the influence of the terminal redundancies (R) present at the 5' and 3' ends of HIV1 RNA were also examined. The first transfer is optimal in the presence of intact NCp7 and necessitates R on both the 5' and 3' RNAs. Sequencing of full length viral DNA products reveals approximately 40% misincorporations at the first nucleotide beyond the transfer point. If such base misincorporations occur during proviral DNA synthesis with possible homologous recombinations it may well contribute to the high level of genetic variability of HIV.

[Cloning and bioinformatic analysis and expression analysis of beta-glucuronidase in Scutellaria baicalensis].

PubMed

Guo, Shuang-shuang; Cheng, Lin; Yang, Li-min; Han, Mei

2015-11-01

The β-Glucuronidase gene (sbGUS) cDNA firstly from Scutellari abaicalensis leaf was cloned by RT-PCR, with GenBank accession number KR364726. The full length cDNA of sbGUS was 1 584 bp with an open reading frame (ORF), encoding an unstable protein with 527 amino acids. The bioinformatic analysis showed that the sbGUS encoding protein had isoelectric point (pI) of 5.55 and a calculated molecular weight about 58.724 8 kDa, with a transmembrane regions and signal peptide, had conserved domains of glycoside hydrolase super family and unintegrated trans-glycosidase catalytic structure. In the secondary structure, the percentage of alpha helix, extended strand, β-extended and random coil were 25.62%, 28.84%, 13.28% and 32.26%, respectively. The homologous analysis indicated the nucleotide sequence 98.93% similarity and the amino acid sequence 98.29% similarity with S. baicalensis (BAA97804.1), in the nine positions were different. The expression level of sGUS was the highest in root based on a real-time PCR analysis, followed by flower and stem, and the lowest was in stem. The results provide a foundation for exploring the molecular function of sbGUS involved in baicalcin biosynthesis based on synthetic biology approach in S. baicalensis plants.

Combined effect of Vacc-4x, recombinant human granulocyte macrophage colony-stimulating factor vaccination, and romidepsin on the HIV-1 reservoir (REDUC): a single-arm, phase 1B/2A trial.

PubMed

Leth, Steffen; Schleimann, Mariane H; Nissen, Sara K; Højen, Jesper F; Olesen, Rikke; Graversen, Mette E; Jørgensen, Sofie; Kjær, Anne Sofie; Denton, Paul W; Mørk, Alejandra; Sommerfelt, Maja A; Krogsgaard, Kim; Østergaard, Lars; Rasmussen, Thomas A; Tolstrup, Martin; Søgaard, Ole Schmeltz

2016-10-01

Immune priming before reversal of latency might be a component of a functional HIV cure. To assess this concept, we assessed if therapeutic HIV immunisation followed by latency reversal would affect measures of viral transcription, plasma viraemia, and reservoir size in patients with HIV on suppressive antiretroviral therapy. In this single-arm, phase 1B/2A trial, we recruited adults treated at the Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark (aged ≥18 years) with successfully treated HIV-1 with plasma RNA loads of less than 50 copies per mL for the previous year and CD4 counts of at least 500 cells per μL. Exclusion criteria included CD4 counts of less than 200 cells per μL within the past 2 years, active hepatitis B or C infections, and clinically significant cardiac disease, including QTc prolongation. Participants received six therapeutic intradermal HIV-1 immunisations with 12 mg/mL Vacc-4x and 0·6 mg/mL rhuGM-CSF over 12 weeks (at 0 weeks, 1 week, 2 weeks, 3 weeks, 11 weeks, and 12 weeks) before receiving 5 mg/m(2) intravenous romidepsin once a week for 3 weeks. This procedure was followed by analytical treatment interruption. Coprimary outcomes were changes in copies of HIV-1 DNA (total and integrated) per million CD4 T cells and infectious units per million (IUPM) resting memory CD4 T cells established by viral outgrowth, assessed in all patients receiving at least one dose of active treatment with assessable data. We assessed total HIV-1 DNA at screening, before romidepsin treatment, and 6 weeks after romidepsin treatment. We assessed integrated viral DNA at baseline, before romidepsin treatment, and 8 weeks after romidepsin treatment. We assessed IUPM at screening, 2 weeks before romidepsin treatment, and 6 weeks after romidepsin treatment. This trial is registered at ClinicalTrials.gov, number NCT02092116. Between May 19, 2014, and Oct 8, 2014, we enrolled 20 individuals, of whom 17 completed all Vacc-4x and rhuGM-CSF administrations and romidepsin infusions. 16 of 17 had assessable total HIV-1 DNA, 15 of 17 had assessable integrated HIV-1 DNA, and six of 17 had assessable IUPM at baseline and at one or more timepoints after study treatment. Total HIV-1 DNA declined from screening to 6 weeks after romidepsin treatment (mean reduction 39·7%, 95% CI -59·7 to -11·5; p=0·012). The decrease in integrated HIV-1 DNA from baseline to 8 weeks after romidepsin treatment was not significant (19·2%, -38·6 to 6·3; p=0·123). Among the six assessable participants, the mean reduction in IUPM from screening to 6 weeks after romidepsin treatment was 38·0% (95% CI -67·0 to -8·0; p=0·019). Of 141 adverse events, 134 (95%) were grade 1 and seven (5%) were grade 2-3. This in-vivo combinatorial approach provides the first evidence for the feasibility of a combined shock and kill strategy, but also emphasises that further optimisation of this strategy is needed to achieve a sizeable effect on the latent reservoir that will translate into clinically measurable benefits for people living with HIV-1. Bionor Pharma, the Research Council of Norway, and SkatteFUNN. Copyright © 2016 Elsevier Ltd. All rights reserved.

Diversion of HIV-1 Vaccine-induced Immunity by gp41-Microbiota Cross-reactive Antibodies

PubMed Central

Williams, Wilton B; Liao, Hua-Xin; Moody, M. Anthony; Kepler, Thomas B.; Alam, S Munir; Gao, Feng; Wiehe, Kevin; Trama, Ashley M.; Jones, Kathryn; Zhang, Ruijun; Song, Hongshuo; Marshall, Dawn J; Whitesides, John F; Sawatzki, Kaitlin; Hua, Axin; Liu, Pinghuang; Tay, Matthew Z; Seaton, Kelly; Shen, Xiaoying; Foulger, Andrew; Lloyd, Krissey E.; Parks, Robert; Pollara, Justin; Ferrari, Guido; Yu, Jae-Sung; Vandergrift, Nathan; Montefiori, David C.; Sobieszczyk, Magdalena E; Hammer, Scott; Karuna, Shelly; Gilbert, Peter; Grove, Doug; Grunenberg, Nicole; McElrath, Julie; Mascola, John R.; Koup, Richard A; Corey, Lawrence; Nabel, Gary J.; Morgan, Cecilia; Churchyard, Gavin; Maenza, Janine; Keefer, Michael; Graham, Barney S.; Baden, Lindsey R.; Tomaras, Georgia D.; Haynes, Barton F.

2015-01-01

A HIV-1 DNA prime-recombinant Adenovirus Type 5 (rAd5) boost vaccine failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1-reactive plasma Ab titers were higher to Env gp41 than gp120, and repertoire analysis demonstrated that 93% of HIV-1-reactive Abs from memory B cells was to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies (mAbs) were non-neutralizing, and frequently polyreactive with host and environmental antigens including intestinal microbiota (IM). Next generation sequencing of an IGHV repertoire prior to vaccination revealed an Env-IM cross-reactive Ab that was clonally-related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy. PMID:26229114

HIV RNA testing in the context of nonoccupational postexposure prophylaxis.

PubMed

Roland, Michelle E; Elbeik, Tarek A; Kahn, James O; Bamberger, Joshua D; Coates, Thomas J; Krone, Melissa R; Katz, Mitchell H; Busch, Michael P; Martin, Jeffrey N

2004-08-01

The specificity and positive predictive value of human immunodeficiency virus (HIV) RNA assays have not been evaluated in the setting of postexposure prophylaxis (PEP). Plasma from subjects enrolled in a nonoccupational PEP study was tested with 2 branched-chain DNA (bDNA) assays, 2 polymerase chain reaction (PCR) assays, and a transcription-mediated amplification (TMA) assay. Assay specificity and positive predictive value were determined for subjects who remained negative for HIV antibody for >or=3 months. In 329 subjects examined, the lowest specificities (90.1%-93.7%) were seen for bDNA testing performed in real time. The highest specificities were seen with batched bDNA version 3.0 (99.1%), standard PCR (99.4%), ultrasensitive PCR (100%), and TMA (99.6%) testing. Only the 2 assays with the highest specificities had positive predictive values >40%. For the bDNA assays, increasing the cutoff point at which a test is called positive (e.g., from 50 copies/mL to 500 copies/mL for version 3.0) increased both specificity and positive predictive values to 100%. The positive predictive value of HIV RNA assays in individuals presenting for PEP is unacceptably low for bDNA-based testing and possibly acceptable for PCR- and TMA-based testing. Routine use of HIV RNA assays in such individuals is not recommended.

HIV DNA-Adenovirus Multiclade Envelope Vaccine Induces gp41 Antibody Immunodominance in Rhesus Macaques

PubMed Central

Williams, Wilton B.; Saunders, Kevin O.; Seaton, Kelly E.; Wiehe, Kevin J.; Vandergrift, Nathan; Von Holle, Tarra A.; Trama, Ashley M.; Parks, Robert J.; Luo, Kan; Gurley, Thaddeus C.; Kepler, Thomas B.; Marshall, Dawn J.; Montefiori, David C.; Sutherland, Laura L.; Alam, Munir S.; Whitesides, John F.; Bowman, Cindy M.; Permar, Sallie R.; Graham, Barney S.; Mascola, John R.; Seed, Patrick C.; Van Rompay, Koen K. A.; Tomaras, Georgia D.; Moody, M. Anthony

2017-01-01

ABSTRACT Dominant antibody responses in vaccinees who received the HIV-1 multiclade (A, B, and C) envelope (Env) DNA/recombinant adenovirus virus type 5 (rAd5) vaccine studied in HIV-1 Vaccine Trials Network (HVTN) efficacy trial 505 (HVTN 505) targeted Env gp41 and cross-reacted with microbial antigens. In this study, we asked if the DNA/rAd5 vaccine induced a similar antibody response in rhesus macaques (RMs), which are commonly used as an animal model for human HIV-1 infections and for testing candidate HIV-1 vaccines. We also asked if gp41 immunodominance could be avoided by immunization of neonatal RMs during the early stages of microbial colonization. We found that the DNA/rAd5 vaccine elicited a higher frequency of gp41-reactive memory B cells than gp120-memory B cells in adult and neonatal RMs. Analysis of the vaccine-induced Env-reactive B cell repertoire revealed that the majority of HIV-1 Env-reactive antibodies in both adult and neonatal RMs were targeted to gp41. Interestingly, a subset of gp41-reactive antibodies isolated from RMs cross-reacted with host antigens, including autologous intestinal microbiota. Thus, gp41-containing DNA/rAd5 vaccine induced dominant gp41-microbiota cross-reactive antibodies derived from blood memory B cells in RMs as observed in the HVTN 505 vaccine efficacy trial. These data demonstrated that RMs can be used to investigate gp41 immunodominance in candidate HIV-1 vaccines. Moreover, colonization of neonatal RMs occurred within the first week of life, and immunization of neonatal RMs during this time also induced a dominant gp41-reactive antibody response. IMPORTANCE Our results are critical to current work in the HIV-1 vaccine field evaluating the phenomenon of gp41 immunodominance induced by HIV-1 Env gp140 in RMs and humans. Our data demonstrate that RMs are an appropriate animal model to study this phenomenon and to determine the immunogenicity in new HIV-1 Env trimer vaccine designs. The demonstration of gp41 immunodominance in memory B cells of both adult and neonatal RMs indicated that early vaccination could not overcome gp41 dominant responses. PMID:28794027

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication.

PubMed

Herzig, Eytan; Voronin, Nickolay; Kucherenko, Nataly; Hizi, Amnon

2015-08-01

The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Drug-Based Lead Discovery: The Novel Ablative Antiretroviral Profile of Deferiprone in HIV-1-Infected Cells and in HIV-Infected Treatment-Naive Subjects of a Double-Blind, Placebo-Controlled, Randomized Exploratory Trial

PubMed Central

Saxena, Deepti; Spino, Michael; Tricta, Fernando; Connelly, John; Cracchiolo, Bernadette M.; Hanauske, Axel-Rainer; D’Alliessi Gandolfi, Darlene; Mathews, Michael B.; Karn, Jonathan; Holland, Bart; Park, Myung Hee; Pe’ery, Tsafi; Palumbo, Paul E.; Hanauske-Abel, Hartmut M.

2016-01-01

Antiretrovirals suppress HIV-1 production yet spare the sites of HIV-1 production, the HIV-1 DNA-harboring cells that evade immune detection and enable viral resistance on-drug and viral rebound off-drug. Therapeutic ablation of pathogenic cells markedly improves the outcome of many diseases. We extend this strategy to HIV-1 infection. Using drug-based lead discovery, we report the concentration threshold-dependent antiretroviral action of the medicinal chelator deferiprone and validate preclinical findings by a proof-of-concept double-blind trial. In isolate-infected primary cultures, supra-threshold concentrations during deferiprone monotherapy caused decline of HIV-1 RNA and HIV-1 DNA; did not allow viral breakthrough for up to 35 days on-drug, indicating resiliency against viral resistance; and prevented, for at least 87 days off-drug, viral rebound. Displaying a steep dose-effect curve, deferiprone produced infection-independent deficiency of hydroxylated hypusyl-eIF5A. However, unhydroxylated deoxyhypusyl-eIF5A accumulated particularly in HIV-infected cells; they preferentially underwent apoptotic DNA fragmentation. Since the threshold, ascertained at about 150 μM, is achievable in deferiprone-treated patients, we proceeded from cell culture directly to an exploratory trial. HIV-1 RNA was measured after 7 days on-drug and after 28 and 56 days off-drug. Subjects who attained supra-threshold concentrations in serum and completed the protocol of 17 oral doses, experienced a zidovudine-like decline of HIV-1 RNA on-drug that was maintained off-drug without statistically significant rebound for 8 weeks, over 670 times the drug’s half-life and thus clearance from circulation. The uniform deferiprone threshold is in agreement with mapping of, and crystallographic 3D-data on, the active site of deoxyhypusyl hydroxylase (DOHH), the eIF5A-hydroxylating enzyme. We propose that deficiency of hypusine-containing eIF5A impedes the translation of mRNAs encoding proline cluster (‘polyproline’)-containing proteins, exemplified by Gag/p24, and facilitated by the excess of deoxyhypusine-containing eIF5A, releases the innate apoptotic defense of HIV-infected cells from viral blockade, thus depleting the cellular reservoir of HIV-1 DNA that drives breakthrough and rebound. Trial Registration: ClinicalTrial.gov NCT02191657 PMID:27191165

Expansion and productive HIV-1 infection of Foxp3 positive CD4 T cells at pleural sites of HIV/TB co-infection

PubMed Central

Hirsch, Christina S; Baseke, Joy; Kafuluma, John Lusiba; Nserko, Mary; Mayanja-Kizza, Harriet; Toossi, Zahra

2016-01-01

Background CD4 T-cells expressing Foxp3 are expanded systemically during active tuberculosis (TB) regardless of HIV-1 co-infection. Foxp3+ CD4 T cells are targets of HIV-1 infection. However, expansion of HIV-1 infected Foxp3+ CD4 T cells at sites of HIV/TB co-infection, and whether they contribute to promotion of HIV-1 viral activity is not known. Methods Pleural fluid mononuclear cells (PFMC) from HIV/TB co-infected patients with pleural TB were characterized by immune-staining and FACS analysis for surface markers CD4, CD127, CCR5, CXCR4, HLA-DR and intracellular expression of Foxp3, HIVp24, IFN-γ and Bcl-2. Whole PFMC and bead separated CD4+CD25+CD127− T cells were assessed for HIV-1 LTR strong stop (SS) DNA by real-time PCR, which represents viral DNA post cell entry and initiation of reverse transcription. Results High numbers of HIV-1 p24 positive Foxp3+ and Foxp3+CD127− CD4 T cells were identified in PFMC from HIV/TB co-infected subjects. CD4+Foxp3+CD127− T cells displayed high expression of the cellular activation marker, HLA-DR. Further, expression of the HIV-1 co-receptors, CCR5 and CXCR4, were higher on CD4+Foxp3+T cells compared to CD4+Foxp3− T cells. Purified CD4+CD25+CD127− T cells isolated from PFMC of HIV/TB co-infected patients, were over 90% CD4+Foxp3+T cells, and exhibited higher HIV-1 SS DNA as compared to whole PFMC, and as compared to CD4+CD25+CD127− T cells from an HIV-infected subject with pleural mesothelioma. HIV-1 p24+ Foxp3+ CD4+T cells from HIV/TB patients higher in Bcl-2 expression as compared to both HIV-1 p24+ Foxp3− CD4 T cells, and Foxp3+ CD4+T cells without HIV-p24 expression. Conclusion Foxp3+ CD4 T cells in PFMC from HIV/TB co-infected subjects are predisposed to productive HIV-1 infection and have survival advantage as compared to Foxp3 negative CD4 T cells. PMID:28124031

HIV-1 VACCINES. Diversion of HIV-1 vaccine-induced immunity by gp41-microbiota cross-reactive antibodies.

PubMed

Williams, Wilton B; Liao, Hua-Xin; Moody, M Anthony; Kepler, Thomas B; Alam, S Munir; Gao, Feng; Wiehe, Kevin; Trama, Ashley M; Jones, Kathryn; Zhang, Ruijun; Song, Hongshuo; Marshall, Dawn J; Whitesides, John F; Sawatzki, Kaitlin; Hua, Axin; Liu, Pinghuang; Tay, Matthew Z; Seaton, Kelly E; Shen, Xiaoying; Foulger, Andrew; Lloyd, Krissey E; Parks, Robert; Pollara, Justin; Ferrari, Guido; Yu, Jae-Sung; Vandergrift, Nathan; Montefiori, David C; Sobieszczyk, Magdalena E; Hammer, Scott; Karuna, Shelly; Gilbert, Peter; Grove, Doug; Grunenberg, Nicole; McElrath, M Juliana; Mascola, John R; Koup, Richard A; Corey, Lawrence; Nabel, Gary J; Morgan, Cecilia; Churchyard, Gavin; Maenza, Janine; Keefer, Michael; Graham, Barney S; Baden, Lindsey R; Tomaras, Georgia D; Haynes, Barton F

2015-08-14

An HIV-1 DNA prime vaccine, with a recombinant adenovirus type 5 (rAd5) boost, failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1-reactive plasma Ab titers were higher to Env gp41 than to gp120, and repertoire analysis demonstrated that 93% of HIV-1-reactive Abs from memory B cells responded to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies were non-neutralizing and frequently polyreactive with host and environmental antigens, including intestinal microbiota (IM). Next-generation sequencing of an immunoglobulin heavy chain variable region repertoire before vaccination revealed an Env-IM cross-reactive Ab that was clonally related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy. Copyright © 2015, American Association for the Advancement of Science.

DNA-MVA-protein vaccination of rhesus macaques induces HIV-specific immunity in mucosal-associated lymph nodes and functional antibodies.

PubMed

Chege, Gerald K; Burgers, Wendy A; Müller, Tracey L; Gray, Clive M; Shephard, Enid G; Barnett, Susan W; Ferrari, Guido; Montefiori, David; Williamson, Carolyn; Williamson, Anna-Lise

2017-02-07

Successful future HIV vaccines are expected to generate an effective cellular and humoral response against the virus in both the peripheral blood and mucosal compartments. We previously reported the development of DNA-C and MVA-C vaccines based on HIV-1 subtype C and demonstrated their immunogenicity when given in a DNA prime-MVA boost combination in a nonhuman primate model. In the current study, rhesus macaques previously vaccinated with a DNA-C and MVA-C vaccine regimen were re-vaccinated 3.5years later with MVA-C followed by a protein vaccine based on HIV-1 subtype C envelope formulated with MF59 adjuvant (gp140Env/MF59), and finally a concurrent boost with both vaccines. A single MVA-C re-vaccination elicited T cell responses in all animals similar to previous peak responses, with 4/7 demonstrating responses >1000 SFU/10 6 PBMC. In contrast to an Env/MF59-only vaccine, concurrent boosting with MVA-C and Env/MF59 induced HIV-specific cellular responses in multiple mucosal associated lymph nodes in 6/7 animals, with high magnitude responses in some animals. Both vaccine regimens induced high titer Env-specific antibodies with ADCC activity, as well as neutralization of Tier 1 viruses and modest Tier 2 neutralization. These data demonstrate the feasibility of inducing HIV-specific immunity in the blood and mucosal sites of viral entry by means of DNA and poxvirus-vectored vaccines, in combination with a HIV envelope-based protein vaccine. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Impact of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy with Sezary syndrome.

PubMed

Rasmussen, Thomas A; McMahon, James; Chang, J Judy; Symons, Jori; Roche, Michael; Dantanarayana, Ashanti; Okoye, Afam; Hiener, Bonnie; Palmer, Sarah; Lee, Wen Shi; Kent, Stephen J; Van Der Weyden, Carrie; Prince, H Miles; Cameron, Paul U; Lewin, Sharon R

2017-08-24

To study the effects of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy (ART) with Sezary syndrome, a rare malignancy of CD4 T cells. Case report. Blood was collected 30 and 18 months prior to presentation with Sezary syndrome, at the time of presentation and during alemtuzumab. T-cell subsets in malignant (CD7-CD26-TCR-VBeta2+) and nonmalignant cells were quantified by flow cytometry. HIV-DNA in total CD4 T cells, in sorted malignant and nonmalignant CD4 T cells, was quantified by PCR and clonal expansion of HIV-DNA assessed by full-length next-generation sequencing. HIV-hepatitis B virus coinfection was diagnosed and antiretroviral therapy initiated 4 years prior to presentation with Sezary syndrome and primary cutaneous anaplastic large cell lymphoma. The patient received alemtuzumab 10 mg three times per week for 4 weeks but died 6 weeks post alemtuzumab. HIV-DNA was detected in nonmalignant but not in malignant CD4 T cells, consistent with expansion of a noninfected CD4 T-cell clone. Full-length HIV-DNA sequencing demonstrated multiple defective viruses but no identical or expanded sequences. Alemtuzumab extensively depleted T cells, including more than 1 log reduction in total T cells and more than 3 log reduction in CD4 T cells. Finally, alemtuzumab decreased HIV-DNA in CD4 T cells by 57% but HIV-DNA remained detectable at low levels even after depletion of nearly all CD4 T cells. Alemtuzumab extensively depleted multiple T-cell subsets and decreased the frequency of but did not eliminate HIV-infected CD4 T cells. Studying the effects on HIV persistence following immune recovery in HIV-infected individuals who require alemtuzumab for malignancy or in animal studies may provide further insights into novel cure strategies.

Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins.

PubMed

Zhang, Mingce; Robinson, Tanya O; Duverger, Alexandra; Kutsch, Olaf; Heath, Sonya L; Cron, Randy Q

2018-03-01

During chronic HIV-1 infection, regulatory CD4 T cells (Tregs) frequently represent the largest subpopulation of CD4 T cell subsets, implying relative resistant to HIV-1. When HIV-1 infection of CD4 T cells was explored in vitro and ex vivo from patient samples, Tregs possessed lower levels of HIV-1 DNA and RNA in comparison with conventional effector and memory CD4 T cells. Moreover, Tregs suppressed HIV-1 expression in other CD4 T cells in an in vitro co-culture system. This suppression was mediated in part via multiple inhibitory surface proteins expressed on Tregs. Antibody blockade of CTLA-4, PD-1, and GARP on Tregs resulted in increased HIV-1 DNA integration and mRNA expression in neighboring CD4 T cells. Moreover, antibody blockade of Tregs inhibitory proteins resulted in increased HIV-1 LTR transcription in co-cultured CD4 T cells. Thus, Tregs inhibit HIV-1 infection of other CD4 T cell subsets via interactions with inhibitory cell surface proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

The distribution of HIV DNA and RNA in cell subsets differs in gut and blood of HIV-positive patients on ART: implications for viral persistence.

PubMed

Yukl, Steven A; Shergill, Amandeep K; Ho, Terence; Killian, Maudi; Girling, Valerie; Epling, Lorrie; Li, Peilin; Wong, Lisa K; Crouch, Pierre; Deeks, Steven G; Havlir, Diane V; McQuaid, Kenneth; Sinclair, Elizabeth; Wong, Joseph K

2013-10-15

Even with optimal antiretroviral therapy, human immunodeficiency virus (HIV) persists in plasma, blood cells, and tissues. To develop new therapies, it is essential to know what cell types harbor residual HIV. We measured levels of HIV DNA, RNA, and RNA/DNA ratios in sorted subsets of CD4+ T cells (CCR7+, transitional memory, and effector memory) and non-CD4+ T leukocytes from blood, ileum, and rectum of 8 ART-suppressed HIV-positive subjects. Levels of HIV DNA/million cells in CCR7+ and effector memory cells were higher in the ileum than blood. When normalized by cell frequencies, most HIV DNA and RNA in the blood were found in CCR7+ cells, whereas in both gut sites, most HIV DNA and RNA were found in effector memory cells. HIV DNA and RNA were observed in non-CD4+ T leukocytes at low levels, particularly in gut tissues. Compared to the blood, the ileum had higher levels of HIV DNA and RNA in both CD4+ T cells and non-CD4+ T leukocytes, whereas the rectum had higher HIV DNA levels in both cell types but lower RNA levels in CD4+ T cells. Future studies should determine whether different mechanisms allow HIV to persist in these distinct reservoirs, and the degree to which different therapies can affect each reservoir.

2B4 expression on natural killer cells increases in HIV-1 infected patients followed prospectively during highly active antiretroviral therapy

PubMed Central

Ostrowski, S R; Ullum, H; Pedersen, B K; Gerstoft, J; Katzenstein, T L

2005-01-01

Human immunodeficiency virus (HIV)-1 infection influences natural killer (NK) cell expression of inhibitory NK receptors and activating natural cytotoxicity receptors. It is unknown whether expression of the co-stimulatory NK cell receptor 2B4 (CD244) on NK cells and CD3+ CD8+ cells are affected by highly active antiretroviral therapy (HAART), low-level viraemia, proviral-DNA or immune activation in HIV-1 infected patients. A total of 101 HAART-treated HIV-1 infected patients with ≤ 200 HIV-RNA copies/ml were followed prospectively for 24 months. HIV-RNA was investigated 3-monthly and 2B4 expression on CD3− CD16+ NK cells and CD3+ CD8+ cells, proviral-DNA and plasma soluble tumour necrosis factor receptor (sTNFr)-II were investigated 6-monthly. For comparison, 2B4 expression was investigated in 20 healthy individuals. The concentration of 2B4+ NK cells was initially reduced in HIV-1 infected patients (P < 0·001) but increased to a normal level during the 24 months’ follow-up. The concentration of CD3+ CD8+ 2B4+ cells in HIV-1 infected patients was normal and did not change during follow-up. The relative fluorescence intensity (RFI) of 2B4 increased on both NK cells and CD3+ CD8+ cells during follow-up (both P < 0·001). Higher levels of proviral-DNA carrying cells and plasma sTNFrII were associated with reductions in the concentration of 2B4+ NK cells (all P < 0·05). HIV-RNA had no effect on 2B4 expression on NK cells or CD3+ CD8+ cells. These findings demonstrate that the concentration of 2B4+ NK cells normalizes during long-term HAART in HIV-1 infected patients. The finding that proviral-DNA and sTNFrII were associated negatively with the concentration of 2B4+ NK cells suggests that immune activation in HIV-1 infected patients receiving HAART influences the target cell recognition by NK cells. PMID:16045743

2B4 expression on natural killer cells increases in HIV-1 infected patients followed prospectively during highly active antiretroviral therapy.

PubMed

Ostrowski, S R; Ullum, H; Pedersen, B K; Gerstoft, J; Katzenstein, T L

2005-09-01

Human immunodeficiency virus (HIV)-1 infection influences natural killer (NK) cell expression of inhibitory NK receptors and activating natural cytotoxicity receptors. It is unknown whether expression of the co-stimulatory NK cell receptor 2B4 (CD244) on NK cells and CD3+ CD8+ cells are affected by highly active antiretroviral therapy (HAART), low-level viraemia, proviral-DNA or immune activation in HIV-1 infected patients. A total of 101 HAART-treated HIV-1 infected patients with < or = 200 HIV-RNA copies/ml were followed prospectively for 24 months. HIV-RNA was investigated 3-monthly and 2B4 expression on CD3- CD16+ NK cells and CD3+ CD8+ cells, proviral-DNA and plasma soluble tumour necrosis factor receptor (sTNFr)-II were investigated 6-monthly. For comparison, 2B4 expression was investigated in 20 healthy individuals. The concentration of 2B4+ NK cells was initially reduced in HIV-1 infected patients (P < 0.001) but increased to a normal level during the 24 months' follow-up. The concentration of CD3+ CD8+ 2B4+ cells in HIV-1 infected patients was normal and did not change during follow-up. The relative fluorescence intensity (RFI) of 2B4 increased on both NK cells and CD3+ CD8+ cells during follow-up (both P < 0.001). Higher levels of proviral-DNA carrying cells and plasma sTNFrII were associated with reductions in the concentration of 2B4+ NK cells (all P < 0.05). HIV-RNA had no effect on 2B4 expression on NK cells or CD3+ CD8+ cells. These findings demonstrate that the concentration of 2B4+ NK cells normalizes during long-term HAART in HIV-1 infected patients. The finding that proviral-DNA and sTNFrII were associated negatively with the concentration of 2B4+ NK cells suggests that immune activation in HIV-1 infected patients receiving HAART influences the target cell recognition by NK cells.

Novel mucosal DNA-MVA HIV vaccination in which DNA-IL-12 plus cholera toxin B subunit (CTB) cooperates to enhance cellular systemic and mucosal genital tract immunity.

PubMed

Maeto, Cynthia; Rodríguez, Ana María; Holgado, María Pía; Falivene, Juliana; Gherardi, María Magdalena

2014-01-01

Induction of local antiviral immune responses at the mucosal portal surfaces where HIV-1 and other viral pathogens are usually first encountered remains a primary goal for most vaccines against mucosally acquired viral infections. Exploring mucosal immunization regimes in order to find optimal vector combinations and also appropriate mucosal adjuvants in the HIV vaccine development is decisive. In this study we analyzed the interaction of DNA-IL-12 and cholera toxin B subunit (CTB) after their mucosal administration in DNA prime/MVA boost intranasal regimes, defining the cooperation of both adjuvants to enhance immune responses against the HIV-1 Env antigen. Our results demonstrated that nasal mucosal DNA/MVA immunization schemes can be effectively improved by the co-delivery of DNA-IL-12 plus CTB inducing elevated HIV-specific CD8 responses in spleen and more importantly in genital tract and genito-rectal draining lymph nodes. Remarkably, these CTL responses were of superior quality showing higher avidity, polyfunctionality and a broader cytokine profile. After IL-12+CTB co-delivery, the cellular responses induced showed an enhanced breadth recognizing with higher efficiency Env peptides from different subtypes. Even more, an in vivo CTL cytolytic assay demonstrated the higher specific CD8 T-cell performance after the IL-12+CTB immunization showing in an indirect manner its potential protective capacity. Improvements observed were maintained during the memory phase where we found higher proportions of specific central memory and T memory stem-like cells T-cell subpopulations. Together, our data show that DNA-IL-12 plus CTB can be effectively employed acting as mucosal adjuvants during DNA prime/MVA boost intranasal vaccinations, enhancing magnitude and quality of HIV-specific systemic and mucosal immune responses.

Quality of Human Immunodeficiency Virus Viral Load Testing in Australia

PubMed Central

Best, Susan J.; Gust, Anthony P.; Johnson, Elizabeth I. M.; McGavin, Catherine H.; Dax, Elizabeth M.

2000-01-01

This study determined the proficiencies of laboratories measuring human immunodeficiency virus type 1 (HIV-1) viral loads and the accuracies of two assays used for HIV-1 viral load measurement in Australia and investigated the variability of the new versions of these assays. Quality assessment program panels containing (i) dilutions of HIV-1 subtype B, (ii) replicates of identical samples of HIV-1 subtype B, and (iii) samples of subtype E and B were tested by laboratories. Total variability (within and between laboratories) was tested with quality control samples. The coefficients of variation (CVs) for the Roche AMPLICOR HIV-1 MONITOR version (v) 1.0 and Chiron Quantiplex bDNA 2.0 assays ranged from 53 to 87% and 22 to 31%, respectively. The widespread occurrence of invalid runs with the AMPLICOR HIV-1 MONITOR 1.0 assay was identified. The CVs of the new versions of the assays were 82 to 86% for the AMPLICOR HIV-1 MONITOR v 1.5 assay and 16 to 23% for the Quantiplex bDNA 3.0 assay. For virus dilution samples, all but 5 of 19 laboratories obtained results within 2 standard deviations of the mean. The Quantiplex bDNA 2.0 assay reported values lower than those reported by the AMPLICOR HIV-1 MONITOR version 1.0 assay for samples containing HIV-1 subtype B, whereas the reverse was true for subtype E. Identification and resolution of the problem of invalid runs markedly improved the quality of HIV-1 viral load testing. The variability observed between laboratories and between assays, even the most recent versions, dictates that monitoring of viral load in an individual should always be by the same laboratory and by the same assay. Results for an individual which differ by less than 0.5 log10 HIV-1 RNA copy number/ml should not be considered clinically significant. PMID:11060062

HIV-1 virological remission lasting more than 12 years after interruption of early antiretroviral therapy in a perinatally infected teenager enrolled in the French ANRS EPF-CO10 paediatric cohort: a case report.

PubMed

Frange, Pierre; Faye, Albert; Avettand-Fenoël, Véronique; Bellaton, Erianna; Descamps, Diane; Angin, Mathieu; David, Annie; Caillat-Zucman, Sophie; Peytavin, Gilles; Dollfus, Catherine; Le Chenadec, Jerome; Warszawski, Josiane; Rouzioux, Christine; Sáez-Cirión, Asier

2016-01-01

Durable HIV-1 remission after interruption of combined antiretroviral therapy (ART) has been reported in some adults who started treatment during primary infection; however, whether long-term remission in vertically infected children is possible was unknown. We report a case of a young adult perinatally infected with HIV-1 with viral remission despite long-term treatment interruption. The patient was identified in the ANRS EPF-CO10 paediatric cohort among 100 children infected with HIV perinatally who started ART before 6 months of age. HIV RNA viral load and CD4 cell counts were monitored from birth. Ultrasensitive HIV RNA, peripheral blood mononuclear cell (PBMC)-associated HIV DNA, HIV-specific T-cell responses (ie, production of cytokines and capacity to suppress HIV infection), reactivation of the CD4 cell reservoir (measured by p24 ELISA and HIV RNA in supernatants upon phytohaemagglutinin activation of purified CD4 cells), and plasma concentrations of antiretroviral drugs were assessed after 10 years of documented control off therapy. The infant was born in 1996 to a woman with uncontrolled HIV-1 viraemia and received zidovudine-based prophylaxis for 6 weeks. HIV RNA and DNA were not detected 3 days and 14 days after birth. HIV DNA was detected at 4 weeks of age. HIV RNA reached 2·17× 10(6) copies per mL at 3 months of age and ART was started. HIV RNA was undetectable 1 month later. ART was discontinued by the family at some point between 5·8 and 6·8 years of age. HIV RNA was undetectable at 6·8 years of age and ART was not resumed. HIV RNA has remained below 50 copies per mL and CD4 cell counts stable through to 18·6 years of age. After 11·5 years of control off treatment, HIV RNA was below 4 copies per mL and HIV DNA was 2·2 log10 copies per 10(6) PBMCs. The HLA genotype showed homozygosity at several loci (A*2301-, B*1503/4101, C*0210/0802, DRB1*1101-, and DQB1*0602-). HIV-specific CD8 T-cell responses and T-cell activation were weak. Findings from this case suggest that long-term HIV-1 remission is possible in perinatally infected children who receive treatment early, with characteristics similar to those reported in adult HIV post-treatment controllers. Further studies are needed to understand the mechanisms associated with HIV remission and whether early treatment of infected children might favour the conditions needed to achieve HIV control after treatment discontinuation. Agence de recherche ANRS (France Recherche Nord & Sud Sida-HIV Hépatites). Copyright © 2016 Elsevier Ltd. All rights reserved.

Dynamics of drug resistance-associated mutations in HIV-1 DNA reverse transcriptase sequence during effective ART.

PubMed

Nouchi, A; Nguyen, T; Valantin, M A; Simon, A; Sayon, S; Agher, R; Calvez, V; Katlama, C; Marcelin, A G; Soulie, C

2018-05-29

To investigate the dynamics of HIV-1 variants archived in cells harbouring drug resistance-associated mutations (DRAMs) to lamivudine/emtricitabine, etravirine and rilpivirine in patients under effective ART free from selective pressure on these DRAMs, in order to assess the possibility of recycling molecules with resistance history. We studied 25 patients with at least one DRAM to lamivudine/emtricitabine, etravirine and/or rilpivirine identified on an RNA sequence in their history and with virological control for at least 5 years under a regimen excluding all drugs from the resistant class. Longitudinal ultra-deep sequencing (UDS) and Sanger sequencing of the reverse transcriptase region were performed on cell-associated HIV-1 DNA samples taken over the 5 years of follow-up. Viral variants harbouring the analysed DRAMs were no longer detected by UDS over the 5 years in 72% of patients, with viruses susceptible to the molecules of interest found after 5 years in 80% of patients with UDS and in 88% of patients with Sanger. Residual viraemia with <50 copies/mL was detected in 52% of patients. The median HIV DNA level remained stable (2.4 at baseline versus 2.1 log10 copies/106 cells 5 years later). These results show a clear trend towards clearance of archived DRAMs to reverse transcriptase inhibitors in cell-associated HIV-1 DNA after a long period of virological control, free from therapeutic selective pressure on these DRAMs, reflecting probable residual replication in some reservoirs of the fittest viruses and leading to persistent evolution of the archived HIV-1 DNA resistance profile.

Prevalence, risk factors, and impact of isolated antibody to hepatitis B core antigen and occult hepatitis B virus infection in HIV-1-infected pregnant women.

PubMed

Khamduang, Woottichai; Ngo-Giang-Huong, Nicole; Gaudy-Graffin, Catherine; Jourdain, Gonzague; Suwankornsakul, Weerapong; Jarupanich, Tapnarong; Chalermpolprapa, Veeradate; Nanta, Sirisak; Puarattana-Aroonkorn, Noossara; Tonmat, Sakchai; Lallemant, Marc; Goudeau, Alain; Sirirungsi, Wasna

2013-06-01

Prevalence and risk factors for isolated antibody to hepatitis B core antigen (anti-HBc) and occult hepatitis B virus (HBV) infection are not well known in human immunodeficiency virus type 1 (HIV-1)-infected pregnant women. It is unclear if women with occult infections are at risk of transmitting HBV to their infants. HIV-1-infected and HBV surface antigen (HBsAg)-negative pregnant women were tested for antibody to HBsAg (anti-HBs) and anti-HBc using enzyme immunoassay. Women with isolated anti-HBc were assessed for occult HBV infection, defined as HBV DNA levels >15 IU/mL, using the Abbott RealTime HBV DNA assay. Infants born to women with isolated anti-HBc and detectable HBV DNA were tested at 4 months of age for HBV DNA. Logistic regression analysis was used to identify factors associated with isolated anti-HBc and occult HBV infection. Among 1812 HIV-infected pregnant women, 1682 were HBsAg negative. Fourteen percent (95% confidence interval [CI], 12%-15%) of HBsAg-negative women had an isolated anti-HBc that was independently associated with low CD4 count, age >35 years, birth in northern Thailand, and positive anti-hepatitis C virus serology. Occult HBV infection was identified in 24% (95% CI, 18%-30%) of women with isolated anti-HBc, representing 2.6% (95% CI, 1.9%-3.5%) of HIV-1-infected pregnant women, and was inversely associated with HIV RNA levels. None of the women with isolated anti-HBc and occult HBV infection transmitted HBV to their infants. HIV-1-infected pregnant women with isolated anti-HBc and occult HBV infection have very low HBV DNA levels and are thus at very low risk to transmit HBV to their infants.

Cross-Clade Ultrasensitive PCR-Based Assays To Measure HIV Persistence in Large-Cohort Studies

PubMed Central

Vandergeeten, Claire; Fromentin, Rémi; Merlini, Esther; Lawani, Mariam B.; DaFonseca, Sandrina; Bakeman, Wendy; McNulty, Amanda; Ramgopal, Moti; Michael, Nelson; Kim, Jerome H.; Ananworanich, Jintanat

2014-01-01

ABSTRACT A small pool of infected cells persists in HIV-infected individuals receiving antiretroviral therapy (ART). Here, we developed ultrasensitive assays to precisely measure the frequency of cells harboring total HIV DNA, integrated HIV DNA, and two long terminal repeat (2-LTR) circles. These assays are performed on cell lysates, which circumvents the labor-intensive step of DNA extraction, and rely on the coquantification of each HIV molecular form together with CD3 gene sequences to precisely measure cell input. Using primary isolates from HIV subtypes A, B, C, D, and CRF01_A/E, we demonstrate that these assays can efficiently quantify low target copy numbers from diverse HIV subtypes. We further used these assays to measure total HIV DNA, integrated HIV DNA, and 2-LTR circles in CD4+ T cells from HIV-infected subjects infected with subtype B. All samples obtained from ART-naive subjects were positive for the three HIV molecular forms (n = 15). Total HIV DNA, integrated HIV DNA, and 2-LTR circles were detected in, respectively, 100%, 94%, and 77% of the samples from individuals in which HIV was suppressed by ART. Higher levels of total HIV DNA and 2-LTR circles were detected in untreated subjects than individuals on ART (P = 0.0003 and P = 0.0004, respectively), while the frequency of CD4+ T cells harboring integrated HIV DNA did not differ between the two groups. These results demonstrate that these novel assays have the ability to quantify very low levels of HIV DNA of multiple HIV subtypes without the need for nucleic acid extraction, making them well suited for the monitoring of viral persistence in large populations of HIV-infected individuals. IMPORTANCE Since the discovery of viral reservoirs in HIV-infected subjects receiving suppressive ART, measuring the degree of viral persistence has been one of the greatest challenges in the field of HIV research. Here, we report the development and validation of ultrasensitive assays to measure HIV persistence in HIV-infected individuals from multiple geographical regions. These assays are relatively inexpensive, do not require DNA extraction, and can be completed in a single day. Therefore, they are perfectly adapted to monitor HIV persistence in large cohorts of HIV-infected individuals and, given their sensitivity, can be used to monitor the efficacy of therapeutic strategies aimed at interfering with HIV persistence after prolonged ART. PMID:25122785

Zinc finger nuclease: a new approach for excising HIV-1 proviral DNA from infected human T cells.

PubMed

Qu, Xiying; Wang, Pengfei; Ding, Donglin; Wang, Xiaohui; Zhang, Gongmin; Zhou, Xin; Liu, Lin; Zhu, Xiaoli; Zeng, Hanxian; Zhu, Huanzhang

2014-09-01

A major reason that Acquired Immune Deficiency Syndrome (AIDS) cannot be completely cured is the human immunodeficiency virus 1 (HIV-1) provirus integrated into the human genome. Though existing therapies can inhibit replication of HIV-1, they cannot eradicate it. A molecular therapy gains popularity due to its specifically targeting to HIV-1 infected cells and effectively removing the HIV-1, regardless of viral genes being active or dormant. Now, we propose a new method which can excellently delete the HIV provirus from the infected human T cell genome. First, we designed zinc-finger nucleases (ZFNs) that target a sequence within the long terminal repeat (LTR) U3 region that is highly conserved in whole clade. Then, we screened out one pair of ZFN and named it as ZFN-U3. We discovered that ZFN-U3 can exactly target and eliminate the full-length HIV-1 proviral DNA after the infected human cell lines treated with it, and the frequency of its excision was about 30 % without cytotoxicity. These results prove that ZFN-U3 can efficiently excise integrated HIV-1 from the human genome in infected cells. This method to delete full length HIV-1 in human genome can therefore provide a novel approach to cure HIV-infected individuals in the future.

Cell-Free (RNA) and Cell-Associated (DNA) HIV-1 and Postnatal Transmission through Breastfeeding

PubMed Central

Bland, Ruth M.; Danaviah, Siva; Thorne, Claire; Van de Perre, Philippe; Newell, Marie-Louise

2012-01-01

Introduction Transmission through breastfeeding remains important for mother-to-child transmission (MTCT) in resource-limited settings. We quantify the relationship between cell-free (RNA) and cell-associated (DNA) shedding of HIV-1 virus in breastmilk and the risk of postnatal HIV-1 transmission in the first 6 months postpartum. Materials and Methods Thirty-six HIV-positive mothers who transmitted HIV-1 by breastfeeding were matched to 36 non-transmitting HIV-1 infected mothers in a case-control study nested in a cohort of HIV-infected women. RNA and DNA were quantified in the same breastmilk sample taken at 6 weeks and 6 months. Cox regression analysis assessed the association between cell-free and cell-associated virus levels and risk of postnatal HIV-1 transmission. Results There were higher median levels of cell-free than cell-associated HIV-1 virus (per ml) in breastmilk at 6 weeks and 6 months. Multivariably, adjusting for antenatal CD4 count and maternal plasma viral load, at 6 weeks, each 10-fold increase in cell-free or cell-associated levels (per ml) was significantly associated with HIV-1 transmission but stronger for cell-associated than cell-free levels [2.47 (95% CI 1.33–4.59) vs. aHR 1.52 (95% CI, 1.17–1.96), respectively]. At 6 months, cell-free and cell-associated levels (per ml) in breastmilk remained significantly associated with HIV-1 transmission but was stronger for cell-free than cell-associated levels [aHR 2.53 (95% CI 1.64–3.92) vs. 1.73 (95% CI 0.94–3.19), respectively]. Conclusions The findings suggest that cell-associated virus level (per ml) is more important for early postpartum HIV-1 transmission (at 6 weeks) than cell-free virus. As cell-associated virus levels have been consistently detected in breastmilk despite antiretroviral therapy, this highlights a potential challenge for resource-limited settings to achieve the UNAIDS goal for 2015 of eliminating vertical transmission. More studies would further knowledge on mechanisms of HIV-1 transmission and help develop more effective drugs during lactation. PMID:23284701

Treatment intensification with maraviroc (CCR5 antagonist) leads to declines in CD16-expressing monocytes in cART-suppressed chronic HIV-infected subjects and is associated with improvements in neurocognitive test performance: implications for HIV-associated neurocognitive disease (HAND).

PubMed

Ndhlovu, Lishomwa C; Umaki, Tracie; Chew, Glen M; Chow, Dominic C; Agsalda, Melissa; Kallianpur, Kalpana J; Paul, Robert; Zhang, Guangxiang; Ho, Erika; Hanks, Nancy; Nakamoto, Beau; Shiramizu, Bruce T; Shikuma, Cecilia M

2014-12-01

HIV-associated neurocognitive disorders (HAND) continues to be prevalent (30-50%) despite plasma HIV-RNA suppression with combination antiretroviral therapy (cART). There is no proven therapy for individuals on suppressive cART with HAND. We have shown that the degree of HIV reservoir burden (HIV DNA) in monocytes appear to be linked to cognitive outcomes. HIV infection of monocytes may therefore be critical in the pathogenesis of HAND. A single arm, open-labeled trial was conducted to examine the effect of maraviroc (MVC) intensification on monocyte inflammation and neuropsychological (NP) performance in 15 HIV subjects on stable 6-month cART with undetectable plasma HIV RNA (<48 copies/ml) and detectable monocyte HIV DNA (>10 copies/10(6) cells). MVC was added to their existing cART regimen for 24 weeks. Post-intensification change in monocytes was assessed using multiparametric flow cytometry, monocyte HIV DNA content by PCR, soluble CD163 (sCD163) by an ELISA, and NP performance over 24 weeks. In 12 evaluable subjects, MVC intensification resulted in a decreased proportion of circulating intermediate (median; 3.06% (1.93, 6.45) to 1.05% (0.77, 2.26)) and nonclassical (5.2% (3.8, 7.9) to 3.2% (1.8, 4.8)) CD16-expressing monocytes, a reduction in monocyte HIV DNA content to zero log10 copies/10(6) cells and in levels of sCD163 of 43% by 24 weeks. This was associated with significant improvement in NP performance among six subjects who entered the study with evidence of mild to moderate cognitive impairment. The results of this study suggest that antiretroviral therapy with potency against monocytes may have efficacy against HAND.

CD4+ T Cells Expressing PD-1, TIGIT and LAG-3 Contribute to HIV Persistence during ART

PubMed Central

Fromentin, Rémi; Bakeman, Wendy; Lawani, Mariam B.; Khoury, Gabriela; Hartogensis, Wendy; DaFonseca, Sandrina; Killian, Marisela; Epling, Lorrie; Hoh, Rebecca; Sinclair, Elizabeth; Hecht, Frederick M.; Bacchetti, Peter; Deeks, Steven G.; Lewin, Sharon R.; Sékaly, Rafick-Pierre; Chomont, Nicolas

2016-01-01

HIV persists in a small pool of latently infected cells despite antiretroviral therapy (ART). Identifying cellular markers expressed at the surface of these cells may lead to novel therapeutic strategies to reduce the size of the HIV reservoir. We hypothesized that CD4+ T cells expressing immune checkpoint molecules would be enriched in HIV-infected cells in individuals receiving suppressive ART. Expression levels of 7 immune checkpoint molecules (PD-1, CTLA-4, LAG-3, TIGIT, TIM-3, CD160 and 2B4) as well as 4 markers of HIV persistence (integrated and total HIV DNA, 2-LTR circles and cell-associated unspliced HIV RNA) were measured in PBMCs from 48 virally suppressed individuals. Using negative binomial regression models, we identified PD-1, TIGIT and LAG-3 as immune checkpoint molecules positively associated with the frequency of CD4+ T cells harboring integrated HIV DNA. The frequency of CD4+ T cells co-expressing PD-1, TIGIT and LAG-3 independently predicted the frequency of cells harboring integrated HIV DNA. Quantification of HIV genomes in highly purified cell subsets from blood further revealed that expressions of PD-1, TIGIT and LAG-3 were associated with HIV-infected cells in distinct memory CD4+ T cell subsets. CD4+ T cells co-expressing the three markers were highly enriched for integrated viral genomes (median of 8.2 fold compared to total CD4+ T cells). Importantly, most cells carrying inducible HIV genomes expressed at least one of these markers (median contribution of cells expressing LAG-3, PD-1 or TIGIT to the inducible reservoir = 76%). Our data provide evidence that CD4+ T cells expressing PD-1, TIGIT and LAG-3 alone or in combination are enriched for persistent HIV during ART and suggest that immune checkpoint blockers directed against these receptors may represent valuable tools to target latently infected cells in virally suppressed individuals. PMID:27415008

Prevalence of human immunodeficiency virus type 1 DNA in hemophilic men and their sex partners. Hemophilia-AIDS Collaborative Study Group.

PubMed

Jason, J; Ou, C Y; Moore, J L; Lawrence, D N; Schochetman, G; Evatt, B L

1989-11-01

Polymerase chain reaction (PCR) was used to detect human immunodeficiency virus (HIV)-1 DNA in peripheral blood mononuclear cells to assess in hemophilic men whether any were HIV-seropositive but uninfected or seronegative but infected and in seronegative sex partners of seropositive hemophilic men whether any were infected. Of 40 seropositive men, 38 (95%) were PCR-positive; one was PCR-indeterminate and one PCR-negative. None of 41 seronegative men who used only donor-screened, virus-inactivated coagulation factor products were PCR-positive. However, two of six who received noninactivated products were PCR-positive; one had low T-helper cell counts and died of unrelated causes and the other had seroconverted 11 mo later. PCR with a second primer pair also detected HIV-1 DNA in these two men. None of 25 seronegative female sex partners of seropositive men, including six men with AIDS and seven with AIDS-related symptoms, were PCR-positive. These data suggest that most seropositive hemophilic men are HIV-infected; whether some are infected with defective virus remains to be resolved as does the infection status of seropositive PCR-negative men. Identification of two seronegative PCR-positive men supports the possibility that HIV-1 DNA can be detected before seroconversion.

Characterization of active reverse transcriptase and nucleoprotein complexes of the yeast retrotransposon Ty3 in vitro.

PubMed

Cristofari, G; Gabus, C; Ficheux, D; Bona, M; Le Grice, S F; Darlix, J L

1999-12-17

Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively. In vitro binding of HIV-1 NCp7 to viral 5' RNA and primer tRNA(3)(Lys) catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid. Nucleocapsid complex formation functions in viral RNA dimerization and tRNA annealing to the primer binding site (PBS). RT is recruited in these nucleoprotein complexes and synthesizes minus-strand cDNA initiated at the PBS. Recent results on yeast Ty3 have shown that the homologous NCp9 promotes annealing of primer tRNA(i)(Met) to a 5'-3' bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5' PBS (). To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3 RNA with the 5'-3' PBS, primer tRNA(i)(Met), NCp9, and for the first time, highly purified Ty3 RT. Here we report that Ty3 RT is as active as retroviral HIV-1 or murine leukemia virus RT using a synthetic template-primer system. Moreover, and in contrast to what was found with retroviral RTs, retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming. We also show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT. These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7.

HIV sequence diversity during the early phase of infection is associated with HIV DNA reductions during antiretroviral therapy.

PubMed

Wang, Nidan; Li, Yijia; Han, Yang; Xie, Jing; Li, Taisheng

2017-06-01

The association between baseline human immunodeficiency virus (HIV) sequence diversity and HIV DNA decay after the initiation of antiretroviral therapy (ART) remains uncharacterized during the early stages of HIV infection. Samples were obtained from a cohort of 17 patients with early HIV infection (<6 months after infection) who initiated ART, and the C2V5 region of the HIV-1 envelope (env) gene was amplified via single genome amplification (SGA) to determine the peripheral plasma HIV quasispecies. We categorized HIV quasispecies into two groups according to baseline viral sequence genetic distance, which was determined by the Poisson-Fitter tool. Total HIV DNA in peripheral blood mononuclear cells (PBMCs), viral load, and T cell subsets were measured prior to and after the initiation of ART. The median SGA sequence number was 17 (range 6-28). At baseline, we identified 7 patients with homogeneous viral populations (designated the Homogeneous group) and 10 patients with heterogeneous viral populations (designated the Heterogeneous group) based on SGA sequences. Both groups exhibited similar HIV DNA decay rates during the first 6 months of ART (P > 0.99), but the Homogenous group experienced more prominent decay than the Heterogeneous group after 6 months (P = 0.037). The Heterogeneous group had higher CD4 cell counts after ART initiation; however, both groups had comparable recovery in terms of CD4/CD8 ratios and CD8 T cell activation levels. Viral population homogeneity upon the initiation of ART is associated with a decrease in HIV DNA levels during ART. J. Med. Virol. 89:982-988, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Randomized phase I trial HIV-CORE 003: Depletion of serum amyloid P component and immunogenicity of DNA vaccination against HIV-1.

PubMed

Borthwick, Nicola J; Lane, Thirusha; Moyo, Nathifa; Crook, Alison; Shim, Jung Min; Baines, Ian; Wee, Edmund G; Hawkins, Philip N; Gillmore, Julian D; Hanke, Tomáš; Pepys, Mark B

2018-01-01

The failure of DNA vaccination in humans, in contrast to its efficacy in some species, is unexplained. Observational and interventional experimental evidence suggests that DNA immunogenicity may be prevented by binding of human serum amyloid P component (SAP). SAP is the single normal DNA binding protein in human plasma. The drug (R)-1-[6-[(R)-2-carboxypyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC, miridesap), developed for treatment of systemic amyloidosis and Alzheimer's disease, depletes circulating SAP by 95-99%. The proof-of-concept HIV-CORE 003 clinical trial tested whether SAP depletion by CPHPC would enhance the immune response in human volunteers to DNA vaccination delivering the HIVconsv immunogen derived from conserved sub-protein regions of HIV-1. Human volunteers received 3 intramuscular immunizations with an experimental DNA vaccine (DDD) expressing HIV-1-derived immunogen HIVconsv, with or without prior depletion of SAP by CPHPC. All subjects were subsequently boosted by simian (chimpanzee) adenovirus (C)- and poxvirus MVA (M)-vectored vaccines delivering the same immunogen. After administration of each vaccine modality, the peak total magnitudes, kinetics, functionality and memory subsets of the T-cell responses to HIVconsv were thoroughly characterized. No differences were observed between the CPHPC treated and control groups in any of the multiple quantitative and qualitative parameters of the T-cell responses to HIVconsv, except that after SAP depletion, there was a statistically significantly greater breadth of T-cell specificities, that is the number of recognized epitopes, following the DDDC vaccination. The protocol used here for SAP depletion by CPHPC prior to DNA vaccination produced only a very modest suggestion of enhanced immunogenicity. Further studies will be required to determine whether SAP depletion might have a practical value in DNA vaccination for other plasmid backbones and/or immunogens. Clinicaltrials.gov NCT02425241.

Intra- and Interlaboratory Variabilities of Results Obtained with the Quantiplex Human Immunodeficiency Virus Type 1 RNA bDNA Assay, Version 3.0

PubMed Central

Kellogg, James A.; Atria, Peter V.; Sanders, Jeffrey C.; Eyster, M. Elaine

2001-01-01

Normal assay variation associated with bDNA tests for human immunodeficiency virus type 1 (HIV-1) RNA performed at two laboratories with different levels of test experience was investigated. Two 5-ml aliquots of blood in EDTA tubes were collected from each patient for whom the HIV-1 bDNA test was ordered. Blood was stored for no more than 4 h at room temperature prior to plasma separation. Plasma was stored at −70°C until transported to the Central Pennsylvania Alliance Laboratory (CPAL; York, Pa.) and to the Hershey Medical Center (Hershey, Pa.) on dry ice. Samples were stored at ≤−70°C at both laboratories prior to testing. Pools of negative (donor), low-HIV-1-RNA-positive, and high-HIV-1-RNA-positive plasma samples were also repeatedly tested at CPAL to determine both intra- and interrun variation. From 11 August 1999 until 14 September 2000, 448 patient specimens were analyzed in parallel at CPAL and Hershey. From 206 samples with results of ≥1,000 copies/ml at CPAL, 148 (72%) of the results varied by ≤0.20 log10 when tested at Hershey and none varied by >0.50 log10. However, of 242 specimens with results of <1,000 copies/ml at CPAL, 11 (5%) of the results varied by >0.50 log10 when tested at Hershey. Of 38 aliquots of HIV-1 RNA pool negative samples included in 13 CPAL bDNA runs, 37 (97%) gave results of <50 copies/ml and 1 (3%) gave a result of 114 copies/ml. Low-positive HIV-1 RNA pool intrarun variation ranged from 0.06 to 0.26 log10 while the maximum interrun variation was 0.52 log10. High-positive HIV-1 RNA pool intrarun variation ranged from 0.04 to 0.32 log10, while the maximum interrun variation was 0.55 log10. In our patient population, a change in bDNA HIV-1 RNA results of ≤0.50 log10 over time most likely represents normal laboratory test variation. However, a change of >0.50 log10, especially if the results are >1,000 copies/ml, is likely to be significant. PMID:11329458

Intra- and interlaboratory variabilities of results obtained with the Quantiplex human immunodeficiency virus type 1 RNA bDNA assay, version 3.0.

PubMed

Kellogg, J A; Atria, P V; Sanders, J C; Eyster, M E

2001-05-01

Normal assay variation associated with bDNA tests for human immunodeficiency virus type 1 (HIV-1) RNA performed at two laboratories with different levels of test experience was investigated. Two 5-ml aliquots of blood in EDTA tubes were collected from each patient for whom the HIV-1 bDNA test was ordered. Blood was stored for no more than 4 h at room temperature prior to plasma separation. Plasma was stored at -70 degrees C until transported to the Central Pennsylvania Alliance Laboratory (CPAL; York, Pa.) and to the Hershey Medical Center (Hershey, Pa.) on dry ice. Samples were stored at < or =-70 degrees C at both laboratories prior to testing. Pools of negative (donor), low-HIV-1-RNA-positive, and high-HIV-1-RNA-positive plasma samples were also repeatedly tested at CPAL to determine both intra- and interrun variation. From 11 August 1999 until 14 September 2000, 448 patient specimens were analyzed in parallel at CPAL and Hershey. From 206 samples with results of > or =1,000 copies/ml at CPAL, 148 (72%) of the results varied by < or =0.20 log(10) when tested at Hershey and none varied by >0.50 log(10). However, of 242 specimens with results of <1,000 copies/ml at CPAL, 11 (5%) of the results varied by >0.50 log(10) when tested at Hershey. Of 38 aliquots of HIV-1 RNA pool negative samples included in 13 CPAL bDNA runs, 37 (97%) gave results of <50 copies/ml and 1 (3%) gave a result of 114 copies/ml. Low-positive HIV-1 RNA pool intrarun variation ranged from 0.06 to 0.26 log(10) while the maximum interrun variation was 0.52 log(10). High-positive HIV-1 RNA pool intrarun variation ranged from 0.04 to 0.32 log(10), while the maximum interrun variation was 0.55 log(10). In our patient population, a change in bDNA HIV-1 RNA results of < or =0.50 log(10) over time most likely represents normal laboratory test variation. However, a change of >0.50 log(10), especially if the results are >1,000 copies/ml, is likely to be significant.

Long Terminal Repeat Circular DNA as Markers of Active Viral Replication of Human T Lymphotropic Virus-1 in Vivo.

PubMed

Fox, James M; Hilburn, Silva; Demontis, Maria-Antonietta; Brighty, David W; Rios Grassi, Maria Fernanda; Galvão-Castro, Bernardo; Taylor, Graham P; Martin, Fabiola

2016-03-14

Clonal expansion of human T-lymphotropic virus type-1 (HTLV-1) infected cells in vivo is well documented. Unlike human immunodeficiency virus type 1 (HIV-1), HTLV-1 plasma RNA is sparse. The contribution of the "mitotic" spread of HTLV-1 compared with infectious spread of the virus to HTLV-1 viral burden in established infection is uncertain. Since extrachromosomal long terminal repeat (LTR) DNA circles are indicators of viral replication in HIV-1 carriers with undetectable plasma HIV RNA, we hypothesised that HTLV-1 LTR circles could indicate reverse transcriptase (RT) usage and infectious activity. 1LTR and 2LTR DNA circles were measured in HTLV-1 cell lines and peripheral blood mononuclear cells (PBMC) of asymptomatic carriers (ACs) and patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukaemia/lymphoma (ATLL). 1LTR DNA circles were detected in 14/20 patients at a mean of 1.38/100 PBMC but did not differentiate disease status nor correlate with HTLV-1 DNA copies. 2LTR DNA circles were detected in 30/31 patients and at higher concentrations in patients with HTLV-1-associated diseases, independent of HTLV-1 DNA load. In an incident case the 2LTR DNA circle concentration increased 2.1 fold at the onset of HAM/TSP compared to baseline. Detectable and fluctuating levels of HTLV-1 DNA circles in patients indicate viral RT usage and virus replication. Our results indicate HTLV-1 viral replication capacity is maintained in chronic infection and may be associated with disease onset.

Increasing procaspase 8 expression using repurposed drugs to induce HIV infected cell death in ex vivo patient cells

PubMed Central

Sampath, Rahul; Cummins, Nathan W.; Natesampillai, Sekar; Bren, Gary D.; Chung, Thomas D.; Baker, Jason; Henry, Keith; Pagliuzza, Amélie; Badley, Andrew D.

2017-01-01

HIV persists because a reservoir of latently infected CD4 T cells do not express viral proteins and are indistinguishable from uninfected cells. One approach to HIV cure suggests that reactivating HIV will activate cytotoxic pathways; yet when tested in vivo, reactivating cells do not die sufficiently to reduce cell-associated HIV DNA levels. We recently showed that following reactivation from latency, HIV infected cells generate the HIV specific cytotoxic protein Casp8p41 which is produced by HIV protease cleaving procaspase 8. However, cell death is prevented, possibly due to low procaspase 8 expression. Here, we tested whether increasing procaspase 8 levels in CD4 T cells will produce more Casp8p41 following HIV reactivation, causing more reactivated cells to die. Screening 1277 FDA approved drugs identified 168 that increased procaspase 8 expression by at least 1.7-fold. Of these 30 were tested for anti-HIV effects in an acute HIVIIIb infection model, and 9 drugs at physiologic relevant levels significantly reduced cell-associated HIV DNA. Primary CD4 T cells from ART suppressed HIV patients were treated with one of these 9 drugs and reactivated with αCD3/αCD28. Four drugs significantly increased Casp8p41 levels following HIV reactivation, and decreased total cell associated HIV DNA levels (flurbiprofen: p = 0.014; doxycycline: p = 0.044; indomethacin: p = 0.025; bezafibrate: P = 0.018) without effecting the viability of uninfected cells. Thus procaspase 8 levels can be increased pharmacologically and, in the context of HIV reactivation, increase Casp8p41 causing death of reactivating cells and decreased HIV DNA levels. Future studies will be required to define the clinical utility of this or similar approaches. PMID:28628632

Concordance of polymerase chain reaction with human immunodeficiency virus antibody detection.

PubMed

Horsburgh, C R; Ou, C Y; Jason, J; Holmberg, S D; Lifson, A R; Moore, J L; Ward, J W; Seage, G R; Mayer, K H; Evatt, B L

1990-08-01

To evaluate the correlation of detection of human immunodeficiency virus (HIV) by polymerase chain reaction (PCR) with detection of HIV antibody, 271 simultaneous serum and peripheral blood mononuclear cell samples were examined from 242 persons whose activities placed them at increased risk for HIV infection: 142 from homosexual men, 86 from hemophilic men, and 43 from heterosexual partners of HIV-infected persons. PCR was performed using the gag region primer pair SK38/39 and the env region primer pairs SK68/69 and CO71/72. Amplified HIV DNA was detected using specific oligomer probes. Of 63 HIV antibody-positive samples, 58 (92%) had HIV DNA by PCR. Of 208 HIV antibody-negative samples, 7 (3.4%) had HIV DNA by PCR. On follow-up, 4 of the latter persons were seropositive when next tested; 2 were well and antibody- and PCR-negative; 1 had died of a stroke before retesting. Thus, PCR detects HIV in most antibody-positive persons; detection is increased by use of multiple primer pairs. PCR-positive antibody-negative specimens may indicate HIV infection in which antibody has not yet developed or may be false-positive PCR results. When PCR is discordant with HIV antibody, testing of additional specimens and clinical follow-up are necessary to assess HIV infection status.

Engineering RENTA, a DNA prime-MVA boost HIV vaccine tailored for Eastern and Central Africa.

PubMed

Nkolola, J P; Wee, E G-T; Im, E-J; Jewell, C P; Chen, N; Xu, X-N; McMichael, A J; Hanke, T

2004-07-01

For the development of human immunodeficiency virus type 1 (HIV-1) vaccines, traditional approaches inducing virus-neutralizing antibodies have so far failed. Thus the effort is now focused on elicitation of cellular immunity. We are currently testing in clinical trials in the United Kingdom and East Africa a T-cell vaccine consisting of HIV-1 clade A Gag-derived immunogen HIVA delivered in a prime-boost regimen by a DNA plasmid and modified vaccinia virus Ankara (MVA). Here, we describe engineering and preclinical development of a second immunogen RENTA, which will be used in combination with the present vaccine in a four-component DNA/HIVA-RENTA prime-MVA/HIVA-RENTA boost formulation. RENTA is a fusion protein derived from consensus HIV clade A sequences of Tat, reverse transcriptase, Nef and gp41. We inactivated the natural biological activities of the HIV components and confirmed immunogenicities of the pTHr.RENTA and MVA.RENTA vaccines in mice. Furthermore, we demonstrated in mice and rhesus monkeys broadening of HIVA-elicited T-cell responses by a parallel induction of HIVA- and RENTA-specific responses recognizing multiple HIV epitopes.

Approaches to Preventative and Therapeutic HIV vaccines

PubMed Central

Gray, Glenda E.; Laher, Fatima; Lazarus, Erica; Ensoli, Barbara; Corey, Lawrence

2016-01-01

Novel strategies are being researched to discover vaccines to prevent and treat HIV-1. Nonefficacious preventative vaccine approaches include bivalent recombinant gp120 alone, HIV gene insertion into an Adenovirus 5 (Ad5) virus vector and the DNA prime/Ad5 boost vaccine regimen. However, the ALVAC-HIV prime/AIDSVAX® B/E gp120 boost regimen showed 31.2% efficacy at 3.5 years, and is being investigated as clade C constructs with an additional boost. Likewise, although multiple therapeutic vaccines have failed in the past, in a non-placebo controlled trial, a Tat vaccine demonstrated immune cell restoration, reduction of immune activation, and reduced HIV-1 DNA viral load. Monoclonal antibodies for passive immunization or treatment show promise, with VRC01 entering advanced clinical trials. PMID:26985884

Optical tweezers reveal how proteins alter replication

NASA Astrophysics Data System (ADS)

Chaurasiya, Kathy

Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic acids. We use single molecule DNA stretching to show that the nucleocapsid protein (NC) of the yeast retrotransposon Ty3, which is likely to be an ancestor of HIV NC, has optimal nucleic acid chaperone activity with only a single zinc finger. We also show that the chaperone activity of the ORF1 protein is responsible for successful replication of the mouse LINE-1 retrotransposon. LINE-1 is also 17% of the human genome, where it generates insertion mutations and alters gene expression. Retrotransposons such as LINE-1 and Ty3 are likely to be ancestors of retroviruses such as HIV. Human APOBEC3G (A3G) inhibits HIV-1 replication via cytidine deamination of the viral ssDNA genome, as well as via a distinct deamination-independent mechanism. Efficient deamination requires rapid on-off binding kinetics, but a slow dissociation rate is required for the proposed deaminase-independent mechanism. We resolve this apparent contradiction with a new quantitative single molecule method, which shows that A3G initially binds ssDNA with fast on-off rates and subsequently converts to a slow binding mode. This suggests that oligomerization transforms A3G from a fast enzyme to a slow binding protein, which is the biophysical mechanism that allows A3G to inhibit HIV replication. A complete understanding of the mechanism of A3G-mediated antiviral activity is required to design drugs that disrupt the viral response to A3G, enhance A3G packaging inside the viral core, and other potential strategies for long-term treatment of HIV infection. We use single molecule biophysics to explore the function of proteins involved in bacterial DNA replication, endogenous retrotransposition of retroelements in eukaryotic hosts such yeast and mice, and HIV replication in human cells. Our quantitative results provide insight into protein function in a range of complex biological systems and have wide-ranging implications for human health.

Nuclear trafficking of the HIV-1 pre-integration complex depends on the ADAM10 intracellular domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Endsley, Mark A., E-mail: maendsle@utmb.edu; Somasunderam, Anoma D., E-mail: asomasun@utmb.edu; Li, Guangyu, E-mail: LIG001@mail.etsu.edu

Previously, we showed that ADAM10 is necessary for HIV-1 replication in primary human macrophages and immortalized cell lines. Silencing ADAM10 expression interrupted the HIV-1 life cycle prior to nuclear translocation of viral cDNA. Furthermore, our data indicated that HIV-1 replication depends on the expression of ADAM15 and γ-secretase, which proteolytically processes ADAM10. Silencing ADAM15 or γ-secretase expression inhibits HIV-1 replication between reverse transcription and nuclear entry. Here, we show that ADAM10 expression also supports replication in CD4{sup +} T lymphocytes. The intracellular domain (ICD) of ADAM10 associates with the HIV-1 pre-integration complex (PIC) in the cytoplasm and immunoprecipitates and co-localizesmore » with HIV-1 integrase, a key component of PIC. Taken together, our data support a model whereby ADAM15/γ-secretase processing of ADAM10 releases the ICD, which then incorporates into HIV-1 PIC to facilitate nuclear trafficking. Thus, these studies suggest ADAM10 as a novel therapeutic target for inhibiting HIV-1 prior to nuclear entry. - Highlights: • Nuclear trafficking of the HIV-1 pre-integration complex depends on ADAM10. • ADAM10 associates with HIV-1 integrase in the pre-integration complex. • HIV-1 replication depends on the expression of ADAM15 and γ-secretase. • Silencing ADAM15 or γ-secretase expression inhibits nuclear import of viral cDNA. • ADAM10 is important for HIV-1 replication in human macrophages and CD4{sup +} T lymphocytes.« less

Occult hepatitis B virus infection in HIV positive patients at a tertiary healthcare unit in eastern India

PubMed Central

Saha, Debraj; Pal, Ananya; Sarkar, Neelakshi; Das, Dipanwita; Blackard, Jason T.; Guha, Subhasish Kamal; Saha, Bibhuti

2017-01-01

Occult HBV infection (OBI), defined by the presence of HBV DNA in absence of hepatitis B surface antigen (HBsAg), is a significant concern in the HIV-infected population. Of 441 HIV+/HBsAg- patients analyzed, the overall prevalence of OBI was 6.3% (28/441). OBI was identified in 21 anti-HBc positives (17.8%), as well as among those who lacked any HBV-specific serological markers (2.2%). Comparison with HIV/HBV co-infection revealed that the levels of CD4, ALT, and HBV DNA were significantly lower during occult infection. Discrete differences were also observed with respect to quasispecies divergence. Additionally, subgenotype D1 was most frequent in occult infection, while D2 was widespread during chronic infection. The majority (~90%) of occult D1 sequences had the sQ129R mutation in the surface gene. This study highlights several distinct features of OBI in India and underscores the need for additional HBV DNA screening in HIV-positive individuals. PMID:28591184

Occult hepatitis B virus infection in HIV positive patients at a tertiary healthcare unit in eastern India.

PubMed

Saha, Debraj; Pal, Ananya; Sarkar, Neelakshi; Das, Dipanwita; Blackard, Jason T; Guha, Subhasish Kamal; Saha, Bibhuti; Chakravarty, Runu

2017-01-01

Occult HBV infection (OBI), defined by the presence of HBV DNA in absence of hepatitis B surface antigen (HBsAg), is a significant concern in the HIV-infected population. Of 441 HIV+/HBsAg- patients analyzed, the overall prevalence of OBI was 6.3% (28/441). OBI was identified in 21 anti-HBc positives (17.8%), as well as among those who lacked any HBV-specific serological markers (2.2%). Comparison with HIV/HBV co-infection revealed that the levels of CD4, ALT, and HBV DNA were significantly lower during occult infection. Discrete differences were also observed with respect to quasispecies divergence. Additionally, subgenotype D1 was most frequent in occult infection, while D2 was widespread during chronic infection. The majority (~90%) of occult D1 sequences had the sQ129R mutation in the surface gene. This study highlights several distinct features of OBI in India and underscores the need for additional HBV DNA screening in HIV-positive individuals.

Broad and Cross-Clade CD4+ T-Cell Responses Elicited by a DNA Vaccine Encoding Highly Conserved and Promiscuous HIV-1 M-Group Consensus Peptides

PubMed Central

Almeida, Rafael Ribeiro; Rosa, Daniela Santoro; Ribeiro, Susan Pereira; Santana, Vinicius Canato; Kallás, Esper Georges; Sidney, John; Sette, Alessandro; Kalil, Jorge; Cunha-Neto, Edecio

2012-01-01

T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4+ T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4+ T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4+ T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IAb and IAd. Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-γ secretion against 11 out of the 27 peptides in BALB/c mice; CD4+ and CD8+ T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4+ and CD8+ T cells, able to simultaneously proliferate and produce IFN-γ and TNF-α, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS. PMID:23028895

Distinct pattern of TP53 mutations in human immunodeficiency virus-related head and neck squamous cell carcinoma.

PubMed

Gleber-Netto, Frederico O; Zhao, Mei; Trivedi, Sanchit; Wang, Jiping; Jasser, Samar; McDowell, Christina; Kadara, Humam; Zhang, Jiexin; Wang, Jing; William, William N; Lee, J Jack; Nguyen, Minh Ly; Pai, Sara I; Walline, Heather M; Shin, Dong M; Ferris, Robert L; Carey, Thomas E; Myers, Jeffrey N; Pickering, Curtis R

2018-01-01

Human immunodeficiency virus-infected individuals (HIVIIs) have a higher incidence of head and neck squamous cell carcinoma (HNSCC), and clinical and histopathological differences have been observed in their tumors in comparison with those of HNSCC patients without a human immunodeficiency virus (HIV) infection. The reasons for these differences are not clear, and molecular differences between HIV-related HNSCC and non-HIV-related HNSCC may exist. This study compared the mutational patterns of HIV-related HNSCC and non-HIV-related HNSCC. The DNA of 20 samples of HIV-related HNSCCs and 32 samples of non-HIV-related HNSCCs was sequenced. DNA libraries covering exons of 18 genes frequently mutated in HNSCC (AJUBA, CASP8, CCND1, CDKN2A, EGFR, FAT1, FBXW7, HLA-A, HRAS, KEAP1, NFE2L2, NOTCH1, NOTCH2, NSD1, PIK3CA, TGFBR2, TP53, and TP63) were prepared and sequenced on an Ion Personal Genome Machine sequencer. DNA sequencing data were analyzed with Ion Reporter software. The human papillomavirus (HPV) status of the tumor samples was assessed with in situ hybridization, the MassARRAY HPV multiplex polymerase chain reaction assay, and p16 immunostaining. Mutation calls were compared among the studied groups. HIV-related HNSCC revealed a distinct pattern of mutations in comparison with non-HIV-related HNSCC. TP53 mutation frequencies were significantly lower in HIV-related HNSCC. Mutations in HIV+ patients tended to be TpC>T nucleotide changes for all mutated genes but especially for TP53. HNSCC in HIVIIs presents a distinct pattern of genetic mutations, particularly in the TP53 gene. HIV-related HNSCC may have a distinct biology, and an effect of the HIV virus on the pathogenesis of these tumors should not be ruled out. Cancer 2018;124:84-94. © 2017 American Cancer Society. © 2017 American Cancer Society.

Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

NASA Astrophysics Data System (ADS)

Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

1990-09-01

Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

HIV RNA and proviral HIV DNA can be detected in semen after 6 months of antiretroviral therapy although HIV RNA is undetectable in blood.

PubMed

Du, Peiwei; Liu, An; Jiao, Yanmei; Liu, Cuie; Jiang, Taiyi; Zhu, Weijun; Zhu, Yunxia; Wu, Hao; Sun, Lijun

2016-03-01

The risk of sexual transmission of HIV is strongly correlated with amounts of genital HIV RNA. Few studies have reported amounts of HIV RNA and HIV DNA in semen in HIV-infected Chinese patients undergoing antiviral treatment (ART). In this observational study, the amounts of HIV RNA and HIV DNA in semen were assessed after six months of ART in HIV-infected Chinese individuals, when HIV RNA was undetectable in blood . This study included 19 HIV-infected Chinese men undergoing ART for six months. Amounts of HIV in paired semen and blood samples were assessed using real-time PCR. The C2-V5 region of the HIV envelope (env) genes was cloned and sequenced and genotype and co-receptor usage predicted based on the sequence. It was found that HIV RNA was undetectable in the plasma of most patients (17/19), whereas HIV RNA could be detected in the semen of most patients (16/19). HIV DNA could be detected in both semen and blood. Genetic diversity of HIV between the seminal and blood compartments was identified. Thus, amounts of HIV RNA and HIV DNA remain high in semen of HIV-infected Chinese patients after six months of ART treatment, even when HIV RNA was undetectable in blood. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

Genetic characterization of the HIV-1 reservoir after Vacc-4x and romidepsin therapy in HIV-1 infected individuals.

PubMed

Winckelmann, Anni; Morcilla, Vincent; Shao, Wei; Schleimann, Mariane H; Højen, Jesper F; Schlub, Timothy E; Denton, Paul W; Østergaard, Lars; Søgaard, Ole S; Tolstrup, Martin; Palmer, Sarah

2018-05-11

Therapeutic HIV-1 immunization followed by latency reversal has been suggested as a strategy to eradicate HIV-1. Here we investigate the phylogenetic composition of the HIV-1 regions targeted by the therapeutic HIV-1 peptide vaccine Vacc-4x in participants in a clinical trial. Seventeen participants on suppressive antiretroviral therapy were vaccinated with six doses of Vacc-4x followed by three doses of romidepsin. Seven study participants were selected for sequencing analysis. All participants underwent an analytical treatment interruption. Single-genome/proviral sequencing of the p24-RT region was performed to genetically characterize proviral DNA, cell-associated (CA) RNA and outgrowth viruses during therapy as well as plasma HIV-1 RNA during an analytical treatment interruption. There were no changes in CA HIV-1 RNA (P = 0.83) and DNA (P = 0.09) diversity over the course of the study and no difference between CA HIV-1 RNA and DNA diversity (P = 0.32). Only one participant showed signs of potential vaccine-related selection in the rebounding plasma virus. In five of seven participants, we identified HLA-specific CTL epitopes containing non-silent mutations in 100% of the sequences. We detected no evidence of selective immune pressure reflected in proviral diversity or by occurrence of specific mutation in the vaccine-targeted epitopes. Pre-existing CTL epitope mutations may affect the potency of this therapeutic vaccine. This highlights the challenges of developing effective HIV-1 therapeutic vaccines.This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0.

Cytotoxic chemotherapy and the evolution of cellular and viral resistance to antiretroviral therapy in HIV- infected individuals with lymphoma.

PubMed

McFaul, Katie; Liptrott, Neill; Cox, Alison; Martin, Phillip; Egan, Deirdre; Owen, Andrew; Kelly, Sarah; Karolia, Zeenat; Shaw, Kate; Bower, Mark; Boffito, Marta

2016-09-01

The use of combination antiretroviral therapy (cART) and cytotoxic chemotherapy for HIV-associated lymphoma runs the risks of inducing HIV drug resistance. This study examined two possible mechanisms: altered expression of membrane drug transporter protein (MTP) and acquisition of mutations in pro-viral DNA. Expression levels of MTP and pro-viral DNA resistance mutation analysis were performed on peripheral blood mononuclear cells (PBMC) before, during, and after chemotherapy. Twenty nine patients completed the three time point estimations. There were no significant variations before, during, and after chemotherapy in the expression of four MTPs: ABCB1, ABCC1, ABCC2, and SLCO3A1 (OATP3A1). Pro-viral DNA sequencing revealed that only one patient developed a new nucleos/tide reverse transcriptase inhibitor-associated mutation (184V) during the course of the study, giving a mutation rate of 0.0027 per person per year. In conclusion, concomitant administration of cytotoxic chemotherapy and cART does not induce expression of MTP. Furthermore, no significant changes in viral resistance were observed pre- and post-chemotherapy, suggesting mutagenic cytotoxic chemotherapy seems not to induce mutations in HIV pro-viral DNA.

Maternal LAMP/p55gagHIV-1 DNA immunization induces in utero priming and a long-lasting immune response in vaccinated neonates.

PubMed

Rigato, Paula Ordonhez; Maciel, Milton; Goldoni, Adriana Letícia; Piubelli, Orlando Guerra; Orii, Noemia Mie; Marques, Ernesto Torres; August, Joseph Thomas; Duarte, Alberto José da Silva; Sato, Maria Notomi

2012-01-01

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+CD25+Foxp3+T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-γ-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods.

Maternal LAMP/p55gagHIV-1 DNA Immunization Induces In Utero Priming and a Long-Lasting Immune Response in Vaccinated Neonates

PubMed Central

Rigato, Paula Ordonhez; Maciel, Milton; Goldoni, Adriana Letícia; Piubelli, Orlando Guerra; Orii, Noemia Mie; Marques, Ernesto Torres; August, Joseph Thomas; Duarte, Alberto José da Silva; Sato, Maria Notomi

2012-01-01

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+CD25+Foxp3+T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-γ-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods. PMID:22355381

Three-Year Durability of Immune Responses Induced by HIV-DNA and HIV-Modified Vaccinia Virus Ankara and Effect of a Late HIV-Modified Vaccinia Virus Ankara Boost in Tanzanian Volunteers.

PubMed

Joachim, Agricola; Munseri, Patricia J; Nilsson, Charlotta; Bakari, Muhammad; Aboud, Said; Lyamuya, Eligius F; Tecleab, Teghesti; Liakina, Valentina; Scarlatti, Gabriella; Robb, Merlin L; Earl, Patricia L; Moss, Bernard; Wahren, Britta; Mhalu, Fred; Ferrari, Guido; Sandstrom, Eric; Biberfeld, Gunnel

2017-08-01

We explored the duration of immune responses and the effect of a late third HIV-modified vaccinia virus Ankara (MVA) boost in HIV-DNA primed and HIV-MVA boosted Tanzanian volunteers. Twenty volunteers who had previously received three HIV-DNA and two HIV-MVA immunizations were given a third HIV-MVA immunization 3 years after the second HIV-MVA boost. At the time of the third HIV-MVA, 90% of the vaccinees had antibodies to HIV-1 subtype C gp140 (median titer 200) and 85% to subtype B gp160 (median titer 100). The majority of vaccinees had detectable antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies, 70% against CRF01_AE virus-infected cells (median titer 239) and 84% against CRF01_AE gp120-coated cells (median titer 499). A high proportion (74%) of vaccinees had IFN-γ ELISpot responses, 63% to Gag and 42% to Env, 3 years after the second HIV-MVA boost. After the third HIV-MVA, there was an increase in Env-binding antibodies and ADCC-mediating antibodies relative to the response seen at the time of the third HIV-MVA vaccination, p < .0001 and p < .05, respectively. The frequency of IFN-γ ELISpot responses increased to 95% against Gag or Env and 90% to both Gag and Env, p = .064 and p = .002, respectively. In conclusion, the HIV-DNA prime/HIV-MVA boost regimen elicited potent antibody and cellular immune responses with remarkable durability, and a third HIV-MVA immunization significantly boosted both antibody and cellular immune responses relative to the levels detected at the time of the third HIV-MVA, but not to higher levels than after the second HIV-MVA.

Detection of HIV-1 p24 Gag in plasma by a nanoparticle-based bio-barcode-amplification method.

PubMed

Kim, Eun-Young; Stanton, Jennifer; Korber, Bette T M; Krebs, Kendall; Bogdan, Derek; Kunstman, Kevin; Wu, Samuel; Phair, John P; Mirkin, Chad A; Wolinsky, Steven M

2008-06-01

Detection of HIV-1 in patients is limited by the sensitivity and selectivity of available tests. The nanotechnology-based bio-barcode-amplification method offers an innovative approach to detect specific HIV-1 antigens from diverse HIV-1 subtypes. We evaluated the efficacy of this protein-detection method in detecting HIV-1 in men enrolled in the Chicago component of the Multicenter AIDS Cohort Study (MACS). The method relies on magnetic microparticles with antibodies that specifically bind the HIV-1 p24 Gag protein and nanoparticles that are encoded with DNA and antibodies that can sandwich the target protein captured by the microparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated to remove the conjugated barcode DNA. The DNA barcodes (hundreds per target) were identified by a nanoparticle-based detection method that does not rely on PCR. Of 112 plasma samples from HIV-1-infected subjects, 111 were positive for HIV-1 p24 Gag protein (range: 0.11-71.5 ng/ml of plasma) by the bio-barcode-amplification method. HIV-1 p24 Gag protein was detected in only 23 out of 112 men by the conventional ELISA. A total of 34 uninfected subjects were negative by both tests. Thus, the specificity of the bio-barcode-amplification method was 100% and the sensitivity 99%. The bio-barcode-amplification method detected HIV-1 p24 Gag protein in plasma from all study subjects with less than 200 CD4(+) T cells/microl of plasma (100%) and 19 out of 20 (95%) HIV-1-infected men who had less than 50 copies/ml of plasma of HIV-1 RNA. In a separate group of 60 diverse international isolates, representative of clades A, B, C and D and circulating recombinant forms CRF01_AE and CRF02_AG, the bio-barcode-amplification method identified the presence of virus correctly. The bio-barcode-amplification method was superior to the conventional ELISA assay for the detection of HIV-1 p24 Gag protein in plasma with a breadth of coverage for diverse HIV-1 subtypes. Because the bio-barcode-amplification method does not require enzymatic amplification, this method could be translated into a robust point-of-care test.

HIV-1 gp120 and Modified Vaccinia Virus Ankara (MVA) gp140 Boost Immunogens Increase Immunogenicity of a DNA/MVA HIV-1 Vaccine.

PubMed

Shen, Xiaoying; Basu, Rahul; Sawant, Sheetal; Beaumont, David; Kwa, Sue Fen; LaBranche, Celia; Seaton, Kelly E; Yates, Nicole L; Montefiori, David C; Ferrari, Guido; Wyatt, Linda S; Moss, Bernard; Alam, S Munir; Haynes, Barton F; Tomaras, Georgia D; Robinson, Harriet L

2017-12-15

An important goal of human immunodeficiency virus (HIV) vaccine design is identification of strategies that elicit effective antiviral humoral immunity. One novel approach comprises priming with DNA and boosting with modified vaccinia virus Ankara (MVA) expressing HIV-1 Env on virus-like particles. In this study, we evaluated whether the addition of a gp120 protein in alum or MVA-expressed secreted gp140 (MVAgp140) could improve immunogenicity of a DNA prime-MVA boost vaccine. Five rhesus macaques per group received two DNA primes at weeks 0 and 8 followed by three MVA boosts (with or without additional protein or MVAgp140) at weeks 18, 26, and 40. Both boost immunogens enhanced the breadth of HIV-1 gp120 and V1V2 responses, antibody-dependent cellular cytotoxicity (ADCC), and low-titer tier 1B and tier 2 neutralizing antibody responses. However, there were differences in antibody kinetics, linear epitope specificity, and CD4 T cell responses between the groups. The gp120 protein boost elicited earlier and higher peak responses, whereas the MVAgp140 boost resulted in improved antibody durability and comparable peak responses after the final immunization. Linear V3 specific IgG responses were particularly enhanced by the gp120 boost, whereas the MVAgp140 boost also enhanced responses to linear C5 and C2.2 epitopes. Interestingly, gp120, but not the MVAgp140 boost, increased peak CD4 + T cell responses. Thus, both gp120 and MVAgp140 can augment potential protection of a DNA/MVA vaccine by enhancing gp120 and V1/V2 antibody responses, whereas potential protection by gp120, but not MVAgp140 boosts, may be further impacted by increased CD4 + T cell responses. IMPORTANCE Prior immune correlate analyses with humans and nonhuman primates revealed the importance of antibody responses in preventing HIV-1 infection. A DNA prime-modified vaccinia virus Ankara (MVA) boost vaccine has proven to be potent in eliciting antibody responses. Here we explore the ability of boosts with recombinant gp120 protein or MVA-expressed gp140 to enhance antibody responses elicited by the GOVX-B11 DNA prime-MVA boost vaccine. We found that both types of immunogen boosts enhanced potentially protective antibody responses, whereas the gp120 protein boosts also increased CD4 + T cell responses. Our data provide important information for HIV vaccine designs that aim for effective and balanced humoral and T cell responses. Copyright © 2017 Shen et al.

HIV-1 Reservoir Association with Immune Activation.

PubMed

Vallejo, Alejandro

2015-09-01

In this issue of EBioMedicine, Ruggiero and colleagues describe immune activation biomarkers associated with the size of the HIV reservoir in a carefully designed cross-sectional study. The cohort consists of a homogeneous sample of HIV-1-infected patients with long-term plasma HIV-1 RNA suppression under antiretroviral treatment (ART). It is crucial to explore the potential utility of biomarkers that are easier (less labor intensive, less expensive) to measure than integrated HIV DNA load, in order to quickly and accurately quantify cellular reservoirs of HIV.

The importance of becoming double-stranded: Innate immunity and the kinetic model of HIV-1 central plus strand synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poeschla, Eric, E-mail: poeschla.eric@mayo.edu

Central initiation of plus strand synthesis is a conserved feature of lentiviruses and certain other retroelements. This complication of the standard reverse transcription mechanism produces a transient “central DNA flap” in the viral cDNA, which has been proposed to mediate its subsequent nuclear import. This model has assumed that the important feature is the flapped DNA structure itself rather than the process that produces it. Recently, an alternative kinetic model was proposed. It posits that central plus strand synthesis functions to accelerate conversion to the double-stranded state, thereby helping HIV-1 to evade single-strand DNA-targeting antiviral restrictions such as APOBEC3 proteins,more » and perhaps to avoid innate immune sensor mechanisms. The model is consistent with evidence that lentiviruses must often synthesize their cDNAs when dNTP concentrations are limiting and with data linking reverse transcription and uncoating. There may be additional kinetic advantages for the artificial genomes of lentiviral gene therapy vectors. - Highlights: • Two main functional models for HIV central plus strand synthesis have been proposed. • In one, a transient central DNA flap in the viral cDNA mediates HIV-1 nuclear import. • In the other, multiple kinetic consequences are emphasized. • One is defense against APOBEC3G, which deaminates single-stranded DNA. • Future questions pertain to antiviral restriction, uncoating and nuclear import.« less

HIV-1 tropism: a comparison between RNA and proviral DNA in routine clinical samples from Chilean patients

PubMed Central

2013-01-01

Background HIV in Chile has a notification rate of 0.01%. Coreceptor antagonists are a family of antiretroviral drugs that are used with the prior knowledge of patients HIV-1 tropism. Viral RNA-based tropism detection requires a plasma viral load ≥1000 copies/mL, while proviral DNA-based detection can be performed regardless of plasma viral load. This test is useful in patients with low or undetectable viral loads and would benefit with a proper therapy. The aim of this study was to determine the correlation between HIV RNA and proviral genotypic DNA tropism tests. Findings Forty three Chilean patients were examined using population-based V3 sequencing, and a geno2pheno false-positive rate (FPR) cutoff values of 5, 5.75, 10 and 20%. With cutoff 5.75% a concordance of 88.4% in tropism prediction was found after a simultaneous comparison between HIV tropism assessment by RNA and DNA. In total, five discrepancies (11.6%) were found, 3 patients were RNA-R5/DNA-X4 and two were RNA-X4/DNA-R5. Proviral DNA enabled the prediction of tropism in patients with a low or undetectable viral load. For cutoff 5 and 5.75% genotypic testing using proviral DNA showed a similar sensitivity for X4 as RNA. We found that the highest sensitivity for detecting the X4 strain occurred with proviral DNA and cutoff of 10 and 20%. Viral loads were higher among X4 strain carriers than among R5 strain carriers (p < 0.05). Conclusions A high degree of concordance was found between tropism testing with RNA and testing with proviral DNA. Our results suggest that proviral DNA-based genotypic tropism testing is a useful option for patients with low or undetectable viral load who require a different therapy. PMID:24165156

A Transient Kinetic Approach to Investigate Nucleoside Inhibitors of Mitochondrial DNA polymerase γ

PubMed Central

Anderson, Karen S.

2010-01-01

Nucleoside analogs play an essential role in treating human immunodeficiency virus (HIV) infection since the beginning of the AIDS epidemic and work by inhibition of HIV-1 reverse transcriptase (RT), a viral polymerase essential for DNA replication. Today, over 90% of all regimens for HIV treatment contain at least one nucleoside. Long-term use of nucleoside analogs has been associated with adverse effects including mitochondrial toxicity due to inhibition of the mitochondrial polymerase, DNA polymerase gamma (mtDNA pol ©). In this review, we describe our efforts to delineate the molecular mechanism of nucleoside inhibition of HIV-1 RT and mtDNA pol © based upon a transient kinetic approach using rapid chemical quench methodology. Using transient kinetic methods, the maximum rate of polymerization (kpol), the dissociation constant for the ground state binding (Kd), and the incorporation efficiency (kpol/Kd) can be determined for the nucleoside analogs and their natural substrates. This analysis allowed us to develop an understanding of the structure activity relationships that allow correlation between the structural and stereochemical features of the nucleoside analog drugs with their mechanistic behavior toward the viral polymerase, RT, and the host cell polymerase, mtDNA pol γ. An in-depth understanding of the mechanisms of inhibition of these enzymes is imperative in overcoming problems associated with toxicity. PMID:20573564

Oral keratinocytes support non-replicative infection and transfer of harbored HIV-1 to permissive cells.

PubMed

Vacharaksa, Anjalee; Asrani, Anil C; Gebhard, Kristin H; Fasching, Claudine E; Giacaman, Rodrigo A; Janoff, Edward N; Ross, Karen F; Herzberg, Mark C

2008-07-17

Oral keratinocytes on the mucosal surface are frequently exposed to HIV-1 through contact with infected sexual partners or nursing mothers. To determine the plausibility that oral keratinocytes are primary targets of HIV-1, we tested the hypothesis that HIV-1 infects oral keratinocytes in a restricted manner. To study the fate of HIV-1, immortalized oral keratinocytes (OKF6/TERT-2; TERT-2 cells) were characterized for the fate of HIV-specific RNA and DNA. At 6 h post inoculation with X4 or R5-tropic HIV-1, HIV-1gag RNA was detected maximally within TERT-2 cells. Reverse transcriptase activity in TERT-2 cells was confirmed by VSV-G-mediated infection with HIV-NL4-3Deltaenv-EGFP. AZT inhibited EGFP expression in a dose-dependent manner, suggesting that viral replication can be supported if receptors are bypassed. Within 3 h post inoculation, integrated HIV-1 DNA was detected in TERT-2 cell nuclei and persisted after subculture. Multiply spliced and unspliced HIV-1 mRNAs were not detectable up to 72 h post inoculation, suggesting that HIV replication may abort and that infection is non-productive. Within 48 h post inoculation, however, virus harbored by CD4 negative TERT-2 cells trans infected co-cultured peripheral blood mononuclear cells (PBMCs) or MOLT4 cells (CD4+ CCR5+) by direct cell-to-cell transfer or by releasing low levels of infectious virions. Primary tonsil epithelial cells also trans infected HIV-1 to permissive cells in a donor-specific manner. Oral keratinocytes appear, therefore, to support stable non-replicative integration, while harboring and transmitting infectious X4- or R5-tropic HIV-1 to permissive cells for up to 48 h.

Epstein-Barr virus and human immunodeficiency virus serological responses and viral burdens in HIV-infected patients treated with HAART

NASA Technical Reports Server (NTRS)

O'Sullivan, Cathal E.; Peng, RongSheng; Cole, Kelly Stefano; Montelaro, Ronald C.; Sturgeon, Timothy; Jenson, Hal B.; Ling, Paul D.; Butel, J. S. (Principal Investigator)

2002-01-01

Epstein-Barr virus (EBV) associated non-Hodgkin lymphoma is recognized as a complication of human immunodeficiency virus (HIV) infection. Little is known regarding the influence of highly active antiretroviral therapy (HAART) on the biology of EBV in this population. To characterize the EBV- and HIV-specific serological responses together with EBV DNA levels in a cohort of HIV-infected adults treated with HAART, a study was conducted to compare EBV and HIV serologies and EBV DNA copy number (DNAemia) over a 12-month period after the commencement of HAART. All patients were seropositive for EBV at baseline. Approximately 50% of patients had detectable EBV DNA at baseline, and 27/30 had detectable EBV DNA at some point over the follow-up period of 1 year. Changes in EBV DNA copy number over time for any individual were unpredictable. Significant increases in the levels of Epstein-Barr nuclear antigen (EBNA) and Epstein-Barr early antigen (EA) antibodies were demonstrated in the 17 patients who had a good response to HAART. Of 29 patients with paired samples tested, four-fold or greater increases in titers were detected for EA in 12/29 (41%), for EBNA in 7/29 (24%), for VCA-IgG in 4/29 (14%); four-fold decreases in titers were detected in 2/29 (7%) for EA and 12/29 (41%) for EBNA. A significant decline in the titer of anti-HIV antibodies was also demonstrated. It was concluded that patients with advanced HIV infection who respond to HAART have an increase in their EBV specific antibodies and a decrease in their HIV-specific antibodies. For the cohort overall, there was a transient increase in EBV DNA levels that had declined by 12 months. Copyright 2002 Wiley-Liss, Inc.

Highly sensitive surface plasmon resonance biosensor for the detection of HIV-related DNA based on dynamic and structural DNA nanodevices.

PubMed

Diao, Wei; Tang, Min; Ding, Shijia; Li, Xinmin; Cheng, Wenbin; Mo, Fei; Yan, Xiaoyu; Ma, Hongmin; Yan, Yurong

2018-02-15

Early detection, diagnosis and treatment of human immune deficiency virus (HIV) infection is the key to reduce acquired immunodeficiency syndrome (AIDS) mortality. In our research, an innovative surface plasmon resonance (SPR) biosensing strategy has been developed for highly sensitive detection of HIV-related DNA based on entropy-driven strand displacement reactions (ESDRs) and double-layer DNA tetrahedrons (DDTs). ESDRs as enzyme-free and label-free signal amplification circuit can be specifically triggered by target DNA, leading to the cyclic utilization of target DNA and the formation of plentiful double-stranded DNA (dsDNA) products. Subsequently, the dsDNA products bind to the immobilized hairpin capture probes and further combine with DDTs nanostructures. Due to the high efficiency of ESDRs and large molecular weight of DDTs, the SPR response signal was enhanced dramatically. The proposed SPR biosensor could detect target DNA sensitively and specifically in a linear range from 1pM to 150nM with a detection limit of 48fM. In addition, the whole detecting process can be accomplished in 60min with high accuracy and duplicability. In particular, the developed SPR biosensor was successfully used to analyze target DNA in complex biological sample, indicating that the developed strategy is promising for rapid and early clinical diagnosis of HIV infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Human immunodeficiency virus bDNA assay for pediatric cases.

PubMed

Avila, M M; Liberatore, D; Martínez Peralta, L; Biglione, M; Libonatti, O; Coll Cárdenas, P; Hodara, V L

2000-01-01

Techniques to quantify plasma HIV-1 RNA viral load (VL) are commercially available, and they are adequate for monitoring adults infected by HIV and treated with antiretroviral drugs. Little experience on HIV VL has been reported in pediatric cases. In Argentina, the evaluation of several assays for VL in pediatrics are now being considered. To evaluate the pediatric protocol for bDNA assay in HIV-infected children, 25 samples from HIV-infected children (according to CDC criteria for pediatric AIDS) were analyzed by using Quantiplex HIV RNA 2.0 Assay (Chiron Corporation) following the manufacturer's recommendations in a protocol that uses 50 microliters of patient's plasma (sensitivity: 10,000 copies/ml). When HIV-RNA was not detected, samples were run with the 1 ml standard bDNA protocol (sensitivity: 500 HIV-RNA c/ml). Nine samples belonged to infants under 12 months of age (group A) and 16 were over 12 months (group B). All infants under one year of age had high HIV-RNA copies in plasma. VL ranged from 30,800 to 2,560,000 RNA copies/ml (median = 362,000 c/ml) for group A and < 10,000 to 554,600 c/ml (median = < 10,000) for group B. Only 25% of children in group B had detectable HIV-RNA. By using the standard test of quantification, none of the patients had non detectable HIV-RNA, ranging between 950 and 226,200 c/ml for group B (median = 23,300 RNA c/ml). The suggested pediatric protocol could be useful in children under 12 months of age, but 1 ml standard protocol must be used for older children. Samples with undetectable results from children under one year of age should be repeated using the standard protocol.

Optimization of HIV-1 Envelope DNA Vaccine Candidates within Three Different Animal Models, Guinea Pigs, Rabbits and Cynomolgus Macaques.

PubMed

Borggren, Marie; Vinner, Lasse; Andresen, Betina Skovgaard; Grevstad, Berit; Repits, Johanna; Melchers, Mark; Elvang, Tara Laura; Sanders, Rogier W; Martinon, Frédéric; Dereuddre-Bosquet, Nathalie; Bowles, Emma Joanne; Stewart-Jones, Guillaume; Biswas, Priscilla; Scarlatti, Gabriella; Jansson, Marianne; Heyndrickx, Leo; Grand, Roger Le; Fomsgaard, Anders

2013-07-19

HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb). We compared three different animal models: guinea pigs, rabbits and cynomolgus macaques. Envelope genes from the prototype isolate HIV-1 Bx08 and two elite neutralizers were included. Codon-optimized genes, encoded secreted gp140 or membrane bound gp150, were modified for expression of stabilized soluble trimer gene products, and delivered individually or mixed. Specific IgG after repeated i.d. inoculations with electroporation confirmed in vivo expression and immunogenicity. Evaluations of rabbits and guinea pigs displayed similar results. The superior DNA construct in rabbits was a trivalent mix of non-modified codon-optimized gp140 envelope genes. Despite NAb responses with some potency and breadth in guinea pigs and rabbits, the DNA vaccinated macaques displayed less bNAb activity. It was concluded that a trivalent mix of non-modified gp140 genes from rationally selected clinical isolates was, in this study, the best option to induce high and broad NAb in the rabbit model, but this optimization does not directly translate into similar responses in cynomolgus macaques.

Optimization of HIV-1 Envelope DNA Vaccine Candidates within Three Different Animal Models, Guinea Pigs, Rabbits and Cynomolgus Macaques

PubMed Central

Borggren, Marie; Vinner, Lasse; Andresen, Betina Skovgaard; Grevstad, Berit; Repits, Johanna; Melchers, Mark; Elvang, Tara Laura; Sanders, Rogier W; Martinon, Frédéric; Dereuddre-Bosquet, Nathalie; Bowles, Emma Joanne; Stewart-Jones, Guillaume; Biswas, Priscilla; Scarlatti, Gabriella; Jansson, Marianne; Heyndrickx, Leo; Le Grand, Roger; Fomsgaard, Anders

2013-01-01

HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb). We compared three different animal models: guinea pigs, rabbits and cynomolgus macaques. Envelope genes from the prototype isolate HIV-1 Bx08 and two elite neutralizers were included. Codon-optimized genes, encoded secreted gp140 or membrane bound gp150, were modified for expression of stabilized soluble trimer gene products, and delivered individually or mixed. Specific IgG after repeated i.d. inoculations with electroporation confirmed in vivo expression and immunogenicity. Evaluations of rabbits and guinea pigs displayed similar results. The superior DNA construct in rabbits was a trivalent mix of non-modified codon-optimized gp140 envelope genes. Despite NAb responses with some potency and breadth in guinea pigs and rabbits, the DNA vaccinated macaques displayed less bNAb activity. It was concluded that a trivalent mix of non-modified gp140 genes from rationally selected clinical isolates was, in this study, the best option to induce high and broad NAb in the rabbit model, but this optimization does not directly translate into similar responses in cynomolgus macaques. PMID:26344115

HIV Type 1 (HIV-1) Proviral Reservoirs Decay Continuously Under Sustained Virologic Control in HIV-1–Infected Children Who Received Early Treatment

PubMed Central

Luzuriaga, Katherine; Tabak, Barbara; Garber, Manuel; Chen, Ya Hui; Ziemniak, Carrie; McManus, Margaret M.; Murray, Danielle; Strain, Matthew C.; Richman, Douglas D.; Chun, Tae-Wook; Cunningham, Coleen K.; Persaud, Deborah

2014-01-01

Background. Early initiation of combination antiretroviral therapy (cART) to human immunodeficiency virus type 1 (HIV-1)–infected infants controls HIV-1 replication and reduces mortality. Methods. Plasma viremia (lower limit of detection, <2 copies/mL), T-cell activation, HIV-1–specific immune responses, and the persistence of cells carrying replication-competent virus were quantified during long-term effective combination antiretroviral therapy (cART) in 4 perinatally HIV-1–infected youth who received treatment early (the ET group) and 4 who received treatment late (the LT group). Decay in peripheral blood mononuclear cell (PBMC) proviral DNA levels was also measured over time in the ET youth. Results. Plasma viremia was not detected in any ET youth but was detected in all LT youth (median, 8 copies/mL; P = .03). PBMC proviral load was significantly lower in ET youth (median, 7 copies per million PBMCs) than in LT youth (median, 181 copies; P = .03). Replication-competent virus was recovered from all LT youth but only 1 ET youth. Decay in proviral DNA was noted in all 4 ET youth in association with limited T-cell activation and with absent to minimal HIV-1–specific immune responses. Conclusions. Initiation of early effective cART during infancy significantly limits circulating levels of proviral and replication-competent HIV-1 and promotes continuous decay of viral reservoirs. Continued cART with reduction in HIV-1 reservoirs over time may facilitate HIV-1 eradication strategies. PMID:24850788

Comparative Performance of Three Viral Load Assays on Human Immunodeficiency Virus Type 1 (HIV-1) Isolates Representing Group M (Subtypes A to G) and Group O: LCx HIV RNA Quantitative, AMPLICOR HIV-1 MONITOR Version 1.5, and Quantiplex HIV-1 RNA Version 3.0

PubMed Central

Swanson, Priscilla; Soriano, Vincent; Devare, Sushil G.; Hackett, John

2001-01-01

The performance of the LCx HIV RNA Quantitative (LCx HIV), AMPLICOR HIV-1 MONITOR version 1.5 (MONITOR v1.5), and Quantiplex HIV-1 RNA version 3.0 (bDNA v3.0) viral load assays was evaluated with 39 viral isolates (3 A, 7 B, 6 C, 4 D, 8 E, 4 F, 1 G, 4 mosaic, and 2 group O). Quantitation across the assay dynamic ranges was assessed using serial fivefold dilutions of the viruses. In addition, sequences of gag-encoded p24 (gag p24), pol-encoded integrase, and env-encoded gp41 were analyzed to assign group and subtype and to assess nucleotide mismatches at primer and probe binding sites. For group M isolates, quantification was highly correlated among all three assays. In contrast, only the LCx HIV assay reliably quantified group O isolates. The bDNA v3.0 assay detected but consistently underquantified group O viruses, whereas the MONITOR v1.5 test failed to detect group O viruses. Analysis of target regions revealed fewer primer or probe mismatches in the LCx HIV assay than in the MONITOR v1.5 test. Consistent with the high level of nucleotide conservation is the ability of the LCx HIV assay to quantify efficiently human immunodeficiency virus type 1 group M and the genetically diverse group O. PMID:11230396

Genome editing strategies: potential tools for eradicating HIV-1/AIDS

PubMed Central

Khalili, Kamel; Gordon, Jennifer; Cosentino, Laura; Hu, Wenhui

2015-01-01

Current therapy for controlling HIV-1 infection and preventing AIDS progression has profoundly decreased viral replication in cells susceptible to HIV-1 infection, but it does not eliminate the low level of viral replication in latently infected cells which contain integrated copies of HIV-1 proviral DNA. There is an urgent need for the development of HIV-1 genome eradication strategies that will lead to a permanent or “sterile” cure of HIV-1/AIDS. In the past few years, novel nuclease-initiated genome editing tools have been developing rapidly, including ZFNs, TALENs, and the CRISPR/Cas9 system. These surgical knives, which can excise any genome, provide a great opportunity to eradicate the HIV-1 genome by targeting highly conserved regions of the HIV-1 long terminal repeats or essential viral genes. Given the time consuming and costly engineering of target-specific ZFNs and TALENs, the RNA-guided endonuclease Cas9 technology has emerged as a simpler and more versatile technology to allow permanent removal of integrated HIV-1 proviral DNA in eukaryotic cells, and hopefully animal models or human patients. The major unmet challenges of this approach at present include inefficient nuclease gene delivery, potential off-target cleavage, and cell-specific genome targeting. Nanoparticle or lentivirus-mediated delivery of next generation Cas9 technologies including nickase or RNA-guided FokI nuclease (RFN) will further improve the potential for genome editing to become a promising approach for curing HIV-1/AIDS. PMID:25716921

Merkel cell polyomavirus IgG antibody levels are associated with progression to AIDS among HIV-infected individuals.

PubMed

Vahabpour, Rouhollah; Nasimi, Maryam; Naderi, Niloofar; Salehi-Vaziri, Mostafa; Mohajel, Nasir; Sadeghi, Farzin; Keyvani, Hossein; Monavari, Seyed Hamidreza

2017-04-01

The association of Merkel cell polyomavirus (MCP y V) with Merkel cell carcinoma (MCC) in immunocompromised individuals has been revealed in a number of surveys. The study of MCP y V specific antibody titers and viral loads in such patients has a great attraction for research groups interested in viral reactivation. In this cross-sectional study to evaluate MCP y V antibody titer, DNA prevalence and viral load in peripheral blood mononuclear cells (PBMCs), we examined 205 HIV-1 infected patients and 100 un-infected controls. The HIV-1 infected patients divided into two groups (HIV/AIDS and non-AIDS) according to their CD4 status. Total IgG antibody titer against MCP y V was analyzed by virus like particle (VLP)-based enzyme linked immunosorbent assay (ELISA). Presence of MCP y V-DNA in subject's PBMCs was examined by quantitative real-time PCR assay. Levels of anti-MCP y V IgG in HIV/AIDS patients were significantly higher than those in non-AIDS HIV-infected and control subjects (p value = <0.001). The prevalence rate of MCP y V-DNA in PBMCs of HIV/AIDS, non-AIDS HIV-infected and un-infected controls were 17%, 16%, and 14% respectively. The MCP y V viral load among the groups ranged between 0.15 to 2.9 copies/10 3 cells (median, 1.9 copies/10 3 cells), with no significant difference between the studied populations (p value = 0.3).

Primary Human Immunodeficiency Virus Type 1 (HIV-1) Infection during HIV-1 Gag Vaccination▿

PubMed Central

Balamurugan, Arumugam; Lewis, Martha J.; Kitchen, Christina M. R.; Robertson, Michael N.; Shiver, John W.; Daar, Eric S.; Pitt, Jacqueline; Ali, Ayub; Ng, Hwee L.; Currier, Judith S.; Yang, Otto O.

2008-01-01

Vaccination for human immunodeficiency virus type 1 (HIV-1) remains an elusive goal. Whether an unsuccessful vaccine might not only fail to provoke detectable immune responses but also could actually interfere with subsequent natural immunity upon HIV-1 infection is unknown. We performed detailed assessment of an HIV-1 gag DNA vaccine recipient (subject 00015) who was previously uninfected but sustained HIV-1 infection before completing a vaccination trial and another contemporaneously acutely infected individual (subject 00016) with the same strain of HIV-1. Subject 00015 received the vaccine at weeks 0, 4, and 8 and was found to have been acutely HIV-1 infected around the time of the third vaccination. Subject 00016 was a previously HIV-1-seronegative sexual contact who had symptoms of acute HIV-1 infection approximately 2 weeks earlier than subject 00015 and demonstrated subsequent seroconversion. Both individuals reached an unusually low level of chronic viremia (<1,000 copies/ml) without treatment. Subject 00015 had no detectable HIV-1-specific cytotoxic T-lymphocyte (CTL) responses until a borderline response was noted at the time of the third vaccination. The magnitude and breadth of Gag-specific CTL responses in subject 00015 were similar to those of subject 00016 during early chronic infection. Viral sequences from gag, pol, and nef confirmed the common source of HIV-1 between these individuals. The diversity and divergence of sequences in subjects 00015 and 00016 were similar, indicating similar immune pressure on these proteins (including Gag). As a whole, the data suggested that while the gag DNA vaccine did not prime detectable early CTL responses in subject 00015, vaccination did not appreciably impair his ability to contain viremia at levels similar to those in subject 00016. PMID:18199650

Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koczor, Christopher A., E-mail: ckoczor@emory.edu; Fields, Earl; Jedrzejczak, Mark J.

This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10 d, 3 mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change > 1.5, p < 0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides formore » calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. - Highlights: • HIV-1 Tat and methamphetamine (METH) alter cardiac gene expression and epigenetics. • METH impacts gene expression or epigenetics more significantly than Tat expression. • METH alters cardiac mitochondrial function and calcium signaling independent of Tat. • METH alters DNA methylation, expression, and protein abundance of CACNA1C (Cav1.2).« less

Extensive virologic and immunologic characterization in an HIV-infected individual following allogeneic stem cell transplant and analytic cessation of antiretroviral therapy: A case study.

PubMed

Cummins, Nathan W; Rizza, Stacey; Litzow, Mark R; Hua, Stephane; Lee, Guinevere Q; Einkauf, Kevin; Chun, Tae-Wook; Rhame, Frank; Baker, Jason V; Busch, Michael P; Chomont, Nicolas; Dean, Patrick G; Fromentin, Rémi; Haase, Ashley T; Hampton, Dylan; Keating, Sheila M; Lada, Steven M; Lee, Tzong-Hae; Natesampillai, Sekar; Richman, Douglas D; Schacker, Timothy W; Wietgrefe, Stephen; Yu, Xu G; Yao, Joseph D; Zeuli, John; Lichterfeld, Mathias; Badley, Andrew D

2017-11-01

Notwithstanding 1 documented case of HIV-1 cure following allogeneic stem cell transplantation (allo-SCT), several subsequent cases of allo-SCT in HIV-1 positive individuals have failed to cure HIV-1 infection. The aim of our study was to describe changes in the HIV reservoir in a single chronically HIV-infected patient on suppressive antiretroviral therapy who underwent allo-SCT for treatment of acute lymphoblastic leukemia. We prospectively collected peripheral blood mononuclear cells (PBMCs) by leukapheresis from a 55-year-old man with chronic HIV infection before and after allo-SCT to measure the size of the HIV-1 reservoir and characterize viral phylogeny and phenotypic changes in immune cells. At day 784 post-transplant, when HIV-1 was undetectable by multiple measures-including PCR measurements of both total and integrated HIV-1 DNA, replication-competent virus measurement by large cell input quantitative viral outgrowth assay, and in situ hybridization of colon tissue-the patient consented to an analytic treatment interruption (ATI) with frequent clinical monitoring. He remained aviremic off antiretroviral therapy until ATI day 288, when a low-level virus rebound of 60 HIV-1 copies/ml occurred, which increased to 1,640 HIV-1 copies/ml 5 days later, prompting reinitiation of ART. Rebounding plasma HIV-1 sequences were phylogenetically distinct from proviral HIV-1 DNA detected in circulating PBMCs before transplantation. The main limitations of this study are the insensitivity of reservoir measurements, and the fact that it describes a single case. allo-SCT led to a significant reduction in the size of the HIV-1 reservoir and a >9-month-long ART-free remission from HIV-1 replication. Phylogenetic analyses suggest that the origin of rebound virus was distinct from the viruses identified pre-transplant in the PBMCs.

Extensive virologic and immunologic characterization in an HIV-infected individual following allogeneic stem cell transplant and analytic cessation of antiretroviral therapy: A case study

PubMed Central

Litzow, Mark R.; Einkauf, Kevin; Rhame, Frank; Busch, Michael P.; Dean, Patrick G.; Hampton, Dylan; Lada, Steven M.; Lee, Tzong-Hae; Natesampillai, Sekar; Schacker, Timothy W.; Yu, Xu G.; Yao, Joseph D.; Zeuli, John; Lichterfeld, Mathias

2017-01-01

Background Notwithstanding 1 documented case of HIV-1 cure following allogeneic stem cell transplantation (allo-SCT), several subsequent cases of allo-SCT in HIV-1 positive individuals have failed to cure HIV-1 infection. The aim of our study was to describe changes in the HIV reservoir in a single chronically HIV-infected patient on suppressive antiretroviral therapy who underwent allo-SCT for treatment of acute lymphoblastic leukemia. Methods and findings We prospectively collected peripheral blood mononuclear cells (PBMCs) by leukapheresis from a 55-year-old man with chronic HIV infection before and after allo-SCT to measure the size of the HIV-1 reservoir and characterize viral phylogeny and phenotypic changes in immune cells. At day 784 post-transplant, when HIV-1 was undetectable by multiple measures—including PCR measurements of both total and integrated HIV-1 DNA, replication-competent virus measurement by large cell input quantitative viral outgrowth assay, and in situ hybridization of colon tissue—the patient consented to an analytic treatment interruption (ATI) with frequent clinical monitoring. He remained aviremic off antiretroviral therapy until ATI day 288, when a low-level virus rebound of 60 HIV-1 copies/ml occurred, which increased to 1,640 HIV-1 copies/ml 5 days later, prompting reinitiation of ART. Rebounding plasma HIV-1 sequences were phylogenetically distinct from proviral HIV-1 DNA detected in circulating PBMCs before transplantation. The main limitations of this study are the insensitivity of reservoir measurements, and the fact that it describes a single case. Conclusions allo-SCT led to a significant reduction in the size of the HIV-1 reservoir and a >9-month-long ART-free remission from HIV-1 replication. Phylogenetic analyses suggest that the origin of rebound virus was distinct from the viruses identified pre-transplant in the PBMCs. PMID:29182633

HIV-DNA content in different CD4+ T-cell subsets correlates with CD4+ cell :  CD8+ cell ratio or length of efficient treatment.

PubMed

Gibellini, Lara; Pecorini, Simone; De Biasi, Sara; Bianchini, Elena; Digaetano, Margherita; Pinti, Marcello; Carnevale, Gianluca; Borghi, Vanni; Guaraldi, Giovanni; Mussini, Cristina; Cossarizza, Andrea; Nasi, Milena

2017-06-19

HIV establishes a latent infection at different degrees within naïve (TN) or central (TCM) and effector memory (TEM) CD4 T cell. Studying patients in whom HIV production was suppressed by combined antiretroviral therapy, our main aim was to find which factors are related or can influence intracellular viral reservoir in different CD4 T-cell subsets. We enrolled 32 HIV patients successfully treated for more than 2 years, with a CD4 T-cell count more than 500 cells/μl and plasma viremia undetectable from at least 1 year. Proviral HIV-DNA, the amount of cells expressing signal-joint T-cell receptor rearrangement excision circles and telomere length were quantified by droplet digital PCR in highly purified, sorted CD4 T-cell subsets; plasma IL-7 and IL-15 were measured by ELISA. HIV-DNA was significantly lower in TN cells compared with TCM or to TEM. Conversely, TN cells contained more signal-joint T-cell receptor rearrangement excision circles compared with TCM or to TEM; no appreciable changes were observed in telomere length. HIV-DNA content was significantly higher in TN and TCM cells, but not in TEM, from patients with shorter time of treatment, or in those with lower CD4 : CD8 ratio. Length of treatment or recovery of CD4 : CD8 ratio significantly influences viral reservoir in both TN and TCM. Measuring HIV-DNA in purified lymphocyte populations allows a better monitoring of HIV reservoir and could be useful for designing future eradication strategies.

Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ordonhez Rigato, Paula; Maciel, Milton; Goldoni, Adriana Leticia

2010-10-10

Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses,more » as measured by the breadth of the Gag peptide-specific IFN-{gamma}, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric LAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory.« less

HIV-1 and HCV viral load cost models for bDNA: 440 Molecular System versus real-time PCR AmpliPrep/TaqMan test.

PubMed

Elbeik, Tarek; Dalessandro, Ralph; Loftus, Richard A; Beringer, Scott

2007-11-01

Comparative cost models were developed to assess cost-per-reportable result and annual costs for HIV-1 and HCV bDNA and AmpliPrep/TaqMan Test (PCR). Model cost components included kit, disposables, platform and related equipment, equipment service plan, equipment maintenance, equipment footprint, waste and labor. Model assessment was most cost-effective when run by bDNA with 36 or more clinical samples and PCR with 30 or fewer clinical samples. Lower costs are attained with maximum samples (84-168) run daily. Highest cost contributors include kit, platform and PCR proprietary disposables. Understanding component costs and the most economic use of HIV-1 and HCV viral load will aid in attaining lowest costs through selection of the appropriate assay and effective negotiations.

DNA and modified vaccinia virus Ankara vaccines encoding multiple cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) are safe but weakly immunogenic in HIV-1-uninfected, vaccinia virus-naive adults.

PubMed

Gorse, Geoffrey J; Newman, Mark J; deCamp, Allan; Hay, Christine Mhorag; De Rosa, Stephen C; Noonan, Elizabeth; Livingston, Brian D; Fuchs, Jonathan D; Kalams, Spyros A; Cassis-Ghavami, Farah L

2012-05-01

We evaluated a DNA plasmid-vectored vaccine and a recombinant modified vaccinia virus Ankara vaccine (MVA-mBN32), each encoding cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) in a randomized, double-blinded, placebo-controlled trial in 36 HIV-1-uninfected adults using a heterologous prime-boost schedule. HIV-1-specific cellular immune responses, measured as interleukin-2 and/or gamma interferon production, were induced in 1 (4%) of 28 subjects after the first MVA-mBN32 immunization and in 3 (12%) of 25 subjects after the second MVA-mBN32 immunization. Among these responders, polyfunctional T-cell responses, including the production of tumor necrosis factor alpha and perforin, were detected. Vaccinia virus-specific antibodies were induced to the MVA vector in 27 (93%) of 29 and 26 (93%) of 28 subjects after the first and second immunizations with MVA-mBN32. These peptide-based vaccines were safe but were ineffective at inducing HIV-1-specific immune responses and induced much weaker responses than MVA vaccines expressing the entire open reading frames of HIV-1 proteins.

DNA-stabilized silver nanoclusters and carbon nanoparticles oxide: A sensitive platform for label-free fluorescence turn-on detection of HIV-DNA sequences.

PubMed

Ye, Yu-Dan; Xia, Li; Xu, Dang-Dang; Xing, Xiao-Jing; Pang, Dai-Wen; Tang, Hong-Wu

2016-11-15

Based on the remarkable difference between the interactions of carbon nanoparticles (CNPs) oxide with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and the fact that fluorescence of DNA-stabilized silver nanoclusters (AgNCs) can be quenched by CNPs oxide, DNA-functionalized AgNCs were applied as label-free fluorescence probes and a novel fluorescence resonance energy transfer (FRET) sensor was successfully constructed for the detection of human immunodeficiency virus (HIV) DNA sequences. CNPs oxide were prepared with the oxidation of candle soot, hence it is simple, time-saving and low-cost. The strategy of dual AgNCs probes was applied to improve the detection sensitivity by using dual- probe capturing the same target DNA in a sandwich mode and as the fluorescence donor, and using CNPs oxide as the acceptor. In the presence of target DNA, a dsDNA hybrid forms, leading to the desorption of the ssDNA-AgNCs probes from CNPs oxide, and the recovering of fluorescence of the AgNCs in a HIV-DNA concentration-dependent manner. The results show that HIV-DNA can be detected in the range of 1-50nM with a detection limit of 0.40nM in aqueous buffer. The method is simple, rapid and sensitive with no need of labeled fluorescent probes, and moreover, the design of fluorescent dual-probe makes full use of the excellent fluorescence property of AgNCs and further improves the detection sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

5-Azacytidine Can Induce Lethal Mutagenesis in Human Immunodeficiency Virus Type 1▿ †

PubMed Central

Dapp, Michael J.; Clouser, Christine L.; Patterson, Steven; Mansky, Louis M.

2009-01-01

Ribonucleosides inhibit human immunodeficiency virus type 1 (HIV-1) replication by mechanisms that have not been fully elucidated. Here, we report the antiviral mechanism for the ribonucleoside analog 5-azacytidine (5-AZC). We hypothesized that the anti-HIV-1 activity of 5-AZC was due to an increase in the HIV-1 mutation rate following its incorporation into viral RNA during transcription. However, we demonstrate that 5-AZC's primary antiviral activity can be attributed to its effect on the early phase of HIV-1 replication. Furthermore, the antiviral activity was associated with an increase in the frequency of viral mutants, suggesting that 5-AZC's primary target is reverse transcription. Sequencing analysis showed an enrichment in G-to-C transversion mutations and further supports the idea that reverse transcription is an antiviral target of 5-AZC. These results indicate that 5-AZC is incorporated into viral DNA following reduction to 5-aza-2′-deoxycytidine. Incorporation into the viral DNA leads to an increase in mutant frequency that is consistent with lethal mutagenesis during reverse transcription as the primary antiviral mechanism of 5-AZC. Antiviral activity and increased mutation frequency were also associated with the late phase of HIV-1 replication; however, 5-AZC's effect on the late phase was less robust. These results reveal that the primary antiviral mechanism of 5-AZC can be attributed to its ability to increase the HIV-1 mutation frequency through viral-DNA incorporation during reverse transcription. Our observations indicate that 5-AZC can affect two steps in HIV-1 replication (i.e., transcription and reverse transcription) but that its primary antiviral activity is due to incorporation during reverse transcription. PMID:19726509

vif-negative human immunodeficiency virus type 1 persistently replicates in primary macrophages, producing attenuated progeny virus.

PubMed Central

Chowdhury, I H; Chao, W; Potash, M J; Sova, P; Gendelman, H E; Volsky, D J

1996-01-01

The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived macrophages (MDM). Viruses carrying missense or deletion mutations in vif were constructed on the background of the monocytotropic recombinant NLHXADA-GP. Using MDM from multiple donors, we found that vif mutants produced in complementing or partially complementing cell lines were approximately 10% as infectious as wild-type virus when assayed for incomplete, complete, and circularized viral DNA molecules by quantitative PCR amplification or for viral core antigen p24 production by enzyme-linked immunosorbent assay. We then determined the structure and infectivity of vif mutant HIV-1 by using MDM exclusively both for virus production and as targets for infection. Biosynthetic labeling and immunoprecipitation analysis of sucrose cushion-purified vif-negative HIV-1 made in MDM revealed that the virus had reduced p24 content compared with wild-type HIV-1. Cell-free MDM-derived vif mutant HIV-1 was infectious in macrophages as determined by the synthesis and maintenance of full-length viral DNA and by the produc- tion of particle-associated viral RNA, but its infectivity was approximately 2,500-fold lower than that of wild-type virus whose titer was determined in parallel by measurement of the viral DNA burden. MDM infected with MDM-derived vif-negative HIV-1 were able to transmit the virus to uninfected MDM by cocultivation, confirming the infectiousness of this virus. We conclude that mutations in vif significantly reduce but do not eliminate the capacity of HIV-1 to replicate and produce infectious progeny virus in primary human macrophages. PMID:8764044

vif-negative human immunodeficiency virus type 1 persistently replicates in primary macrophages, producing attenuated progeny virus.

PubMed

Chowdhury, I H; Chao, W; Potash, M J; Sova, P; Gendelman, H E; Volsky, D J

1996-08-01

The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived macrophages (MDM). Viruses carrying missense or deletion mutations in vif were constructed on the background of the monocytotropic recombinant NLHXADA-GP. Using MDM from multiple donors, we found that vif mutants produced in complementing or partially complementing cell lines were approximately 10% as infectious as wild-type virus when assayed for incomplete, complete, and circularized viral DNA molecules by quantitative PCR amplification or for viral core antigen p24 production by enzyme-linked immunosorbent assay. We then determined the structure and infectivity of vif mutant HIV-1 by using MDM exclusively both for virus production and as targets for infection. Biosynthetic labeling and immunoprecipitation analysis of sucrose cushion-purified vif-negative HIV-1 made in MDM revealed that the virus had reduced p24 content compared with wild-type HIV-1. Cell-free MDM-derived vif mutant HIV-1 was infectious in macrophages as determined by the synthesis and maintenance of full-length viral DNA and by the produc- tion of particle-associated viral RNA, but its infectivity was approximately 2,500-fold lower than that of wild-type virus whose titer was determined in parallel by measurement of the viral DNA burden. MDM infected with MDM-derived vif-negative HIV-1 were able to transmit the virus to uninfected MDM by cocultivation, confirming the infectiousness of this virus. We conclude that mutations in vif significantly reduce but do not eliminate the capacity of HIV-1 to replicate and produce infectious progeny virus in primary human macrophages.

A Directed Molecular Evolution Approach to Improved Immunogenicity of the HIV-1 Envelope Glycoprotein

PubMed Central

Du, Sean X.; Xu, Li; Zhang, Wenge; Tang, Susan; Boenig, Rebecca I.; Chen, Helen; Mariano, Ellaine B.; Zwick, Michael B.; Parren, Paul W. H. I.; Burton, Dennis R.; Wrin, Terri; Petropoulos, Christos J.; Ballantyne, John A.; Chambers, Michael; Whalen, Robert G.

2011-01-01

A prophylactic vaccine is needed to slow the spread of HIV-1 infection. Optimization of the wild-type envelope glycoproteins to create immunogens that can elicit effective neutralizing antibodies is a high priority. Starting with ten genes encoding subtype B HIV-1 gp120 envelope glycoproteins and using in vitro homologous DNA recombination, we created chimeric gp120 variants that were screened for their ability to bind neutralizing monoclonal antibodies. Hundreds of variants were identified with novel antigenic phenotypes that exhibit considerable sequence diversity. Immunization of rabbits with these gp120 variants demonstrated that the majority can induce neutralizing antibodies to HIV-1. One novel variant, called ST-008, induced significantly improved neutralizing antibody responses when assayed against a large panel of primary HIV-1 isolates. Further study of various deletion constructs of ST-008 showed that the enhanced immunogenicity results from a combination of effective DNA priming, an enhanced V3-based response, and an improved response to the constant backbone sequences. PMID:21738594

Vaccine-induced HIV seropositivity/reactivity in noninfected HIV vaccine recipients.

PubMed

Cooper, Cristine J; Metch, Barbara; Dragavon, Joan; Coombs, Robert W; Baden, Lindsey R

2010-07-21

Induction of protective anti-human immunodeficiency virus (HIV) immune responses is the goal of an HIV vaccine. However, this may cause a reactive result in routine HIV testing in the absence of HIV infection. To evaluate the frequency of vaccine-induced seropositivity/reactivity (VISP) in HIV vaccine trial participants. Three common US Food and Drug Administration-approved enzyme immunoassay (EIA) HIV antibody kits were used to determine VISP, and a routine diagnostic HIV algorithm was used to evaluate VISP frequency in healthy, HIV-seronegative adults who completed phase 1 (n = 25) and phase 2a (n = 2) vaccine trials conducted from 2000-2010 in the United States, South America, Thailand, and Africa. Vaccine-induced seropositivity/reactivity, defined as reactive on 1 or more EIA tests and either Western blot-negative or Western blot-indeterminate/atypical positive (profile consistent with vaccine product) and HIV-1-negative by nucleic acid testing. Among 2176 participants free of HIV infection who received a vaccine product, 908 (41.7%; 95% confidence interval [CI], 39.6%-43.8%) had VISP, but the occurrence of VISP varied substantially across different HIV vaccine product types: 399 of 460 (86.7%; 95% CI, 83.3%-89.7%) adenovirus 5 product recipients, 295 of 552 (53.4%; 95% CI, 49.2%-57.7%) recipients of poxvirus alone or as a boost, and 35 of 555 (6.3%; 95% CI, 4.4%-8.7%) of DNA-alone product recipients developed VISP. Overall, the highest proportion of VISP (891/2176 tested [40.9%]) occurred with the HIV 1/2 (rDNA) EIA kit compared with the rLAV EIA (150/700 tested [21.4%]), HIV-1 Plus O Microelisa System (193/1309 tested [14.7%]), and HIV 1/2 Peptide and HIV 1/2 Plus O (189/2150 tested [8.8%]) kits. Only 17 of the 908 participants (1.9%) with VISP tested nonreactive using the HIV 1/2 (rDNA) kit. All recipients of a glycoprotein 140 vaccine (n = 70) had VISP, with 94.3% testing reactive with all 3 EIA kits tested. Among 901 participants with VISP and a Western blot result, 92 (10.2%) had a positive Western blot result (displaying an atypical pattern consistent with vaccine product), and 592 (65.7%) had an indeterminate result. Only 8 participants with VISP received a vaccine not containing an envelope insert. The induction of VISP in HIV vaccine recipients is common, especially with vaccines containing both the HIV-1 envelope and group-specific core antigen gene proteins. Development and detection of VISP appear to be associated with the immunogenicity of the vaccine and the EIA assay used.

The enzymatic activity of CEM15/Apobec-3G is essential for the regulation of the infectivity of HIV-1 virion but not a sole determinant of its antiviral activity.

PubMed

Shindo, Keisuke; Takaori-Kondo, Akifumi; Kobayashi, Masayuki; Abudu, Aierken; Fukunaga, Keiko; Uchiyama, Takashi

2003-11-07

Human immunodeficiency virus, type 1 (HIV-1) Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion. Vif functions to counteract an anti-HIV-1 cellular factor in non-permissive cells, CEM15/Apobec-3G, which shares a cytidine deaminase motif. CEM15/Apobec-3G deaminates dC to dU in the minus strand DNA of HIV-1, resulting in G to A hypermutation in the plus strand DNA. In this study, we have done the mutagenesis analysis on two cytidine deaminase motifs in CEM15/Apobec-3G and examined their antiviral functions as well as the DNA editing activity. Point mutations in the C-terminal active site such as E259Q and C291A almost completely abrogated the antiviral function, while those in the N-terminal active site such as E67Q and C100A retained this activity to a lesser extent as compared with that of the wild type. The DNA editing activities of E67Q and E259Q mutants were both retained but impaired to the same extent. This indicates that the enzymatic activity of this protein is essential but not a sole determinant of the antiviral activity. Furthermore, all the deletion mutants tested in this study lost the antiviral activity because of the loss of the activity for dimerization, suggesting that the entire protein structure is necessary for the antiviral function.

T lymphocytes among HIV-infected and -uninfected infants: CD4/CD8 ratio as a potential tool in diagnosis of infection in infants under the age of 2 years.

PubMed

Zijenah, Lynn S; Katzenstein, David A; Nathoo, Kusum J; Rusakaniko, Simbarashe; Tobaiwa, Ocean; Gwanzura, Christine; Bikoue, Arsene; Nhembe, Margaret; Matibe, Petronella; Janossy, George

2005-02-01

BACKGROUND: Serologic tests for HIV infection in infants less than 18 months do not differentiate exposure and infection since maternally acquired IgG antibodies may be detected in infants. Thus, the gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA or reverse transcriptase polymerase chain reaction. There is an urgent need to evaluate alternative and cost effective laboratory methods for early diagnosis of infant HIV-1 infection as well as identifying infected infants who may benefit from cotrimoxazole prophylaxis and/or initiation of highly active antiretroviral therapy. METHODS: Whole blood was collected in EDTA from 137 infants aged 0 to 18 months. DNA polymerase chain reaction was used as the reference standard for diagnosis of HIV-1 infection. T-cell subset profiles were determined by flow cytometry. RESULTS: Seventy-six infants were DNA PCR positive while 61 were negative. The median CD4 counts of PCR negative infants were significantly higher than those of the PCR positive infants, p < 0.001. The median CD4/CD8 ratio and the %CD4 of the PCR positive infants were both significantly lower than those of the negative infants, p < 0.001. The CD4/CD8 ratio had a >98% sensitivity for diagnosis of HIV-1 infection and a specificity of >98%. CONCLUSION: The CD4/CD8 ratio appears useful in identifying HIV-infected infants. The development of lower cost and more robust flow cytometric methods that provide both CD4/CD8 ratio and %CD4 may be cost-effective for HIV-1 diagnosis and identification of infants for cotrimoxazole prophylaxis and/or highly active antiretroviral therapy.

T lymphocytes among HIV-infected and -uninfected infants: CD4/CD8 ratio as a potential tool in diagnosis of infection in infants under the age of 2 years

PubMed Central

Zijenah, Lynn S; Katzenstein, David A; Nathoo, Kusum J; Rusakaniko, Simbarashe; Tobaiwa, Ocean; Gwanzura, Christine; Bikoue, Arsene; Nhembe, Margaret; Matibe, Petronella; Janossy, George

2005-01-01

Background Serologic tests for HIV infection in infants less than 18 months do not differentiate exposure and infection since maternally acquired IgG antibodies may be detected in infants. Thus, the gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA or reverse transcriptase polymerase chain reaction. There is an urgent need to evaluate alternative and cost effective laboratory methods for early diagnosis of infant HIV-1 infection as well as identifying infected infants who may benefit from cotrimoxazole prophylaxis and/or initiation of highly active antiretroviral therapy. Methods Whole blood was collected in EDTA from 137 infants aged 0 to 18 months. DNA polymerase chain reaction was used as the reference standard for diagnosis of HIV-1 infection. T-cell subset profiles were determined by flow cytometry. Results Seventy-six infants were DNA PCR positive while 61 were negative. The median CD4 counts of PCR negative infants were significantly higher than those of the PCR positive infants, p < 0.001. The median CD4/CD8 ratio and the %CD4 of the PCR positive infants were both significantly lower than those of the negative infants, p < 0.001. The CD4/CD8 ratio had a >98% sensitivity for diagnosis of HIV-1 infection and a specificity of >98%. Conclusion The CD4/CD8 ratio appears useful in identifying HIV-infected infants. The development of lower cost and more robust flow cytometric methods that provide both CD4/CD8 ratio and %CD4 may be cost-effective for HIV-1 diagnosis and identification of infants for cotrimoxazole prophylaxis and/or highly active antiretroviral therapy. PMID:15683549

A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR.

PubMed

McFall, Sally M; Wagner, Robin L; Jangam, Sujit R; Yamada, Douglas H; Hardie, Diana; Kelso, David M

2015-03-01

Early diagnosis and access to treatment for infants with human immunodeficiency virus-1 (HIV-1) is critical to reduce infant mortality. The lack of simple point-of-care tests impedes the timely initiation of antiretroviral therapy. The development of FINA, filtration isolation of nucleic acids, a novel DNA extraction method that can be performed by clinic personnel in less than 2 min has been reported previously. In this report, significant improvements in the DNA extraction and amplification methods are detailed that allow sensitive quantitation of as little as 10 copies of HIV-1 proviral DNA and detection of 3 copies extracted from 100 μl of whole blood. An internal control to detect PCR inhibition was also incorporated. In a preliminary field evaluation of 61 South African infants, the FINA test demonstrated 100% sensitivity and specificity. The proviral copy number of the infant specimens was quantified, and it was established that 100 microliters of whole blood is required for sensitive diagnosis of infants. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Frequency and genotype of human parvovirus B19 among Iranian patients infected with HIV.

PubMed

Azadmanesh, Kayhan; Mohraz, Minoo; Kazemimanesh, Monireh; Aghakhani, Arezoo; Foroughi, Maryam; Banifazl, Mohammad; Eslamifar, Ali; Ramezani, Amitis

2015-07-01

The human parvovirus B19 (B19) usually causes a subclinical infection in immunocompetent individuals. Whereas immunocompromised individuals such as patients infected with HIV are at risk of persistent anemia due to B19 infection. Only few studies have been carried out on distribution and molecular epidemiology of B19 in Iran. We aimed to determine the frequency and genotype of B19 among Iranian patients infected with HIV. We conducted a survey on 99 HIV patients and 64 healthy controls. IgG and IgM antibodies against B19 were detected by ELISA and B19 DNA was assessed by nested PCR. PCR products were subjected to direct sequencing and classified after phylogenetic analysis. The prevalence of B19 immunoglobulin was 11.1% for IgG and 1% for IgM. B19 DNA was detected in 13.1% of cases. The prevalence of B19 IgG, IgM, and DNA in control group was 25%, 1.6%, and 9.4%, respectively. B19 IgG was significantly lower in HIV group than in normal controls. There was no significant difference regarding anemia between cases and controls. All sequenced B19 isolates belonged to genotype 1A with low genetic diversity. Our findings indicated that in the HAART era, the importance of B19 infections in HIV patients may be limited whereas persistent B19 viremia in the circulation of healthy controls raises a potential concern in blood donations. © 2015 Wiley Periodicals, Inc.

Comparative evaluation of the performance of the Abbott RealTime HIV-1 assay for measurement of HIV-1 plasma viral load on genetically diverse samples from Greece

PubMed Central

2011-01-01

Background HIV-1 is characterized by increased genetic heterogeneity which tends to hinder the reliability of detection and accuracy of HIV-1 RNA quantitation assays. Methods In this study, the Abbott RealTime HIV-1 (Abbott RealTime) assay was compared to the Roche Cobas TaqMan HIV-1 (Cobas TaqMan) and the Siemens Versant HIV-1 RNA 3.0 (bDNA 3.0) assays, using clinical samples of various viral load levels and subtypes from Greece, where the recent epidemiology of HIV-1 infection has been characterized by increasing genetic diversity and a marked increase in subtype A genetic strains among newly diagnosed infections. Results A high correlation was observed between the quantitative results obtained by the Abbott RealTime and the Cobas TaqMan assays. Viral load values quantified by the Abbott RealTime were on average lower than those obtained by the Cobas TaqMan, with a mean (SD) difference of -0.206 (0.298) log10 copies/ml. The mean differences according to HIV-1 subtypes between the two techniques for samples of subtype A, B, and non-A/non-B were 0.089, -0.262, and -0.298 log10 copies/ml, respectively. Overall, differences were less than 0.5 log10 for 85% of the samples, and >1 log10 in only one subtype B sample. Similarly, Abbott RealTime and bDNA 3.0 assays yielded a very good correlation of quantitative results, whereas viral load values assessed by the Abbott RealTime were on average higher (mean (SD) difference: 0.160 (0.287) log10 copies/ml). The mean differences according to HIV-1 subtypes between the two techniques for subtype A, B and non-A/non-B samples were 0.438, 0.105 and 0.191 log10 copies/ml, respectively. Overall, the majority of samples (86%) differed by less than 0.5 log10, while none of the samples showed a deviation of more than 1.0 log10. Conclusions In an area of changing HIV-1 subtype pattern, the Abbott RealTime assay showed a high correlation and good agreement of results when compared both to the Cobas TaqMan and bDNA 3.0 assays, for all HIV-1 subtypes tested. All three assays could determine viral load from samples of different HIV-1 subtypes adequately. However, assay variation should be taken into account when viral load monitoring of the same individual is assessed by different systems. PMID:21219667

HIV-1 non-R5 tropism correlates with a larger size of the cellular viral reservoir and a detectable residual viremia in patients under suppressive ART.

PubMed

Lombardi, Francesca; Belmonti, Simone; Rapone, Lucrezia; Borghetti, Alberto; Ciccullo, Arturo; Gagliardini, Roberta; Baldin, Gianmaria; Montagnani, Francesca; Moschese, Davide; Emiliozzi, Arianna; Rossetti, Barbara; De Luca, Andrea; Di Giambenedetto, Simona

2018-06-01

The influence of HIV-1 co-receptor usage on the course of therapy in subjects fully responding to ART has been poorly investigated. To explore the relationship between co-receptor tropism and cellular reservoir size, residual viremia and subsequent virological outcome in ART-treated patients with HIV-1 RNA stable <50 copies/mL. Viral co-receptor usage was predicted by viral env DNA sequencing with geno2pheno interpretation (FPR20%) and classified as R5 and non-R5. Total blood-associated HIV-1 DNA levels (log 10 copies/10 6 leukocytes) were measured by qRT-PCR (5'LTR). Residual plasma viremia was categorized as detectable (1-49 cps/mL) or undetectable (<1 copy/mL). Virological rebounds (any HIV-1 RNA >50 copies/mL) were evaluated over 96 weeks. The study included 116 subjects. Patients with R5 virus (n = 59) and non-R5 virus (n = 57) were homogeneous for the main characteristics except for the lower nadir CD4 cell count in the non-R5 group. Patients with non-R5 variants showed higher levels of HIV-1 DNA as compared to patients with R5 virus: mean 2.47 (95% CI 2.37-2.56) vs 2.17 (2.08-2.26) (p < 0.001). Moreover, a higher proportion of patients in the non-R5 group displayed detectable residual viremia with respect to the R5-group (54.4% vs 32.2%, p = .016). Detectable residual viremia was found to be significantly associated with viral rebounds. The presence of non-R5 viral DNA variants is related to a higher probability of residual viremia and to a larger size of the cellular viral reservoir in this setting. These data highlight a potential role of viral tropism in the monitoring of HIV-1 infection in virologically controlled subject. Copyright © 2018 Elsevier B.V. All rights reserved.

Compartmentalized Cytomegalovirus Replication and Transmission in the Setting of Maternal HIV-1 Infection

PubMed Central

Slyker, Jennifer; Farquhar, Carey; Atkinson, Claire; Ásbjörnsdóttir, Kristjana; Roxby, Alison; Drake, Alison; Kiarie, James; Wald, Anna; Boeckh, Michael; Richardson, Barbra; Odem-Davis, Katherine; John-Stewart, Grace; Emery, Vincent

2014-01-01

Background. Cytomegalovirus (CMV) infection is associated with adverse outcomes in human immunodeficiency virus (HIV)–exposed infants. Determinants of vertical CMV transmission in the setting of maternal HIV-1 infection are not well-defined. Methods. CMV and HIV-1 levels were measured in plasma, cervical secretions, and breast milk of 147 HIV-1–infected women to define correlates of maternal CMV replication and infant CMV acquisition. Results. Although few women had detectable CMV in plasma (4.8%), the majority had detectable CMV DNA in cervical secretions (66%) and breast milk (99%). There was a strong association between cervical CMV detection during pregnancy and later breast milk levels (β = 0.47; P = .005). Plasma HIV-1 level and CD4 counts were associated with CMV in the cervix and breast milk. However HIV-1 levels within the cervix and breast milk were not associated with CMV within these compartments. Maternal breast milk CMV levels (hazard ratio [HR], 1.4; P = .003) and maternal CD4 < 450 cells/mm3 (HR, 1.8; P = .008) were independently associated with infant CMV acquisition; each log10 increase in breast milk CMV was associated with a 40% increase in infant infection. The breast milk CMV level required to attain a 50% probability of CMV transmission increased with higher maternal CD4 counts, increasing from 3.55 log10 CMV DNA copies/mL at a CD4 count of 350 cells/mm3 to 5.50 log10 CMV DNA copies/mL at a CD4 count of 1000 cells/mm3. Conclusions. Breast milk CMV levels and maternal CD4 count are major determinants of CMV transmission in the setting of maternal HIV-1. Maternal immune reconstitution or lowering breast milk CMV levels may reduce vertical CMV transmission. PMID:24192386

Persistence of Viral Reservoirs in Multiple Tissues after Antiretroviral Therapy Suppression in a Macaque RT-SHIV Model

PubMed Central

Franks, Tamera; Kiser, Rebecca; Coalter, Vicky; Smedley, Jeremy; Piatak, Michael; Mellors, John W.; Lifson, Jeffrey D.; Ambrose, Zandrea

2013-01-01

Although antiretroviral therapy (ART) can suppress HIV-1 replication sufficiently to eliminate measurable plasma viremia, infected cells remain and ensure viral recrudescence after discontinuation of ART. We used a macaque model of HIV-1/AIDS to evaluate the location of infected cells during ART. Twelve macaques were infected with RT-SHIVmne, a SIV containing HIV-1 reverse transcriptase, conferring sensitivity to non-nucleoside reverse transcriptase inhibitors (NNRTIs). Ten to fourteen weeks post-infection, 6 animals were treated with 3 or 4 antiretroviral drugs for 17-20 weeks; 6 control animals remained untreated. Viral DNA (vDNA) and RNA (vRNA) were measured in peripheral blood mononuclear cells (PBMC) and at necropsy in multiple tissues by quantitative PCR and RT-PCR. The majority of virally infected cells were located in lymphoid tissues with variable levels in the gastrointestinal tract of both treated and untreated animals. Tissue viral DNA levels correlated with week 1 plasma viremia, suggesting that tissues that harbor proviral DNA are established within the first week of infection. PBMC vDNA levels did not correlate with plasma viremia or tissue levels of vDNA. vRNA levels were high in lymphoid and gastrointestinal tissues of the untreated animals; animals on ART had little vRNA expressed in tissues and virus could not be cultured from lymph node resting CD4+ cells after 17-20 weeks on ART, indicating little or no ongoing viral replication. Strategies for eradication of HIV-1 will need to target residual virus in ART suppressed individuals, which may not be accurately reflected by frequencies of infected cells in blood. PMID:24367650

DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1.

PubMed

Bower, Joseph F; Green, Thomas D; Ross, Ted M

2004-10-25

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d3) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d3. In addition, both sCD4-gp120 and sCD4-gp120-mC3d3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d3 or sCD4-gp120-mC3d3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d3-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.

Autoantibodies and human immunodeficiency viruses infection: a case-control study.

PubMed

Chretien, P; Monier, J C; Oksman, F; San Marco, M; Escande, A; Goetz, J; Cohen, J; Baquey, A; Humbel, R L; Sibilia, J

2003-01-01

To determine the prevalence of organ-specific and non-specific autoantibodies in HIV-infected patients. A multicentric collaborative case-control study including 105 HIV patients and 100 sex- and age-matched HIV-negative healthy volunteers. Antinuclear, anti-ds DNA, anti-histone, anti-Sm, rheumatoid factor(IgM), anti-beta 2 glycoprotein 1, antineutrophil cytoplasmic, anti-LKM1, anti-LCA1, anti-gastric parietal cell, antiplatelet, anti-intermediate filament, anti-mitotic spindle apparatus, anti-Golgi, anti-ribosome and anti-thyroid autoantibodies were screened in six European laboratories. Only IgG and IgM anticardiolipin, IgG antiplatelet, anti-smooth muscle and anti-thyroglobulin antibodies were statistically more frequent in HIV patients. There was no correlation with the numbers of CD4+ cells except in the case of anti-smooth muscle antibodies. We were unable to find specific autoantibodies such as anti-ds DNA, anti-Sm, AMA, anti-LKM1, anti-LCA1 or anti-beta 2 GP1 antibodies in these patients. Our results indicate that the autoantibody profile of HIV infections is comparable to those of other chronic viral infections. HIV does not seem to be more autoimmunogenic than other viruses.

Thalassiolins A-C: new marine-derived inhibitors of HIV cDNA integrase.

PubMed

Rowley, David C; Hansen, Mark S T; Rhodes, Denise; Sotriffer, Christoph A; Ni, Haihong; McCammon, J Andrew; Bushman, Frederic D; Fenical, William

2002-11-01

Human immunodeficiency virus (HIV) replication requires integration of viral cDNA into the host genome, a process mediated by the viral enzyme integrase. We describe a new series of HIV integrase inhibitors, thalassiolins A-C (1-3), isolated from the Caribbean sea grass Thalassia testudinum. The thalassiolins are distinguished from other flavones previously studied by the substitution of a sulfated beta-D-glucose at the 7-position, a substituent that imparts increased potency against integrase in biochemical assays. The most active of these molecules, thalassiolin A (1), displays in vitro inhibition of the integrase catalyzed strand transfer reaction (IC50=0.4 microM) and an antiviral IC50 of 30 microM. Molecular modeling studies indicate a favorable binding mode is probable at the catalytic core domain of HIV-1 integrase.

Selective Regulation of Human Immunodeficiency Virus-Infected CD4+ Lymphocytes by a Synthetic Immunomodulator Leads to Potent Virus Suppression In Vitro and in hu-PBL-SCID Mice

PubMed Central

Bahr, George M.; Darcissac, Edith C. A.; Castéran, Nathalie; Amiel, Corinne; Cocude, Cécile; Truong, Marie-José; Dewulf, Joëlle; Capron, André; Mouton, Yves

2001-01-01

We have previously observed that the synthetic immunomodulator Murabutide inhibits human immunodeficiency virus type 1 (HIV-1) replication at multiple levels in macrophages and dendritic cells. The present study was designed to profile the activity of Murabutide on CD8-depleted phytohemagglutinin-activated lymphocytes from HIV-1-infected subjects and on the outcome of HIV-1 infection in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID mice). Maintaining cultures of CD8-depleted blasts from 36 patients in the presence of Murabutide produced dramatically reduced levels of viral p24 protein in the supernatants. This activity correlated with reduced viral transcripts and proviral DNA, was evident in cultures harboring R5, X4-R5, or X4 HIV-1 isolates, was not linked to inhibition of cellular DNA synthesis, and did not correlate with β-chemokine release. Moreover, c-myc mRNA expression was down-regulated in Murabutide-treated cells, suggesting potential interference of the immunomodulator with the nuclear transport of viral preintegration complexes. On the other hand, daily treatment of HIV-1-infected hu-PBL-SCID mice with Murabutide significantly reduced the viral loads in plasma and the proviral DNA content in human peritoneal cells. These results are the first to demonstrate that a clinically acceptable synthetic immunomodulator with an ability to enhance the host's nonspecific immune defense mechanisms against infections can directly regulate cellular factors in infected lymphocytes, leading to controlled HIV-1 replication. PMID:11435574

Tracking Human Immunodeficiency Virus-1 Infection in the Humanized DRAG Mouse Model

PubMed Central

Kim, Jiae; Peachman, Kristina K.; Jobe, Ousman; Morrison, Elaine B.; Allam, Atef; Jagodzinski, Linda; Casares, Sofia A.; Rao, Mangala

2017-01-01

Humanized mice are emerging as an alternative model system to well-established non-human primate (NHP) models for studying human immunodeficiency virus (HIV)-1 biology and pathogenesis. Although both NHP and humanized mice have their own strengths and could never truly reflect the complex human immune system and biology, there are several advantages of using the humanized mice in terms of using primary HIV-1 for infection instead of simian immunodeficiency virus or chimera simian/HIV. Several different types of humanized mice have been developed with varying levels of reconstitution of human CD45+ cells. In this study, we utilized humanized Rag1KO.IL2RγcKO.NOD mice expressing HLA class II (DR4) molecule (DRAG mice) infused with HLA-matched hematopoietic stem cells from umbilical cord blood to study early events after HIV-1 infection, since the mucosal tissues of these mice are highly enriched for human lymphocytes and express the receptors and coreceptors needed for HIV-1 entry. We examined the various tissues on days 4, 7, 14, and 21 after an intravaginal administration of a single dose of purified primary HIV-1. Plasma HIV-1 RNA was detected as early as day 7, with 100% of the animals becoming plasma RNA positive by day 21 post-infection. Single cells were isolated from lymph nodes, bone marrow, spleen, gut, female reproductive tissue, and brain and analyzed for gag RNA and strong stop DNA by quantitative (RT)-PCR. Our data demonstrated the presence of HIV-1 viral RNA and DNA in all of the tissues examined and that the virus was replication competent and spread rapidly. Bone marrow, gut, and lymph nodes were viral RNA positive by day 4 post-infection, while other tissues and plasma became positive typically between 7 and 14 days post-infection. Interestingly, the brain was the last tissue to become HIV-1 viral RNA and DNA positive by day 21 post-infection. These data support the notion that humanized DRAG mice could serve as an excellent model for studying the trafficking of HIV-1 to the various tissues, identification of cells harboring the virus, and thus could serve as a model system for HIV-1 pathogenesis and reservoir studies. PMID:29163484

UV-induced DNA damage is an intermediate step in UV-induced expression of human immunodeficiency virus type 1, collagenase, c-fos, and metallothionein.

PubMed Central

Stein, B; Rahmsdorf, H J; Steffen, A; Litfin, M; Herrlich, P

1989-01-01

UV irradiation of human and murine cells enhances the transcription of several genes. Here we report on the primary target of relevant UV absorption, on pathways leading to gene activation, and on the elements receiving the UV-induced signal in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, in the gene coding for collagenase, and in the cellular oncogene fos. In order to induce the expression of genes. UV radiation needs to be absorbed by DNA and to cause DNA damage of the kind that cannot be repaired by cells from patients with xeroderma pigmentosum group A. UV-induced activation of the three genes is mediated by the major enhancer elements (located between nucleotide positions -105 and -79 of HIV-1, between positions -72 and -65 of the collagenase gene, and between positions -320 and -299 of fos). These elements share no apparent sequence motif and bind different trans-acting proteins; a member of the NF kappa B family binds to the HIV-1 enhancer, the heterodimer of Jun and Fos (AP-1) binds to the collagenase enhancer, and the serum response factors p67 and p62 bind to fos. DNA-binding activities of the factors recognizing the HIV-1 and collagenase enhancers are augmented in extracts from UV-treated cells. The increase in activity is due to posttranslational modification. While AP-1 resides in the nucleus and must be modulated there, NF kappa B is activated in the cytoplasm, indicating the existence of a cytoplasmic signal transduction pathway triggered by UV-induced DNA damage. In addition to activation, new synthesis of AP-1 is induced by UV radiation. Images PMID:2557547

Co-expression of HIV-1 virus-like particles and granulocyte-macrophage colony stimulating factor by GEO-D03 DNA vaccine.

PubMed

Hellerstein, Michael; Xu, Yongxian; Marino, Tracie; Lu, Shan; Yi, Hong; Wright, Elizabeth R; Robinson, Harriet L

2012-11-01

Here, we report on GEO-D03, a DNA vaccine that co-expresses non-infectious HIV-1 virus-like particles (VLPs) and the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). The virus-like particles display the native gp160 form of the HIV-1 Envelope glycoprotein (Env) and are designed to elicit antibody against the natural form of Env on virus and virus-infected cells. The DNA-expressed HIV Gag, Pol and Env proteins also have the potential to elicit virus-specific CD4 and CD8 T cells. The purpose of the co-expressed GM-CSF is to target a cytokine that recruits, expands and differentiates macrophages and dendritic cells to the site of VLP expression. The GEO-D03 DNA vaccine is currently entered into human trials as a prime for a recombinant modified vaccinia Ankara (MVA) boost. In preclinical studies in macaques using an SIV prototype vaccine, this vaccination regimen elicited both anti-viral T cells and antibody, and provided 70% protection against acquisition during 12 weekly rectal exposures with a heterologous SIV. Higher avidity of the Env-specific Ab for the native form of the Env in the challenge virus correlated with lower likelihood of SIV infection.

Major groove binding track residues of the connection subdomain of human immunodeficiency virus type 1 reverse transcriptase enhance cDNA synthesis at high temperatures.

PubMed

Matamoros, Tania; Barrioluengo, Verónica; Abia, David; Menéndez-Arias, Luis

2013-12-23

At high temperatures, RNA denaturation can improve the efficiency and specificity of reverse transcription. Refined structures and molecular models of HIV-1 reverse transcriptases (RTs) from phylogenetically distant clades (i.e., group M subtype B and group O) revealed a major interaction between the template-primer and the Arg³⁵⁸-Gly³⁵⁹-Ala³⁶⁰ triad in the large subunit of HIV-1M/B RT. However, fewer contacts were predicted for the equivalent Lys³⁵⁸-Ala³⁵⁹-Ser³⁶⁰ triad of HIV-1O RT and the nucleic acid. An engineered HIV-1O K358R/A359G/S360A RT showed increased cDNA synthesis efficiency above 68 °C, as determined by qualitative and quantitative reverse transcription polymerase chain reactions. In comparison with wild-type HIV-1O RT, the mutant enzyme showed higher thermal stability but retained wild-type RNase H activity. Mutations that increased the accuracy of HIV-1M/B RTs were tested in combination with the K358R/A359G/S360A triple mutation. Some of them (e.g., F61A, K65R, K65R/V75I, and V148I) had a negative effect on reverse transcription efficiency above 65 °C. RTs with improved DNA binding affinities also showed higher cDNA synthesis efficiencies at elevated temperatures. Two of the most thermostable RTs (i.e., mutants T69SSG/K358R/A359G/S360A and K358R/A359G/S360A/E478Q) showed moderately increased fidelity in forward mutation assays. Our results demonstrate that the triad of Arg³⁵⁸, Gly³⁵⁹, and Ala³⁶⁰ in the major groove binding track of HIV-1 RT is a major target for RT stabilization, and most relevant for improving reverse transcription efficiency at high temperatures.

A Paper and Plastic Device for Performing Recombinase Polymerase Amplification of HIV DNA

PubMed Central

Rohrman, Brittany A.; Richards-Kortum, Rebecca R.

2013-01-01

Despite the importance of early diagnosis and treatment of HIV, only a small fraction of HIV-exposed infants in low- and middle-income countries are tested for the disease. The gold standard for early infant diagnosis, DNA PCR, requires resources that are unavailable in poor settings, and no point-of-care HIV DNA test is currently available. We have developed a device constructed of layers of paper, glass fiber, and plastic that is capable of performing isothermal, enzymatic amplification of HIV DNA. The device is inexpensive, small, light-weight, and easy to assemble. The device stores lyophilized enzymes, facilitates mixing of reaction components, and supports recombinase polymerase amplification in five steps of operation. Using commercially available lateral flow strips as a detection method, we demonstrate the ability of our device to amplify 10 copies of HIV DNA to detectable levels in 15 minutes. Our results suggest that our device, which is designed to be used after DNA extraction from dried-blood spots, may serve in conjunction with lateral flow strips as part of a point-of-care HIV DNA test to be used in low resource settings. PMID:22733333

A paper and plastic device for performing recombinase polymerase amplification of HIV DNA.

PubMed

Rohrman, Brittany A; Richards-Kortum, Rebecca R

2012-09-07

Despite the importance of early diagnosis and treatment of HIV, only a small fraction of HIV-exposed infants in low- and middle-income countries are tested for the disease. The gold standard for early infant diagnosis, DNA PCR, requires resources that are unavailable in poor settings, and no point-of-care HIV DNA test is currently available. We have developed a device constructed of layers of paper, glass fiber, and plastic that is capable of performing isothermal, enzymatic amplification of HIV DNA. The device is inexpensive, small, light-weight, and easy to assemble. The device stores lyophilized enzymes, facilitates mixing of reaction components, and supports recombinase polymerase amplification in five steps of operation. Using commercially available lateral flow strips as a detection method, we demonstrate the ability of our device to amplify 10 copies of HIV DNA to detectable levels in 15 min. Our results suggest that our device, which is designed to be used after DNA extraction from dried-blood spots, may serve in conjunction with lateral flow strips as part of a point-of-care HIV DNA test to be used in low resource settings.

Multiple nucleotide preferences determine cleavage-site recognition by the HIV-1 and M-MuLV RNases H.

PubMed

Schultz, Sharon J; Zhang, Miaohua; Champoux, James J

2010-03-19

The RNase H activity of reverse transcriptase is required during retroviral replication and represents a potential target in antiviral drug therapies. Sequence features flanking a cleavage site influence the three types of retroviral RNase H activity: internal, DNA 3'-end-directed, and RNA 5'-end-directed. Using the reverse transcriptases of HIV-1 (human immunodeficiency virus type 1) and Moloney murine leukemia virus (M-MuLV), we evaluated how individual base preferences at a cleavage site direct retroviral RNase H specificity. Strong test cleavage sites (designated as between nucleotide positions -1 and +1) for the HIV-1 and M-MuLV enzymes were introduced into model hybrid substrates designed to assay internal or DNA 3'-end-directed cleavage, and base substitutions were tested at specific nucleotide positions. For internal cleavage, positions +1, -2, -4, -5, -10, and -14 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV significantly affected RNase H cleavage efficiency, while positions -7 and -12 for HIV-1 and positions -4, -9, and -11 for M-MuLV had more modest effects. DNA 3'-end-directed cleavage was influenced substantially by positions +1, -2, -4, and -5 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV. Cleavage-site distance from the recessed end did not affect sequence preferences for M-MuLV reverse transcriptase. Based on the identified sequence preferences, a cleavage site recognized by both HIV-1 and M-MuLV enzymes was introduced into a sequence that was otherwise resistant to RNase H. The isolated RNase H domain of M-MuLV reverse transcriptase retained sequence preferences at positions +1 and -2 despite prolific cleavage in the absence of the polymerase domain. The sequence preferences of retroviral RNase H likely reflect structural features in the substrate that favor cleavage and represent a novel specificity determinant to consider in drug design. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Effect of potentially interfering substances on the measurement of HIV-1 viral load by the bDNA assay.

PubMed

Alonso, R; García de Viedma, D; Rodríguez-Creixems, M; Bouza, E

1999-03-01

As high heterogeneity of plasma composition may be responsible for interference with HIV-1 viral load determination by the bDNA assay, the potential interference caused by a number of plasma components was examined. Among the biochemical substances assayed, cholesterol, bilirubin, and triglycerides did not affect viral load quantification. Hemoglobin did not interfere with the assay at concentrations lower than or equal to 14 g/dl. Above this concentration, measurements decreased by up to 0.78 log, but these hemoglobin levels do not usually occur in the clinical setting. None of the antiretroviral drugs assayed (AZT, dDC, d4T, 3TC and Indinavir) interfered with the measurement. HIV bDNA is a robust assay even in those frequent circumstances in which plasma composition differs notably from normal.

Impact of early cART on HIV blood and semen compartments at the time of primary infection.

PubMed

Chéret, Antoine; Durier, Christine; Mélard, Adeline; Ploquin, Mickaël; Heitzmann, Julia; Lécuroux, Camille; Avettand-Fenoël, Véronique; David, Ludivine; Pialoux, Gilles; Chennebault, Jean-Marie; Müller-Trutwin, Michaela; Goujard, Cécile; Rouzioux, Christine; Meyer, Laurence

2017-01-01

HIV-infected cells in semen facilitate viral transmission. We studied the establishment of HIV reservoirs in semen and blood during PHI, along with systemic immune activation and the impact of early cART. Patients in the ANRS-147-OPTIPRIM trial received two years of early cART. Nineteen patients of the trial were analyzed, out of which 8 had acute PHI (WB ≤1 Ab). We quantified total cell-associated (ca) HIV-DNA in blood and semen and HIV-RNA in blood and semen plasma samples, collected during PHI and at 24 months of treatment. At enrollment, HIV-RNA load was higher in blood than in semen (median 5.66 vs 4.22 log10 cp/mL, p<0.0001). Semen HIV-RNA load correlated strongly with blood HIV-RNA load (r = 0.81, p = 0.02, the CD4 cell count (r = -0.98, p<0.0001), and the CD4/CD8 ratio (r = -0.85, p<0.01) in acute infection but not in later stages of PHI. Median blood and seminal cellular HIV-DNA levels were 3.59 and 0.31 log10cp/106 cells, respectively. HIV-DNA load peaked in semen later than in blood and then correlated with blood IP10 level (r = 0.62, p = 0.04). HIV-RNA was undetectable in blood and semen after two years of effective cART. Semen HIV-DNA load declined similarly, except in one patient who had persistently high IP-10 and IL-6 levels and used recreational drugs. HIV reservoir cells are found in semen during PHI, with gradual compartmentalization. Its size was linked to the plasma IP-10 level. Early treatment purges both the virus and infected cells, reducing the high risk of transmission during PHI. NCT01033760.

Decreased HIV Type 1 Transcription in CCR5-Δ32 Heterozygotes During Suppressive Antiretroviral Therapy

PubMed Central

Wang, Charlene; Abdel-Mohsen, Mohamed; Strain, Matthew C.; Lada, Steven M.; Yukl, Steven; Cockerham, Leslie R.; Pilcher, Christopher D.; Hecht, Frederick M.; Sinclair, Elizabeth; Liegler, Teri; Richman, Douglas D.; Deeks, Steven G.; Pillai, Satish K.

2014-01-01

Individuals who are heterozygous for the CCR5-Δ32 mutation provide a natural model to examine the effects of reduced CCR5 expression on human immunodeficiency virus (HIV) persistence. We evaluated the HIV reservoir in 18 CCR5-Δ32 heterozygotes and 54 CCR5 wild-type individuals during suppressive antiretroviral therapy. Cell-associated HIV RNA levels (P = .035), RNA to DNA transcriptional ratios (P = .013), and frequency of detectable HIV 2–long terminal repeat circular DNA (P = .013) were significantly lower in CD4+ T cells from CCR5-Δ32 heterozygotes. Cell-associated HIV RNA was significantly correlated with CCR5 surface expression on CD4+ T cells (r2 = 0.136; P = .002). Our findings suggest that curative strategies should further explore manipulation of CCR5. PMID:24935955

Abacavir, an anti-HIV-1 drug, targets TDP1-deficient adult T cell leukemia.

PubMed

Tada, Kohei; Kobayashi, Masayuki; Takiuchi, Yoko; Iwai, Fumie; Sakamoto, Takashi; Nagata, Kayoko; Shinohara, Masanobu; Io, Katsuhiro; Shirakawa, Kotaro; Hishizawa, Masakatsu; Shindo, Keisuke; Kadowaki, Norimitsu; Hirota, Kouji; Yamamoto, Junpei; Iwai, Shigenori; Sasanuma, Hiroyuki; Takeda, Shunichi; Takaori-Kondo, Akifumi

2015-04-01

Adult T cell leukemia (ATL) is an aggressive T cell malignancy caused by human T cell leukemia virus type 1 (HTLV-1) and has a poor prognosis. We analyzed the cytotoxic effects of various nucleoside analog reverse transcriptase inhibitors (NRTIs) for HIV-1 on ATL cells and found that abacavir potently and selectively kills ATL cells. Although NRTIs have minimal genotoxicities on host cells, the therapeutic concentration of abacavir induced numerous DNA double-strand breaks (DSBs) in the chromosomal DNA of ATL cells. DSBs persisted over time in ATL cells but not in other cell lines, suggesting impaired DNA repair. We found that the reduced expression of tyrosyl-DNA phosphodiesterase 1 (TDP1), a repair enzyme, is attributable to the cytotoxic effect of abacavir on ATL cells. We also showed that TDP1 removes abacavir from DNA ends in vitro. These results suggest a model in which ATL cells with reduced TDP1 expression are unable to excise abacavir incorporated into genomic DNA, leading to irreparable DSBs. On the basis of the above mechanism, we propose abacavir as a promising chemotherapeutic agent for ATL.

European Mitochondrial DNA Haplogroups are Associated with Cerebrospinal Fluid Biomarkers of Inflammation in HIV Infection

PubMed Central

Samuels, David C.; Kallianpur, Asha R.; Ellis, Ronald J.; Bush, William S.; Letendre, Scott; Franklin, Donald; Grant, Igor; Hulgan, Todd

2017-01-01

Background Mitochondrial DNA (mtDNA) haplogroups are ancestry-related patterns of single-nucleotide polymorphisms that are associated with differential mitochondrial function in model systems, neurodegenerative diseases in HIV-negative populations, and chronic complications of HIV infection, including neurocognitive impairment. We hypothesized that mtDNA haplogroups are associated with neuroinflammation in HIV-infected adults. Methods CNS HIV Antiretroviral Therapy Effects Research (CHARTER) is a US-based observational study of HIV-infected adults who underwent standardized neurocognitive assessments. Participants who consented to DNA collection underwent whole blood mtDNA sequencing, and a subset also underwent lumbar puncture. IL-6, IL-8, TNF-α (high-sensitivity), and IP-10 were measured in cerebrospinal fluid (CSF) by immunoassay. Multivariable regression of mtDNA haplogroups and log-transformed CSF biomarkers were stratified by genetic ancestry using whole-genome nuclear DNA genotyping (European [EA], African [AA], or Hispanic ancestry [HA]), and adjusted for age, sex, antiretroviral therapy (ART), detectable CSF HIV RNA, and CD4 nadir. A total of 384 participants had both CSF cytokine measures and genetic data (45% EA, 44% AA, 11% HA, 22% female, median age 43 years, 74% on ART). Results In analyses stratified by the 3 continental ancestry groups, no haplogroups were significantly associated with the 4 biomarkers. In the subgroup of participants with undetectable plasma HIV RNA on ART, European haplogroup H participants had significantly lower CSF TNF-α (P = 0.001). Conclusions Lower CSF TNF-α may indicate lower neuroinflammation in the haplogroup H participants with well-controlled HIV on ART. PMID:28317034

A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice.

PubMed

Tomusange, Khamis; Wijesundara, Danushka; Gummow, Jason; Garrod, Tamsin; Li, Yanrui; Gray, Lachlan; Churchill, Melissa; Grubor-Bauk, Branka; Gowans, Eric J

2016-06-30

DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans.

Similarities and differences in the nucleic acid chaperone activity of HIV-2 and HIV-1 nucleocapsid proteins in vitro.

PubMed

Pachulska-Wieczorek, Katarzyna; Stefaniak, Agnieszka K; Purzycka, Katarzyna J

2014-07-03

The nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein. We have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (-) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity. Our data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5'UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may influence the efficiency of reverse transcription and other key steps of the HIV-2 replication cycle.

Mitochondrial DNA and retroviral RNA analyses of archival oral polio vaccine (OPV CHAT) materials: evidence of macaque nuclear sequences confirms substrate identity.

PubMed

Berry, Neil; Jenkins, Adrian; Martin, Javier; Davis, Clare; Wood, David; Schild, Geoffrey; Bottiger, Margareta; Holmes, Harvey; Minor, Philip; Almond, Neil

2005-02-25

Inoculation of live experimental oral poliovirus vaccines (OPV CHAT) during the 1950s in central Africa has been proposed to account for the introduction of HIV into human populations. For this to have occurred, it would have been necessary for chimpanzee rather than macaque kidney epithelial cells to have been included in the preparation of early OPV materials. Theoretically, this could have led to contamination with a progenitor of HIV-1 derived from a related simian immunodeficiency virus of chimpanzees (SIVCPZ). In this article we present further detailed analyses of two samples of OPV, CHAT 10A-11 and CHAT 6039/Yugo, which were used in early human trials of poliovirus vaccination. Recovery of poliovirus by culture techniques confirmed the biological viability of the vaccines and sequence analysis of poliovirus RNA specifically identified the presence of the CHAT strain. Independent nested sets of oligonucleotide primers specific for HIV-1/SIVCPZ and HIV-2/SIVMAC/SIVSM phylogenetic lineages, respectively, indicated no evidence of HIV/SIV RNA in either vaccine preparation, at a sensitivity of 100 RNA equivalents/ml. Analysis of cellular substrate by the amplification of two distinct regions of mitochondrial DNA (D-loop control region and 12S ribosomal sequences) revealed no evidence of chimpanzee cellular sequences. However, this approach positively identified rhesus and cynomolgus macaque DNA for the CHAT 10A-11 and CHAT 6039/Yugo vaccine preparations, respectively. Analysis of multiple clones of mtDNA 12S rDNA indicated a relatively high number of nuclear mitochondrial DNA sequences (numts) in the CHAT 10A-11 material, but confirmed the macaque origin of cellular substrate used in vaccine preparation. These data reinforce earlier findings on this topic providing no evidence to support the contention that poliovirus vaccination was responsible for the introduction of HIV into humans and sparking the AIDS pandemic.

Semi-NLO production of Higgs bosons in the framework of kt-factorization using KMR unintegrated parton distributions

NASA Astrophysics Data System (ADS)

Modarres, M.; Masouminia, M. R.; Aminzadeh Nik, R.; Hosseinkhani, H.; Olanj, N.

2018-01-01

The cross-section for the production of the Standard Model Higgs boson has been calculated using a mixture of LO and NLO partonic diagrams and the unintegrated parton distribution functions (UPDF) of the Kimber-Martin-Ryskin (KMR) from the kt-factorization framework. The UPDF are prepared using the phenomenological libraries of Martin-Motylinski-Harland Lang-Thorne (MMHT 2014). The results are compared against the existing experimental data from the CMS and the ATLAS collaborations and available pQCD calculation. It is shown that, while the present calculation is in agreement with the experimental data, it is comparable with the pQCD results. It is also concluded that the K-factor approximation is comparable with the semi-NLOkt-factorization predictions.

Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells.

PubMed

Liao, Hsin-Kai; Gu, Ying; Diaz, Arturo; Marlett, John; Takahashi, Yuta; Li, Mo; Suzuki, Keiichiro; Xu, Ruo; Hishida, Tomoaki; Chang, Chan-Jung; Esteban, Concepcion Rodriguez; Young, John; Izpisua Belmonte, Juan Carlos

2015-03-10

To combat hostile viruses, bacteria and archaea have evolved a unique antiviral defense system composed of clustered regularly interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated genes (Cas). The CRISPR/Cas9 system develops an adaptive immune resistance to foreign plasmids and viruses by creating site-specific DNA double-stranded breaks (DSBs). Here we adapt the CRISPR/Cas9 system to human cells for intracellular defense against foreign DNA and viruses. Using HIV-1 infection as a model, our results demonstrate that the CRISPR/Cas9 system disrupts latently integrated viral genome and provides long-term adaptive defense against new viral infection, expression and replication in human cells. We show that engineered human-induced pluripotent stem cells stably expressing HIV-targeted CRISPR/Cas9 can be efficiently differentiated into HIV reservoir cell types and maintain their resistance to HIV-1 challenge. These results unveil the potential of the CRISPR/Cas9 system as a new therapeutic strategy against viral infections.

Formation of stable and functional HIV-1 nucleoprotein complexes in vitro.

PubMed

Tanchou, V; Gabus, C; Rogemond, V; Darlix, J L

1995-10-06

HIV genomic RNA resides within the nucleocapsid, in the interior of the virus, which serves to protect the RNA against nuclease degradation and to promote its reverse transcription. To investigate the role of nucleocapsid protein (NCp7) in the stability and replication of genomic RNA within the nucleocapsid, we used NCp7, reverse transcriptase (RT) and RNAs representing the 5' and 3' regions of the genome to reconstitute functional HIV-1 nucleocapsids. The nucleoprotein complexes generated in vitro were found to be stable, which, according to biochemical and genetic data, probably results from the tight binding of NCp7 molecules to the RNA and strong NCp7/NCp7 interactions. The nucleoprotein complexes efficiently protected viral RNA against RNase degradation and, at the same time, promoted viral DNA synthesis by RT. DNA strand transfer from the 5' to the 3' RNA template was very efficient in nucleoprotein complexes formed in the presence of both RNAs, but not when the RNAs were in separate complexes. These results indicate that the in vitro reconstituted HIV-1 nucleoprotein complexes function like virion nucleocapsids and thus provide a way to study at the molecular level this viral substructure and the synthesis of proviral DNA, and to search for new anti-HIV agents.

Reliability at the lower limits of HIV-1 RNA quantification in clinical samples: a comparison of RT-PCR versus bDNA assays.

PubMed

Lubelchek, Ronald J; Max, Blake; Sandusky, Caroline J; Hota, Bala; Barker, David E

2009-06-23

To explore whether an assay change was responsible for an increasing proportion of patients with undetectable HIV viral loads at our urban HIV clinic, we selected highly stable patients, examining their viral loads before and after changing assays. We compared the proportion with detectable viremia during RT-PCR vs. bDNA periods. We selected patients with > or =1 viral loads assessed during both RT-PCR and bDNA periods. We included patients with stable CD4 counts, excluding patients with viral loads > or =1,000 copies/ml or any significant changes in therapy. Out of 4500 clinic patients, 419 patients (1588 viral loads) were included. 39% of viral loads were reported as detectable by RT-PCR vs. 5% reported as detectable by bDNA. The mean coefficient of variation was higher before vs. after assay change. We found an odds' ratio of 16.7 for having a viral load >75 copies/ml during the RT-PCR vs. bDNA periods. These data support previous reports, suggesting that bDNA may more reliably discriminate between viral suppression and low level viremia in stable patients on therapy. Low-level viremia, noted more with RT-PCR, may promote unneeded testing, while differences in viral load reliability may impact antiretroviral trial and quality assurance endpoints. Commonly used plasma separator tubes may differentially affect RT-PCR and bDNA results.

The HIV-1 reservoir in eight patients on long-term suppressive antiretroviral therapy is stable with few genetic changes over time

PubMed Central

Josefsson, Lina; von Stockenstrom, Susanne; Faria, Nuno R.; Sinclair, Elizabeth; Bacchetti, Peter; Killian, Maudi; Epling, Lorrie; Tan, Alice; Ho, Terence; Lemey, Philippe; Shao, Wei; Hunt, Peter W.; Somsouk, Ma; Wylie, Will; Douek, Daniel C.; Loeb, Lisa; Custer, Jeff; Hoh, Rebecca; Poole, Lauren; Deeks, Steven G.; Hecht, Frederick; Palmer, Sarah

2013-01-01

The source and dynamics of persistent HIV-1 during long-term combinational antiretroviral therapy (cART) are critical to understanding the barriers to curing HIV-1 infection. To address this issue, we isolated and genetically characterized HIV-1 DNA from naïve and memory T cells from peripheral blood and gut-associated lymphoid tissue (GALT) from eight patients after 4–12 y of suppressive cART. Our detailed analysis of these eight patients indicates that persistent HIV-1 in peripheral blood and GALT is found primarily in memory CD4+ T cells [CD45RO+/CD27(+/−)]. The HIV-1 infection frequency of CD4+ T cells from peripheral blood and GALT was higher in patients who initiated treatment during chronic compared with acute/early infection, indicating that early initiation of therapy results in lower HIV-1 reservoir size in blood and gut. Phylogenetic analysis revealed an HIV-1 genetic change between RNA sequences isolated before initiation of cART and intracellular HIV-1 sequences from the T-cell subsets after 4–12 y of suppressive cART in four of the eight patients. However, evolutionary rate analyses estimated no greater than three nucleotide substitutions per gene region analyzed during all of the 4–12 y of suppressive therapy. We also identified a clearly replication-incompetent viral sequence in multiple memory T cells in one patient, strongly supporting asynchronous cell replication of a cell containing integrated HIV-1 DNA as the source. This study indicates that persistence of a remarkably stable population of infected memory cells will be the primary barrier to a cure, and, with little evidence of viral replication, this population could be maintained by homeostatic cell proliferation or other processes. PMID:24277811

A novel acute HIV infection staging system based on 4th generation immunoassay.

PubMed

Ananworanich, Jintanat; Fletcher, James L K; Pinyakorn, Suteeraporn; van Griensven, Frits; Vandergeeten, Claire; Schuetz, Alexandra; Pankam, Tippawan; Trichavaroj, Rapee; Akapirat, Siriwat; Chomchey, Nitiya; Phanuphak, Praphan; Chomont, Nicolas; Michael, Nelson L; Kim, Jerome H; de Souza, Mark

2013-05-29

Fourth generation (4thG) immunoassay (IA) is becoming the standard HIV screening method but was not available when the Fiebig acute HIV infection (AHI) staging system was proposed. Here we evaluated AHI staging based on a 4thG IA (4thG staging). Screening for AHI was performed in real-time by pooled nucleic acid testing (NAT, n=48,828 samples) and sequential enzyme immunoassay (EIA, n=3,939 samples) identifying 63 subjects with non-reactive 2nd generation EIA (Fiebig stages I (n=25), II (n=7), III (n=29), IV (n=2)). The majority of samples tested (n=53) were subtype CRF_01AE (77%). NAT+ subjects were re-staged into three 4thG stages: stage 1 (n=20; 4th gen EIA-, 3rd gen EIA-), stage 2 (n=12; 4th gen EIA+, 3rd gen EIA-), stage 3 (n=31; 4th gen EIA+, 3rd gen EIA+, Western blot-/indeterminate). 4thG staging distinguishes groups of AHI subjects by time since presumed HIV exposure, pattern of CD8+ T, B and natural killer cell absolute numbers, and HIV RNA and DNA levels. This staging system further stratified Fiebig I subjects: 18 subjects in 4thG stage 1 had lower HIV RNA and DNA levels than 7 subjects in 4thG stage 2. Using 4th generation IA as part of AHI staging distinguishes groups of patients by time since exposure to HIV, lymphocyte numbers and HIV viral burden. It identifies two groups of Fiebig stage I subjects who display different levels of HIV RNA and DNA, which may have implication for HIV cure. 4th generation IA should be incorporated into AHI staging systems.

A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen

PubMed Central

Vijayan, Aneesh; García-Arriaza, Juan; C. Raman, Suresh; Conesa, José Javier; Chichón, Francisco Javier; Santiago, César; Sorzano, Carlos Óscar S.; Carrascosa, José L.; Esteban, Mariano

2015-01-01

In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive and memory T-cell immune responses, as well as humoral responses. This novel HIV-1 gp120-14K immunogen might be considered as an HIV vaccine candidate for broad T and B-cell immune responses. PMID:26208356

Transgene vaccination using Ulex europaeus agglutinin I (UEA-1) for targeted mucosal immunization against HIV-1 envelope.

PubMed

Wang, Xinhai; Kochetkova, Irina; Haddad, Asmahan; Hoyt, Teri; Hone, David M; Pascual, David W

2005-05-31

Receptor-mediated gene transfer using an M cell ligand has been shown to be an efficient method for mucosal DNA immunization. To investigate further into alternative M cell ligands, the plant lectin, Ulex europaeus agglutinin I (UEA-1), was tested. UEA-1 binds to human intestinal Caco-2 cells, and these cells can be transfected with poly-l-lysine (PL)-conjugated UEA-1 for expression of reporter cDNAs. When tested in vivo, mice nasally immunized with UEA-1-PL complexed to plasmid encoding HIV-1 envelope showed elevated systemic and mucosal antibody responses, and these were supported by tissue antibody-forming cells. Likewise, elevated envelope-specific CTLs were induced. Thus, UEA-1 mediated DNA delivery represents an alternative mucosal formulation for inducing humoral and cellular immunity against HIV-1.

Comprehensive comparison of the VERSANT HIV-1 RNA 3.0 (bDNA) and COBAS AMPLICOR HIV-1 MONITOR 1.5 assays on 1,000 clinical specimens.

PubMed

Galli, Rick; Merrick, Linda; Friesenhahn, Michel; Ziermann, Rainer

2005-12-01

Plasma human immunodeficiency virus type 1 (HIV-1) RNA level is an important parameter for patient management, yet viral load assays from different manufacturers are not standardized. In this study, we evaluated the concordance between test results obtained for 1,000 plasma specimens collected from HIV-1-infected individuals measured with the VERSANT HIV-1 RNA 3.0 assay (bDNA) and the COBAS AMPLICOR HIV-1 MONITOR 1.5 test (PCR). We compared viral load values obtained by each of these assays throughout their dynamic ranges, with particular focus on samples with low viral load (i.e. 50-250 copies/mL), and calculated the estimated distribution of distinct plasma viral load levels for the entire study population modeled from the data observed in the study. We found that these two assays show excellent agreement, with a correlation (R(2)) of 0.957 and a slope of 1.004. The mean difference in viral load values between the two assays was less than 0.10-log(10) throughout the dynamic range and 98.2% of all samples had bDNA and PCR results within 0.5-log(10) of each other, a difference that is within the range considered to be a minimal change in plasma viremia. Moreover, the two assays show very similar results across all assay ranges tested. The estimated prevalence of samples with results <50 copies/mL, 50-250 copies/mL, and 250-500,000 copies/mL were 41.6%, 7.7%, and 49.7%, respectively, by the bDNA assay, and 42.4%, 6.9%, and 50.7%, respectively, by the PCR assay. Based on our findings from 1,000 clinical specimens, we do not see the need to re-establish a baseline value or apply a conversion factor when switching from one assay to the other. Since the majority of our patient population likely is infected with subtype B virus, it is unclear if our findings will apply to other patient populations with a greater incidence of infection with non-B subtypes.

Superior induction of T cell responses to conserved HIV-1 regions by electroporated alphavirus replicon DNA compared to that with conventional plasmid DNA vaccine.

PubMed

Knudsen, Maria L; Mbewe-Mvula, Alice; Rosario, Maximillian; Johansson, Daniel X; Kakoulidou, Maria; Bridgeman, Anne; Reyes-Sandoval, Arturo; Nicosia, Alfredo; Ljungberg, Karl; Hanke, Tomás; Liljeström, Peter

2012-04-01

Vaccination using "naked" DNA is a highly attractive strategy for induction of pathogen-specific immune responses; however, it has been only weakly immunogenic in humans. Previously, we constructed DNA-launched Semliki Forest virus replicons (DREP), which stimulate pattern recognition receptors and induce augmented immune responses. Also, in vivo electroporation was shown to enhance immune responses induced by conventional DNA vaccines. Here, we combine these two approaches and show that in vivo electroporation increases CD8(+) T cell responses induced by DREP and consequently decreases the DNA dose required to induce a response. The vaccines used in this study encode the multiclade HIV-1 T cell immunogen HIVconsv, which is currently being evaluated in clinical trials. Using intradermal delivery followed by electroporation, the DREP.HIVconsv DNA dose could be reduced to as low as 3.2 ng to elicit frequencies of HIV-1-specific CD8(+) T cells comparable to those induced by 1 μg of a conventional pTH.HIVconsv DNA vaccine, representing a 625-fold molar reduction in dose. Responses induced by both DREP.HIVconsv and pTH.HIVconsv were further increased by heterologous vaccine boosts employing modified vaccinia virus Ankara MVA.HIVconsv and attenuated chimpanzee adenovirus ChAdV63.HIVconsv. Using the same HIVconsv vaccines, the mouse observations were supported by an at least 20-fold-lower dose of DNA vaccine in rhesus macaques. These data point toward a strategy for overcoming the low immunogenicity of DNA vaccines in humans and strongly support further development of the DREP vaccine platform for clinical evaluation.

An advanced BLT-humanized mouse model for extended HIV-1 cure studies.

PubMed

Lavender, Kerry J; Pace, Craig; Sutter, Kathrin; Messer, Ronald J; Pouncey, Dakota L; Cummins, Nathan W; Natesampillai, Sekar; Zheng, Jim; Goldsmith, Joshua; Widera, Marek; Van Dis, Erik S; Phillips, Katie; Race, Brent; Dittmer, Ulf; Kukolj, George; Hasenkrug, Kim J

2018-01-02

Although bone marrow, liver, thymus (BLT)-humanized mice provide a robust model for HIV-1 infection and enable evaluation of cure strategies dependent on endogenous immune responses, most mice develop graft versus host disease (GVHD), limiting their utility for extended HIV cure studies. This study aimed to: evaluate the GVHD-resistant C57 black 6 (C57BL/6) recombination activating gene 2 (Rag2)γcCD47 triple knockout (TKO)-BLT mouse as a model to establish HIV-1 latency. Determine whether TKO-BLT mice could be maintained on antiretroviral therapy (ART) for extended periods of time. Assess the rapidity of viral rebound following therapy interruption. TKO-BLT mice were HIV-1 infected, treated with various ART regimens over extended periods of time and assayed for viral rebound following therapy interruption. Daily subcutaneous injection and oral ART-mediated suppression of HIV-1 infection was tested at various doses in TKO-BLT mice. Mice were monitored for suppression of viremia and cellular HIV-1 RNA and DNA prior to and following therapy interruption. Mice remained healthy for 45 weeks posthumanization and could be treated with ART for up to 18 weeks. Viremia was suppressed to less than 200 copies/ml in the majority of mice with significant reductions in cellular HIV-1 RNA and DNA. Treatment interruption resulted in rapid viral recrudescence. HIV-1 latency can be maintained in TKO-BLT mice over extended periods on ART and rapid viral rebound occurs following therapy removal. The additional 15-18 weeks of healthy longevity compared with other BLT models provides sufficient time to examine the decay kinetics of the latent reservoir as well as observe delays in recrudescence in HIV-1 cure studies.

Signal-boosted qualitative ultrasensitive p24 antigen assay for diagnosis of subtype C HIV-1 infection in infants under the age of 2 years.

PubMed

Zijenah, Lynn S; Tobaiwa, Ocean; Rusakaniko, Simbarashe; Nathoo, Kusum J; Nhembe, Margaret; Matibe, Petronella; Katzenstein, David A

2005-08-01

The gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA or reverse transcriptase polymerase chain reaction. However, these tests are expensive and therefore not available in resource-limited countries. With the increasing availability of antiretroviral drugs for prevention of mother-to-child transmission of HIV and treatment of AIDS in resource-poor countries, there is an urgent need to develop cheaper, alternative, and cost-effective laboratory methods for early diagnosis of infant HIV-1 infection that will be useful in identifying infected infants who may benefit from early cotrimoxazole prophylaxis or commencement of antiretroviral therapy. We evaluated an alternative method, the enzyme-linked immunosorbent assay-based qualitative ultrasensitive p24 antigen assay for diagnosis of subtype C HIV-1 infection in infants under the age of 2 years using DNA polymerase chain reaction as the reference method. The assay showed a sensitivity of 96.7% (95% CI: 93.0-100) for detection of HIV-1 infection among infants 0-18 months of age with a specificity of 96.1% (95% CI: 91.7-100). These evaluated parameters were not statistically different between infants aged 0-6 and 7-18 months. The ultrasensitive p24 antigen assay is a useful diagnostic test for detection of HIV-1 infection among infants aged 0-18 months.

Bicistronic DNA vaccines simultaneously encoding HIV, HSV and HPV antigens promote CD8⁺ T cell responses and protective immunity.

PubMed

Santana, Vinicius C; Diniz, Mariana O; Cariri, Francisco A M O; Ventura, Armando M; Cunha-Neto, Edécio; Almeida, Rafael R; Campos, Marco A; Lima, Graciela K; Ferreira, Luís C S

2013-01-01

Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.

The role of phenylalanine-119 of the reverse transcriptase of mouse mammary tumour virus in DNA synthesis, ribose selection and drug resistance.

PubMed

Entin-Meer, Michal; Sevilya, Ziv; Hizi, Amnon

2002-10-15

Phe-119 in the reverse transcriptase (RT) of mouse mammary tumour virus (MMTV) is homologous with Tyr-115 in HIV type 1 (HIV-1) RT and to Phe-155 in murine leukaemia virus (MLV) RT. By mutating these residues in HIV-1 and MLV RTs (which are strict DNA polymerases) the enzymes were shown to function also as RNA polymerases. Owing to the uniqueness of MMTV as a type B retrovirus, we have generated a Phe-119-Val mutant of MMTV RT to study the involvement of this residue in affecting the catalytic features of this RT. The data presented here show that the mutant MMTV RT can incorporate both deoxyribonucleosides and ribonucleosides while copying either RNA or DNA. In addition, this mutant RT shows resistance to nucleoside analogues and an enhanced fidelity of DNA synthesis; all relative to the wild-type enzyme. The Phe-119-Val mutant is also different from the wild-type enzyme in its preference for most template primers tested and in its ability to synthesize DNA under non-processive and processive conditions. Overall, it is likely that the aromatic side chain of Phe-119 is located at the dNTP-binding site of MMTV RT and thus might be part of a putative "steric gate" that prevents the incorporation of nucleoside triphosphates. Since the only three-dimensional structures of RTs published so far are those of HIV-1 and MLV, it is likely that MMTV RT folds quite similarly to these RTs.

Mitochondrial DNA Haplogroups and Neurocognitive Impairment During HIV Infection

PubMed Central

Hulgan, Todd; Samuels, David C.; Bush, William; Ellis, Ronald J.; Letendre, Scott L.; Heaton, Robert K.; Franklin, Donald R.; Straub, Peter; Murdock, Deborah G.; Clifford, David B.; Collier, Ann C.; Gelman, Benjamin B.; Marra, Christina M.; McArthur, Justin C.; McCutchan, J. Allen; Morgello, Susan; Simpson, David M.; Grant, Igor; Kallianpur, Asha R.

2015-01-01

Background. Neurocognitive impairment (NCI) remains an important complication in persons infected with human immunodeficiency virus (HIV). Ancestry-related mitochondrial DNA (mtDNA) haplogroups have been associated with outcomes of HIV infection and combination antiretroviral therapy (CART), and with neurodegenerative diseases. We hypothesize that mtDNA haplogroups are associated with NCI in HIV-infected adults and performed a genetic association study in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) cohort. Methods. CHARTER is an observational study of ambulatory HIV-infected adults. Haplogroups were assigned using mtDNA sequence, and principal components were derived from ancestry-informative nuclear DNA variants. Outcomes were cross-sectional global deficit score (GDS) as a continuous measure, GDS impairment (GDS ≥ 0.50), and HIV-associated neurocognitive disorder (HAND) using international criteria. Multivariable models were adjusted for comorbidity status (incidental vs contributing), current CART, plasma HIV RNA, reading ability, and CD4 cell nadir. Results. Haplogroups were available from 1027 persons; median age 43 years, median CD4 nadir 178 cells/mm3, 72% on CART, and 46% with HAND. The 102 (9.9%) persons of genetically determined admixed Hispanic ancestry had more impairment by GDS or HAND than persons of European or African ancestry (P < .001 for all). In multivariate models including persons of admixed Hispanic ancestry, those with haplogroup B had lower GDS (β = −0.34; P = .008) and less GDS impairment (odds ratio = 0.16; 95% confidence interval, .04, .63; P = .009) than other haplogroups. There were no significant haplogroup associations among persons of European or African ancestry. Conclusions. In these mostly CART-treated persons, mtDNA haplogroup B was associated with less NCI among persons of genetically determined Hispanic ancestry. mtDNA variation may represent an ancestry-specific factor influencing NCI in HIV-infected persons. PMID:26129753

FBI-1 can stimulate HIV-1 Tat activity and is targeted to a novel subnuclear domain that includes the Tat-P-TEFb-containing nuclear speckles.

PubMed

Pendergrast, P Shannon; Wang, Chen; Hernandez, Nouria; Huang, Sui

2002-03-01

FBI-1 is a cellular POZ-domain-containing protein that binds to the HIV-1 LTR and associates with the HIV-1 transactivator protein Tat. Here we show that elevated levels of FBI-1 specifically stimulate Tat activity and that this effect is dependent on the same domain of FBI-1 that mediates Tat-FBI-1 association in vivo. FBI-1 also partially colocalizes with Tat and Tat's cellular cofactor, P-TEFb (Cdk9 and cyclin T1), at the splicing-factor-rich nuclear speckle domain. Further, a less-soluble population of FBI-1 distributes in a novel peripheral-speckle pattern of localization as well as in other nuclear regions. This distribution pattern is dependent on the FBI-1 DNA binding domain, on the presence of cellular DNA, and on active transcription. Taken together, these results suggest that FBI-1 is a cellular factor that preferentially associates with active chromatin and that can specifically stimulate Tat-activated HIV-1 transcription.

FBI-1 Can Stimulate HIV-1 Tat Activity and Is Targeted to a Novel Subnuclear Domain that Includes the Tat-P-TEFb—containing Nuclear Speckles

PubMed Central

Pendergrast, P. Shannon; Wang, Chen; Hernandez, Nouria; Huang, Sui

2002-01-01

FBI-1 is a cellular POZ-domain–containing protein that binds to the HIV-1 LTR and associates with the HIV-1 transactivator protein Tat. Here we show that elevated levels of FBI-1 specifically stimulate Tat activity and that this effect is dependent on the same domain of FBI-1 that mediates Tat-FBI-1 association in vivo. FBI-1 also partially colocalizes with Tat and Tat's cellular cofactor, P-TEFb (Cdk9 and cyclin T1), at the splicing-factor–rich nuclear speckle domain. Further, a less-soluble population of FBI-1 distributes in a novel peripheral-speckle pattern of localization as well as in other nuclear regions. This distribution pattern is dependent on the FBI-1 DNA binding domain, on the presence of cellular DNA, and on active transcription. Taken together, these results suggest that FBI-1 is a cellular factor that preferentially associates with active chromatin and that can specifically stimulate Tat-activated HIV-1 transcription. PMID:11907272

Cytomegalovirus Replication in Semen Is Associated with Higher Levels of Proviral HIV DNA and CD4+ T Cell Activation during Antiretroviral Treatment

PubMed Central

Massanella, Marta; Richman, Douglas D.; Little, Susan J.; Spina, Celsa A.; Vargas, Milenka V.; Lada, Steven M.; Daar, Eric S.; Dube, Michael P.; Haubrich, Richard H.; Morris, Sheldon R.; Smith, Davey M.

2014-01-01

ABSTRACT Asymptomatic cytomegalovirus (CMV) replication occurs frequently in the genital tract in untreated HIV-infected men and is associated with increased immune activation and HIV disease progression. To determine the connections between CMV-associated immune activation and the size of the viral reservoir, we evaluated the interactions between (i) asymptomatic seminal CMV replication, (ii) levels of T cell activation and proliferation in blood, and (iii) the size and transcriptional activity of the HIV DNA reservoir in blood from 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. We found that asymptomatic CMV shedding in semen was associated with significantly higher levels of proliferating and activated CD4+ T cells in blood (P < 0.01). Subjects with detectable CMV in semen had approximately five times higher average levels of HIV DNA in blood CD4+ T cells than subjects with no CMV. There was also a trend for CMV shedders to have increased cellular (multiply spliced) HIV RNA transcription (P = 0.068) compared to participants without CMV, but it is unclear if this transcription pattern is associated with residual HIV replication. In multivariate analysis, the presence of seminal plasma CMV (P = 0.04), detectable 2-long terminal repeat (2-LTR), and lower nadir CD4+ (P < 0.01) were independent predictors of higher levels of proviral HIV DNA in blood. Interventions aimed at reducing seminal CMV and associated immune activation may be important for HIV curative strategies. Future studies of anti-CMV therapeutics will help to establish causality and determine the mechanisms underlying these described associations. IMPORTANCE Almost all individuals infected with HIV are also infected with cytomegalovirus (CMV), and the replication dynamics of the two viruses likely influence each other. This study investigated interactions between asymptomatic CMV replication within the male genital tract, levels of inflammation in blood, and the size of the HIV DNA reservoir in 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. In support of our primary hypothesis, shedding of CMV DNA in semen was associated with increased activation and proliferation of T cells in blood and also significantly higher levels of HIV DNA in blood cells. These results suggest that CMV reactivation might play a role in the maintenance of the HIV DNA reservoir during suppressive ART and that it could be a target of pharmacologic intervention in future studies. PMID:24789781

The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro.

PubMed

Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

2008-06-01

The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44-61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties.

The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro

PubMed Central

Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

2008-01-01

The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44–61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties. PMID:18442994

Nedd4-mediated increase in HIV-1 Gag and Env proteins and immunity following DNA-vaccination of BALB/c mice.

PubMed

Lewis, Brad; Whitney, Stephen; Hudacik, Lauren; Galmin, Lindsey; Huaman, Maria Cecilia; Cristillo, Anthony D

2014-01-01

The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation.

DNA Physical Properties and Nucleosome Positions Are Major Determinants of HIV-1 Integrase Selectivity

PubMed Central

Naughtin, Monica; Haftek-Terreau, Zofia; Xavier, Johan; Meyer, Sam; Silvain, Maud; Jaszczyszyn, Yan; Levy, Nicolas; Miele, Vincent; Benleulmi, Mohamed Salah; Ruff, Marc; Parissi, Vincent; Vaillant, Cédric; Lavigne, Marc

2015-01-01

Retroviral integrases (INs) catalyse the integration of the reverse transcribed viral DNA into the host cell genome. This process is selective, and chromatin has been proposed to be a major factor regulating this step in the viral life cycle. However, the precise underlying mechanisms are still under investigation. We have developed a new in vitro integration assay using physiologically-relevant, reconstituted genomic acceptor chromatin and high-throughput determination of nucleosome positions and integration sites, in parallel. A quantitative analysis of the resulting data reveals a chromatin-dependent redistribution of the integration sites and establishes a link between integration sites and nucleosome positions. The co-activator LEDGF/p75 enhanced integration but did not modify the integration sites under these conditions. We also conducted an in cellulo genome-wide comparative study of nucleosome positions and human immunodeficiency virus type-1 (HIV-1) integration sites identified experimentally in vivo. These studies confirm a preferential integration in nucleosome-covered regions. Using a DNA mechanical energy model, we show that the physical properties of DNA probed by IN binding are important in determining IN selectivity. These novel in vitro and in vivo approaches confirm that IN has a preference for integration into a nucleosome, and suggest the existence of two levels of IN selectivity. The first depends on the physical properties of the target DNA and notably, the energy required to fit DNA into the IN catalytic pocket. The second depends on the DNA deformation associated with DNA wrapping around a nucleosome. Taken together, these results indicate that HIV-1 IN is a shape-readout DNA binding protein. PMID:26075397

Analysis of HIV using a high resolution melting (HRM) diversity assay: automation of HRM data analysis enhances the utility of the assay for analysis of HIV incidence.

PubMed

Cousins, Matthew M; Swan, David; Magaret, Craig A; Hoover, Donald R; Eshleman, Susan H

2012-01-01

HIV diversity may be a useful biomarker for discriminating between recent and non-recent HIV infection. The high resolution melting (HRM) diversity assay was developed to quantify HIV diversity in viral populations without sequencing. In this assay, HIV diversity is expressed as a single numeric HRM score that represents the width of a melting peak. HRM scores are highly associated with diversity measures obtained with next generation sequencing. In this report, a software package, the HRM Diversity Assay Analysis Tool (DivMelt), was developed to automate calculation of HRM scores from melting curve data. DivMelt uses computational algorithms to calculate HRM scores by identifying the start (T1) and end (T2) melting temperatures for a DNA sample and subtracting them (T2 - T1 =  HRM score). DivMelt contains many user-supplied analysis parameters to allow analyses to be tailored to different contexts. DivMelt analysis options were optimized to discriminate between recent and non-recent HIV infection and to maximize HRM score reproducibility. HRM scores calculated using DivMelt were compared to HRM scores obtained using a manual method that is based on visual inspection of DNA melting curves. HRM scores generated with DivMelt agreed with manually generated HRM scores obtained from the same DNA melting data. Optimal parameters for discriminating between recent and non-recent HIV infection were identified. DivMelt provided greater discrimination between recent and non-recent HIV infection than the manual method. DivMelt provides a rapid, accurate method of determining HRM scores from melting curve data, facilitating use of the HRM diversity assay for large-scale studies.

Analysis of HIV Using a High Resolution Melting (HRM) Diversity Assay: Automation of HRM Data Analysis Enhances the Utility of the Assay for Analysis of HIV Incidence

PubMed Central

Cousins, Matthew M.; Swan, David; Magaret, Craig A.; Hoover, Donald R.; Eshleman, Susan H.

2012-01-01

Background HIV diversity may be a useful biomarker for discriminating between recent and non-recent HIV infection. The high resolution melting (HRM) diversity assay was developed to quantify HIV diversity in viral populations without sequencing. In this assay, HIV diversity is expressed as a single numeric HRM score that represents the width of a melting peak. HRM scores are highly associated with diversity measures obtained with next generation sequencing. In this report, a software package, the HRM Diversity Assay Analysis Tool (DivMelt), was developed to automate calculation of HRM scores from melting curve data. Methods DivMelt uses computational algorithms to calculate HRM scores by identifying the start (T1) and end (T2) melting temperatures for a DNA sample and subtracting them (T2–T1 = HRM score). DivMelt contains many user-supplied analysis parameters to allow analyses to be tailored to different contexts. DivMelt analysis options were optimized to discriminate between recent and non-recent HIV infection and to maximize HRM score reproducibility. HRM scores calculated using DivMelt were compared to HRM scores obtained using a manual method that is based on visual inspection of DNA melting curves. Results HRM scores generated with DivMelt agreed with manually generated HRM scores obtained from the same DNA melting data. Optimal parameters for discriminating between recent and non-recent HIV infection were identified. DivMelt provided greater discrimination between recent and non-recent HIV infection than the manual method. Conclusion DivMelt provides a rapid, accurate method of determining HRM scores from melting curve data, facilitating use of the HRM diversity assay for large-scale studies. PMID:23240016

HIV-DNA priming alters T-cell responses to HIV-adenovirus vaccine even when responses to DNA are undetectable1

PubMed Central

De Rosa, Stephen C.; Thomas, Evan P.; Bui, John; Huang, Yunda; deCamp, Allan; Morgan, Cecilia; Kalams, Spyros; Tomaras, Georgia D.; Akondy, Rama; Ahmed, Rafi; Lau, Chuen-Yen; Graham, Barney S.; Nabel, Gary J.; McElrath, M. Juliana

2011-01-01

Many candidate HIV vaccines are designed to primarily elicit T-cell responses. Although repeated immunization with the same vaccine boosts antibody responses, the benefit for T-cell responses is ill-defined. We compared two immunization regimens that include the same recombinant adenoviral serotype 5 (rAd5) boost. Repeated homologous rAd5 immunization fails to increase T-cell responses, but increases gp140 antibody responses ten-fold. DNA prime, as compared with rAd5 prime, directs long-term memory CD8+ T cells toward a terminally differentiated effector memory phenotype with cytotoxic potential. Based on the kinetics of activated cells measured directly ex vivo, the DNA vaccination primes for both CD4+ and CD8+ T cells, despite the lack of detection of the latter until after the boost. These results suggest that heterologous prime-boost combinations have distinct immunological advantages over homologous prime-boosts, and suggest that the effect of DNA on subsequent boosting may not be easily detectable directly after the DNA vaccination. PMID:21844392

Expression of Human Immunodeficiency Virus type 1 (HIV-1) coat protein genes in plants using cotton leaf curl Multan betasatellite-based vector.

PubMed

Ataie Kachoie, Elham; Behjatnia, Seyed Ali Akbar; Kharazmi, Sara

2018-01-01

It has already been demonstrated that a betasatellite associated with cotton leaf curl Multan virus (CLCuMB) can be used as a plant and animal gene delivery vector to plants. To examine the ability of CLCuMB as a tool to transfer coat protein genes of HIV-1 to plants, two recombinant CLCuMB constructs in which the CLCuMB βC1 ORF was replaced with two HIV-1 genes fractions including a 696 bp DNA fragment related to the HIV-1 p24 gene and a 1501 bp DNA fragment related to the HIV-1 gag gene were constructed. Gag is the HIV-1 coat protein gene and p24 is a component of the particle capsid. Gag and p24 are used for vaccine production. Recombinant constructs were inoculated to Nicotiana glutinosa and N. benthamiana plants in the presence of an Iranian isolate of Tomato yellow leaf curl virus (TYLCV-[Ab]) as a helper virus. PCR analysis of inoculated plants indicated that p24 gene was successfully replicated in inoculated plants, but the gag gene was not. Real-time PCR and ELISA analysis of N. glutinosa and N. benthamiana plants containing the replicative forms of recombinant construct of CLCuMB/p24 indicated that p24 was expressed in these plants. This CLCuMB-based expression system offers the possibility of mass production of recombinant HIV-1 p24 protein in plants.

Performance of three commercial viral load assays, Versant human immunodeficiency virus type 1 (HIV-1) RNA bDNA v3.0, Cobas AmpliPrep/Cobas TaqMan HIV-1, and NucliSens HIV-1 EasyQ v1.2, testing HIV-1 non-B subtypes and recombinant variants.

PubMed

Holguín, Africa; López, Marisa; Molinero, Mar; Soriano, Vincent

2008-09-01

Monitoring antiretroviral therapy requires that human immunodeficiency virus type 1 (HIV-1) viremia assays are applicable to all distinct variants. This study evaluates the performance of three commercial viral load assays-Versant HIV-1 RNA bDNA v3.0, Cobas AmpliPrep/Cobas TaqMan HIV-1, and NucliSens HIV-1 EasyQ v1.2-in testing 83 plasma specimens from patients carrying HIV-1 non-B subtypes and recombinants previously defined by phylogenetic analysis of the pol gene. All 28 specimens from patients under treatment presented viremia values below the detection limit with the three methods. In the remaining 55 specimens from naive individuals viremia could not be detected in 32.7, 20, and 14.6% using the NucliSens, Versant, or TaqMan tests, respectively, suggesting potential viral load underestimation of some samples by all techniques. Only 32 (58.2%) samples from naive subjects were quantified by the three methods; the NucliSens test provided the highest HIV RNA values (mean, 4.87 log copies/ml), and the Versant test provided the lowest (mean, 4.16 log copies/ml). Viremia differences of greater than 1 log were seen in 8 (14.5%) of 55 specimens, occurring in 10.9, 7.3, and 5.4%, respectively, of the specimens in comparisons of Versant versus NucliSens, Versant versus TaqMan, and TaqMan versus NucliSens. Differences greater than 0.5 log, considered significant for clinicians, occurred in 45.5, 27.3, and 29% when the same assays were compared. Some HIV-1 strains, such as subtype G and CRF02_AG, showed more discrepancies in distinct quantification methods than others. In summary, an adequate design of primers and probes is needed for optimal quantitation of plasma HIV-RNA in non-B subtypes. Our data emphasize the need to use the same method for monitoring patients on therapy and also the convenience of HIV-1 subtyping.

HIV-1 protease has a genetic T-cell adjuvant effect which is negatively regulated by proteolytic activity.

PubMed

Kim, Kwang Soon; Jin, Dong Bin; Ahn, So Shin; Park, Ki Seok; Seo, Sang Hwan; Suh, You Suk; Sung, Young Chul

2010-08-01

HIV protease (PR) mediates the processing of human immunodeficiency virus (HIV) polyproteins and is necessary for the viral production. Recently, HIV PR was shown to possess both cytotoxic and chaperone like activity. We demonstrate here that HIV PR can serve as a genetic adjuvant that enhances the HIV Env and human papillomavirus (HPV) DNA vaccine-induced T-cell response in a dose-dependent manner, only when codelivered with DNA vaccine. Interestingly, the T-cell adjuvant effects of HIV PR were increased by introducing several mutations that inhibited its proteolytic activity, indicating that the adjuvant properties were inversely correlated with its proteolytic activity. Conversely, the introduction of a mutation in the flap region of HIV PR limiting the access to the core domain of HIV PR inhibited the T-cell adjuvant effect, suggesting that the HIV PR chaperone like activity may play a role in mediating T-cell adjuvant properties. A similar adjuvant effect was also observed in adenovirus vaccine, indicating vaccine type independency. These findings suggest that HIV PR can modulate T-cell responses elicited by a gene-based vaccine positively by inherent chaperone like activity and negatively by its proteolytic activity.

A cognitive perspective on object relations, drive development and ego structure in the second and third years of life.

PubMed

Posener, J A

1989-01-01

This paper extends a recent line of research by correlating Piaget's theory of cognitive development with several psychoanalytic perspectives on development during the second and third years of life. The concrete, imagistic, unintegrated nature of mental representations associated by Mahler and Kernberg with this period, along with the mental operation of splitting, are related to preconceptual representation, a cognitive mode described by Piaget. Psychoanalytic perspectives on the body ego and object world associated with the anal period are also seen to involve concrete, unintegrated representations which show correspondence with preconceptual cognition. Parallels are explored between cognitive stages and the psychoanalytic understanding of ego and superego development. While psychoanalysis is not a cognitive psychology, aspects of its theory are concerned with cognitive structure and are enriched by a consideration of cognitive development.

Subclinical herpesvirus shedding among HIV-1-infected men on antiretroviral therapy.

PubMed

Agudelo-Hernandez, Arcadio; Chen, Yue; Bullotta, Arlene; Buchanan, William G; Klamar-Blain, Cynthia R; Borowski, Luann; Riddler, Sharon A; Rinaldo, Charles R; Macatangay, Bernard J C

2017-09-24

We evaluated the subclinical shedding of six different herpesviruses in antiretroviral drug-treated HIV-positive [HIV(+)] MSM, and determined how this is associated with markers of inflammation and immune activation. We obtained blood, semen, throat washing, urine, and stool from 15 antiretroviral-treated HIV-1-infected MSM with CD4 T-cell reconstitution, and 12 age-matched HIV-negative [HIV (-)] MSM from the Multicenter AIDS Cohort Study at four timepoints over 24 weeks to measure DNA levels of cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus 1 and 2, human herpesvirus 6 (HHV6), and HHV8. T-cell activation and plasma levels of soluble markers of inflammation and activation were also measured at the corresponding timepoints. HIV(+) participants had a trend for higher total herpesvirus shedding rate. HIV(+) participants also had a significantly higher rate of shedding EBV and CMV compared with the HIV(-) group. Herpesvirus shedding was mostly seen in throat washings. In the HIV(+) group, herpesvirus shedding rate inversely correlated with plasma levels of interferon γ-induced protein 10 and soluble CD163. CMV DNA levels negatively correlated with levels of T-cell activation. There was a trend for a positive correlation between EBV shedding rate and plasma soluble CD14. HHV6 shedding rate negatively correlated with plasma levels of interleukin-6, soluble CD163, and interferon gamma-induced protein 10. Correlations were not observed among HIV(-) individuals. Among treated HIV-infected MSM, there are higher subclinical shedding rates of some herpesviruses that occur in different body compartments and negatively correlate with levels of inflammation and immune activation.

Cyclophilin A as a potential genetic adjuvant to improve HIV-1 Gag DNA vaccine immunogenicity by eliciting broad and long-term Gag-specific cellular immunity in mice.

PubMed

Hou, Jue; Zhang, Qicheng; Liu, Zheng; Wang, Shuhui; Li, Dan; Liu, Chang; Liu, Ying; Shao, Yiming

2016-01-01

Previous research has shown that host Cyclophilin A (CyPA) can promote dendritic cell maturation and the subsequent innate immune response when incorporated into an HIV-1 Gag protein to circumvent the resistance of dendritic cells to HIV-1 infection. This led us to hypothesize that CyPA may improve HIV-1 Gag-specific vaccine immunogenicity via binding with Gag antigen. The adjuvant effect of CyPA was evaluated using a DNA vaccine with single or dual expression cassettes. Mouse studies indicated that CyPA specifically and markedly promoted HIV-1 Gag-specific cellular immunity but not an HIV-1 Env-specific cellular response. The Gag/CyPA dual expression cassettes stimulated a greater Gag-specific cellular immune response, than Gag immunization alone. Furthermore, CyPA induced a broad Gag-specific T cell response and strong cellular immunity that lasted up to 5 months. In addition, CyPA skewed to cellular rather than humoral immunity. To investigate the mechanisms of the adjuvant effect, site-directed mutagenesis in CyPA, including active site residues H54Q and F60A resulted in mutants that were co-expressed with Gag in dual cassettes. The immune response to this vaccine was analyzed in vivo. Interestingly, the wild type CyPA markedly increased Gag cellular immunity, but the H54Q and F60A mutants drastically reduced CyPA adjuvant activation. Therefore, we suggest that the adjuvant effect of CyPA was based on Gag-CyPA-specific interactions. Herein, we report that Cyclophilin A can augment HIV-1 Gag-specific cellular immunity as a genetic adjuvant in multiplex DNA immunization strategies, and that activity of this adjuvant is specific, broad, long-term, and based on Gag-CyPA interaction.

Comparative Analysis of Measures of Viral Reservoirs in HIV-1 Eradication Studies

PubMed Central

Lysenko, Elena S.; Bosch, Ronald J.; Lai, Jun; Chioma, Stanley; Emad, Fatemeh; Abdel-Mohsen, Mohamed; Hoh, Rebecca; Hecht, Frederick; Hunt, Peter; Somsouk, Ma; Wong, Joseph; Johnston, Rowena; Siliciano, Robert F.; Richman, Douglas D.; O'Doherty, Una; Palmer, Sarah; Deeks, Steven G.; Siliciano, Janet D.

2013-01-01

HIV-1 reservoirs preclude virus eradication in patients receiving highly active antiretroviral therapy (HAART). The best characterized reservoir is a small, difficult-to-quantify pool of resting memory CD4+ T cells carrying latent but replication-competent viral genomes. Because strategies targeting this latent reservoir are now being tested in clinical trials, well-validated high-throughput assays that quantify this reservoir are urgently needed. Here we compare eleven different approaches for quantitating persistent HIV-1 in 30 patients on HAART, using the original viral outgrowth assay for resting CD4+ T cells carrying inducible, replication-competent viral genomes as a standard for comparison. PCR-based assays for cells containing HIV-1 DNA gave infected cell frequencies at least 2 logs higher than the viral outgrowth assay, even in subjects who started HAART during acute/early infection. This difference may reflect defective viral genomes. The ratio of infected cell frequencies determined by viral outgrowth and PCR-based assays varied dramatically between patients. Although strong correlations with the viral outgrowth assay could not be formally excluded for most assays, correlations achieved statistical significance only for integrated HIV-1 DNA in peripheral blood mononuclear cells and HIV-1 RNA/DNA ratio in rectal CD4+ T cells. Residual viremia was below the limit of detection in many subjects and did not correlate with the viral outgrowth assays. The dramatic differences in infected cell frequencies and the lack of a precise correlation between culture and PCR-based assays raise the possibility that the successful clearance of latently infected cells may be masked by a larger and variable pool of cells with defective proviruses. These defective proviruses are detected by PCR but may not be affected by reactivation strategies and may not require eradication to accomplish an effective cure. A molecular understanding of the discrepancy between infected cell frequencies measured by viral outgrowth versus PCR assays is an urgent priority in HIV-1 cure research. PMID:23459007

High level of APOBEC3F/3G editing in HIV-2 DNA vif and pol sequences from antiretroviral-naive patients.

PubMed

Bertine, Mélanie; Charpentier, Charlotte; Visseaux, Benoit; Storto, Alexandre; Collin, Gilles; Larrouy, Lucile; Damond, Florence; Matheron, Sophie; Brun-Vézinet, Françoise; Descamps, Diane

2015-04-24

In HIV-1, hypermutation introduced by APOBEC3F/3G cytidine deaminase activity leads to defective viruses. In-vivo impact of APOBEC3F/3G editing on HIV-2 sequences remains unknown. The objective of this study was to assess the level of APOBEC3F/3G editing in HIV-2-infected antiretroviral-naive patients. Direct sequencing of vif and pol regions was performed on HIV-2 proviral DNA from antiretroviral-naive patients included in the French Agence Nationale de Recherches sur le SIDA et les hépatites virales CO5 HIV-2 cohort. Hypermutated sequences were identified using Hypermut2.0 program. HIV-1 proviral sequences from Genbank were also assessed. Among 82 antiretroviral-naive HIV-2-infected patients assessed, 15 (28.8%) and five (16.7%) displayed Vif proviral defective sequences in HIV-2 groups A and B, respectively. A lower proportion of defective sequences was observed in protease-reverse transcriptase region. A higher median number of G-to-A mutations was observed in HIV-2 group B than in group A, both in Vif and protease-reverse transcriptase regions (P = 0.02 and P = 0.006, respectively). Compared with HIV-1 Vif sequences, a higher number of Vif defective sequences was observed in HIV-2 group A (P = 0.00001) and group B sequences (P = 0.013). We showed for the first time a high level of APOBEC3F/3G editing in HIV-2 sequences from antiretroviral-naive patients. Our study reported a group effect with a significantly higher level of APOBEC3F/3G editing in HIV-2 group B than in group A sequences.

Reliability at the Lower Limits of HIV-1 RNA Quantification in Clinical Samples: A Comparison of RT-PCR versus bDNA Assays

PubMed Central

Lubelchek, Ronald J.; Max, Blake; Sandusky, Caroline J.; Hota, Bala; Barker, David E.

2009-01-01

Introduction To explore whether an assay change was responsible for an increasing proportion of patients with undetectable HIV viral loads at our urban HIV clinic, we selected highly stable patients, examining their viral loads before and after changing assays. We compared the proportion with detectable viremia during RT-PCR vs. bDNA periods. Methodology/Principal Findings We selected patients with ≥1 viral loads assessed during both RT-PCR and bDNA periods. We included patients with stable CD4 counts, excluding patients with viral loads ≥1,000 copies/ml or any significant changes in therapy. Out of 4500 clinic patients, 419 patients (1588 viral loads) were included. 39% of viral loads were reported as detectable by RT-PCR vs. 5% reported as detectable by bDNA. The mean coefficient of variation was higher before vs. after assay change. We found an odds' ratio of 16.7 for having a viral load >75 copies/ml during the RT-PCR vs. bDNA periods. Discussion These data support previous reports, suggesting that bDNA may more reliably discriminate between viral suppression and low level viremia in stable patients on therapy. Low-level viremia, noted more with RT-PCR, may promote unneeded testing, while differences in viral load reliability may impact antiretroviral trial and quality assurance endpoints. Commonly used plasma separator tubes may differentially affect RT-PCR and bDNA results. PMID:19547711

Placental Mitochondrial Toxicity, Oxidative Stress, Apoptosis, and Adverse Perinatal Outcomes in HIV Pregnancies Under Antiretroviral Treatment Containing Zidovudine.

PubMed

Hernández, Sandra; Catalán-García, Marc; Morén, Constanza; García-Otero, Laura; López, Marta; Guitart-Mampel, Mariona; Milisenda, José; Coll, Oriol; Cardellach, Francesc; Gratacós, Eduard; Miró, Òscar; Garrabou, Glòria

2017-08-01

To determine whether mitochondrial, oxidative, and apoptotic abnormalities in placenta derived from HIV and combined antiretroviral therapy (cART) containing zidovudine (AZT) could be associated with adverse perinatal outcome. Cross-sectional, controlled, observational study. We studied obstetric results and mitochondrial, oxidative, and apoptotic state in placenta of 24 treated HIV-infected and 32 -uninfected pregnant women. We measured mitochondrial DNA (mtDNA) content by quantitative reverse transcriptase-polymerase chain reaction (mtND2/n18SrRNA), oxidative stress by the spectrophotometric quantification of lipid peroxidation and apoptosis by Western blot analysis of active caspase-3 respect to β-actin content and analysis of the terminal deoxynucleotidyl transferase dUTP nick end labeling. Global adverse perinatal outcome (defined as preterm delivery or/and small newborns for gestational age) was significantly increased in HIV pregnancies [or 6.7 (1.3-33.2); P < 0.05]. mtDNA content in HIV-infected women was significantly depleted (39.20% ± 2.78%) with respect to controls (0.59 ± 0.03 vs. 0.97 ± 0.07; P < 0.001). A significant 29.50% ± 9.14% increase in oxidative stress was found in placentas of HIV-infected women (23.23 ± 1.64 vs. 17.94 ± 1.03; P < 0.01). A trend toward 41.18% ± 29.41% increased apoptosis active caspase-3/β-actin was found in HIV patients (0.48 ± 0.10 vs. 0.34 ± 0.05; P = not significant), confirmed by transferase dUTP nick end labeling assay. Adverse perinatal outcome did not correlate mitochondrial, oxidative, or apoptotic findings. Placentas of HIV-infected pregnant women under AZT cART showed evidence of mtDNA depletion, increased oxidative stress levels, and apoptosis suggestive of secondary mitochondrial failure, potential base of associated adverse perinatal outcome. Despite the fact that further demonstration of causality would need new approaches and bigger sample sizes, AZT-sparing cART should be considered in the context of pregnancy.

The Effect of Antiretroviral Combination Treatment on Epstein-Barr Virus (EBV) Genome Load in HIV-Infected Patients

PubMed Central

Friis, Anna M. C.; Gyllensten, Katarina; Aleman, Anna; Ernberg, Ingemar; Åkerlund, Börje

2010-01-01

We evaluated the effect of combination anti-retroviral treatment (cART) on the host control of EBV infection in moderately immunosuppressed HIV-1 patients. Twenty HIV-1 infected individuals were followed for five years with repeated measurements of EBV DNA load in peripheral blood lymphocytes in relation to HIV-RNA titers and CD4+ cell counts. Individuals with optimal response, i.e. durable non-detectable HIV-RNA, showed a decline of EBV load to the level of healthy controls. Individuals with non-optimal HIV-1 control did not restore their EBV control. Long-lasting suppression of HIV-replication after early initiation of cART is a prerequisite for re-establishing the immune control of EBV. PMID:21994658

Suboptimal Doses of Raltegravir Cause Aberrant HIV Integrations | Center for Cancer Research

Cancer.gov

When a cell is infected with HIV, a DNA copy of the HIV genome is inserted into that cell’s chromosomal DNA. This insertion reaction is carried out by the viral enzyme integrase (IN) and involves two distinct steps: removal of two nucleotides from each 3’ end of the viral DNA, followed by the strand transfer reaction, in which the viral DNA ends are inserted into the host

CD32-Expressing CD4 T Cells Are Phenotypically Diverse and Can Contain Proviral HIV DNA.

PubMed

Martin, Genevieve E; Pace, Matthew; Thornhill, John P; Phetsouphanh, Chansavath; Meyerowitz, Jodi; Gossez, Morgane; Brown, Helen; Olejniczak, Natalia; Lwanga, Julianne; Ramjee, Gita; Kaleebu, Pontiano; Porter, Kholoud; Willberg, Christian B; Klenerman, Paul; Nwokolo, Nneka; Fox, Julie; Fidler, Sarah; Frater, John

2018-01-01

Efforts to both characterize and eradicate the HIV reservoir have been limited by the rarity of latently infected cells and the absence of a specific denoting biomarker. CD32a (FcγRIIa) has been proposed to be a marker for an enriched CD4 T cell HIV reservoir, but this finding remains controversial. Here, we explore the expression of CD32 on CD3 + CD4 + cells in participants from two primary HIV infection studies and identify at least three distinct phenotypes (CD32 low , CD32 + CD14 + , and CD32 high ). Of note, CD4 negative enrichment kits remove the majority of CD4 + CD32 + T cells, potentially skewing subsequent analyses if used. CD32 high CD4 T cells had higher levels of HLA-DR and HIV co-receptor expression than other subsets, compatible with their being more susceptible to infection. Surprisingly, they also expressed high levels of CD20, TCRαβ, IgD, and IgM (but not IgG), markers for both T cells and naïve B cells. Compared with other populations, CD32 low cells had a more differentiated memory phenotype and high levels of immune checkpoint receptors, programmed death receptor-1 (PD-1), Tim-3, and TIGIT. Within all three CD3 + CD4 + CD32 + phenotypes, cells could be identified in infected participants, which contained HIV DNA. CD32 expression on CD4 T cells did not correlate with HIV DNA or cell-associated HIV RNA (both surrogate measures of overall reservoir size) or predict time to rebound viremia following treatment interruption, suggesting that it is not a dominant biomarker for HIV persistence. Our data suggest that while CD32 + T cells can be infected with HIV, CD32 is not a specific marker of the reservoir although it might identify a population of HIV enriched cells in certain situations.

Overt and occult hepatitis B virus infection in adult Sudanese HIV patients.

PubMed

Mudawi, Hatim; Hussein, Waleed; Mukhtar, Maowia; Yousif, Mukhlid; Nemeri, Omer; Glebe, Dieter; Kramvis, Anna

2014-12-01

Human immunodeficiency virus (HIV) infection in Sub-Saharan Africa is complicated by co-infection with hepatitis B and C viruses (HBV and HCV), which share similar transmission routes. The aims of this study were to determine the prevalence of hepatitis B surface antigen (HBsAg)-positive and HBsAg-negative HBV infection and of HCV infection among HIV-infected patients. A cross-sectional study was conducted among treatment-naïve HIV-positive adults in Khartoum State. HBV, HCV, and HIV infections were detected using immunoassays for HBsAg, hepatitis B core antibodies (anti-HBc), hepatitis C antibodies (anti-HCV), and HIV antibodies (anti-HIV), while real-time PCR was used to measure HBV DNA. The mean age of the 358 patients was 35.2±9.3 years and the male to female ratio was 1.3:1.0. The mean alanine aminotransferase (ALT) level was 10.9±18.0 U/l. Evidence of 23, current or past HBV infection was detected in 62.8% of the patients. HBV DNA was detected in 96 patients (26.8%), 42 HBsAg-positive (11.7%) and 54 (15.1%) HBsAg-negative, indicating occult hepatitis B infection. Anti-HCV was detected in 1.7%. Evidence of HBV infection was detected in 26.8% of HIV patients with HBsAg-negative infection, with viraemia detected in 15.1% of the patients. All HIV-infected patients should be screened carefully for HBV infection with HBsAg and anti-HBc IgG antibodies prior to starting antiretroviral therapy. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bower, Joseph F.; Green, Thomas D.; Ross, Ted M.

2004-10-25

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d{sub 3}) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d{sub 3}. In addition, bothmore » sCD4-gp120 and sCD4-gp120-mC3d{sub 3} bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d{sub 3} or sCD4-gp120-mC3d{sub 3} elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d{sub 3}-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d{sub 3} had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.« less

Detection of HIV-1 by digoxigenin-labelled PCR and microtitre plate solution hybridisation assay and prevention of PCR carry-over by uracil-N-glycosylase.

PubMed

King, J A; Ball, J K

1993-09-01

An extremely sensitive and convenient microtiter plate solution hybridisation assay for the detection of HIV-1 PCR products was developed. The PCR product is labelled by direct incorporation of digoxigenin-dUTP and after denaturation is captured by a microtitre plate coated with a streptavidin-linked biotinylated probe. The PCR/probe hybrids are reacted with an alkaline phosphate conjugated anti-digoxigenin antibody and detected using an alkaline phosphatase enzyme amplification system. The use of uracil-N-glycosylase and dUTP instead of dTTP in the PCR is used to effectively control carry-over from previous PCR products. The assay can detect single HIV-1 DNA molecules in a background DNA of 0.75 microgram.

The yeast Ty3 retrotransposon contains a 5'-3' bipartite primer-binding site and encodes nucleocapsid protein NCp9 functionally homologous to HIV-1 NCp7.

PubMed Central

Gabus, C; Ficheux, D; Rau, M; Keith, G; Sandmeyer, S; Darlix, J L

1998-01-01

Retroviruses, including HIV-1 and the distantly related yeast retroelement Ty3, all encode a nucleoprotein required for virion structure and replication. During an in vitro comparison of HIV-1 and Ty3 nucleoprotein function in RNA dimerization and cDNA synthesis, we discovered a bipartite primer-binding site (PBS) for Ty3 composed of sequences located at opposite ends of the genome. Ty3 cDNA synthesis requires the 3' PBS for primer tRNAiMet annealing to the genomic RNA, and the 5' PBS, in cis or in trans, as the reverse transcription start site. Ty3 RNA alone is unable to dimerize, but formation of dimeric tRNAiMet bound to the PBS was found to direct dimerization of Ty3 RNA-tRNAiMet. Interestingly, HIV-1 nucleocapsid protein NCp7 and Ty3 NCp9 were interchangeable using HIV-1 and Ty3 RNA template-primer systems. Our findings impact on the understanding of non-canonical reverse transcription as well as on the use of Ty3 systems to screen for anti-NCp7 drugs. PMID:9707446

The yeast Ty3 retrotransposon contains a 5'-3' bipartite primer-binding site and encodes nucleocapsid protein NCp9 functionally homologous to HIV-1 NCp7.

PubMed

Gabus, C; Ficheux, D; Rau, M; Keith, G; Sandmeyer, S; Darlix, J L

1998-08-17

Retroviruses, including HIV-1 and the distantly related yeast retroelement Ty3, all encode a nucleoprotein required for virion structure and replication. During an in vitro comparison of HIV-1 and Ty3 nucleoprotein function in RNA dimerization and cDNA synthesis, we discovered a bipartite primer-binding site (PBS) for Ty3 composed of sequences located at opposite ends of the genome. Ty3 cDNA synthesis requires the 3' PBS for primer tRNAiMet annealing to the genomic RNA, and the 5' PBS, in cis or in trans, as the reverse transcription start site. Ty3 RNA alone is unable to dimerize, but formation of dimeric tRNAiMet bound to the PBS was found to direct dimerization of Ty3 RNA-tRNAiMet. Interestingly, HIV-1 nucleocapsid protein NCp7 and Ty3 NCp9 were interchangeable using HIV-1 and Ty3 RNA template-primer systems. Our findings impact on the understanding of non-canonical reverse transcription as well as on the use of Ty3 systems to screen for anti-NCp7 drugs.

Stimulation of the human immunodeficiency virus type 1 enhancer by the human T-cell leukemia virus type I tax gene product involves the action of inducible cellular proteins.

PubMed

Böhnlein, E; Siekevitz, M; Ballard, D W; Lowenthal, J W; Rimsky, L; Bogérd, H; Hoffman, J; Wano, Y; Franza, B R; Greene, W C

1989-04-01

The human immunodeficiency virus type 1 (HIV-1) preferentially infects CD4+ T lymphocytes and may exist as a latent provirus within these cells for extended periods. The transition to a productive retroviral infection results in T-cell death and clinically may lead to the acquired immune deficiency syndrome. Accelerated production of infectious HIV-1 virions appears to be closely linked to a heightened state of T-cell activation. The transactivator (Tax) protein of the type I human T-cell leukemia virus (HTLV-I) can produce such an activated T-cell phenotype and augments activity of the HIV-1 long terminal repeat. One Tax-responsive region within the HIV-1 long terminal repeat has been mapped to a locus composed of two 10-base-pair direct repeats sharing homology with the binding site for the eucaryotic transcription factor NF-kappaB (GGGACTTTCC). Tax-expressing Jurkat T cells contain one or more inducible cellular proteins that specifically associate with the HIV-1 enhancer at these binding sites. Microscale DNA affinity precipitation assays identified a Tax-inducible 86-kilodalton protein, HIVEN86A, as one of these HIV-1 enhancer-binding factors. The interaction of HIVEN86A, and presumably other cellular proteins, with the HIV-1 enhancer appears functionally important as oligonucleotides corresponding to this enhancer were sufficient to impart Tax inducibility to an unresponsive heterologous promoter. These findings suggest that the Tax-inducible cellular protein HIVEN86A plays an important role in the transcriptional activation of the HIV-1 enhancer. These specific protein-DNA interactions may also be important for the transition of HIV-1 from a latent to a productive mode of infection. Furthermore, these findings highlight an intriguing biological interplay between HTLV-1 and HIV-1 through a cellular transcriptional pathway that is normally involved in T-cell activation and growth.

The Anti-Human Immunodeficiency Virus Drug Tenofovir, a Reverse Transcriptase Inhibitor, Also Targets the Herpes Simplex Virus DNA Polymerase.

PubMed

Andrei, Graciela; Gillemot, Sarah; Topalis, Dimitrios; Snoeck, Robert

2018-02-14

Genital herpes is an important cofactor for acquisition of human immunodeficiency virus (HIV) infection, and effective prophylaxis is a helpful strategy to halt both HIV and herpes simplex virus (HSV) transmission. The antiretroviral agent tenofovir, formulated as a vaginal microbicide gel, was shown to reduce the risk of HIV and HSV type 2 (HSV-2) acquisition. HSV type 1 (HSV-1) and HSV-2 mutants were selected for resistance to tenofovir and PMEO-DAPy (6-phosphonylmethoxyethoxy-2,4-diaminopyrimidine, an acyclic nucleoside phosphonate with dual anti-HSV and anti-HIV activity) by stepwise dose escalation. Several plaque-purified viruses were characterized phenotypically (drug resistance profiling) and genotypically (sequencing of the viral DNA polymerase gene). Tenofovir resistant and PMEO-DAPy-resistant viruses harbored specific amino acid substitutions associated with resistance not only to tenofovir and PMEO-DAPy but also to acyclovir and foscarnet. These amino acid changes (A719V, S724N, and L802F [HSV-1] and M789T and A724V [HSV-2]) were also found in clinical isolates recovered from patients refractory to acyclovir and/or foscarnet therapy or in laboratory-derived strains. A total of 10 (HSV-1) and 18 (HSV-2) well-characterized DNA polymerase mutants had decreased susceptibility to tenofovir and PMEO-DAPy. Tenofovir and PMEO-DAPy target the HSV DNA polymerase, and clinical isolates with DNA polymerase mutations emerging under acyclovir and/or foscarnet therapy showed cross-resistance to tenofovir and PMEO-DAPy. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Inhibition of HIV Expression and Integration in Macrophages by Methylglyoxal-Bis-Guanylhydrazone

PubMed Central

Jin, Xia

2015-01-01

ABSTRACT Macrophages are a target for infection with HIV and represent one of the viral reservoirs that are relatively resistant to current antiretroviral drugs. Here we demonstrate that methylglyoxal-bis-guanylhydrazone (MGBG), a polyamine analog and potent S-adenosylmethionine decarboxylase inhibitor, decreases HIV expression in monocytes and macrophages. MGBG is selectively concentrated by these cells through a mechanism consistent with active transport by the polyamine transporter. Using a macrophage-tropic reporter virus tagged with the enhanced green fluorescent protein, we demonstrate that MGBG decreases the frequency of HIV-infected cells. The effect is dose dependent and correlates with the production of HIV p24 in culture supernatants. This anti-HIV effect was further confirmed using three macrophage-tropic primary HIV isolates. Viral life cycle mapping studies show that MGBG inhibits HIV DNA integration into the cellular DNA in both monocytes and macrophages. IMPORTANCE Our work demonstrates for the first time the selective concentration of MGBG by monocytes/macrophages, leading to the inhibition of HIV-1 expression and a reduction in proviral load within macrophage cultures. These results suggest that MGBG may be useful in adjunctive macrophage-targeted therapy for HIV infection. PMID:26223636

Inhibition of HIV Expression and Integration in Macrophages by Methylglyoxal-Bis-Guanylhydrazone.

PubMed

Jin, Xia; McGrath, Michael S; Xu, Hua

2015-11-01

Macrophages are a target for infection with HIV and represent one of the viral reservoirs that are relatively resistant to current antiretroviral drugs. Here we demonstrate that methylglyoxal-bis-guanylhydrazone (MGBG), a polyamine analog and potent S-adenosylmethionine decarboxylase inhibitor, decreases HIV expression in monocytes and macrophages. MGBG is selectively concentrated by these cells through a mechanism consistent with active transport by the polyamine transporter. Using a macrophage-tropic reporter virus tagged with the enhanced green fluorescent protein, we demonstrate that MGBG decreases the frequency of HIV-infected cells. The effect is dose dependent and correlates with the production of HIV p24 in culture supernatants. This anti-HIV effect was further confirmed using three macrophage-tropic primary HIV isolates. Viral life cycle mapping studies show that MGBG inhibits HIV DNA integration into the cellular DNA in both monocytes and macrophages. Our work demonstrates for the first time the selective concentration of MGBG by monocytes/macrophages, leading to the inhibition of HIV-1 expression and a reduction in proviral load within macrophage cultures. These results suggest that MGBG may be useful in adjunctive macrophage-targeted therapy for HIV infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Baseline characteristics of HIV & hepatitis B virus (HIV/HBV) co-infected patients from Kolkata, India.

PubMed

Sarkar, Jayeeta; Saha, Debraj; Bandyopadhyay, Bhaswati; Saha, Bibhuti; Kedia, Deepika; Guha Mazumder, D N; Chakravarty, Runu; Guha, Subhasish Kamal

2016-05-01

Hepatitis B virus (HBV) and HIV co-infection has variable prevalence worldwide. In comparison to HBV mono-infection, the course of chronic HBV infection is accelerated in HIV/HBV co-infected patients. the present study was carried out to analyse the baseline characteristics (clinical, biochemical, serological and virological) of treatment naïve HIV/HBV co-infected and HIV mono-infected patients. Between July 2011 and January 2013, a total number of 1331 HIV-seropositive treatment naïve individuals, enrolled in the ART Centre of Calcutta School of Tropical Medicine, Kolkata, India, were screened for hepatitis B surface antigen (HBsAg). A total of 1253 HIV mono-infected and 78 HIV/HBV co-infected patients were characterized. The co-infected patients were evaluated for HBeAg and anti-HBe antibody by ELISA. HIV RNA was quantified for all co-infected patients. HBV DNA was detected and quantified by real time-PCR amplification followed by HBV genotype determination. HIV/HBV co-infected patients had proportionately more advanced HIV disease (WHO clinical stage 3 and 4) than HIV mono-infected individuals (37.1 vs. 19.9%). The co-infected patients had significantly higher serum bilirubin, alanine aminotransferase (ALT), alkaline phosphatase and ALT/platelet ratio index (APRI). CD4 count was non-significantly lower in co-infected patients. Majority (61.5%) were HBeAg positive with higher HIV RNA (P<0.05), HBV DNA (p<0.001) and APRI (p<0.05) compared to those who were HBeAg negative. HBV/D was the predominant genotype (73.2%) and D2 (43.7%) was the commonest subgenotype. HIV/HBV co-infected patients had significantly higher serum bilirubin, ALT, alkaline phosphatase and lower platelet count. HBeAg positive co-infected patients had higher HIV RNA and HBV DNA compared to HBeAg negative co-infected patients. Prior to initiation of antiretroviral treatment (ART) all patients should be screened for HBsAg to initiate appropriate ART regimen.

Virological and immunological outcome of treatment interruption in HIV-1-infected subjects vaccinated with MVA-B

PubMed Central

Noguera-Julian, Marc; Bellido, Rocío; Puertas, Maria C.; Carrillo, Jorge; Rodriguez, C.; Perez-Alvarez, Núria; Cobarsí, Patricia; Gomez, Carmen E.; Esteban, Mariano; Jímenez, Jose Luis; García, Felipe; Blanco, Julià; Martinez-Picado, Javier; Paredes, Roger

2017-01-01

The most relevant endpoint in therapeutic HIV vaccination is the assessment of time to viral rebound or duration of sustained control of low-level viremia upon cART treatment cessation. Structured treatment interruptions (STI) are however not without risk to the patient and reliable predictors of viral rebound/control after therapeutic HIV-1 vaccination are urgently needed to ensure patient safety and guide therapeutic vaccine development. Here, we integrated immunological and virological parameters together with viral rebound dynamics after STI in a phase I therapeutic vaccine trial of a polyvalent MVA-B vaccine candidate to define predictors of viral control. Clinical parameters, proviral DNA, host HLA genetics and measures of humoral and cellular immunity were evaluated. A sieve effect analysis was conducted comparing pre-treatment viral sequences to breakthrough viruses after STI. Our results show that a reduced proviral HIV-1 DNA at study entry was independently associated with two virological parameters, delayed HIV-1 RNA rebound (p = 0.029) and lower peak viremia after treatment cessation (p = 0.019). Reduced peak viremia was also positively correlated with a decreased number of HLA class I allele associated polymorphisms in Gag sequences in the rebounding virus population (p = 0.012). Our findings suggest that proviral DNA levels and the number of HLA-associated Gag polymorphisms may have an impact on the clinical outcome of STI. Incorporation of these parameters in future therapeutic vaccine trials may guide refined immunogen design and help conduct safer STI approaches. PMID:28953921

Reverse Transcription Quantitative Polymerase Chain Reaction for Detection of and Differentiation Between RNA and DNA of HIV-1-Based Lentiviral Vectors.

PubMed

Pavlovic, Melanie; Koehler, Nina; Anton, Martina; Dinkelmeier, Anna; Haase, Maren; Stellberger, Thorsten; Busch, Ulrich; Baiker, Armin E

2017-08-01

The purpose of the described method is the detection of and differentiation between RNA and DNA of human immunodeficiency virus (HIV)-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering, operations methods for the detection of the HIV-1-based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here, a quantitative polymerase chain reaction method targeting the long terminal repeat U5 subunit and the start sequence of the packaging signal ψ is described. Numerous controls are included in order to monitor the technical procedure.

The impact of nevirapine- versus protease inhibitor-based regimens on virological markers of HIV-1 persistence during seemingly suppressive ART.

PubMed

Kiselinova, Maja; Anna, Maria; Malatinkova, Eva; Vervish, Karen; Beloukas, Apostolos; Messiaen, Peter; Bonczkowski, Pawel; Trypsteen, Wim; Callens, Steven; Verhofstede, Chris; De Spiegelaere, Ward; Vandekerckhove, Linos

2014-01-01

The source and significance of residual plasma HIV-1 RNA detection during suppressive ART remain controversial. It has been proposed that nevirapine (NVP)-based regimens achieve a greater HIV-1 RNA suppression than regimens containing a protease inhibitor (PI). The aim of this study was to compare the effect of receiving NVP- vs PI-based ART on the virological markers of HIV persistence in peripheral blood. The study population comprised 161 HIV-1 infected patients receiving either NVP-based (n=81) or PI-based (n=80) ART and showing a HIV-1 RNA load stably suppressed <40 copies/mL for median of 5.2 years (IQR 2.2-8.0). Residual viraemia was detected by real-time PCR with 50% and 95% detection thresholds of 1 and 3 HIV-1 RNA copies/mL, respectively. Cell-associated (CA) unspliced HIV-1 RNA, total HIV-1 DNA and 2 LTR circles were quantified in peripheral blood mononuclear cells (PBMCs) using droplet digital PCR. Groups were compared by standard non-parametric tests; factors associated with HIV-1 detection were analyzed by univariate regression analysis and generalized linear models (SPSS® V22 and Rstudio). Plasma HIV-1 RNA was detected in 37/81 (45.7%) and 47/80 (58.8%) subjects on NVP- and PI-based ART, with median (IQR) levels of 5 (3-6) and 5 (3-8) copies/mL, respectively. HIV-1 RNA detection was associated with shorter duration of suppressive ART regardless of treatment arm (p=0.007), and lower CD4 nadir (p=0.015). HIV-1 DNA levels were median 282 (120-484) and 213 (87-494) copies/106 PBMCs in the two groups respectively, and were lowest (<100 copies/106 PBMCs) in subjects with lower plasma HIV-1 RNA (p=0.049), CA unspliced HIV-1 RNA (p=0.0001), 2 LTR circles (p=0.005) and pre-ART HIV-1 RNA load (p=0.0001). In this comprehensive characterization of patients on long-term suppressive ART, we did not observe evidence for a greater suppressive activity of NVP-based over PI-based therapy on plasma and intracellular markers of virus persistence. Overall excellent correlation was observed between the markers, allowing the identification of a subset of treated patients with low HIV-1 expression as an important cohort for future HIV cure studies.

Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen.

PubMed

Dong, Huahuang; Liu, Jianli; Zhu, Hong; Ou, Chin-Yih; Xing, Wenge; Qiu, Maofeng; Zhang, Guiyun; Xiao, Yao; Yao, Jun; Pan, Pinliang; Jiang, Yan

2012-08-31

HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen. A pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3-4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV.

Rapid, Point-of-Care Extraction of Human Immunodeficiency Virus Type 1 Proviral DNA from Whole Blood for Detection by Real-Time PCR ▿

PubMed Central

Jangam, Sujit R.; Yamada, Douglas H.; McFall, Sally M.; Kelso, David M.

2009-01-01

PCR detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA is the method recommended for use for the diagnosis of HIV-1 infection in infants in limited-resource settings. Currently, testing must be performed in central laboratories, which are usually located some distance from health care facilities. While the collection and transportation of samples, such as dried blood spots, has improved test accessibility, the results are often not returned for several weeks. To enable PCR to be performed at the point of care while the mothers wait, we have developed a vertical filtration method that uses a separation membrane and an absorbent pad to extract cellular DNA from whole blood in less than 2 min. Cells are trapped in the separation membrane as the specimen is collected, and then a lysis buffer is added. The membrane retains the DNA, while the buffer washes away PCR inhibitors, which get wicked into the absorbent blotter pad. The membrane containing the entrapped DNA is then added to the PCR mixture without further purification. The method demonstrates a high degree of reproducibility and analytical sensitivity and allows the quantification of as few as 20 copies of HIV-1 proviral DNA from 100 μl of blood. In a blinded study with 182 longitudinal samples from infants (ages, 0 to 72 weeks) obtained from the Women and Infants Transmission Study, our assay demonstrated a sensitivity of 99% and a specificity of 100%. PMID:19644129

Elucidating the Burden of HIV in Tissues Using Multiplexed Immunofluorescence and In Situ Hybridization: Methods for the Single-Cell Phenotypic Characterization of Cells Harboring HIV In Situ.

PubMed

Vasquez, Joshua J; Hussien, Rajaa; Aguilar-Rodriguez, Brandon; Junger, Henrik; Dobi, Dejan; Henrich, Timothy J; Thanh, Cassandra; Gibson, Erica; Hogan, Louise E; McCune, Joseph; Hunt, Peter W; Stoddart, Cheryl A; Laszik, Zoltan G

2018-06-01

Persistent tissue reservoirs of HIV present a major barrier to cure. Defining subsets of infected cells in tissues is a major focus of HIV cure research. Herein, we describe a novel multiplexed in situ hybridization (ISH) (RNAscope) protocol to detect HIV-DNA (vDNA) and HIV-RNA (vRNA) in formalin-fixed paraffin-embedded (FFPE) human tissues in combination with immunofluorescence (IF) phenotyping of the infected cells. We show that multiplexed IF and ISH (mIFISH) is suitable for quantitative assessment of HIV vRNA and vDNA and that multiparameter IF phenotyping allows precise identification of the cellular source of the ISH signal. We also provide semi-quantitative data on the impact of various tissue fixatives on the detectability of vDNA and vRNA with RNAscope technology. Finally, we describe methods to quantitate the ISH signal on whole-slide digital images and validation of the quantitative ISH data with quantitative real-time PCR for vRNA. It is our hope that this approach will provide insight into the biology of HIV tissue reservoirs and to inform strategies aimed at curing HIV.

In silico design of a DNA-based HIV-1 multi-epitope vaccine for Chinese populations

PubMed Central

Yang, Yi; Sun, Weilai; Guo, Jingjing; Zhao, Guangyu; Sun, Shihui; Yu, Hong; Guo, Yan; Li, Jungfeng; Jin, Xia; Du, Lanying; Jiang, Shibo; Kou, Zhihua; Zhou, Yusen

2015-01-01

The development of an HIV-1 vaccine that is capable of inducing effective and broadly cross-reactive humoral and cellular immune responses remains a challenging task because of the extensive diversity of HIV-1, the difference of virus subtypes (clades) in different geographical regions, and the polymorphism of human leukocyte antigens (HLA). We performed an in silico design of 3 DNA vaccines, designated pJW4303-MEG1, pJW4303-MEG2 and pJW4303-MEG3, encoding multi-epitopes that are highly conserved within the HIV-1 subtypes most prevalent in China and can be recognized through HLA alleles dominant in China. The pJW4303-MEG1-encoded protein consisted of one Th epitope in Env, and one, 2, and 6 epitopes in Pol, Env, and Gag proteins, respectively, with a GGGS linker sequence between epitopes. The pJW4303-MEG2-encoded protein contained similar epitopes in a different order, but with the same linker as pJW4303-MEG1. The pJW4303-MEG3-encoded protein contained the same epitopes in the same order as that of pJW4303-MEG2, but with a different linker sequence (AAY). To evaluate immunogenicity, mice were immunized intramuscularly with these DNA vaccines. Both pJW4303-MEG1 and pJW4303-MEG2 vaccines induced equally potent humoral and cellular immune responses in the vaccinated mice, while pJW4303-MEG3 did not induce immune responses. These results indicate that both epitope and linker sequences are important in designing effective epitope-based vaccines against HIV-1 and other viruses. PMID:25839222

Role and evolution of viral tropism in patients with advanced HIV disease receiving intensified initial regimen in the ANRS 130 APOLLO trial.

PubMed

Charpentier, Charlotte; Joly, Véronique; Larrouy, Lucile; Fagard, Catherine; Visseaux, Benoit; de Verdière, Nathalie Colin; Raffi, François; Yeni, Patrick; Descamps, Diane

2013-03-01

The aims of the study were to assess in patients with advanced HIV disease receiving antiretroviral therapy (ART) intensification with enfuvirtide (i) resistance at virological failure (VF), (ii) impact of baseline tropism on immunovirological response, and (iii) HIV-1 DNA tropism evolution during ART. The ANRS 130 APOLLO randomized trial evaluated in naive patients the immunovirological impact of standard ART without (control arm) or with enfuvirtide. Tropism was determined on RNA and DNA by V3-loop sequencing interpreted using the Geno2Pheno algorithm. At baseline the median CD4 cell count was 30 cells/mm(3). Among the 170 patients assessable in this virological substudy, HIV-1 RNA tropism was as follows: 60% of viruses were R5 and 40% were R5X4/X4. HIV-1 DNA tropism was as follows: 54% were R5 and 46% were R5X4/X4. At week 24, 39% and 49% of patients experienced VF in the enfuvirtide and control arms, respectively. In the enfuvirtide arm, only resistance-associated mutations to enfuvirtide were detected. In the control arm, two patients displayed drug-resistant viruses at the time of VF. No impact of baseline tropism was observed on immunovirological response, regardless of the study arm. Among the 25 patients experiencing DNA tropism switch between baseline and week 24, 16 (64%) switched from R5 to R5X4/X4. These latter were mostly successfully suppressed patients receiving enfuvirtide and exhibiting poorer immunological response. Baseline RNA tropism had no impact on the immunovirological response. Drug resistance mutations were only detected for the fusion inhibitor. Finally, the mechanism of replenishment of the viral cellular reservoir with X4 viruses observed needs to be further analysed.

FISHing Out the Hidden Enemy: Advances in Detecting and Measuring Latent HIV-Infected Cells.

PubMed

Prasad, Vinayaka R; Kalpana, Ganjam V

2017-09-19

The indomitable aspect of HIV-1 infection is not that HIV-1 proviral DNA is integrated into host DNA but that it can also turn itself off, remaining invisible to drug or immune surveillance. Thus, the goals of eradication include ways to precisely excise HIV-1 DNA or wake up the silent HIV-1 provirus and eliminate the infected cells thus identified. Methods to identify and fish out the latently infected cells or to delineate their characteristics are being rapidly developed. In 2016, Baxter et al. (A. E. Baxter, J. Niessl, R. Fromentin, J. Richard, F. Porichis, R. Charlebois, M. Massanella, N. Brassard, N. Alsahafi, G. G. Delgado, J. P. Routy, B. D. Walker, A. Finzi, N. Chomont, and D. E. Kaufmann, Cell Host Microbe 20:368-380, 2016, https://doi.org/10.1016/j.chom.2016.07.015) and Martrus et al. (G. Martrus, A. Niehrs, R. Cornelis, A. Rechtien, W. García-Beltran, M. Lütgehetmann, C. Hoffmann, and M. Altfeld, J Virol 90:9018-9028, 2016, https://doi.org/10.1128/JVI.01448-16) reported using the fluorescence in situ hybridization-flow cytometry technique to identify and quantify cells expressing HIV-1 RNA and Gag protein, as well as bearing unique cell surface markers. In a recent article in mBio , Grau-Expósito et al. (J. Grau-Expósito, C. Serra-Peinado, L. Miguel, J. Navarro, A. Curran, J. Burgos, I. Ocaña, E. Ribera, A. Torrella, B. Planas, R. Badía, J. Castellví, V. Falcó, M. Crespo, and M. J. Buzon, mBio 8:e00876-17, 2017, https://doi.org/10.1128/mBio.00876-17) reported a similar method that they claim to be more sensitive. With these methods, researchers are one step closer to measuring latent reservoirs and eliminating critical barriers to HIV eradication. Copyright © 2017 Prasad and Kalpana.

Walleye dermal sarcoma virus: expression of a full-length clone or the rv-cyclin (orf a) gene is cytopathic to the host and human tumor cells.

PubMed

Xu, Kun; Zhang, Ting Ting; Wang, Ling; Zhang, Cun Fang; Zhang, Long; Ma, Li Xia; Xin, Ying; Ren, Chong Hua; Zhang, Zhi Qiang; Yan, Qiang; Martineau, Daniel; Zhang, Zhi Ying

2013-02-01

Walleye dermal sarcoma virus (WDSV) is etiologically associated with a skin tumor, walleye dermal sarcoma (WDS), which develops in the fall and regresses in the spring. WDSV genome contains, in addition to gag, pol and env, three open reading frames (orfs) designated orf a (rv-cyclin), orf b and orf c. Unintegrated linear WDSV provirus DNA isolated from infected tumor cells was used to construct a full-length WDSV provirus clone pWDSV, while orf a was cloned into pSVK3 to construct the expression vector porfA. Stable co-transfection of a walleye cell line (W12) with pWDSV and pcDNA3 generated fewer and smaller G418-resistant colonies compared to the control. By Northern blot analysis, several small transcripts (2.8, 1.8, 1.2, and 0.8 kb) were detected using a WDSV LTR-specific probe. By RT-PCR and Southern blot analysis, three cDNAs (2.4, 1.6 and 0.8 kb) were identified, including both orf a and orf b messenger. Furthermore stable co-transfection of both a human lung adenocarcinoma cell line (SPC-A-1) and a cervical cancer cell line (HeLa) with pcDNA3 and ether porfA or pWDSV also generated fewer and smaller G418-resistant colonies. We conclude that expression of the full-length WDSV clone or the orf a gene inhibits the host fish and human tumor cell growth, and Orf A protein maybe a potential factor which contributes to the seasonal tumor development and regression. This is the first fish provirus clone that has been expressed in cell culture system, which will provide a new in vitro model for tumor research and oncotherapy study.

Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation.

PubMed Central

Garcia, J A; Harrich, D; Soultanakis, E; Wu, F; Mitsuyasu, R; Gaynor, R B

1989-01-01

The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP-1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide-directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat-activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem-loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide-directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat-activation. This indicated a significant role for the stem-loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat-activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted this stem and loop mutation relative to the HIV LTR mRNA start site resulted in even larger decreases in tat-activation. This suggests that multiple determinants, including protein binding, the loop sequence, and RNA or DNA secondary structure, are important in tat-activation and suggests that tat may interact with cellular proteins binding to DNA to increase HIV gene expression. Images PMID:2721501

Detection of Leishmania DNA in saliva among patients with HIV/AIDS in Trang Province, southern Thailand.

PubMed

Pandey, Netranapha; Siripattanapipong, Suradej; Leelayoova, Saovanee; Manomat, Jipada; Mungthin, Mathirut; Tan-Ariya, Peerapan; Bualert, Lertwut; Naaglor, Tawee; Siriyasatien, Padet; Phumee, Atchara; Piyaraj, Phunlerd

2018-06-08

Leishmaniasis is a neglected tropical disease causing opportunistic infection among patients with HIV/AIDS. The fatal form of this disease is visceral leishmaniasis (VL). DNA of Leishmania can be detected in saliva, for which the collection is noninvasive and requires little expertise. This study aimed to evaluate the sensitivity and specificity of a nested-PCR to amplify the Internal Transcribed Spacer 1 (ITS1) to detect Leishmania DNA in paired saliva and buffy coat samples of 305 Thai patients with HIV/AIDS in Trang Hospital, Trang Province, southern Thailand. For asymptomatic Leishmania infection among Thai patients with HIV/AIDS, the sensitivity and specificity of the nested-PCR-ITS1 in buffy coat were 73.9 and 100%, respectively. However, the sensitivity in saliva was 26.1% and specificity was 100%. Using the nested-PCR-ITS1, saliva and buffy coat samples showed positive agreement in only 52.0% of patients. Saliva tested results with the nested-PCR-ITS1 showed positive agreement with the Direct Agglutination Test (DAT) in 46.5% of patients. Only 12.1% of the samples showed positive agreement for Leishmania infection among all the three tests: saliva, buffy coat and DAT results. Using nucleotide sequencing, at least three species of Leishmania infection were identified in saliva, i.e., L. siamensis (n = 28), L. martiniquensis (n = 9), and L. donovani complex (n = 1). As a result, buffy coat still appears to be a better specimen to diagnose asymptomatic VL infection among individuals with HIV. However, the use of both buffy coat and saliva together as clinical specimens would increase the sensitivity of Leishmania detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Small heat shock protein 27: An effective adjuvant for enhancement of HIV-1 Nef antigen-specific immunity.

PubMed

Milani, Alireza; Bolhassani, Azam; Shahbazi, Sepideh; Motevalli, Fatemeh; Sadat, Seyed Mehdi; Soleymani, Sepehr

2017-11-01

Novel vaccine modalities have been designed to improve the efficiency of vaccines against HIV infections. In this way, the HIV-1 Nef protein has been known as an attractive antigenic candidate in therapeutic vaccine development. Moreover, the endogenous adjuvants such as heat shock proteins (HSPs) and high mobility group box 1 protein (HMGB1) have been suggested effectively to induce antigen-specific humoral and cellular immune responses. In this study, different Nef DNA and protein constructs were produced in eukaryotic and prokaryotic expression systems, and their immunostimulatory properties were evaluated using small heat shock protein 27 (Hsp27) and the HMGB1-derived peptide (Hp91) in a mouse model. Generally, our results indicated that the Hsp27-Nef fusion DNA or protein could significantly elicit higher humoral and cellular immune responses than Nef DNA or protein, respectively. Analysis of the immune responses demonstrated that the Hsp27-Nef fusion protein, and also the mixture of Nef and Hp91 significantly enhanced the Nef-specific T cell responses. Indeed, these regimens induced high levels of IgG2a and IFN-γ directed toward Th1 responses and also Granzyme B secretion as compared to other immunization strategies. The immunostimulatory properties of Freund's adjuvant were significantly less than Hsp27 and Hp91 peptide in various immunization strategies. These findings showed that the use of Hsp27 and Hp91 in protein strategy could improve HIV-1 Nef-specific B- and T-cell immune responses, and also represent a promising HIV-1 vaccine candidate in future. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells

PubMed Central

Orlova-Fink, Nina; Einkauf, Kevin; Chowdhury, Fatema Z.; Sun, Xiaoming; Harrington, Sean; Kuo, Hsiao-Hsuan; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Reddy, Kavidha; Dong, Krista; Ndung’u, Thumbi; Walker, Bruce D.; Rosenberg, Eric S.; Yu, Xu G.

2017-01-01

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells. PMID:28628034

Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells.

PubMed

Lee, Guinevere Q; Orlova-Fink, Nina; Einkauf, Kevin; Chowdhury, Fatema Z; Sun, Xiaoming; Harrington, Sean; Kuo, Hsiao-Hsuan; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Reddy, Kavidha; Dong, Krista; Ndung'u, Thumbi; Walker, Bruce D; Rosenberg, Eric S; Yu, Xu G; Lichterfeld, Mathias

2017-06-30

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.

A Single HIV-1 Cluster and a Skewed Immune Homeostasis Drive the Early Spread of HIV among Resting CD4+ Cell Subsets within One Month Post-Infection

PubMed Central

Avettand-Fenoël, Véronique; Nembot, Georges; Mélard, Adeline; Blanc, Catherine; Lascoux-Combe, Caroline; Slama, Laurence; Allegre, Thierry; Allavena, Clotilde; Yazdanpanah, Yazdan; Duvivier, Claudine; Katlama, Christine; Goujard, Cécile; Seksik, Bao Chau Phung; Leplatois, Anne; Molina, Jean-Michel; Meyer, Laurence; Autran, Brigitte; Rouzioux, Christine

2013-01-01

Optimizing therapeutic strategies for an HIV cure requires better understanding the characteristics of early HIV-1 spread among resting CD4+ cells within the first month of primary HIV-1 infection (PHI). We studied the immune distribution, diversity, and inducibility of total HIV-DNA among the following cell subsets: monocytes, peripheral blood activated and resting CD4 T cells, long-lived (naive [TN] and central-memory [TCM]) and short-lived (transitional-memory [TTM] and effector-memory cells [TEM]) resting CD4+T cells from 12 acutely-infected individuals recruited at a median 36 days from infection. Cells were sorted for total HIV-DNA quantification, phylogenetic analysis and inducibility, all studied in relation to activation status and cell signaling. One month post-infection, a single CCR5-restricted viral cluster was massively distributed in all resting CD4+ subsets from 88% subjects, while one subject showed a slight diversity. High levels of total HIV-DNA were measured among TN (median 3.4 log copies/million cells), although 10-fold less (p = 0.0005) than in equally infected TCM (4.5), TTM (4.7) and TEM (4.6) cells. CD3−CD4+ monocytes harbored a low viral burden (median 2.3 log copies/million cells), unlike equally infected resting and activated CD4+ T cells (4.5 log copies/million cells). The skewed repartition of resting CD4 subsets influenced their contribution to the pool of resting infected CD4+T cells, two thirds of which consisted of short-lived TTM and TEM subsets, whereas long-lived TN and TCM subsets contributed the balance. Each resting CD4 subset produced HIV in vitro after stimulation with anti-CD3/anti-CD28+IL-2 with kinetics and magnitude varying according to subset differentiation, while IL-7 preferentially induced virus production from long-lived resting TN cells. In conclusion, within a month of infection, a clonal HIV-1 cluster is massively distributed among resting CD4 T-cell subsets with a flexible inducibility, suggesting that subset activation and skewed immune homeostasis determine the conditions of viral dissemination and early establishment of the HIV reservoir. PMID:23691172

Nedd4-Mediated Increase in HIV-1 Gag and Env Proteins and Immunity following DNA-Vaccination of BALB/c Mice

PubMed Central

Lewis, Brad; Whitney, Stephen; Hudacik, Lauren; Galmin, Lindsey; Huaman, Maria Cecilia; Cristillo, Anthony D.

2014-01-01

The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation. PMID:24614057

Viral load and genomic integration of HPV 16 in cervical samples from HIV-1-infected and uninfected women in Burkina Faso.

PubMed

Rousseau, Marie-Noelle Didelot; Costes, Valérie; Konate, Issouf; Nagot, Nicolas; Foulongne, Vincent; Ouedraogo, Abdoulaye; Van de Perre, Philippe; Mayaud, Philippe; Segondy, Michel

2007-06-01

The relationships between human papillomavirus type 16 (HPV 16) viral load, HPV 16 integration status, human immunodeficiency virus type 1 (HIV-1) status, and cervical cytology were studied among women enrolled in a cohort of female sex workers in Burkina Faso. The study focused on 24 HPV 16-infected women. The HPV 16 viral load in cervical samples was determined by real-time PCR. Integration ratio was estimated as the ratio between E2 and E6 genes DNA copy numbers. Integrated HPV16 viral load was defined as the product of HPV 16 viral load by the integration ratio. High HPV 16 viral load and high integration ratio were more frequent among women with squamous intraepithelial lesions compared with women with normal cytology (33% vs. 11%, and 33% vs. 0%, respectively), and among women with high-grade squamous intraepithelial lesions compared with women without high-grade squamous intraepithelial lesions (50% vs. 17%, and 50% vs. 11%, respectively). High HPV 16 DNA load, but not high integration ratio, was also more frequent among HIV-1-positive women (39% vs. 9%; and 23% vs. 18%, respectively). The absence of statistical significance of these differences might be explained by the small study sample size. High-integrated HPV 16 DNA load was significantly associated with the presence of high-grade squamous intraepithelial lesions (50% vs. 5%, P = 0.03) in univariate and multivariate analysis (adjusted odds-ratio: 19.05; 95% confidence interval (CI), 1.11-328.3, P = 0.03), but not with HIV-1 or other high-risk HPV types (HR-HPV). Integrated HPV 16 DNA load may be considered as a useful marker of high-grade cervical lesions in HPV 16-infected women. (c) 2007 Wiley-Liss, Inc.

Vpx overcomes a SAMHD1-independent block to HIV reverse transcription that is specific to resting CD4 T cells.

PubMed

Baldauf, Hanna-Mari; Stegmann, Lena; Schwarz, Sarah-Marie; Ambiel, Ina; Trotard, Maud; Martin, Margarethe; Burggraf, Manja; Lenzi, Gina M; Lejk, Helena; Pan, Xiaoyu; Fregoso, Oliver I; Lim, Efrem S; Abraham, Libin; Nguyen, Laura A; Rutsch, Frank; König, Renate; Kim, Baek; Emerman, Michael; Fackler, Oliver T; Keppler, Oliver T

2017-03-07

Early after entry into monocytes, macrophages, dendritic cells, and resting CD4 T cells, HIV encounters a block, limiting reverse transcription (RT) of the incoming viral RNA genome. In this context, dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) has been identified as a restriction factor, lowering the concentration of dNTP substrates to limit RT. The accessory lentiviral protein X (Vpx) proteins from the major simian immunodeficiency virus of rhesus macaque, sooty mangabey, and HIV-2 (SIVsmm/SIVmac/HIV-2) lineage packaged into virions target SAMHD1 for proteasomal degradation, increase intracellular dNTP pools, and facilitate HIV cDNA synthesis. We find that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages, and, unexpectedly, acted in the absence of SAMHD1 degradation, dNTP pool elevation, or changes in SAMHD1 phosphorylation. Vpx rcm/mnd-2 virion incorporation resulted in a dramatic increase of HIV-1 RT intermediates and viral cDNA in infected resting CD4 T cells. These analyses also revealed a barrier limiting HIV-1 infection of resting CD4 T cells at the level of nuclear import. Single amino acid changes in the SAMHD1-degrading Vpx mac239 allowed it to enhance early postentry steps in a Vpx rcm/mnd-2-like fashion. Moreover, Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. These results indicate that Vpx, in addition to SAMHD1, overcomes a previously unappreciated restriction for lentiviruses at the level of RT that acts independently of dNTP concentrations and is specific to resting CD4 T cells.

Fc Receptor-Mediated Activities of Env-Specific Human Monoclonal Antibodies Generated from Volunteers Receiving the DNA Prime-Protein Boost HIV Vaccine DP6-001.

PubMed

Costa, Matthew R; Pollara, Justin; Edwards, Regina Whitney; Seaman, Michael S; Gorny, Miroslaw K; Montefiori, David C; Liao, Hua-Xin; Ferrari, Guido; Lu, Shan; Wang, Shixia

2016-11-15

HIV-1 is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years of infection, and therefore, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that moderate protection is possible and that this protection may correlate with antibody-dependent cellular cytotoxicity (ADCC) activity. Our previous studies demonstrated that in an HIV vaccine phase I trial, the DP6-001 trial, a polyvalent Env DNA prime-protein boost formulation could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities. Here we report on the production and analysis of HIV-1 Env-specific human monoclonal antibodies (hMAbs) isolated from vaccinees in the DP6-001 trial. For this initial report, 13 hMAbs from four vaccinees in the DP6-001 trial showed broad binding to gp120 proteins of diverse subtypes both autologous and heterologous to vaccine immunogens. Equally cross-reactive Fc receptor-mediated functional activities, including ADCC and antibody-dependent cellular phagocytosis (ADCP) activities, were present with both immune sera and isolated MAbs, confirming the induction of nonneutralizing functional hMAbs by the DNA prime-protein boost vaccination. Elicitation of broadly reactive hMAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV vaccine design. The roles of Fc receptor-mediated protective antibody responses are gaining more attention due to their potential contribution to the low-level protection against HIV-1 infection that they provided in the RV144 trial. At the same time, information about hMabs from other human HIV vaccine studies is very limited. In the current study, both immune sera and monoclonal antibodies from vaccinated humans showed not only high-level ADCC and ADCP activities but also cross-subtype ADCC and ADCP activities when a polyvalent DNA prime-protein boost vaccine formulation was used. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

HIV integration sites in latently infected cell lines: evidence of ongoing replication.

PubMed

Symons, Jori; Chopra, Abha; Malatinkova, Eva; De Spiegelaere, Ward; Leary, Shay; Cooper, Don; Abana, Chike O; Rhodes, Ajantha; Rezaei, Simin D; Vandekerckhove, Linos; Mallal, Simon; Lewin, Sharon R; Cameron, Paul U

2017-01-13

Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position. We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells. Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.

Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells

NASA Technical Reports Server (NTRS)

Plymale, D. R.; Tang, D. S.; Comardelle, A. M.; Fermin, C. D.; Lewis, D. E.; Garry, R. F.

1999-01-01

BACKGROUND: Data currently available on HIV-1-induced cytopathology is unclear regarding the mechanism of cell killing. OBJECTIVE: To clarify the extent to which apoptosis or necrosis is involved in HIV-1-induced cell death in view of conflicting existing data. METHODS: T lymphoblastoid cells or peripheral blood mononuclear cells were infected by various strains of HIV-1 and the numbers of apoptotic or necrotic cells were quantified at various times after infection using video-image analysis techniques; the results were compared with the amount of fragmented DNA using a quantitative method. Measurement of mitochondrial transmembrane potential (deltapsi(m)) and intracellular calcium concentrations [Ca2+]i was performed with fluorescent probes and fluorescence concentration analysis (FCA). RESULTS: Although lymphoblastoid and monocytoid cells acutely infected by HIV-1 had increased levels of fragmented DNA, a marker of apoptotic cell death, few (<12%) had condensed chromatin and fragmented nuclei, the morphological features of apoptosis. The predominant alterations in acutely infected cells were distended endoplasmic reticulum and abnormal mitochondria; these ultrastructural changes are consistent with necrosis, although some infected cells simultaneously displayed features of both necrosis and apoptosis. Viability of cells persistently infected by HIV-1 was only minimally reduced from that of uninfected cells. This reduction was accounted for by an increased propensity of the persistently infected cells to die by apoptosis. Alterations in [Ca2+]i and deltapsi(m) occurred in both acutely and persistently infected cells. CONCLUSION: Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells.

[Comparison of commercial HIV-1 viral load tests by using proficiency test results in China, 2013- 2015].

PubMed

Zhang, L; Jin, C; Jiang, Z; Tang, T; Jiang, Y; Pan, P L

2017-09-10

Objective: To compare the bio-equivalence among commercial HIV-1 viral load tests, including EasyQ HIV-1 v2.0 (EasyQ) from bioMerieux NucliSens of France; VERSANT HIV-1 RNA 3.0 assay (bDNA) from Siemens Healthcare Diagnostics of USA; COBAS AmpliPrep/COBAS TaqMan HIV-1 test (Taqman) from Roche Molecular Diagnosis of USA; Abbott Real Time HIV-1 Kit (M2000) from Abbott Molecular of USA and two domestic HIV-1 viral load test kits (domestic kit) from DaAn Gene Company of Sun Yat-Sen University and Liaoning Bio-Pharmaceutical company of Northeast pharmaceutical group, by using proficiency test results in China from 2013 to 2015. Methods: A total of 2 954 proficiency test results, obtained from 22 positive samples of 6 proficiency tests in 155 laboratories conducted by China CDC were analyzed during 2013-2015. The results from each sample were first logarithmic transformed and then grouped according to the method used, the mean value of logarithmic results was calculated. Subsequently, 22 clusters of mean values were analyzed by Bland-Altman analysis for the consistency, and linear regression analysis for the interdependency. Results: The results indicated that, by taking Taqman as the reference, EasyQ, M2000, bDNA and domestic kit had good consistency (90 % -100 % ) and interdependency. Conclusion: All the viral load tests were bio-equivalent. Moreover, according to the conversion formula derived from domestic proficiency test results, all the viral load results could be converted, which is critical for epidemiological analysis.

Possible roles of HIV-1 nucleocapsid protein in the specificity of proviral DNA synthesis and in its variability.

PubMed

Lapadat-Tapolsky, M; Gabus, C; Rau, M; Darlix, J L

1997-05-02

Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it coats the dimeric RNA genome. Due to its nucleic acid binding and annealing activities, NC protein directs the annealing of the tRNA primer to the primer binding site and greatly facilitates minus strand DNA elongation and transfer while protecting the nucleic acids against nuclease degradation. To understand the role of NCp7 in viral DNA synthesis, we examined the influence of NCp7 on self-primed versus primer-specific reverse transcription. The results show that HIV-1 NCp7 can extensively inhibit self-primed reverse transcription of viral and cellular RNAs while promoting primer-specific synthesis of proviral DNA. The role of NCp7 vis-a-vis the presence of mutations in the viral DNA during minus strand elongation was examined. NCp7 maximized the annealing between a cDNA(-) primer containing one to five consecutive errors and an RNA representing the 3' end of the genome. The ability of reverse transcriptase (RT) in the presence of NCp7 to subsequently extend the mutated primers depended upon the position of the mismatch within the primer:template complex. When the mutations were at the polymerisation site, primer extension by RT in the presence of NCp7 was very high, about 40% for one mismatch and 3% for five consecutive mismatches. Mutations within the DNA primer or at its 5' end had little effect on the extension of viral DNA by RT. Taken together these results indicate that NCp7 plays major roles in proviral DNA synthesis within the virion core due to its ability to promote prime-specific proviral DNA synthesis while concurrently inhibiting non-specific reverse transcription of viral and cellular RNAs. Moreover, the observation that NCp7 enhances the incorporation of mutations during minus strand DNA elongation favours the notion that NCp7 is a factor contributing to the high mutation rate of HIV-1.

Interaction of HIV-1 Gag protein components with single DNA molecules

NASA Astrophysics Data System (ADS)

Cruceanu, Margareta; Gorelick, Robert J.; Williams, Mark C.

2003-03-01

The Gag protein of the HIV-1 retrovirus is cleaved into three major proteins as part of viral maturation: nucleocapsid (NC), capsid, and matrix. NC is the first of these proteins to be cleaved, and it is cleaved in three stages into NCp15, followed by NCp9, and finally NCp7. In this study, we use optical tweezers to investigate the capability of these NC proteins to alter the helix-coil transition of single DNA molecules. We have previously shown that the capability to alter the DNA helix-coil transition is an excellent probe of the nucleic acid chaperone activity of NC proteins, in which the secondary structure of nucleic acids is rearranged to facilitate reverse transcription. By examining the capability of NCp15, NCp9, and NCp7 to alter DNA stretching, the current studies will test the role of proteolytic cleavage of Gag in regulating the nucleic acid chaperone activity of NC. Whereas binding studies suggest that NCp9 and NCp15 bind more strongly to DNA than NCp7, our DNA stretching results indicate that these proteins all have similar effects on DNA stretching.

Prevalence of hepatitis B and delta according to HIV-type: a multi-country cross-sectional survey in West Africa.

PubMed

Coffie, Patrick A; Tchounga, Boris K; Bado, Guillaume; Kabran, Mathieu; Minta, Daouda K; Wandeler, Gilles; Gottlieb, Geoffrey S; Dabis, François; Eholie, Serge P; Ekouevi, Didier K

2017-07-04

In West Africa where HIV-1 and HIV-2 co-circulate, the co-infection with hepatitis B virus (HBV) and hepatitis Delta virus (HDV) is not well described. This study aimed at estimating the prevalence of HBV and HBV/HDV co-infection according to HIV types and risk factors for HBV infection among West African HIV-infected patients. A cross-sectional survey was conducted within the IeDEA West Africa cohort from March to December 2012 in Côte d'Ivoire (three sites), Burkina Faso and Mali (one site each). All HIV-infected adult patients on antiretroviral therapy (ART) or not who attended one of the participating HIV clinics during the study period and agreed to participate were included. Blood samples were collected and re-tested for HIV type discrimination, HBV and HDV serology as well as HBV viral load. Logistic regression was used to identify risk factors for HBV infection. A total of 791 patients were included: 192 HIV-1, 447 HIV-2 and 152 HIV-1&2 dually reactive. At time of sampling, 555 (70.2%) were on ART and median CD4+ cell count was 472/mm 3 (inter-quartile range [IQR]: IQR: 294-644). Sixty-seven (8.5%, 95% CI 6.6-10.6) patients were HBsAg positive without any difference according to HIV type (7.9% in HIV-1, 7.2% in HIV-1&2 dually reactive and 9.4% in HIV-2; p = 0.61). In multivariate logistic analysis, age ≤ 30 years old (adjusted odds ratio [aOR] 5.00, 95% CI 1.96-12.76), age between 31 and 49 years old (aOR 1.78, 95% CI 1.00-2.21) and male gender (aOR 2.15, 95% CI 1.25-3.69) were associated with HBsAg positivity. HBV DNA testing was performed in 36 patients with blood sample available (25 on ART) and 8 (22.2%) had detectable HBV DNA. Among the HBsAg-positive individuals, 14.9% (95% CI 7.4-25.7) were also positive for anti-HDV antibody without any difference according to HIV type (28.6% in HIV-1, 14.3% in HIV-2 and 0.0% in HIV-1&2 dually reactive; p = 0.15). HBV and HBV/HDV co-infection are common in West Africa, irrespective of HIV type. Therefore, screening for both viruses should be systematically performed to allow a better management of HIV-infected patients. Follow-up studies are necessary to determine the impact of these two viruses on HIV infection.

Low Plasma Zinc Is Associated with Higher Mitochondrial Oxidative Stress and Faster Liver Fibrosis Development in the Miami Adult Studies in HIV Cohort.

PubMed

Martinez, Sabrina S; Campa, Adriana; Li, Yinghui; Fleetwood, Christina; Stewart, Tiffanie; Ramamoorthy, Venkataraghavan; Baum, Marianna K

2017-04-01

Background: Oxidative stress and reduced antioxidants may be a trigger for liver fibrogenesis. Reducing oxidative stress through higher antioxidant concentration may be a potential antifibrotic target. Objective: We aimed to investigate longitudinally whether plasma zinc, an antioxidant, is related to mitochondrial oxidative stress and the progression of liver fibrosis in the Miami Adult Studies in HIV (MASH) cohort. Methods: A prospective observational cohort study was conducted in 487 predominantly African American HIV-monoinfected and HIV/hepatitis C virus (HCV)-coinfected adults with a mean ± SD age of 47.08 ± 7.67 y from the MASH cohort and followed for a median of 34 mo. Blood was collected for plasma zinc and measures were used to calculate the fibrosis-4 (FIB-4) score (aspartate amino transferase, alanine aminotransferase, and platelets). Plasma zinc deficiency was defined as <0.75 mg/L. Total DNA was extracted from peripheral blood mononuclear cells and mitochondrial DNA (mtDNA) 8-hydroxyguanosine (8-oxo-dG) was determined. Adjusted mixed models were used to assess the relations between zinc, stage of liver disease, and oxidative stress over time and compared between HIV and HIV/HCV groups. Results: Zinc concentrations (β: -0.368, SE = 0.172; P = 0.033) and deficiency were associated with lower FIB-4 scores over time (β: 0.381, SE = 0.118; P = 0.001). Compared with those who were not zinc deficient, zinc-deficient participants had an increased risk of having more-progressed liver disease (OR: 1.91; 95% CI: 1.15, 3.16; P = 0.012). Higher mtDNA 8-oxo-dG was associated with zinc deficiency (β: 0.049, SE = 0.024; P = 0.044) and higher FIB-4 scores over time (β: 0.597, SE = 0.168, P < 0.001). Conclusions: Lower plasma zinc concentrations were associated with liver fibrosis progression and mitochondrial oxidative stress in the HIV and HIV/HCV groups. Zinc may play a role in the impact of liver disease outcomes. © 2017 American Society for Nutrition.

Low Plasma Zinc Is Associated with Higher Mitochondrial Oxidative Stress and Faster Liver Fibrosis Development in the Miami Adult Studies in HIV Cohort1234

PubMed Central

Martinez, Sabrina S; Campa, Adriana; Li, Yinghui; Fleetwood, Christina; Stewart, Tiffanie; Ramamoorthy, Venkataraghavan; Baum, Marianna K

2017-01-01

Background: Oxidative stress and reduced antioxidants may be a trigger for liver fibrogenesis. Reducing oxidative stress through higher antioxidant concentration may be a potential antifibrotic target. Objective: We aimed to investigate longitudinally whether plasma zinc, an antioxidant, is related to mitochondrial oxidative stress and the progression of liver fibrosis in the Miami Adult Studies in HIV (MASH) cohort. Methods: A prospective observational cohort study was conducted in 487 predominantly African American HIV-monoinfected and HIV/hepatitis C virus (HCV)–coinfected adults with a mean ± SD age of 47.08 ± 7.67 y from the MASH cohort and followed for a median of 34 mo. Blood was collected for plasma zinc and measures were used to calculate the fibrosis-4 (FIB-4) score (aspartate amino transferase, alanine aminotransferase, and platelets). Plasma zinc deficiency was defined as <0.75 mg/L. Total DNA was extracted from peripheral blood mononuclear cells and mitochondrial DNA (mtDNA) 8-hydroxyguanosine (8-oxo-dG) was determined. Adjusted mixed models were used to assess the relations between zinc, stage of liver disease, and oxidative stress over time and compared between HIV and HIV/HCV groups. Results: Zinc concentrations (β: −0.368, SE = 0.172; P = 0.033) and deficiency were associated with lower FIB-4 scores over time (β: 0.381, SE = 0.118; P = 0.001). Compared with those who were not zinc deficient, zinc-deficient participants had an increased risk of having more-progressed liver disease (OR: 1.91; 95% CI: 1.15, 3.16; P = 0.012). Higher mtDNA 8-oxo-dG was associated with zinc deficiency (β: 0.049, SE = 0.024; P = 0.044) and higher FIB-4 scores over time (β: 0.597, SE = 0.168, P < 0.001). Conclusions: Lower plasma zinc concentrations were associated with liver fibrosis progression and mitochondrial oxidative stress in the HIV and HIV/HCV groups. Zinc may play a role in the impact of liver disease outcomes. PMID:28228506

Immunogenicity of a novel Clade B HIV-1 vaccine combination: Results of phase 1 randomized placebo controlled trial of an HIV-1 GM-CSF-expressing DNA prime with a modified vaccinia Ankara vaccine boost in healthy HIV-1 uninfected adults

PubMed Central

Grunenberg, Nicole A.; Sanchez, Brittany J.; Seaton, Kelly E.; Ferrari, Guido; Moody, M. Anthony; Frahm, Nicole; Montefiori, David C.; Hay, Christine M.; Goepfert, Paul A.; Baden, Lindsey R.; Robinson, Harriet L.; Yu, Xuesong; Gilbert, Peter B.; McElrath, M. Juliana; Huang, Yunda; Tomaras, Georgia D.

2017-01-01

Background A phase 1 trial of a clade B HIV vaccine in HIV-uninfected adults evaluated the safety and immunogenicity of a DNA prime co-expressing GM-CSF (Dg) followed by different numbers and intervals of modified vaccinia Ankara Boosts (M). Both vaccines produce virus-like particles presenting membrane-bound Env. Methods Four US sites randomized 48 participants to receiving 1/10th the DNA dose as DgDgMMM given at 0, 2, 4, 6 and 8 months, or full dose DgDgM_M or DgDgMM_M regimens, given at 0, 2, 4, and 8 months, and 0, 2, 4, 6, and 10 months, respectively. Peak immunogenicity was measured 2 weeks post-last vaccination. Results All regimens were well tolerated and safe. Full dose DgDgM_M and DgDgMM_M regimens generated Env-specific IgG to HIV-1 Env in >90%, IgG3 in >80%, and IgA in <20% of participants. Responses to gp140 and gp41 targets were more common and of higher magnitude than to gp120 and V1V2. The gp41 antibody included reactivity to the conserved immunodominant region with specificities known to mediate virus capture and phagocytosis and did not cross-react with a panel of intestinal flora antigens. The 3rd dose of MVA increased the avidity of elicited antibody (7.5% to 39%), the ADCC response to Bal gp120 (14% to 64%), and the one-year durability of the IgG3 responses to gp41 by 4-fold (13% vs. 3.5% retention of peak response). The co-expressed GM-CSF did not enhance responses over those in trials testing this vaccine without GM-CSF. Conclusion This DNA/MVA prime-boost regimen induced durable, functional humoral responses that included ADCC, high antibody avidity, and Env IgG1 and IgG3 binding responses to the immunodominant region of gp41. The third, spaced MVA boost improved the overall quality of the antibody response. These products without co-expressed GM-CSF but combined with protein boosts will be considered for efficacy evaluation. Trial registration ClinicalTrials.gov NCT01571960 PMID:28727817

Immunogenicity of a novel Clade B HIV-1 vaccine combination: Results of phase 1 randomized placebo controlled trial of an HIV-1 GM-CSF-expressing DNA prime with a modified vaccinia Ankara vaccine boost in healthy HIV-1 uninfected adults.

PubMed

Buchbinder, Susan P; Grunenberg, Nicole A; Sanchez, Brittany J; Seaton, Kelly E; Ferrari, Guido; Moody, M Anthony; Frahm, Nicole; Montefiori, David C; Hay, Christine M; Goepfert, Paul A; Baden, Lindsey R; Robinson, Harriet L; Yu, Xuesong; Gilbert, Peter B; McElrath, M Juliana; Huang, Yunda; Tomaras, Georgia D

2017-01-01

A phase 1 trial of a clade B HIV vaccine in HIV-uninfected adults evaluated the safety and immunogenicity of a DNA prime co-expressing GM-CSF (Dg) followed by different numbers and intervals of modified vaccinia Ankara Boosts (M). Both vaccines produce virus-like particles presenting membrane-bound Env. Four US sites randomized 48 participants to receiving 1/10th the DNA dose as DgDgMMM given at 0, 2, 4, 6 and 8 months, or full dose DgDgM_M or DgDgMM_M regimens, given at 0, 2, 4, and 8 months, and 0, 2, 4, 6, and 10 months, respectively. Peak immunogenicity was measured 2 weeks post-last vaccination. All regimens were well tolerated and safe. Full dose DgDgM_M and DgDgMM_M regimens generated Env-specific IgG to HIV-1 Env in >90%, IgG3 in >80%, and IgA in <20% of participants. Responses to gp140 and gp41 targets were more common and of higher magnitude than to gp120 and V1V2. The gp41 antibody included reactivity to the conserved immunodominant region with specificities known to mediate virus capture and phagocytosis and did not cross-react with a panel of intestinal flora antigens. The 3rd dose of MVA increased the avidity of elicited antibody (7.5% to 39%), the ADCC response to Bal gp120 (14% to 64%), and the one-year durability of the IgG3 responses to gp41 by 4-fold (13% vs. 3.5% retention of peak response). The co-expressed GM-CSF did not enhance responses over those in trials testing this vaccine without GM-CSF. This DNA/MVA prime-boost regimen induced durable, functional humoral responses that included ADCC, high antibody avidity, and Env IgG1 and IgG3 binding responses to the immunodominant region of gp41. The third, spaced MVA boost improved the overall quality of the antibody response. These products without co-expressed GM-CSF but combined with protein boosts will be considered for efficacy evaluation. ClinicalTrials.gov NCT01571960.

Polyoma virus JC DNA detection by polymerase chain reaction in CSF of HIV infected patients with suspected progressive multifocal leukoencephalopathy.

PubMed

Giri, J A; Gregoresky, J; Silguero, P; García Messina, O; Planes, N

2001-01-01

Several studies had previously demonstrated the high sensitivity and specificity of JCV DNA detection in CSF by PCR. This paper reported the implementation of a simple PCR procedure to detect JCV in the CSF in a cohort of HIV-1 infected patients from Argentina. Years ago, the confirmatory diagnosis of this disease was made by in-situ hybridization or immunohistochemistry techniques on brain biopsies. The PCR procedure described here improves the diagnosis of PML because it is simple and noninvasive, and allows the differential diagnosis of PML from other neurological syndromes associated with AIDS. Many recent studies report a significant benefit of combined antiretroviral therapy on the survival of HIV patients without clear neurological improvements. A negative correlation has been described between the concentration of JCV in the CSF and survival time in HIV-1 infected patients, and the level of immune depression may influence JCV replication. This suggests that a single CSF JCV viral load determination during the course of PML disease progression may be of prognostic value for managing HIV patients.

Interaction of HIV-1 reverse transcriptase ribonuclease H with an acylhydrazone inhibitor.

PubMed

Gong, Qingguo; Menon, Lakshmi; Ilina, Tatiana; Miller, Lena G; Ahn, Jinwoo; Parniak, Michael A; Ishima, Rieko

2011-01-01

HIV-1 reverse transcriptase is a bifunctional enzyme, having both DNA polymerase (RNA- and DNA-dependent) and ribonuclease H activities. HIV-1 reverse transcriptase has been an exceptionally important target for antiretroviral therapeutic development, and nearly half of the current clinically used antiretrovirals target reverse transcriptase DNA polymerase. However, no inhibitors of reverse transcriptase ribonuclease H are on the market or in preclinical development. Several drug-like small molecule inhibitors of reverse transcriptase ribonuclease H have been described, but little structural information is available about the interactions between reverse transcriptase ribonuclease H and inhibitors that exhibit antiviral activity. In this report, we describe NMR studies of the interaction of a new ribonuclease H inhibitor, BHMP07, with a catalytically active HIV-1 reverse transcriptase ribonuclease H domain fragment. We carried out solution NMR experiments to identify the interaction interface of BHMP07 with the ribonuclease H domain fragment. Chemical shift changes of backbone amide signals at different BHMP07 concentrations clearly demonstrate that BHMP07 mainly recognizes the substrate handle region in the ribonuclease H fragment. Using ribonuclease H inhibition assays and reverse transcriptase mutants, the binding specificity of BHMP07 was compared with another inhibitor, dihydroxy benzoyl naphthyl hydrazone. Our results provide a structural characterization of the ribonuclease H inhibitor interaction and are likely to be useful for further improvements of the inhibitors. © 2010 John Wiley & Sons A/S.

PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1.

PubMed

Derrington, Edmund; Gabus, Caroline; Leblanc, Pascal; Chnaidermann, Jonas; Grave, Linda; Dormont, Dominique; Swietnicki, Wieslaw; Morillas, Manuel; Marck, Daniel; Nandi, Pradip; Darlix, Jean-Luc

2002-01-01

The function of the cellular prion protein (PrPC) remains obscure. Studies suggest that PrPC functions in several processes including signal transduction and Cu2+ metabolism. PrPC has also been established to bind nucleic acids. Therefore we investigated the properties of PrPC as a putative nucleic acid chaperone. Surprisingly, PrPC possesses all the nucleic acid chaperoning properties previously specific to retroviral nucleocapsid proteins. PrPC appears to be a molecular mimic of NCP7, the nucleocapsid protein of HIV-1. Thus PrPC, like NCP7, chaperones the annealing of tRNA(Lys) to the HIV-1 primer binding site, the initial step of retrovirus replication. PrPC also chaperones the two DNA strand transfers required for production of a complete proviral DNA with LTRs. Concerning the functions of NCP7 during budding, PrPC also mimices NCP7 by dimerizing the HIV-1 genomic RNA. These data are unprecedented because, although many cellular proteins have been identified as nucleic acid chaperones, none have the properties of retroviral nucleocapsid proteins.

Systematic Review of the Performance of HIV Viral Load Technologies on Plasma Samples

PubMed Central

Sollis, Kimberly A.; Smit, Pieter W.; Fiscus, Susan; Ford, Nathan; Vitoria, Marco; Essajee, Shaffiq; Barnett, David; Cheng, Ben; Crowe, Suzanne M.; Denny, Thomas; Landay, Alan; Stevens, Wendy; Habiyambere, Vincent; Perrins, Jos; Peeling, Rosanna W.

2014-01-01

Background Viral load (VL) monitoring is the standard of care in developing country settings for detecting HIV treatment failure. Since 2010 the World Health Organization has recommended a phase-in approach to VL monitoring in resource-limited settings. We conducted a systematic review of the accuracy and precision of HIV VL technologies for treatment monitoring. Methods and Findings A search of Medline and Embase was conducted for studies evaluating the accuracy or reproducibility of commercially available HIV VL assays. 37 studies were included for review including evaluations of the Amplicor Monitor HIV-1 v1.5 (n = 25), Cobas TaqMan v2.0 (n = 11), Abbott RealTime HIV-1 (n = 23), Versant HIV-1 RNA bDNA 3.0 (n = 15), Versant HIV-1 RNA kPCR 1.0 (n = 2), ExaVir Load v3 (n = 2), and NucliSens EasyQ v2.0 (n = 1). All currently available HIV VL assays are of sufficient sensitivity to detect plasma virus levels at a lower detection limit of 1,000 copies/mL. Bias data comparing the Abbott RealTime HIV-1, TaqMan v2.0 to the Amplicor Monitor v1.5 showed a tendency of the Abbott RealTime HIV-1 to under-estimate results while the TaqMan v2.0 overestimated VL counts. Compared to the Amplicor Monitor v1.5, 2–26% and 9–70% of results from the Versant bDNA 3.0 and Abbott RealTime HIV-1 differed by greater than 0.5log10. The average intra and inter-assay variation of the Abbott RealTime HIV-1 were 2.95% (range 2.0–5.1%) and 5.44% (range 1.17–30.00%) across the range of VL counts (2log10–7log10). Conclusions This review found that all currently available HIV VL assays are of sufficient sensitivity to detect plasma VL of 1,000 copies/mL as a threshold to initiate investigations of treatment adherence or possible treatment failure. Sources of variability between VL assays include differences in technology platform, plasma input volume, and ability to detect HIV-1 subtypes. Monitoring of individual patients should be performed on the same technology platform to ensure appropriate interpretation of changes in VL. Prospero registration # CD42013003603. PMID:24558359

Compartmentalization of HIV-1 within the female genital tract is due to monotypic and low-diversity variants not distinct viral populations.

PubMed

Bull, Marta; Learn, Gerald; Genowati, Indira; McKernan, Jennifer; Hitti, Jane; Lockhart, David; Tapia, Kenneth; Holte, Sarah; Dragavon, Joan; Coombs, Robert; Mullins, James; Frenkel, Lisa

2009-09-22

Compartmentalization of HIV-1 between the genital tract and blood was noted in half of 57 women included in 12 studies primarily using cell-free virus. To further understand differences between genital tract and blood viruses of women with chronic HIV-1 infection cell-free and cell-associated virus populations were sequenced from these tissues, reasoning that integrated viral DNA includes variants archived from earlier in infection, and provides a greater array of genotypes for comparisons. Multiple sequences from single-genome-amplification of HIV-1 RNA and DNA from the genital tract and blood of each woman were compared in a cross-sectional study. Maximum likelihood phylogenies were evaluated for evidence of compartmentalization using four statistical tests. Genital tract and blood HIV-1 appears compartmentalized in 7/13 women by >/=2 statistical analyses. These subjects' phylograms were characterized by low diversity genital-specific viral clades interspersed between clades containing both genital and blood sequences. Many of the genital-specific clades contained monotypic HIV-1 sequences. In 2/7 women, HIV-1 populations were significantly compartmentalized across all four statistical tests; both had low diversity genital tract-only clades. Collapsing monotypic variants into a single sequence diminished the prevalence and extent of compartmentalization. Viral sequences did not demonstrate tissue-specific signature amino acid residues, differential immune selection, or co-receptor usage. In women with chronic HIV-1 infection multiple identical sequences suggest proliferation of HIV-1-infected cells, and low diversity tissue-specific phylogenetic clades are consistent with bursts of viral replication. These monotypic and tissue-specific viruses provide statistical support for compartmentalization of HIV-1 between the female genital tract and blood. However, the intermingling of these clades with clades comprised of both genital and blood sequences and the absence of tissue-specific genetic features suggests compartmentalization between blood and genital tract may be due to viral replication and proliferation of infected cells, and questions whether HIV-1 in the female genital tract is distinct from blood.

The prion protein has RNA binding and chaperoning properties characteristic of nucleocapsid protein NCP7 of HIV-1.

PubMed

Gabus, C; Derrington, E; Leblanc, P; Chnaiderman, J; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W K; Marc, D; Nandi, P; Darlix, J L

2001-06-01

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases associated with the accumulation of a protease-resistant form of the prion protein (PrP). Although PrP is conserved in vertebrates, its function remains to be identified. In vitro PrP binds large nucleic acids causing the formation of nucleoprotein complexes resembling human immunodeficiency virus type 1 (HIV-1) nucleocapsid-RNA complexes and in vivo MuLV replication accelerates the scrapie infectious process, suggesting possible interactions between retroviruses and PrP. Retroviruses, including HIV-1 encode a major nucleic acid binding protein (NC protein) found within the virus where 2000 NC protein molecules coat the dimeric genome. NC is required in virus assembly and infection to chaperone RNA dimerization and packaging and in proviral DNA synthesis by reverse transcriptase (RT). In HIV-1, 5'-leader RNA/NC interactions appear to control these viral processes. This prompted us to compare and contrast the interactions of human and ovine PrP and HIV-1 NCp7 with HIV-1 5'-leader RNA. Results show that PrP has properties characteristic of NCp7 with respect to viral RNA dimerization and proviral DNA synthesis by RT. The NC-like properties of huPrP map to the N-terminal region of huPrP. Interestingly, PrP localizes in the membrane and cytoplasm of PrP-expressing cells. These findings suggest that PrP is a multifunctional protein possibly participating in nucleic acid metabolism.

HIV-1 and herpes simplex virus type-2 genital shedding among co-infected women using self-collected swabs in Chiang Rai, Thailand.

PubMed

Forhan, S E; Dunne, E F; Sternberg, M R; Whitehead, S J; Leelawiwat, W; Thepamnuay, S; Chen, C; Evans-Strickfaden, Tt; McNicholl, J M; Markowitz, L E

2012-08-01

We analysed 528 genital self-collected swabs (SCS) from 67 HIV-1 and herpes simplex virus type-2 (HSV-2) co-infected women collected during the placebo month of a randomized crossover clinical trial of suppressive acyclovir in Chiang Rai, Thailand. In this first longitudinal study of HIV-1 and HSV-2 co-infected women using genital SCS specimens, we found frequent mucosal HIV-1 shedding. Overall, 372 (70%) swabs had detectable HIV-1 RNA with median HIV-1 viral load of 2.61 log(10) copies/swab. We found no statistically significant association between detectable HIV-1 RNA and HSV-2 DNA in the same SCS specimen (adjusted odds ratio [aOR] 1.40; 95% confidence intervals [CI], 0.78-2.60, P = 0.25). Only baseline HIV-1 plasma viral load was independently associated with genital HIV-1 RNA shedding (aOR, 7.6; 95% CI, 3.3-17.2, P < 0.0001). SCS may be useful for future HIV-1 and HSV-2 studies because this method allows for frequent genital sampling, and inclusion of genital sites other than the cervix.

Cloning of an SNF2/SWI2-related protein that binds specifically to the SPH motifs of the SV40 enhancer and to the HIV-1 promoter.

PubMed

Sheridan, P L; Schorpp, M; Voz, M L; Jones, K A

1995-03-03

We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/inhibitor region of the human immunodeficiency virus (HIV)-1 promoter. The predicted HIP116 protein is related to the yeast SNF2/SWI2 transcription factor and to other members of this extended family and contains seven domains similar to those found in the vaccinia NTP1 ATPase. Interestingly, HIP116 also contains a C3HC4 zinc-binding motif (RING finger) interspersed between the ATPase motifs in an arrangement similar to that found in the yeast RAD5 and RAD16 proteins. The HIP116 amino terminus is unique among the members of this family, and houses a specific DNA-binding domain. Antiserum raised against HIP116 recognizes a 116-kDa nuclear protein in Western blots and specifically supershifts SV40 and HIV-1 protein-DNA complexes in gel shift experiments. The binding site for HIP116 on the SV40 enhancer directly overlaps the site for TEF-1, and like TEF-1, binding of HIP116 to the SV40 enhancer is destroyed by mutations that inhibit SPH enhancer activity in vivo. Purified fractions of HIP116 display strong ATPase activity that is preferentially stimulated by SPH DNA and can be inhibited specifically by antibodies to HIP116. These findings suggest that HIP116 might affect transcription, directly or indirectly, by acting as a DNA binding site-specific ATPase.

Utilization of a DNA enzyme immunoassay for the detection of proviral DNA of human immunodeficiency virus type 1 by polymerase chain reaction.

PubMed

Zella, D; Cavicchini, A; Cattaneo, E; Cimarelli, A; Bertazzoni, U

1995-02-01

The detection of proviral DNA by Polymerase Chain Reaction (PCR) is regarded as an important tool in the diagnosis of HIV-1 infection, specially among adults at risk of AIDS and children born to seropositive mothers. However, application of PCR in routine testing is hampered by the need to use radioactive probes. In this study, a non-radioactive test based on a microtiter plate (DNA Enzyme ImmunoAssay, DEIA) was used for the detection of proviral sequences of HIV-1 in peripheral blood cells of different patients. The results of the PCR-DEIA assay were compared to those obtained by liquid hybridization (PCR-LH), virus isolation (VI) and Western blot (WB). The study population included 92 patients belonging to three different groups: seropositive subjects with a well-defined clinical status and WB profile; adults at risk of infection with negative or indeterminate WB; children born to seropositive mothers with still unestablished HIV-1 infection. In the seropositive subjects, both PCR-LH and PCR-DEIA confirmed infection and gave the same results as WB. In adults at risk of infection, PCR with both methods anticipated the seroconversion in one patient with indeterminate WB and confirmed the absence of infection among seronegative and other indeterminate patients. In children born to seropositive mothers, both PCR systems as well as VI permitted an early diagnosis of infection, as confirmed by the clinical follow-up. This study has shown that in subjects at risk of AIDS and in children born to seropositive mothers, the non-isotopic DEIA method presents the same sensitivity and specificity for the detection of HIV-1 infection as the radioactive procedure. The DEIA method appears to be particularly useful for the detection of PCR products in routine diagnostic analyses.

[Association between inflammatory markers and microbial translocation in patients with human immunodeficiency virus infection taking antiretroviral treatment].

PubMed

Reus Bañuls, Sergio; Portilla Sogorb, Joaquín; Sanchez-Paya, José; Boix Martínez, Vicente; Giner Oncina, Livia; Frances, Rubén; Such, José; Merino Lucas, Esperanza; Gimeno Gascón, Adelina

2014-01-21

Inflammatory biomarkers are increased in patients with human immunodeficiency virus (HIV) infection. Antiretroviral treatment (ART) improves some parameters but do not normalize them. The aim of this study is to determine those factors (including microbial translocation) associated with higher inflammation in HIV treated patients. Transversal observational study. HIV patients receiving ART with an HIV viral load (VL)<400 copies/mL. Selection of patients: consecutively between November 2011 and January 2012. Main variable: plasma levels of interleukin 6 (IL-6) and tumour necrosis factor α (TNF-α). Main explanatory variable: microbial translocation markers (16S ribosomal DNA and sCD14). Patients with IL-6 or TNF-α levels above percentile 75 (group 1) were compared with the rest of patients (group 2). Odds ratio (OR) were determined. Eighty-one patients were included (73% male, median age 45 years, 48% stage C). Twenty-six percent had chronic hepatitis C. Median CD4 cell was 493/mm(3) and 30% had detectable HIV VL. 16S ribosomal DNA was detected in 21% of patients. Factors associated with the higher levels of inflammatory markers were 16S ribosomal DNA (OR 77, P<.0001), sCD14 levels (P<.0001) and history of cardiovascular disease (OR 15, P<.01). In multivariate analysis, associations remained for 16S ribosomal DNA (OR 62, P<.0001) and previous cardiovascular disease (OR 25, P<.01). In patients with HIV infection receiving treatment, the higher levels of inflammatory markers are associated with microbial translocation and past cardiovascular events. Copyright © 2013 Elsevier España, S.L. All rights reserved.

Human immunodeficiency virus/human parvovirus B19 co-infection in blood donors and AIDS patients in Sichuan, China

PubMed Central

He, Miao; Zhu, Jiang; Yin, Huimin; Ke, Ling; Gao, Lei; Pan, Zhihong; Yang, Xiuhua; Li, Wuping

2012-01-01

Background Human parvovirus B19 (B19) is a common pathogen which causes a variety of diseases. Persistent B19 infection is related to the degree of host immunodeficiency in patients with human immunodeficiency virus (HIV) infection. However, the existence, loading, virus evolution and distribution of B19 in Chinese HIV-positive patients have not been determined. Materials and methods. We investigated 573 HIV-positive blood donors and AIDS patients in Sichuan, China in the last two decades. Bl9-specific serology and quantitative polymerase chain reaction were used to determine the prevalence of B19/HIV co-infection. Viral genome fragments were subjected to phylogeny and haplotype analysis. Results B19 genomic DNA was found in 26 of 573 (4.5%) HIV-positive individuals, a higher prevalence than in blood donors. DNA levels ranged from 5.3×102–1.1×105 copies/mL. The seroprevalence of IgG was significantly lower in HIV-positive samples than in HIV-negative blood donors, indicating deficient production of B19-specific IgG in the former. The B19 isolates were genotype-1 subtype B19-1A which formed a monophyletic group; seven distinct haplotypes were discovered with 60% of the B19/HIV co-infected variants sharing one central haplotype. Discussion. This study on the prevalence, phylogeny and distribution of human parvovirus B19 in Sichuan, China, demonstrates the persistence of B19 in the circulation of both immunocompetent and immunocompromised subjects, with implications for blood safety. PMID:22790259

Human immunodeficiency virus type 1 Nef protein inhibits NF-kappa B induction in human T cells.

PubMed Central

Niederman, T M; Garcia, J V; Hastings, W R; Luria, S; Ratner, L

1992-01-01

Human immunodeficiency virus type 1 (HIV-1) can establish a persistent and latent infection in CD4+ T lymphocytes (W. C. Greene, N. Engl. J. Med. 324:308-317, 1991; S. M. Schnittman, M. C. Psallidopoulos, H. C. Lane, L. Thompson, M. Baseler, F. Massari, C. H. Fox, N. P. Salzman, and A. S. Fauci, Science 245:305-308, 1989). Production of HIV-1 from latently infected cells requires host cell activation by T-cell mitogens (T. Folks, D. M. Powell, M. M. Lightfoote, S. Benn, M. A. Martin, and A. S. Fauci, Science 231:600-602, 1986; D. Zagury, J. Bernard, R. Leonard, R. Cheynier, M. Feldman, P. S. Sarin, and R. C. Gallo, Science 231:850-853, 1986). This activation is mediated by the host transcription factor NF-kappa B [G. Nabel and D. Baltimore, Nature (London) 326:711-717, 1987]. We report here that the HIV-1-encoded Nef protein inhibits the induction of NF-kappa B DNA-binding activity by T-cell mitogens. However, Nef does not affect the DNA-binding activity of other transcription factors implicated in HIV-1 regulation, including SP-1, USF, URS, and NF-AT. Additionally, Nef inhibits the induction of HIV-1- and interleukin 2-directed gene expression, and the effect on HIV-1 transcription depends on an intact NF-kappa B-binding site. These results indicate that defective recruitment of NF-kappa B may underlie Nef's negative transcriptional effects on the HIV-1 and interleukin 2 promoters. Further evidence suggests that Nef inhibits NF-kappa B induction by interfering with a signal derived from the T-cell receptor complex. Images PMID:1527859

Mechanism of Enhanced HIV Restriction by Virion Coencapsidated Cytidine Deaminases APOBEC3F and APOBEC3G.

PubMed

Ara, Anjuman; Love, Robin P; Follack, Tyson B; Ahmed, Khawaja A; Adolph, Madison B; Chelico, Linda

2017-02-01

The APOBEC3 (A3) enzymes, A3G and A3F, are coordinately expressed in CD4 + T cells and can become coencapsidated into HIV-1 virions, primarily in the absence of the viral infectivity factor (Vif). A3F and A3G are deoxycytidine deaminases that inhibit HIV-1 replication by inducing guanine-to-adenine hypermutation through deamination of cytosine to form uracil in minus-strand DNA. The effect of the simultaneous presence of both A3G and A3F on HIV-1 restriction ability is not clear. Here, we used a single-cycle infectivity assay and biochemical analyses to determine if coencapsidated A3G and A3F differ in their restriction capacity from A3G or A3F alone. Proviral DNA sequencing demonstrated that compared to each A3 enzyme alone, A3G and A3F, when combined, had a coordinate effect on hypermutation. Using size exclusion chromatography, rotational anisotropy, and in vitro deamination assays, we demonstrate that A3F promotes A3G deamination activity by forming an A3F/G hetero-oligomer in the absence of RNA which is more efficient at deaminating cytosines. Further, A3F caused the accumulation of shorter reverse transcripts due to decreasing reverse transcriptase efficiency, which would leave single-stranded minus-strand DNA exposed for longer periods of time, enabling more deamination events to occur. Although A3G and A3F are known to function alongside each other, these data provide evidence for an A3F/G hetero-oligomeric A3 with unique properties compared to each individual counterpart. The APOBEC3 enzymes APOBEC3F and APOBEC3G act as a barrier to HIV-1 replication in the absence of the HIV-1 Vif protein. After APOBEC3 enzymes are encapsidated into virions, they deaminate cytosines in minus-strand DNA, which forms promutagenic uracils that induce transition mutations or proviral DNA degradation. Even in the presence of Vif, footprints of APOBEC3-catalyzed deaminations are found, demonstrating that APOBEC3s still have discernible activity against HIV-1 in infected individuals. We undertook a study to better understand the activity of coexpressed APOBEC3F and APOBEC3G. The data demonstrate that an APOBEC3F/APOBEC3G hetero-oligomer can form that has unique properties compared to each APOBEC3 alone. This hetero-oligomer has increased efficiency of virus hypermutation, raising the idea that we still may not fully realize the antiviral mechanisms of endogenous APOBEC3 enzymes. Hetero-oligomerization may be a mechanism to increase their antiviral activity in the presence of Vif. Copyright © 2017 American Society for Microbiology.

Cocaine evokes a profile of oxidative stress and impacts innate antiviral response pathways in astrocytes.

PubMed

Cisneros, Irma E; Erdenizmenli, Mert; Cunningham, Kathryn A; Paessler, Slobodan; Dineley, Kelly T

2018-06-01

HIV-1 and Zika virus (ZIKV) represent RNA viruses with neurotropic characteristics. Infected individuals suffer neurocognitive disorders aggravated by environmental toxins, including drugs of abuse such as cocaine, exacerbating HIV-associated neurocognitive disorders through a combination of astrogliosis, oxidative stress and innate immune signaling; however, little is known about how cocaine impacts the progression of ZIKV neural perturbations. Impaired innate immune signaling is characterized by weakened antiviral activation of interferon signaling and alterations in inflammatory signaling, factors contributing to cognitive sequela associated with cocaine in HIV-1/ZIKV infection. We employed cellular/molecular biology techniques to test if cocaine suppresses the efficacy of astrocytes to initiate a Type 1 interferon response to HIV-1/ZIKV, in vitro. We found cocaine activated antiviral signaling pathways and type I interferon in the absence of inflammation. Cocaine pre-exposure suppressed antiviral responses to HIV-1/ZIKV, triggering antiviral signaling and phosphorylation of interferon regulatory transcription factor 3 to stimulate type I interferon gene transcription. Our data indicate that oxidative stress is a major driver of cocaine-mediated astrocyte antiviral immune responses. Although astrocyte antiviral signaling is activated following detection of foreign pathogenic material, oxidative stress and increased cytosolic double-stranded DNA (dsDNA) can drive antiviral signaling via stimulation of pattern recognition receptors. Pretreatment with the glial modulators propentofylline (PPF) or pioglitazone (PIO) reversed cocaine-mediated attenuation of astrocyte responses to HIV-1/ZIKV. Both PPF/PIO protected against cocaine-mediated generation of reactive oxygen species (ROS), increased dsDNA, antiviral signaling pathways and increased type I interferon, indicating that cocaine induces astrocyte type I interferon signaling in the absence of virus and oxidative stress is a major driver of cocaine-mediated astrocyte antiviral immunity. Lastly, PPF and PIO have therapeutic potential to ameliorate cocaine-mediated dysregulation of astrocyte antiviral immunity possibly via a myriad of protective actions including decreases in reactive phenotype and damaging immune factors. Published by Elsevier Ltd.

Quantitative Analysis of HIV-1 Preintegration Complexes

PubMed Central

Engelman, Alan; Oztop, Ilker; Vandegraaff, Nick; Raghavendra, Nidhanapati K.

2009-01-01

Retroviral replication proceeds through the formation of a provirus, an integrated DNA copy of the viral RNA genome. The linear cDNA product of reverse transcription is the integration substrate and two different integrase activities, 3′ processing and DNA strand transfer, are required for provirus formation. Integrase nicks the cDNA ends adjacent to phylogenetically-conserved CA dinucleotides during 3′ processing. After nuclear entry and locating a suitable chromatin acceptor site, integrase joins the recessed 3′-OHs to the 5′-phosphates of a double-stranded staggered cut in the DNA target. Integrase functions in the context of a large nucleoprotein complex, called the preintegration complex (PIC), and PICs are analyzed to determine levels of integrase 3′ processing and DNA strand transfer activities that occur during acute virus infection. Denatured cDNA end regions are monitored by indirect end-labeling to measure the extent of 3′ processing. Native PICs can efficiently integrate their viral cDNA into exogenously added target DNA in vitro, and Southern blotting or nested PCR assays are used to quantify the resultant DNA strand transfer activity. This study details HIV-1 infection, PIC extraction, partial purification, and quantitative analyses of integrase 3′ processing and DNA strand transfer activities. PMID:19233280

Genotypic tropism testing of proviral DNA to guide maraviroc initiation in aviraemic subjects: 48-week analysis of results from the PROTEST study.

PubMed

Poveda, E; Hernández-Quero, J; Pérez-Elías, M J; Ribas, M A; Martínez-Madrid, O J; Flores, J; Navarro, J; Gutiérrez, F; García-Deltoro, M; Imaz, A; Ocampo, A; Artero, A; Blanco, F; Bernal, E; Pasquau, J; Mínguez-Gallego, C; Pérez, N; Aiestaran, A; García, F; Paredes, R

2017-08-01

Maraviroc (MVC) is a suitable drug for aviraemic subjects on antiretroviral treatment (ART) developing toxicity. Its prescription requires prior tropism testing. It is unknown if proviral DNA genotypic tropism testing is reliable for guiding MVC initiation in aviraemic subjects, so this study was carried out to address this issue. PROTEST was a phase 4, prospective, single-arm clinical trial carried out in 24 HIV care centres in Spain. MVC-naïve HIV-1-infected patients with HIV-1 RNA < 50 copies/mL on stable ART during the previous 6 months who required an ART change because of toxicity and who had R5 HIV, as determined by proviral DNA genotypic tropism testing, initiated MVC with two nucleoside reverse transcriptase inhibitors (NRTIs) and were followed for 48 weeks. Virological failure was defined as two consecutive viral load measurements > 50 copies/mL. Tropism results were available for 141 of 175 (80.6%) subjects screened: 60% had R5 and 85% of these (n = 74) were finally included in the study. Previous ART included protease inhibitors (PIs) in 62% of subjects, nonnucleoside reverse transcriptase inhibitors (NNRTIs) in 36%, and integrase inhibitors (INIs) in 2%. Main reasons for treatment change were dyslipidaemia (42%), gastrointestinal symptoms (22%) and liver toxicity (15%). MVC was given alongside tenofovir (TDF)/emtricitabine (FTC) (54%) and abacavir (ABC)/lamivudine (3TC) (40%) in most patients. Eighty-four per cent of patients maintained a viral load < 50 copies/mL to week 48, whereas 16% discontinued treatment: two withdrew informed consent, one had an R5 to X4 shift between screening and baseline, one was lost to follow-up, one developed an adverse event (rash), two died from non-study-related causes, and five developed protocol-defined virological failure. Initiation of MVC plus two NRTIs in aviraemic subjects based on genotypic tropism testing of proviral HIV-1 DNA is associated with low rates of virological failure for up to 1 year. © 2016 British HIV Association.

SUN1 Regulates HIV-1 Nuclear Import in a Manner Dependent on the Interaction between the Viral Capsid and Cellular Cyclophilin A.

PubMed

Luo, Xinlong; Yang, Wei; Gao, Guangxia

2018-07-01

Human immunodeficiency virus type 1 (HIV-1) can infect nondividing cells via passing through the nuclear pore complex. The nuclear membrane-imbedded protein SUN2 was recently reported to be involved in the nuclear import of HIV-1. Whether SUN1, which shares many functional similarities with SUN2, is involved in this process remained to be explored. Here we report that overexpression of SUN1 specifically inhibited infection by HIV-1 but not that by simian immunodeficiency virus (SIV) or murine leukemia virus (MLV). Overexpression of SUN1 did not affect reverse transcription but led to reduced accumulation of the 2-long-terminal-repeat (2-LTR) circular DNA and integrated viral DNA, suggesting a block in the process of nuclear import. HIV-1 CA was mapped as a determinant for viral sensitivity to SUN1. Treatment of SUN1-expressing cells with cyclosporine (CsA) significantly reduced the sensitivity of the virus to SUN1, and an HIV-1 mutant containing CA-G89A, which does not interact with cyclophilin A (CypA), was resistant to SUN1 overexpression. Downregulation of endogenous SUN1 inhibited the nuclear entry of the wild-type virus but not that of the G89A mutant. These results indicate that SUN1 participates in the HIV-1 nuclear entry process in a manner dependent on the interaction of CA with CypA. IMPORTANCE HIV-1 infects both dividing and nondividing cells. The viral preintegration complex (PIC) can enter the nucleus through the nuclear pore complex. It has been well known that the viral protein CA plays an important role in determining the pathways by which the PIC enters the nucleus. In addition, the interaction between CA and the cellular protein CypA has been reported to be important in the selection of nuclear entry pathways, though the underlying mechanisms are not very clear. Here we show that both SUN1 overexpression and downregulation inhibited HIV-1 nuclear entry. CA played an important role in determining the sensitivity of the virus to SUN1: the regulatory activity of SUN1 toward HIV-1 relied on the interaction between CA and CypA. These results help to explain how SUN1 is involved in the HIV-1 nuclear entry process. Copyright © 2018 American Society for Microbiology.

Boosting of HIV-1 Neutralizing Antibody Responses by a Distally Related Retroviral Envelope Protein

PubMed Central

Uchtenhagen, Hannes; Schiffner, Torben; Bowles, Emma; Heyndrickx, Leo; LaBranche, Celia; Applequist, Steven E.; Jansson, Marianne; De Silva, Thushan; Back, Jaap Willem; Achour, Adnane; Scarlatti, Gabriella; Fomsgaard, Anders; Montefiori, David; Stewart-Jones, Guillaume; Spetz, Anna-Lena

2014-01-01

Our knowledge of the binding sites for neutralizing antibodies (NAbs) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B-cell responses to vulnerable conserved sites within the HIV-1 envelope glycoprotein (Env). Here we report an immunization strategy composed of a trivalent HIV-1 (clade B envs) DNA prime, followed by a SIVmac239 gp140 Env protein boost that aimed to focus the immune response to structurally conserved parts of the HIV-1 and SIV Envs. Heterologous NAb titres, primarily to tier 1 HIV-1 isolates, elicited during the trivalent HIV-1 env prime, were significantly increased by the SIVmac239 gp140 protein boost in rabbits. Epitope mapping of antibody binding reactivity revealed preferential recognition of the C1, C2, V2, V3 and V5 regions. These results provide a proof of concept that a distally related retroviral SIV Env protein boost can increase pre-existing NAb responses against HIV-1. PMID:24829409

Baseline characteristics of HIV & hepatitis B virus (HIV/HBV) co-infected patients from Kolkata, India

PubMed Central

Sarkar, Jayeeta; Saha, Debraj; Bandyopadhyay, Bhaswati; Saha, Bibhuti; Kedia, Deepika; Guha Mazumder, D.N.; Chakravarty, Runu; Guha, Subhasish Kamal

2016-01-01

Background & objectives: Hepatitis B virus (HBV) and HIV co-infection has variable prevalence worldwide. In comparison to HBV mono-infection, the course of chronic HBV infection is accelerated in HIV/HBV co-infected patients. The present study was carried out to analyse the baseline characteristics (clinical, biochemical, serological and virological) of treatment naïve HIV/HBV co-infected and HIV mono-infected patients. Methods: Between July 2011 and January 2013, a total number of 1331 HIV-seropositive treatment naïve individuals, enrolled in the ART Centre of Calcutta School of Tropical Medicine, Kolkata, India, were screened for hepatitis B surface antigen (HBsAg). A total of 1253 HIV mono-infected and 78 HIV/HBV co-infected patients were characterized. The co-infected patients were evaluated for HBeAg and anti-HBe antibody by ELISA. HIV RNA was quantified for all co-infected patients. HBV DNA was detected and quantified by real time-PCR amplification followed by HBV genotype determination. Results: HIV/HBV co-infected patients had proportionately more advanced HIV disease (WHO clinical stage 3 and 4) than HIV mono-infected individuals (37.1 vs. 19.9%). The co-infected patients had significantly higher serum bilirubin, alanine aminotransferase (ALT), alkaline phosphatase and ALT/platelet ratio index (APRI). CD4 count was non-significantly lower in co-infected patients. Majority (61.5%) were HBeAg positive with higher HIV RNA (P<0.05), HBV DNA (P<0.001) and APRI (P<0.05) compared to those who were HBeAg negative. HBV/D was the predominant genotype (73.2%) and D2 (43.7%) was the commonest subgenotype. Interpretation & conclusions: HIV/HBV co-infected patients had significantly higher serum bilirubin, ALT, alkaline phosphatase and lower platelet count. HBeAg positive co-infected patients had higher HIV RNA and HBV DNA compared to HBeAg negative co-infected patients. Prior to initiation of antiretroviral treatment (ART) all patients should be screened for HBsAg to initiate appropriate ART regimen. PMID:27488008

Frequency and subtype of BK virus infection in Iranian patients infected with HIV.

PubMed

Akhgari, Shahla; Mohraz, Minoo; Azadmanesh, Kayhan; Vahabpour, Rouhollah; Kazemimanesh, Monireh; Aghakhani, Arezoo; Jozpanahi, Manizheh; Banifazl, Mohammad; Bavand, Anahita; Ramezani, Amitis

2016-02-01

Human polyomavirus BK virus (BKV) is a double-stranded DNA virus that infects approximately 90 % of the general population as a subclinical or mild infection. In immunosuppressed patients, such as HIV cases, BKV may be reactivated resulting hemorrhagic cystitis and tubulointerstitial nephritis. However, there are limited studies on prevalence and molecular epidemiology of BKV in Iran. We therefore aimed to evaluate the prevalence and subtypes of BKV in Iranian HIV patients. A total of 99 patients with HIV infection were enrolled in the study. Presence of BKV DNA in plasma was evaluated by nested PCR. PCR products were sequenced directly, and phylogenetic analysis was performed. BKV DNA was detected in 8.08 % of HIV patients. BKV viremia presented in 4 out of 25 patients (16 %) not receiving antiretroviral therapy in comparison with 4 out 74 of HAART-treated patients (5.4 %) (P = 0.023). In patients with CD4 counts ≥200 cells/mm(3), viremia was found more commonly (7/80 = 8.8 %) than in those with lower counts (1/19 = 5.2 %) (not significant). All sequenced BKV isolates belonged to subtype Ib-2. Our findings indicated that the prevalence of BKV viremia is relatively prevalent in patients with HIV infection and significantly higher in naïve than HAART-treated cases. Therefore, HAART can eliminate BKV infection from plasma and reduce viremia although the actual implication of BKV viremia in HIV patients is not clear.

Induction of HIV Neutralizing Antibodies against the MPER of the HIV Envelope Protein by HA/gp41 Chimeric Protein-Based DNA and VLP Vaccines

PubMed Central

Ye, Ling; Wen, Zhiyuan; Dong, Ke; Wang, Xi; Bu, Zhigao; Zhang, Huizhong; Compans, Richard W.; Yang, Chinglai

2011-01-01

Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy. PMID:21625584

Multi-spectroscopic and molecular docking studies on the interaction of darunavir, a HIV protease inhibitor with calf thymus DNA

NASA Astrophysics Data System (ADS)

Shi, Jie-Hua; Zhou, Kai-Li; Lou, Yan-Yue; Pan, Dong-Qi

2018-03-01

Molecular interaction of darunavir (DRV), a HIV protease inhibitor with calf thymus deoxyribonucleic acid (ct-DNA) was studied in physiological buffer (pH 7.4) by multi-spectroscopic approaches hand in hand with viscosity measurements and molecular docking technique. The UV absorption and fluorescence results together revealed the formation of a DRV-ct-DNA complex having binding affinities of the order of 103 M- 1, which was more in keeping with the groove binding. The results that DRV bound to ct-DNA via groove binding mode was further evidenced by KI quenching studies, viscosity measurements, competitive binding investigations with EB and Rhodamine B and CD spectral analysis. The effect of ionic strength indicated the negligible involvement of electrostatic interaction between DRV and ct-DNA. The thermodynamic parameters regarding the binding interaction of DRV with ct-DNA in terms of enthalpy change (ΔH0) and entropy change (ΔS0) were - 63.19 kJ mol- 1 and - 141.92 J mol- 1 K- 1, indicating that hydrogen bonds and van der Waals forces played a predominant role in the binding process. Furthermore, molecular simulation studies suggested that DRV molecule was prone to bind in the A-T rich region of the minor groove of DNA.

Risk factors for anal human papillomavirus infection type 16 among HIV-positive men who have sex with men in San Francisco

PubMed Central

Hernandez, Alexandra L.; Efird, Jimmy T.; Holly, Elizabeth A.; Berry, J. Michael; Jay, Naomi; Palefsky, Joel M.

2015-01-01

Background and Objective HIV-positive men who have sex with men (MSM) are at high risk of anal cancer compared with the general population. Human papillomavirus (HPV) infection, particularly HPV 16, is causally associated with anal cancer. However, risk factors for anal HPV 16 infection are poorly understood. We determined the prevalence and risk factors for anal HPV 16 infection in a population of HIV-positive MSM, most of whom were being treated with antiretroviral therapy. Design Cross-sectional data from the baseline visit of a 4-year prospective cohort study. Methods 348 HIV-positive MSM were recruited in San Francisco and received a detailed sexual behavior risk-factor questionnaire. An anal swab was used to collect specimens for HPV type-specific DNA testing using L1 HPV DNA PCR. We used log-binomial multivariable models to determine risk factors for anal HPV 16 infection. Results 92% of HIV-positive MSM had at least one anal HPV type, 80% had at least one oncogenic HPV type and 42% had HPV 16. Non-Hispanic white race and higher level of education were associated with a decreased risk of HPV 16 infection. A higher number of total male partners was associated with HPV 16 (RR: 1.6, 95%CI 1.1–2.4, p=0.01) for 201–1000 partners compared with 1–200. Injection drug use (IDU) was independently associated with anal HPV 16 infection (RR: 1.5, 95%CI 1.2–1.9, p=0.003). Conclusions The prevalence of anal HPV infection, including HPV 16, is high in HIV-positive MSM. HIV-positive MSM should be counseled about the risk associated with increased partners and IDU. PMID:23614994

Early infant diagnosis of HIV infection using DNA-PCR at a referral center: an 8 years retrospective analysis.

PubMed

Olana, Tolessa; Bacha, Tigist; Worku, Walelign; Tadesse, Birkneh Tilahun

2016-01-01

Over the last decade, Ethiopia adopted different strategies of prevention of mother to child transmission of HIV (PMTCT). Prior to implementation of Option A in 2011, there was no provision of prophylaxis for PMTCT. With 'Option A', PMTCT interventions relied on maternal CD4 count. In early 2013, ''Option B+'' has been started; with this option, antiretroviral therapy is started and continued for life to any HIV positive pregnant mother irrespective of CD4 count with an enhanced treatment for the baby. Though there are a number of studies which evaluated the effectiveness of PMTCT interventions, the current study assessed the real-world effectiveness of PMTCT options in a setting where there is limitation of resources. This study tried to address three questions: what proportion of babies tested by DNA-PCR are HIV infected in the first 2 months of life? How does the type of PMTCT intervention affect presence of HIV infection at this age? What are the factors affecting HIV transmission, after controlling for type of PMCT-HIV intervention? We assessed records of 624 registered HIV exposed infants and 412 mothers who were delivered at Bishoftu Hospital from May 2006 to August 2014. Presence of HIV infection at 6-8 weeks of age was assessed from the records. Maternal and infant risk factors for infection at this age were analyzed. Data were collected using standard data abstraction format and were analyzed using SPSS version 20. Among all the infants who were delivered at the hospital during the study period, 624/936 (66.7 %) had undergone early infant diagnosis at 6-8 weeks. Twenty-seven (4.3 %) were positive for HIV DNA PCR at the age of 6-8 weeks. None of the infants who received ''Option B+'' had a positive HIV DNA PCR result. HIV infection rate was highest among those who took either no prophylaxis or single dose Nevirapine (11.5 and 11.1 % respectively). Those who took single dose Nevirapine and Zidovudine had HIV positivity rate of 3.9 %. Many of the covariates which were shown to be predictors on bivariate analysis were found not to be independent predictors on multivariate analysis. PMTCT ''Option B+'' resulted in zero HIV infection rates among the included infants. There was a high loss to follow up rate at 6-8 weeks of age. The authors recommend that a better strategy of linkage to care and treatment should be devised for HIV exposed infants.

Induction of long-lasting multi-specific CD8+ T cells by a four-component DNA-MVA/HIVA-RENTA candidate HIV-1 vaccine in rhesus macaques.

PubMed

Im, Eung-Jun; Nkolola, Joseph P; di Gleria, Kati; McMichael, Andrew J; Hanke, Tomás

2006-10-01

As a part of a long-term effort to develop vaccine against HIV-1 clade A inducing protective T cell responses in humans, we run mutually complementing studies in humans and non-human primates (NHP) with the aim to maximize vaccine immunogenicity. The candidate vaccine under development has four components, pTHr.HIVA and pTH.RENTA DNA, and modified vaccinia virus Ankara (MVA).HIVA and MVA.RENTA, delivered in a heterologous DNA prime-MVA boost regimen. While the HIVA (Gag/epitopes) components have been tested in NHP and over 300 human subjects, we plan to test in humans the RENTA (reverse transcriptase, gp41, Nef, Tat) vaccines designed to broaden HIVA-induced responses in year 2007. Here, we investigated the four-component vaccine long-term immunogenicity in Mamu-A*01-positive rhesus macaques and demonstrated that the vaccine-induced T cells were multi-specific, multi-functional, readily proliferated to recall peptides and were circulating in the peripheral blood of vaccine recipients over 1 year after vaccine administration. The consensus clade A-elicited T cells recognized 50% of tested epitope variants from other HIV-1 clades. Thus, the DNA-MVA/HIVA-RENTA vaccine induced memory T cells of desirable characteristics and similarities to those induced in humans by HIVA vaccines alone; however, single-clade vaccines may not elicit sufficiently cross-reactive responses.

Virus-producing cells determine the host protein profiles of HIV-1 virion cores

PubMed Central

2012-01-01

Background Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized. Results We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of incorporation of some RNA binding (RHA and HELIC2) and DNA binding proteins (MCM5 and Ku80) in the viral cores from T cells was higher than in the cores from both mMΦ and mMN and did not correlate with the abundance of these proteins in virus producing cells. Conclusions Profiles of host proteins packaged in the cores of HIV-1 virions depend on the type of virus producing cell. The pool of proteins present in the cores of all virions is likely to contain factors important for viral functions. Incorporation ratio of certain RNA- and DNA-binding proteins suggests their more efficient, non-random packaging into virions in T cells than in mMΦ and mMN. PMID:22889230

Molecular Dynamics Study of HIV-1 RT-DNA-Nevirapine Complexes Explains NNRTI Inhibition, and Resistance by Connection Mutations

PubMed Central

Vijayan, R.S.K.; Arnold, Eddy; Das, Kalyan

2015-01-01

HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that is targeted by nucleoside analogs (NRTIs) and nonnucleoside inhibitors (NNRTIs). NNRTIs are allosteric inhibitors of RT, and constitute an integral part of the highly active antiretroviral therapy (HAART) regimen. Under selective pressure, HIV-1 acquires resistance against NNRTIs primarily by selecting mutations around the NNRTI pocket. Complete RT sequencing of clinical isolates revealed that spatially distal mutations arising in connection and the RNase H domain also confer NNRTI resistance and contribute to NRTI resistance. However, the precise structural mechanism by which the connection domain mutations confer NNRTI resistance is poorly understood. We performed 50-ns MD simulations, followed by essential dynamics, free-energy landscape analyses and network analyses of RT-DNA, RT-DNA-nevirapine, and N348I/T369I mutant RT-DNA-nevirapine complexes. MD simulation studies revealed altered global motions and restricted conformational landscape of RT upon nevirapine binding. Analysis of protein structure network parameters demonstrated a dissortative hub pattern in the RT-DNA complex and an assortative hub pattern in the RT-DNA-nevirapine complex suggesting enhanced rigidity of RT upon nevirapine binding. The connection subdomain mutations N348I/T369I did not induce any significant structural change; rather, these mutations modulate the conformational dynamics and alter the long-range allosteric communication network between the connection subdomain and NNRTI pocket. Insights from the present study provide a structural basis for the biochemical and clinical findings on drug resistance caused by the connection and RNase H mutations. PMID:24174331

2012 HIV Diagnostics Conference: the molecular diagnostics perspective.

PubMed

Branson, Bernard M; Pandori, Mark

2013-04-01

2012 HIV Diagnostic Conference Atlanta, GA, USA, 12-14 December 2012. This report highlights the presentations and discussions from the 2012 National HIV Diagnostic Conference held in Atlanta (GA, USA), on 12-14 December 2012. Reflecting changes in the evolving field of HIV diagnostics, the conference provided a forum for evaluating developments in molecular diagnostics and their role in HIV diagnosis. In 2010, the HIV Diagnostics Conference concluded with the proposal of a new diagnostic algorithm which included nucleic acid testing to resolve discordant screening and supplemental antibody test results. The 2012 meeting, picking up where the 2010 meeting left off, focused on scientific presentations that assessed this new algorithm and the role played by RNA testing and new developments in molecular diagnostics, including detection of total and integrated HIV-1 DNA, detection and quantification of HIV-2 RNA, and rapid formats for detection of HIV-1 RNA.

Detection of polyomavirus simian virus 40 tumor antigen DNA in AIDS-related systemic non-Hodgkin lymphoma

NASA Technical Reports Server (NTRS)

Vilchez, Regis A.; Lednicky, John A.; Halvorson, Steven J.; White, Zoe S.; Kozinetz, Claudia A.; Butel, Janet S.

2002-01-01

Systemic non-Hodgkin lymphoma (S-NHL) is a common malignancy during HIV infection, and it is hypothesized that infectious agents may be involved in the etiology. Epstein-Barr virus DNA is found in <40% of patients with AIDS-related S-NHL, suggesting that other oncogenic viruses, such as polyomaviruses, may play a role in pathogenesis. We analyzed AIDS-related S-NHL samples, NHL samples from HIV-negative patients, peripheral blood leukocytes from HIV-infected and -uninfected patients without NHL, and lymph nodes without tumors from HIV-infected patients. Specimens were examined by polymerase chain reaction analysis with use of primers specific for an N-terminal region of the oncoprotein large tumor antigen ( T-ag ) gene conserved among all three polyomaviruses (simian virus 40 [SV40], JC virus, and BK virus). Polyomavirus T-ag DNA sequences, proven to be SV40-specific, were detected more frequently in AIDS-related S-NHL samples (6 of 26) than in peripheral blood leukocytes from HIV-infected patients (6 of 26 vs. 0 of 69; p =.0001), NHL samples from HIV-negative patients (6 of 26 vs. 0 of 10; p =.09), or lymph nodes (6 of 26 vs. 0 of 7; p =.16). Sequences of C-terminal T-ag DNA from SV40 were amplified from two AIDS-related S-NHL samples. Epstein-Barr virus DNA sequences were detected in 38% (10 of 26) AIDS-related S-NHL samples, 50% (5 of 10) HIV-negative S-NHL samples, and 57% (4 of 7) lymph nodes. None of the S-NHL samples were positive for both Epstein-Barr virus DNA and SV40 DNA. Further studies of the possible role of SV40 in the pathogenesis of S-NHL are warranted.

Functional interactions of nucleocapsid protein of feline immunodeficiency virus and cellular prion protein with the viral RNA.

PubMed

Moscardini, Mila; Pistello, Mauro; Bendinelli, M; Ficheux, Damien; Miller, Jennifer T; Gabus, Caroline; Le Grice, Stuart F J; Surewicz, Witold K; Darlix, Jean-Luc

2002-04-19

All lentiviruses and oncoretroviruses examined so far encode a major nucleic-acid binding protein (nucleocapsid or NC* protein), approximately 2500 molecules of which coat the dimeric RNA genome. Studies on HIV-1 and MoMuLV using in vitro model systems and in vivo have shown that NC protein is required to chaperone viral RNA dimerization and packaging during virus assembly, and proviral DNA synthesis by reverse transcriptase (RT) during infection. The human cellular prion protein (PrP), thought to be the major component of the agent causing transmissible spongiform encephalopathies (TSE), was recently found to possess a strong affinity for nucleic acids and to exhibit chaperone properties very similar to HIV-1 NC protein in the HIV-1 context in vitro. Tight binding of PrP to nucleic acids is proposed to participate directly in the prion disease process. To extend our understanding of lentiviruses and of the unexpected nucleic acid chaperone properties of the human prion protein, we set up an in vitro system to investigate replication of the feline immunodeficiency virus (FIV), which is functionally and phylogenetically distant from HIV-1. The results show that in the FIV model system, NC protein chaperones viral RNA dimerization, primer tRNA(Lys,3) annealing to the genomic primer-binding site (PBS) and minus strand DNA synthesis by the homologous FIV RT. FIV NC protein is able to trigger specific viral DNA synthesis by inhibiting self-priming of reverse transcription. The human prion protein was found to mimic the properties of FIV NC with respect to primer tRNA annealing to the viral RNA and chaperoning minus strand DNA synthesis. Copyright 2002 Elsevier Science Ltd.

Decreased mutation frequencies among immunoglobulin G variable region genes during viremic HIV-1 infection.

PubMed

Bowers, Elisabeth; Scamurra, Ronald W; Asrani, Anil; Beniguel, Lydie; MaWhinney, Samantha; Keays, Kathryne M; Thurn, Joseph R; Janoff, Edward N

2014-01-01

HIV-1 infection is complicated by high rates of opportunistic infections against which specific antibodies contribute to immune defense. Antibody function depends on somatic hypermutation (SHM) of variable regions of immunoglobulin heavy chain genes (VH-D-J). We characterized the frequency of SHM in expressed IgG mRNA immunoglobulin transcripts from control and HIV-1-infected patients. We compared utilization of genes in the most prominent VH family (VH3) and mutation frequencies and patterns of cDNA from VH3-IgG genes from 10 seronegative control subjects and 21 patients with HIV-1 infection (6 without and 15 patients with detectable plasma viremia). Unique IgG VH3 family cDNA sequences (n = 1,565) were PCR amplified, cloned, and sequenced from blood. Sequences were analyzed using online (Vbase) and in-house immunoglobulin alignment resources. Mutation frequencies in the antigen-binding hypervariable complementarity determining regions (CDR1/2) of IgG class-switched B cells were lower among viremic HIV-1-infected patients vs. controls for nucleotides (CDR1/2: 10±5% vs. 13.5±6%, p = 0.03) and amino acids (CDR: 20%±10 vs. 25%±12, p = 0.02) and in structural framework regions. Mutation patterns were similar among groups. The most common VH3 gene, VH3-23, was utilized less frequently among viremic HIV-1-infected patients (p = 0.03), and overall, mutation frequencies were decreased in nearly all VH3 genes compared with controls. B cells from HIV-1-infected patients show decreased mutation frequencies, especially in antigen-binding VH3 CDR genes, and selective defects in gene utilization. Similar mutation patterns suggest defects in the quantity, but not quality, of mutator activity. Lower levels of SHM in IgG class-switched B cells from HIV-1-infected patients may contribute to the increased risk of opportunistic infections and impaired humoral responses to preventative vaccines.

Early diagnosis of HIV in children below 18 months using DNA PCR test--assessment of the effectiveness of PMTCT interventions and challenges in early initiation of ART in a resource-limited setting.

PubMed

Gupta, Anil; Singh, Girraj; Kaushik, Pratap; Joshi, Bipin; Kalra, Kamlesh; Chakraborty, Sabyasachi

2013-04-01

This study was conducted to assess efficacy of the current Indian Prevention of Mother-to-Child Transmission (PMTCT) protocol in 217 HIV-exposed infants, and to assess challenges in the early initiation of antiretroviral treatment (ART) in 18 (8.3%) infants with HIV, as determined by the HIV-1 DNA polymerase chain reaction (PCR) test at ≥ 6 weeks to <18 months of age. The mother-to-child transmission (MTCT) rate in 154 mother-baby pairs fully compliant with the PMTCT protocol was 5.2%. However, if 25 pairs who were positive using dried blood spot (DBS) DNA PCR and who did not undergo whole blood testing are included in the analysis, then the overall MTCT rate would be 19.8%. The current protocol is 50% effective considering an MTCT rate of 35-40% without any intervention. ART was initiated in 10 (55.6%) HIV-infected children at a mean ± standard deviation (SD) age of 10.45 ± 4.9 (range: 4-17.5) months; delay resulted in opportunistic infections in one-third of the children. A single-dose nevirapine PMTCT regimen should be replaced by a triple antiretroviral regimen; DBS DNA PCR-positive infants may be given ART, and simultaneously a whole blood specimen should be taken to determine whether ART should be continued.

Rational design based synthetic polyepitope DNA vaccine for eliciting HIV-specific CD8+ T cell responses.

PubMed

Bazhan, S I; Karpenko, L I; Ilyicheva, T N; Belavin, P A; Seregin, S V; Danilyuk, N K; Antonets, D V; Ilyichev, A A

2010-04-01

Advances in defining HIV-1 CD8+ T cell epitopes and understanding endogenous MHC class I antigen processing enable the rational design of polyepitope vaccines for eliciting broadly targeted CD8+ T cell responses to HIV-1. Here we describe the construction and comparison of experimental DNA vaccines consisting of ten selected HLA-A2 epitopes from the major HIV-1 antigens Env, Gag, Pol, Nef, and Vpr. The immunogenicity of designed gene constructs was assessed after double DNA prime, single vaccinia virus boost immunization of HLA-A2 transgenic mice. We compared a number of parameters including different strategies for fusing ubiquitin to the polyepitope and including spacer sequences between epitopes to optimize proteasome liberation and TAP transport. It was demonstrated that the vaccine construct that induced in vitro the largest number of [peptide-MHC class I] complexes was also the most immunogenic in the animal experiments. This most immunogenic vaccine construct contained the N-terminal ubiquitin for targeting the polyepitope to the proteasome and included both proteasome liberation and TAP transport optimized spacer sequences that flanked the epitopes within the polyepitope construct. The immunogenicity of determinants was strictly related to their affinities for HLA-A2. Our finding supports the concept of rational vaccine design based on detailed knowledge of antigen processing. Copyright 2010 Elsevier Ltd. All rights reserved.

Long-Term Resolution of Viral Breakthrough after Changing HIV Viral Load Assay.

PubMed

Obeid, Karam M; Sural, Preethi; Szpunar, Susan; Johnson, Leonard B

2011-01-01

Viral load (VL) measurement assays differ in their sensitivity with polymerase chain reaction assays (PCR) being more sensitive than branched DNA (bDNA) assays. We evaluated virologic outcomes of patients and physicians' response to increased VL after a switch from bDNA to PCR assay. Retrospective, case-control study on 65 HIV+ patients receiving highly active antiretroviral therapy (HAART). Cases included patients with undetectable VL by bDNA that became detectable after the switch; controls were patients that remained undetectable. Records were reviewed up to 1 year after the switch. A total of 58.5% patients had detectable VL after the switch. Repeat VL testing and resistance testing were ordered in 15.4% and 23.1% of these patients, respectively. By 1 year, VL was undetectable in 82.8% of cases and 92% of controls (P = .30), without change in HAART. Transient viremia after changing VL assay reflects the different sensitivity of these assays with no impact on patients' outcomes compared to controls.

Targeted DNA Mutagenesis for the Cure of Chronic Viral Infections

PubMed Central

Schiffer, Joshua T.; Aubert, Martine; Weber, Nicholas D.; Mintzer, Esther; Stone, Daniel

2012-01-01

Human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and herpes simplex virus (HSV) have been incurable to date because effective antiviral therapies target only replicating viruses and do not eradicate latently integrated or nonreplicating episomal viral genomes. Endonucleases that can target and cleave critical regions within latent viral genomes are currently in development. These enzymes are being engineered with high specificity such that off-target binding of cellular DNA will be absent or minimal. Imprecise nonhomologous-end-joining (NHEJ) DNA repair following repeated cleavage at the same critical site may permanently disrupt translation of essential viral proteins. We discuss the benefits and drawbacks of three types of DNA cleavage enzymes (zinc finger endonucleases, transcription activator-like [TAL] effector nucleases [TALENs], and homing endonucleases [also called meganucleases]), the development of delivery vectors for these enzymes, and potential obstacles for successful treatment of chronic viral infections. We then review issues regarding persistence of HIV-1, HBV, and HSV that are relevant to eradication with genome-altering approaches. PMID:22718830

Structural determinants of HIV-1 nucleocapsid protein for cTAR DNA binding and destabilization, and correlation with inhibition of self-primed DNA synthesis.

PubMed

Beltz, Hervé; Clauss, Céline; Piémont, Etienne; Ficheux, Damien; Gorelick, Robert J; Roques, Bernard; Gabus, Caroline; Darlix, Jean-Luc; de Rocquigny, Hugues; Mély, Yves

2005-05-20

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is formed of two highly conserved CCHC zinc fingers flanked by small basic domains. NC is required for the two obligatory strand transfers in viral DNA synthesis through its nucleic acid chaperoning properties. The first DNA strand transfer relies on NC's ability to bind and destabilize the secondary structure of complementary transactivation response region (cTAR) DNA, to inhibit self-priming, and to promote the annealing of cTAR to TAR RNA. To further investigate NC chaperone properties, our aim was to identify by fluorescence spectroscopy and gel electrophoresis, the NC structural determinants for cTAR binding and destabilization, and for the inhibition of self-primed DNA synthesis on a model system using a series of NC mutants and HIV-1 reverse transcriptase. NC destabilization and self-priming inhibition properties were found to be supported by the two fingers in their proper context and the basic (29)RAPRKKG(35) linker. The strict requirement of the native proximal finger suggests that its hydrophobic platform (Val13, Phe16, Thr24 and Ala25) is crucial for binding, destabilization and inhibition of self-priming. In contrast, only partial folding of the distal finger is required, probably for presenting the Trp37 residue in an appropriate orientation. Also, Trp37 and the hydrophobic residues of the proximal finger appear to be essential for the propagation of the melting from the cTAR ends up to the middle of the stem. Finally, both N-terminal and C-terminal basic domains contribute to cTAR binding but not to its destabilization.

Potent Cell-Intrinsic Immune Responses in Dendritic Cells Facilitate HIV-1-Specific T Cell Immunity in HIV-1 Elite Controllers.

PubMed

Martin-Gayo, Enrique; Buzon, Maria Jose; Ouyang, Zhengyu; Hickman, Taylor; Cronin, Jacqueline; Pimenova, Dina; Walker, Bruce D; Lichterfeld, Mathias; Yu, Xu G

2015-06-01

The majority of HIV-1 elite controllers (EC) restrict HIV-1 replication through highly functional HIV-1-specific T cell responses, but mechanisms supporting the evolution of effective HIV-1-specific T cell immunity in these patients remain undefined. Cytosolic immune recognition of HIV-1 in conventional dendritic cells (cDC) can facilitate priming and expansion of HIV-1-specific T cells; however, HIV-1 seems to be able to avoid intracellular immune recognition in cDCs in most infected individuals. Here, we show that exposure of cDCs from EC to HIV-1 leads to a rapid and sustained production of type I interferons and upregulation of several interferon-stimulated effector genes. Emergence of these cell-intrinsic immune responses was associated with a reduced induction of SAMHD1 and LEDGF/p75, and an accumulation of viral reverse transcripts, but inhibited by pharmacological blockade of viral reverse transcription or siRNA-mediated silencing of the cytosolic DNA sensor cGAS. Importantly, improved cell-intrinsic immune recognition of HIV-1 in cDCs from elite controllers translated into stronger abilities to stimulate and expand HIV-1-specific CD8 T cell responses. These data suggest an important role of cell-intrinsic type I interferon secretion in dendritic cells for the induction of effective HIV-1-specific CD8 T cells, and may be helpful for eliciting functional T cell immunity against HIV-1 for preventative or therapeutic clinical purposes.

Oral human Papillomavirus DNA detection in HIV-positive men: prevalence, predictors, and co-occurrence at anal site.

PubMed

Vergori, Alessandra; Garbuglia, Anna Rosa; Piselli, Pierluca; Del Nonno, Franca; Sias, Catia; Lupi, Federico; Lapa, Daniele; Baiocchini, Andrea; Cimaglia, Claudia; Gentile, Marco; Antinori, Andrea; Capobianchi, Maria; Ammassari, Adriana

2018-01-08

HIV-positive patients carry an increased risk of HPV infection and associated cancers. Therefore, prevalence and patterns of HPV infection at different anatomical sites, as well as theoretical protection of nonavalent vaccine should be investigated. Aim was to describe prevalence and predictors of oral HPV detection in HIV-positive men, with attention to nonavalent vaccine-targeted HPV types. Further, co-occurrence of HPV DNA at oral cavity and at anal site was assessed. This cross-sectional, clinic-based study included 305 HIV-positive males (85.9% MSM; median age 44.7 years; IQR: 37.4-51.0), consecutively observed within an anal cancer screening program, after written informed consent. Indication for anal screening was given by the HIV physician during routine clinic visit. Paired oral rinse and anal samples were processed for the all HPV genotypes with QIASYMPHONY and a PCR with MY09/MY11 primers for the L1 region. At the oral cavity, HPV DNA was detected in 64 patients (20.9%), and in 28.1% of these cases multiple HPV infections were found. Prevalence of oral HPV was significantly lower than that observed at the anal site (p < 0.001), where HPV DNA was found in 199 cases (85.2%). Oral HPV tended to be more frequent in patients with detectable anal HPV than in those without (p = 0.08). Out of 265 HPV DNA-positive men regardless anatomic site, 59 cases (19.3%) had detectable HPV at both sites, and 51 of these showed completely different HPV types. At least one nonavalent vaccine-targeted HPV type was found in 17/64 (26.6%) of patients with oral and 199/260 (76.5%) with anal infection. At multivariable analysis, factors associated with positive oral HPV were: CD4 cells <200/μL (versus CD4 cells >200/μL, p = 0.005) and >5 sexual partners in the previous 12 months (versus 0-1 partner, p = 0.008). In this study on Italian HIV-positive men (predominantly MSM), oral HPV DNA was detected in approximately one fifth of tested subjects, but prevalence was significantly lower than that observed at anal site. Low CD4 cell count and increasing number of recent sexual partners significantly increased the odds of positive oral HPV. The absence of co-occurrence at the two anatomical sites may suggest different routes or timing of infection.

A Sensitive Branched DNA HIV-1 Signal Amplification Viral Load Assay with Single Day Turnaround

PubMed Central

Baumeister, Mark A.; Zhang, Nan; Beas, Hilda; Brooks, Jesse R.; Canchola, Jesse A.; Cosenza, Carlo; Kleshik, Felix; Rampersad, Vinod; Surtihadi, Johan; Battersby, Thomas R.

2012-01-01

Branched DNA (bDNA) is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA) (“Versant Assay”) currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16–18 h to 2.5 h, composition of only the “Lysis Diluent” solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available) was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results) were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135) showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements. PMID:22479381

A sensitive branched DNA HIV-1 signal amplification viral load assay with single day turnaround.

PubMed

Baumeister, Mark A; Zhang, Nan; Beas, Hilda; Brooks, Jesse R; Canchola, Jesse A; Cosenza, Carlo; Kleshik, Felix; Rampersad, Vinod; Surtihadi, Johan; Battersby, Thomas R

2012-01-01

Branched DNA (bDNA) is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA) ("Versant Assay") currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16-18 h to 2.5 h, composition of only the "Lysis Diluent" solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available) was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results) were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135) showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements.

EV02: a Phase I trial to compare the safety and immunogenicity of HIV DNA-C prime-NYVAC-C boost to NYVAC-C alone.

PubMed

McCormack, Sheena; Stöhr, Wolfgang; Barber, Tristan; Bart, Pierre-Alexandre; Harari, Alexandre; Moog, Christiane; Ciuffreda, Donatella; Cellerai, Cristina; Cowen, Miranda; Gamboni, Romilda; Burnet, Séverine; Legg, Ken; Brodnicki, Elizabeth; Wolf, Hans; Wagner, Ralf; Heeney, Jonathan; Frachette, Marie-Joëlle; Tartaglia, Jim; Babiker, Abdel; Pantaleo, Giuseppe; Weber, Jonathan

2008-06-13

The aim of this randomised controlled trial was to see if the addition of 4 mg/ml DNA-C priming given by the intramuscular route at weeks 0 and 4 to NYVAC-C at weeks 20 and 24, safely increased the proportion of participants with HIV-specific T-cell responses measured by the interferon (IFN)-gamma ELISpot assay at weeks 26 and/or 28 compared to NYVAC-C alone. Although 2 individuals discontinued after the first DNA-C due to adverse events (1 vaso-vagal; 1 transient, asymptomatic elevation in alanine transaminase), the vaccines were well tolerated. Three others failed to complete the regimen (1 changed her mind; 2 lost to follow-up). Of the 35 that completed the regimen 90% (18/20) in the DNA-C group had ELISpot responses compared to 33% (5/15) that received NYVAC-C alone (p=0.001). Responses were to envelope in the majority (21/23). Of the 9 individuals with responses to envelope and other peptides, 8 were in the DNA-C group. These promising results suggest that DNA-C was an effective priming agent, that merits further investigation.

Structure-Based Mutational Analysis of the C-Terminal DNA-Binding Domain of Human Immunodeficiency Virus Type 1 Integrase: Critical Residues for Protein Oligomerization and DNA Binding

PubMed Central

Lutzke, Ramon A. Puras; Plasterk, Ronald H. A.

1998-01-01

The C-terminal domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a dimer that binds to DNA in a nonspecific manner. The structure of the minimal region required for DNA binding (IN220–270) has been solved by nuclear magnetic resonance spectroscopy. The overall fold of the C-terminal domain of HIV-1 IN is similar to those of Src homology region 3 domains. Based on the structure of IN220–270, we studied the role of 15 amino acid residues potentially involved in DNA binding and oligomerization by mutational analysis. We found that two amino acid residues, arginine 262 and leucine 234, contribute to DNA binding in the context of IN220–270, as indicated by protein-DNA UV cross-link analysis. We also analyzed mutant proteins representing portions of the full-length IN protein. Amino acid substitution of residues located in the hydrophobic dimer interface, such as L241A and L242A, results in the loss of oligomerization of IN; consequently, the levels of 3′ processing, DNA strand transfer, and intramolecular disintegration are strongly reduced. These results suggest that dimerization of the C-terminal domain of IN is important for correct multimerization of IN. PMID:9573250

Short Communication: Transplacental Nucleoside Analogue Exposure and Mitochondrial Parameters in HIV-Uninfected Children

PubMed Central

Brogly, Susan B.; DiMauro, Salvatore; Van Dyke, Russell B.; Williams, Paige L.; Naini, Ali; Libutti, Daniel E.; Choi, Julia; Chung, Michelle

2011-01-01

Abstract Transplacental nucleoside analogue exposure can affect infant mitochondrial DNA (mtDNA). We evaluated mitochondria in peripheral blood mononuclear cells of children with and without clinical signs of mitochondrial dysfunction (MD) and antiretroviral (ARV) exposure. We previously identified 20 children with signs of MD (cases) among 1037 HIV-uninfected children born to HIV-infected women. We measured mtDNA copies/cell and oxidative phosphorylation (OXPHOS) NADH dehydrogenase (complex I) and cytochrome c oxidase (complex IV) protein levels and enzyme activities, determined mtDNA haplogroups and deletions in 18 of 20 cases with stored samples and in sex- and age-matched HIV-uninfected children, both ARV exposed and unexposed, (1) within 18 months of birth and (2) at the time of presentation of signs of MD. In specimens drawn within 18 months of birth, mtDNA levels were higher and OXPHOS protein levels and enzyme activities lower in cases than controls. In contrast, at the time of MD presentation, cases and ARV-exposed controls had lower mtDNA levels, 214 and 215 copies/cell, respectively, than ARV-unexposed controls, 254 copies/cell. OXPHOS protein levels and enzyme activities were lower in cases than exposed controls, and higher in cases than unexposed controls, except for complex IV activity, which was higher in cases. Haplotype H was less frequent among cases (6%) than controls (31%). No deletions were found. The long-term significance of these small but potentially important alterations should continue to be studied as these children enter adolescence and adulthood. PMID:21142587

Impact of Maternal HIV Seroconversion during Pregnancy on Early Mother to Child Transmission of HIV (MTCT) Measured at 4-8 Weeks Postpartum in South Africa 2011-2012: A National Population-Based Evaluation

PubMed Central

Dinh, Thu-Ha; Delaney, Kevin P.; Goga, Ameena; Jackson, Debra; Lombard, Carl; Woldesenbet, Selamawit; Mogashoa, Mary; Pillay, Yogan; Shaffer, Nathan

2015-01-01

Background Mother-to-child transmission of HIV (MTCT) depends on the timing of HIV infection. We estimated HIV-seroconversion during pregnancy (HSP) after having a HIV-negative result antenatally, and its contribution to early MTCT in South Africa (SA). Methods and Findings Between August 2011 and March 2012, we recruited a nationally representative sample of mother-infant pairs with infants aged 4-to-8 weeks from 578 health facilities. Data collection included mother interviews, child health-card reviews, and infant dried-blood-spots sample (iDBS). iDBS were tested for HIV antibodies and HIV-deoxyribonucleic-acid (HIV-DNA). HSP was defined as maternal self-report of an HIV-negative test during this pregnancy, no documented use of antiretroviral drugs and a matched HIV sero-positive iDBS. We used 20 imputations from a uniform distribution for time from reported antenatal HIV-negative result to delivery to estimate time of HSP. Early MTCT was defined based on detection of HIV-DNA in iDBS. Estimates were adjusted for clustering, nonresponse, and weighted by SA’s 2011 live-births. Results Of 9802 mother-infant pairs, 2738 iDBS were HIV sero-positive, including 212 HSP, resulting in a nationally weighted estimate of 3.3% HSP (95% Confidence Interval: 2.8%-3.8%). Median time of HIV-seroconversion was 32.8weeks gestation;28.3% (19.7%- 36.9%) estimated to be >36 weeks. Early MTCT was 10.7% for HSP (6.2%-16.8%) vs. 2.2% (1.7%-2.8%) for mothers with known HIV-positive status. Although they represent 2.2% of all mothers and 6.7% of HIV-infected mothers, HSP accounted for 26% of early MTCT. Multivariable analysis indicated the highest risk for HSP was among women who knew the baby’s father was HIV-infected (adjusted-hazard ratio (aHR) 4.71; 1.49-14.99), or who had been screened for tuberculosis (aHR 1.82; 1.43-2.32). Conclusions HSP risk is high and contributes significantly to early MTCT. Identification of HSP by repeat-testing at 32 weeks gestation, during labor, 6 weeks postpartum, in tuberculosis-exposed women, and in discordant couples might reduce MTCT. PMID:25942423

Genital warts and infection with human immunodeficiency virus in high-risk women in Burkina Faso: a longitudinal study

PubMed Central

2011-01-01

Background Human papillomaviruses are the most common sexually transmitted infections, and genital warts, caused by HPV-6 and 11, entail considerable morbidity and cost. The natural history of genital warts in relation to HIV-1 infection has not been described in African women. We examined risk factors for genital warts in a cohort of high-risk women in Burkina Faso, in order to further describe their epidemiology. Methods A prospective study of 765 high-risk women who were followed at 4-monthly intervals for 27 months in Burkina Faso. Logistic and Cox regression were used to identify factors associated with prevalent, incident and persistent genital warts, including HIV-1 serostatus, CD4+ count, and concurrent sexually transmitted infections. In a subset of 306 women, cervical HPV DNA was tested at enrolment. Results Genital wart prevalence at baseline was 1.6% (8/492) among HIV-uninfected and 7.0% (19/273) among HIV-1 seropositive women. Forty women (5.2%) experienced at least one incident GW episode. Incidence was 1.1 per 100 person-years among HIV-uninfected women, 7.4 per 100 person-years among HIV-1 seropositive women with a nadir CD4+ count >200 cells/μL and 14.6 per 100 person-years among HIV-1 seropositive women with a nadir CD4+ count ≤200 cells/μL. Incident genital warts were also associated with concurrent bacterial vaginosis, and genital ulceration. Antiretroviral therapy was not protective against incident or persistent genital warts. Detection of HPV-6 DNA and abnormal cervical cytology were strongly associated with incident genital warts. Conclusions Genital warts occur much more frequently among HIV-1 infected women in Africa, particularly among those with low CD4+ counts. Antiretroviral therapy did not reduce the incidence or persistence of genital warts in this population. PMID:21251265

Genital warts and infection with human immunodeficiency virus in high-risk women in Burkina Faso: a longitudinal study.

PubMed

Low, Andrea J; Clayton, Tim; Konate, Issouf; Nagot, Nicolas; Ouedraogo, Abdoulaye; Huet, Charlotte; Didelot-Rousseau, Marie-Noelle; Segondy, Michel; Van de Perre, Philippe; Mayaud, Philippe

2011-01-20

Human papillomaviruses are the most common sexually transmitted infections, and genital warts, caused by HPV-6 and 11, entail considerable morbidity and cost. The natural history of genital warts in relation to HIV-1 infection has not been described in African women. We examined risk factors for genital warts in a cohort of high-risk women in Burkina Faso, in order to further describe their epidemiology. A prospective study of 765 high-risk women who were followed at 4-monthly intervals for 27 months in Burkina Faso. Logistic and Cox regression were used to identify factors associated with prevalent, incident and persistent genital warts, including HIV-1 serostatus, CD4+ count, and concurrent sexually transmitted infections. In a subset of 306 women, cervical HPV DNA was tested at enrollment. Genital wart prevalence at baseline was 1.6% (8/492) among HIV-uninfected and 7.0% (19/273) among HIV-1 seropositive women. Forty women (5.2%) experienced at least one incident GW episode. Incidence was 1.1 per 100 person-years among HIV-uninfected women, 7.4 per 100 person-years among HIV-1 seropositive women with a nadir CD4+ count >200 cells/μL and 14.6 per 100 person-years among HIV-1 seropositive women with a nadir CD4+ count ≤ 200 cells/μL. Incident genital warts were also associated with concurrent bacterial vaginosis, and genital ulceration. Antiretroviral therapy was not protective against incident or persistent genital warts. Detection of HPV-6 DNA and abnormal cervical cytology were strongly associated with incident genital warts. Genital warts occur much more frequently among HIV-1 infected women in Africa, particularly among those with low CD4+ counts. Antiretroviral therapy did not reduce the incidence or persistence of genital warts in this population.

Initiation of antiretroviral therapy before detection of colonic infiltration by HIV reduces viral reservoirs, inflammation and immune activation

PubMed Central

Crowell, Trevor A; Fletcher, James LK; Sereti, Irini; Pinyakorn, Suteeraporn; Dewar, Robin; Krebs, Shelly J; Chomchey, Nitiya; Rerknimitr, Rungsun; Schuetz, Alexandra; Michael, Nelson L; Phanuphak, Nittaya; Chomont, Nicolas; Ananworanich, Jintanat

2016-01-01

Introduction Colonic infiltration by HIV occurs soon after infection, establishing a persistent viral reservoir and a barrier to cure. We investigated virologic and immunologic correlates of detectable colonic HIV RNA during acute HIV infection (AHI) and their response to antiretroviral treatment (ART). Methods From 49,458 samples screened for HIV, 74 participants were enrolled during AHI and 41 consented to optional sigmoidoscopy, HIV RNA was categorized as detectable (≥50 copies/mg) or undetectable in homogenized colon biopsy specimens. Biomarkers and HIV burden in blood, colon and cerebrospinal fluid were compared between groups and after 24 weeks of ART. Results Colonic HIV RNA was detectable in 31 participants (76%) and was associated with longer duration since HIV exposure (median 16 vs. 11 days, p=0.02), higher median plasma levels of cytokines and inflammatory markers (CXCL10 476 vs. 148 pg/mL, p=0.02; TNF-RII 1036 vs. 649 pg/mL, p<0.01; neopterin 2405 vs. 1368 pg/mL, p=0.01) and higher levels of CD8+ T cell activation in the blood (human leukocyte antigen - antigen D related (HLA-DR)/CD38 expression 14.4% vs. 7.6%, p <0.01) and colon (8.9% vs. 4.5%, p=0.01). After 24 weeks of ART, participants with baseline detectable colonic HIV RNA demonstrated persistent elevations in total HIV DNA in colonic mucosal mononuclear cells (CMMCs) (median 61 vs. 0 copies/106 CMMCs, p=0.03) and a trend towards higher total HIV DNA in peripheral blood mononuclear cells (PBMC) (41 vs. 1.5 copies/106 PBMCs, p=0.06). There were no persistent differences in immune activation and inflammation. Conclusions The presence of detectable colonic HIV RNA at the time of ART initiation during AHI is associated with higher levels of proviral DNA after 24 weeks of treatment. Seeding of HIV in the gut may have long-lasting effects on the size of persistent viral reservoirs and may represent an important therapeutic target in eradication strategies. PMID:27637172

Multifaceted counter-APOBEC3G mechanisms employed by HIV-1 Vif.

PubMed

Britan-Rosich, Elena; Nowarski, Roni; Kotler, Moshe

2011-07-29

In the absence of human immunodeficiency virus type 1 (HIV-1) Vif protein, the host antiviral deaminase apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (A3G) restricts the production of infectious HIV-1 by deamination of dC residues in the negative single-stranded DNA produced by reverse transcription. The Vif protein averts the lethal threat of deamination by precluding the packaging of A3G into assembling virions by mediating proteasomal degradation of A3G. In spite of this robust Vif activity, residual A3G molecules that escape degradation and incorporate into newly assembled virions are potentially deleterious to the virus. We hypothesized that virion-associated Vif inhibits A3G enzymatic activity and therefore prevents lethal mutagenesis of the newly synthesized viral DNA. Here, we show that (i) Vif-proficient HIV-1 particles released from H9 cells contain A3G with lower specific activity compared with Δvif-virus-associated A3G, (ii) encapsidated HIV-1 Vif inhibits the deamination activity of recombinant A3G, and (iii) purified HIV-1 Vif protein and the Vif-derived peptide Vif25-39 inhibit A3G activity in vitro at nanomolar concentrations in an uncompetitive manner. Our results manifest the potentiality of Vif to control the deamination threat in virions or in the pre-integration complexes following entry to target cells. Hence, virion-associated Vif could serve as a last line of defense, protecting the virus against A3G antiviral activity. Copyright © 2011 Elsevier Ltd. All rights reserved.

Performance of an Early Infant Diagnostic Test, AmpliSens DNA-HIV-FRT, Using Dried Blood Spots Collected from Children Born to Human Immunodeficiency Virus-Infected Mothers in Ukraine

PubMed Central

Shanmugam, Vedapuri; Azarskova, Marianna; Nguyen, Shon; Hurlston, Mackenzie; Sabatier, Jennifer; Zhang, Guoqing; Osmanov, Saladin; Ellenberger, Dennis; Yang, Chunfu; Vitek, Charles; Liulchuk, Maria; Nizova, Natalya

2015-01-01

An accurate accessible test for early infant diagnosis (EID) is crucial for identifying HIV-infected infants and linking them to treatment. To improve EID services in Ukraine, dried blood spot (DBS) samples obtained from 237 HIV-exposed children (≤18 months of age) in six regions in Ukraine in 2012 to 2013 were tested with the AmpliSens DNA-HIV-FRT assay, the Roche COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) HIV-1 Qual test, and the Abbott RealTime HIV-1 Qualitative assay. In comparison with the paired whole-blood results generated from AmpliSens testing at the oblast HIV reference laboratories in Ukraine, the sensitivity was 0.99 (95% confidence interval [CI], 0.95 to 1.00) for the AmpliSens and Roche CAP/CTM Qual assays and 0.96 (95% CI, 0.90 to 0.98) for the Abbott Qualitative assay. The specificity was 1.00 (95% CI, 0.97 to 1.00) for the AmpliSens and Abbott Qualitative assays and 0.99 (95% CI, 0.96 to 1.00) for the Roche CAP/CTM Qual assay. McNemar analysis indicated that the proportions of positive results for the tests were not significantly different (P > 0.05). Cohen's kappa (0.97 to 0.99) indicated almost perfect agreement among the three tests. These results indicated that the AmpliSens DBS and whole-blood tests performed equally well and were comparable to the two commercially available EID tests. More importantly, the performance characteristics of the AmpliSens DBS test meets the World Health Organization EID test requirements; implementing AmpliSens DBS testing might improve EID services in resource-limited settings. PMID:26447114

Insight into HIV-2 latency may disclose strategies for a cure for HIV-1 infection.

PubMed

Saleh, Suha; Vranckx, Lenard; Gijsbers, Rik; Christ, Frauke; Debyser, Zeger

2017-01-01

HIV-1 and HIV-2 originate from two distinct zoonotic transmissions of simian immunodeficiency viruses from primate to human. Although both share similar modes of transmission and can result in the development of AIDS with similar clinical manifestations, HIV-2 infection is generally milder and less likely to progress to AIDS. HIV is currently incurable due to the presence of HIV provirus integrated into the host DNA of long-lived memory cells of the immune system without active replication. As such, the latent virus is immunologically inert and remains insensitive to the administered antiviral drugs targeting active viral replication steps. Recent evidence suggests that persistent HIV replication may occur in anatomical sanctuaries such as the lymphoid tissue due to low drug penetration. At present, different strategies are being evaluated either to completely eradicate the virus from the patient (sterilising cure) or to allow treatment interruption without viral rebound (functional cure). Because HIV-2 is naturally less pathogenic and displays a more latent phenotype than HIV-1, it may represent a valuable model that provides elementary information to cure HIV-1 infection. Insight into the viral and cellular determinants of HIV-2 replication may therefore pave the way for alternative strategies to eradicate HIV-1 or promote viral remission.

Recombinant Salmonella Bacteria Vectoring HIV/AIDS Vaccines

PubMed Central

Chin’ombe, Nyasha; Ruhanya, Vurayai

2013-01-01

HIV/AIDS is an important public health problem globally. An affordable, easy-to-deliver and protective HIV vaccine is therefore required to curb the pandemic from spreading further. Recombinant Salmonella bacteria can be harnessed to vector HIV antigens or DNA vaccines to the immune system for induction of specific protective immunity. These are capable of activating the innate, humoral and cellular immune responses at both mucosal and systemic compartments. Several studies have already demonstrated the utility of live recombinant Salmonella in delivering expressed foreign antigens as well as DNA vaccines to the host immune system. This review gives an overview of the studies in which recombinant Salmonella bacteria were used to vector HIV/AIDS antigens and DNA vaccines. Most of the recombinant Salmonella-based HIV/AIDS vaccines developed so far have only been tested in animals (mainly mice) and are yet to reach human trials. PMID:24478808

Metabarcoding analysis of eukaryotic microbiota in the gut of HIV-infected patients.

PubMed

Hamad, Ibrahim; Abou Abdallah, Rita; Ravaux, Isabelle; Mokhtari, Saadia; Tissot-Dupont, Hervé; Michelle, Caroline; Stein, Andreas; Lagier, Jean-Christophe; Raoult, Didier; Bittar, Fadi

2018-01-01

Research on the relationship between changes in the gut microbiota and human disease, including AIDS, is a growing field. However, studies on the eukaryotic component of the intestinal microbiota have just begun and have not yet been conducted in HIV-infected patients. Moreover, eukaryotic community profiling is influenced by the use of different methodologies at each step of culture-independent techniques. Herein, initially, four DNA extraction protocols were compared to test the efficiency of each method in recovering eukaryotic DNA from fecal samples. Our results revealed that recovering eukaryotic components from fecal samples differs significantly among DNA extraction methods. Subsequently, the composition of the intestinal eukaryotic microbiota was evaluated in HIV-infected patients and healthy volunteers through clone sequencing, high-throughput sequencing of nuclear ribosomal internal transcribed spacers 1 (ITS1) and 2 (ITS2) amplicons and real-time PCRs. Our results revealed that not only richness (Chao-1 index) and alpha diversity (Shannon diversity) differ between HIV-infected patients and healthy volunteers, depending on the molecular strategy used, but also the global eukaryotic community composition, with little overlapping taxa found between techniques. Moreover, our results based on cloning libraries and ITS1/ITS2 metabarcoding sequencing showed significant differences in fungal composition between HIV-infected patients and healthy volunteers, but without distinct clusters separating the two groups. Malassezia restricta was significantly more prevalent in fecal samples of HIV-infected patients, according to cloning libraries, whereas operational taxonomic units (OTUs) belonging to Candida albicans and Candida tropicalis were significantly more abundant in fecal samples of HIV-infected patients compared to healthy subjects in both ITS subregions. Finally, real-time PCR showed the presence of Microsporidia, Giardia lamblia, Blastocystis and Hymenolepis diminuta in different proportions in fecal samples from HIV patients as compared to healthy individuals. Our work revealed that the use of different sequencing approaches can impact the perceived eukaryotic diversity results of the human gut. We also provide a more comprehensive view of the eukaryotic community in the gut of HIV-infected patients through the complementarity of the different molecular techniques used. Combining these various methodologies may provide a gold standard for a more complete characterization of the eukaryotic microbiome in future studies.

Docking of anti-HIV-1 oxoquinoline-acylhydrazone derivatives as potential HSV-1 DNA polymerase inhibitors

NASA Astrophysics Data System (ADS)

Yoneda, Julliane Diniz; Albuquerque, Magaly Girão; Leal, Kátia Zaccur; Santos, Fernanda da Costa; Batalha, Pedro Netto; Brozeguini, Leonardo; Seidl, Peter R.; de Alencastro, Ricardo Bicca; Cunha, Anna Cláudia; de Souza, Maria Cecília B. V.; Ferreira, Vitor F.; Giongo, Viveca A.; Cirne-Santos, Cláudio; Paixão, Izabel C. P.

2014-09-01

Although there are many antiviral drugs available for the treatment of herpes simplex virus (HSV) infections, still the synthesis of new anti-HSV candidates is an important strategy to be pursued, due to the emergency of resistant HSV strains mainly in human immunodeficiency virus (HIV) co-infected patients. Some 1,4-dihydro-4-oxoquinolines, such as PNU-183792 (1), show a broad spectrum antiviral activity against human herpes viruses, inhibiting the viral DNA polymerase (POL) without affecting the human POLs. Thus, on an ongoing antiviral research project, our group has synthesized ribonucleosides containing the 1,4-dihydro-4-oxoquinoline (quinolone) heterocyclic moiety, such as the 6-Cl derivative (2), which is a dual antiviral agent (HSV-1 and HIV-1). Molecular dynamics simulations of the complexes of 1 and 2 with the HSV-1 POL suggest that structural modifications of 2 should increase its experimental anti-HSV-1 activity, since its ribosyl and carboxyl groups are highly hydrophilic to interact with a hydrophobic pocket of this enzyme. Therefore, in this work, comparative molecular docking simulations of 1 and three new synthesized oxoquinoline-acylhydrazone HIV-1 inhibitors (3-5), which do not contain those hydrophilic groups, were carried out, in order to access these modifications in the proposition of new potential anti-HSV-1 agents, but maintaining the anti-HIV-1 activity. Among the docked compounds, the oxoquinoline-acylhydrazone 3 is the best candidate for an anti-HSV-1 agent, and, in addition, it showed anti-HIV-1 activity (EC50 = 3.4 ± 0.3 μM). Compounds 2 and 3 were used as templates in the design of four new oxoquinoline-acylhydrazones (6-9) as potential anti-HSV-1 agents to increase the antiviral activity of 2. Among the docked compounds, oxoquinoline-acylhydrazone 7 was selected as the best candidate for further development of dual anti-HIV/HSV activity.

Hypochondria as an actual neurosis.

PubMed

Nissen, Bernd

2017-09-27

Freud defined hypochondria as an actual neurosis. In this paper the actual neurosis will be interpreted as unbound traumatic elements which threaten the self. In severe hypochondria, breakdowns have occurred, as outlined by Winnicott. The nameless traumatic elements of the breakdown have been encapsulated. The moment these encapsulated elements are liberated, an actual dynamic takes place which threatens the self with annihilation. Projective identification is not possible because no idea of containment exists. The self tries to evacuate these elements projectively, thus triggering a disintegrative regression. However, the object of this projection, which becomes a malign introject, is felt to remove the remaining psychical elements, forcing the worthless residue back into the self. In a final re-introjection, the self is threatened by unintegration. To save the self, these elements are displaced into an organ which becomes hypochondriacal, an autistoid object, protecting itself against unintegration and decomposition. An autistoid dynamic develops between the hypochondriac organ, the ego and the introject. Two short clinical vignettes illustrate the regressive dynamical and metapsychological considerations. Copyright © 2017 Institute of Psychoanalysis.

Structural insights into the cTAR DNA recognition by the HIV-1 nucleocapsid protein: role of sugar deoxyriboses in the binding polarity of NC

PubMed Central

Bazzi, Ali; Zargarian, Loussiné; Chaminade, Françoise; Boudier, Christian; De Rocquigny, Hughes; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

2011-01-01

An essential step of the reverse transcription of the HIV-1 genome is the first strand transfer that requires the annealing of the TAR RNA hairpin to the cTAR DNA hairpin. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. Using nuclear magnetic resonance and gel retardation assays, we investigated the interaction between NC and the top half of the cTAR DNA (mini-cTAR). We show that NC(11-55) binds the TGG sequence in the lower stem that is destabilized by the adjacent internal loop. The 5′ thymine interacts with residues of the N-terminal zinc knuckle and the 3′ guanine is inserted in the hydrophobic plateau of the C-terminal zinc knuckle. The TGG sequence is preferred relative to the apical and internal loops containing unpaired guanines. Investigation of the DNA–protein contacts shows the major role of hydrophobic interactions involving nucleobases and deoxyribose sugars. A similar network of hydrophobic contacts is observed in the published NC:DNA complexes, whereas NC contacts ribose differently in NC:RNA complexes. We propose that the binding polarity of NC is related to these contacts that could be responsible for the preferential binding to single-stranded nucleic acids. PMID:21227929

HIV Tat/P-TEFb Interaction: A Potential Target for Novel Anti-HIV Therapies.

PubMed

Asamitsu, Kaori; Fujinaga, Koh; Okamoto, Takashi

2018-04-17

Transcription is a crucial step in the life cycle of the human immunodeficiency virus type 1 (HIV 1) and is primarily involved in the maintenance of viral latency. Both viral and cellular transcription factors, including transcriptional activators, suppressor proteins and epigenetic factors, are involved in HIV transcription from the proviral DNA integrated within the host cell genome. Among them, the virus-encoded transcriptional activator Tat is the master regulator of HIV transcription. Interestingly, unlike other known transcriptional activators, Tat primarily activates transcriptional elongation and initiation by interacting with the cellular positive transcriptional elongation factor b (P-TEFb). In this review, we describe the molecular mechanism underlying how Tat activates viral transcription through interaction with P-TEFb. We propose a novel therapeutic strategy against HIV replication through blocking Tat action.

Boosting of HIV-1 neutralizing antibody responses by a distally related retroviral envelope protein.

PubMed

Uchtenhagen, Hannes; Schiffner, Torben; Bowles, Emma; Heyndrickx, Leo; LaBranche, Celia; Applequist, Steven E; Jansson, Marianne; De Silva, Thushan; Back, Jaap Willem; Achour, Adnane; Scarlatti, Gabriella; Fomsgaard, Anders; Montefiori, David; Stewart-Jones, Guillaume; Spetz, Anna-Lena

2014-06-15

Our knowledge of the binding sites for neutralizing Abs (NAb) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B cell responses to vulnerable conserved sites within the HIV-1 envelope glycoprotein (Env). In this article, we report an immunization strategy composed of a trivalent HIV-1 (clade B envs) DNA prime, followed by a SIVmac239 gp140 Env protein boost that aimed to focus the immune response to structurally conserved parts of the HIV-1 and simian immunodeficiency virus (SIV) Envs. Heterologous NAb titers, primarily to tier 1 HIV-1 isolates, elicited during the trivalent HIV-1 env prime, were significantly increased by the SIVmac239 gp140 protein boost in rabbits. Epitope mapping of Ab-binding reactivity revealed preferential recognition of the C1, C2, V2, V3, and V5 regions. These results provide a proof of concept that a distally related retroviral SIV Env protein boost can increase pre-existing NAb responses against HIV-1. Copyright © 2014 by The American Association of Immunologists, Inc.

Replication Competent Molecular Clones of HIV-1 Expressing Renilla Luciferase Facilitate the Analysis of Antibody Inhibition in PBMC

PubMed Central

Edmonds, Tara G.; Ding, Haitao; Yuan, Xing; Wei, Qing; Smith, Kendra S.; Conway, Joan A.; Wieczorek, Lindsay; Brown, Bruce; Polonis, Victoria; West, John T.; Montefiori, David C.; Kappes, John C.; Ochsenbauer, Christina

2010-01-01

Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy. PMID:20863545

Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC.

PubMed

Edmonds, Tara G; Ding, Haitao; Yuan, Xing; Wei, Qing; Smith, Kendra S; Conway, Joan A; Wieczorek, Lindsay; Brown, Bruce; Polonis, Victoria; West, John T; Montefiori, David C; Kappes, John C; Ochsenbauer, Christina

2010-12-05

Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy. Copyright © 2010 Elsevier Inc. All rights reserved.

Suboptimal Doses of Raltegravir Cause Aberrant HIV Integrations | Center for Cancer Research

Cancer.gov

When a cell is infected with HIV, a DNA copy of the HIV genome is inserted into that cell’s chromosomal DNA. This insertion reaction is carried out by the viral enzyme integrase (IN) and involves two distinct steps: removal of two nucleotides from each 3’ end of the viral DNA, followed by the strand transfer reaction, in which the viral DNA ends are inserted into the host chromosomal DNA. Integration is essential for viral replication, making it an important target for antiviral therapy. Raltegravir, and the other approved integrase inhibitor, Elvitegravir, are called integrase strand transfer inhibitors (INSTIs), because they bind to the active site of IN and block the strand transfer reaction.      

Improved detection of CXCR4-using HIV by V3 genotyping: application of population-based and "deep" sequencing to plasma RNA and proviral DNA.

PubMed

Swenson, Luke C; Moores, Andrew; Low, Andrew J; Thielen, Alexander; Dong, Winnie; Woods, Conan; Jensen, Mark A; Wynhoven, Brian; Chan, Dennison; Glascock, Christopher; Harrigan, P Richard

2010-08-01

Tropism testing should rule out CXCR4-using HIV before treatment with CCR5 antagonists. Currently, the recombinant phenotypic Trofile assay (Monogram) is most widely utilized; however, genotypic tests may represent alternative methods. Independent triplicate amplifications of the HIV gp120 V3 region were made from either plasma HIV RNA or proviral DNA. These underwent standard, population-based sequencing with an ABI3730 (RNA n = 63; DNA n = 40), or "deep" sequencing with a Roche/454 Genome Sequencer-FLX (RNA n = 12; DNA n = 12). Position-specific scoring matrices (PSSMX4/R5) (-6.96 cutoff) and geno2pheno[coreceptor] (5% false-positive rate) inferred tropism from V3 sequence. These methods were then independently validated with a separate, blinded dataset (n = 278) of screening samples from the maraviroc MOTIVATE trials. Standard sequencing of HIV RNA with PSSM yielded 69% sensitivity and 91% specificity, relative to Trofile. The validation dataset gave 75% sensitivity and 83% specificity. Proviral DNA plus PSSM gave 77% sensitivity and 71% specificity. "Deep" sequencing of HIV RNA detected >2% inferred-CXCR4-using virus in 8/8 samples called non-R5 by Trofile, and <2% in 4/4 samples called R5. Triplicate analyses of V3 standard sequence data detect greater proportions of CXCR4-using samples than previously achieved. Sequencing proviral DNA and "deep" V3 sequencing may also be useful tools for assessing tropism.

Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method.

PubMed

McFall, Sally M; Neto, Mário F; Reed, Jennifer L; Wagner, Robin L

2016-08-06

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 µl whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

Multi-spectroscopic and molecular docking studies on the interaction of darunavir, a HIV protease inhibitor with calf thymus DNA.

PubMed

Shi, Jie-Hua; Zhou, Kai-Li; Lou, Yan-Yue; Pan, Dong-Qi

2018-03-15

Molecular interaction of darunavir (DRV), a HIV protease inhibitor with calf thymus deoxyribonucleic acid (ct-DNA) was studied in physiological buffer (pH7.4) by multi-spectroscopic approaches hand in hand with viscosity measurements and molecular docking technique. The UV absorption and fluorescence results together revealed the formation of a DRV-ct-DNA complex having binding affinities of the order of 10 3 M -1 , which was more in keeping with the groove binding. The results that DRV bound to ct-DNA via groove binding mode was further evidenced by KI quenching studies, viscosity measurements, competitive binding investigations with EB and Rhodamine B and CD spectral analysis. The effect of ionic strength indicated the negligible involvement of electrostatic interaction between DRV and ct-DNA. The thermodynamic parameters regarding the binding interaction of DRV with ct-DNA in terms of enthalpy change (ΔH 0 ) and entropy change (ΔS 0 ) were -63.19kJ mol -1 and -141.92J mol -1 K -1 , indicating that hydrogen bonds and van der Waals forces played a predominant role in the binding process. Furthermore, molecular simulation studies suggested that DRV molecule was prone to bind in the A-T rich region of the minor groove of DNA. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular dynamics study of HIV-1 RT-DNA-nevirapine complexes explains NNRTI inhibition and resistance by connection mutations.

PubMed

Vijayan, R S K; Arnold, Eddy; Das, Kalyan

2014-05-01

HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that is targeted by nucleoside analogs (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). NNRTIs are allosteric inhibitors of RT, and constitute an integral part of several highly active antiretroviral therapy regimens. Under selective pressure, HIV-1 acquires resistance against NNRTIs primarily by selecting mutations around the NNRTI pocket. Complete RT sequencing of clinical isolates revealed that spatially distal mutations arising in connection and the RNase H domain also confer NNRTI resistance and contribute to NRTI resistance. However, the precise structural mechanism by which the connection domain mutations confer NNRTI resistance is poorly understood. We performed 50-ns molecular dynamics (MD) simulations, followed by essential dynamics, free-energy landscape analyses, and network analyses of RT-DNA, RT-DNA-nevirapine (NVP), and N348I/T369I mutant RT-DNA-NVP complexes. MD simulation studies revealed altered global motions and restricted conformational landscape of RT upon NVP binding. Analysis of protein structure network parameters demonstrated a dissortative hub pattern in the RT-DNA complex and an assortative hub pattern in the RT-DNA-NVP complex suggesting enhanced rigidity of RT upon NVP binding. The connection subdomain mutations N348I/T369I did not induce any significant structural change; rather, these mutations modulate the conformational dynamics and alter the long-range allosteric communication network between the connection subdomain and NNRTI pocket. Insights from the present study provide a structural basis for the biochemical and clinical findings on drug resistance caused by the connection and RNase H mutations. Copyright © 2013 Wiley Periodicals, Inc.

A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.

PubMed

Gatto, Francesca; Cassina, Giulia; Broccolo, Francesco; Morreale, Giuseppe; Lanino, Edoardo; Di Marco, Eddi; Vardas, Efthiya; Bernasconi, Daniela; Buttò, Stefano; Principi, Nicola; Esposito, Susanna; Scarlatti, Gabriella; Lusso, Paolo; Malnati, Mauro S

2011-12-01

Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Cellular HIV-1 Inhibition by Truncated Old World Primate APOBEC3A Proteins Lacking a Complete Deaminase Domain

PubMed Central

Katuwal, Miki; Wang, Yaqiong; Schmitt, Kimberly; Guo, Kejun; Halemano, Kalani; Santiago, Mario L.; Stephens, Edward B.

2014-01-01

The APOBEC3 (A3) deaminases are retrovirus restriction factors that were proposed as inhibitory components of HIV-1 gene therapy vectors. However, A3 mutational activity may induce undesired genomic damage and enable HIV-1 to evade drugs and immune responses. Here, we show that A3A protein from Colobus guereza (colA3A) can restrict HIV-1 replication in producer cells in a deaminase-independent manner without inducing DNA damage. Neither HIV-1 reverse transcription nor integration were significantly affected by colA3A, but capsid protein synthesis was inhibited. The determinants for colA3A restriction mapped to the N-terminal region. These properties extend to A3A from mandrills and De Brazza’s monkeys. Surprisingly, truncated colA3A proteins expressing only the N-terminal 100 amino acids effectively exclude critical catalytic regions but retained potent cellular restriction activity. These highlight a unique mechanism of cellular HIV-1 restriction by several Old World monkey A3A proteins that may be exploited for functional HIV-1 cure strategies. PMID:25262471

Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments

NASA Technical Reports Server (NTRS)

Ojha, R. P.; Dhingra, M. M.; Sarma, M. H.; Myer, Y. P.; Setlik, R. F.; Shibata, M.; Kazim, A. L.; Ornstein, R. L.; Rein, R.; Turner, C. J.;

HIV‑1 Integrase Strand Transfer Inhibitors with Reduced Susceptibility to Drug Resistant Mutant Integrases | Center for Cancer Research

Cancer.gov

On the cover: Mutant forms of HIV-1 IN reduce the therapeutic effectiveness of integrase strand transfer inhibitors (INSTIs). The cover figure shows the IN of prototype foamy virus complexed to a novel INSTI (gold) that retains potency against resistant mutants of HIV-1 IN. Overlain are the host and viral DNA substrates (blue and green, respectively), showing substrate mimicry

Virtual photon impact factors with exact gluon kinematics

NASA Astrophysics Data System (ADS)

Bialas, A.; Navelet, H.; Peschanski, R.

2001-06-01

An explicit analytic formula for the transverse and longitudinal impact factors ST, L( N, γ) of the photon using kT factorization with exact gluon kinematics is given. Applications to the QCD dipole model and the extraction of the unintegrated gluon structure function from data are proposed.

Requirement for an intact T-cell actin and tubulin cytoskeleton for efficient assembly and spread of human immunodeficiency virus type 1.

PubMed

Jolly, Clare; Mitar, Ivonne; Sattentau, Quentin J

2007-06-01

Human immunodeficiency virus type 1 (HIV-1) infection of CD4(+) T cells leads to the production of new virions that assemble at the plasma membrane. Gag and Env accumulate in the context of lipid rafts at the inner and outer leaflets of the plasma membrane, respectively, forming polarized domains from which HIV-1 buds. HIV-1 budding can result in either release of cell-free virions or direct cell-cell spread via a virological synapse (VS). The recruitment of Gag and Env to these plasma membrane caps in T cells is poorly understood but may require elements of the T-cell secretory apparatus coordinated by the cytoskeleton. Using fixed-cell immunofluorescence labeling and confocal microscopy, we observed a high percentage of HIV-1-infected T cells with polarized Env and Gag in capped, lipid raft-like assembly domains. Treatment of infected T cells with inhibitors of actin or tubulin remodeling disrupted Gag and Env compartmentalization within the polarized raft-like domains. Depolymerization of the actin cytoskeleton reduced Gag release and viral infectivity, and actin and tubulin inhibitors reduced Env incorporation into virions. Live- and fixed-cell confocal imaging and assay of de novo DNA synthesis by real-time PCR allowed quantification of HIV-1 cell-cell transfer. Inhibition of actin and tubulin remodeling in infected cells interfered with cell-cell spread across a VS and reduced new viral DNA synthesis. Based on these data, we propose that HIV-1 requires both actin and tubulin components of the T-cell cytoskeleton to direct its assembly and budding and to elaborate a functional VS.

Nevirapine-Resistant HIV-1 DNA in Breast Milk After Single-Dose Nevirapine With or Without Zidovudine for Prevention of Mother-to-Child Transmission.

PubMed

Gantt, Soren; Payant, Rachel; Carlsson, Jacquelyn; Micek, Mark A; Blanco, Ana Judith; Beck, Ingrid A; Matunha, Laurinda; Montoya, Pablo; Matediana, Eduardo; Gloyd, Stephen; Frenkel, Lisa M

2012-09-01

Among 30 human immunodeficiency virus type 1 (HIV-1)-infected women who received single-dose nevirapine (NVP), 17 (57%) had NVP-resistant HIV-1 detected in breast milk. NVP resistance in breast milk persisted for at least 8 months postpartum and was apparently transmitted to at least 1 infant. NVP resistance was detected less often in women who also received zidovudine. © The Author 2012. Published by Oxford University Press on behalf of the Pediatric Infectious Diseases Society. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The proton FL dipole approximation in the KMR and the MRW unintegrated parton distribution functions frameworks

NASA Astrophysics Data System (ADS)

Modarres, M.; Masouminia, M. R.; Hosseinkhani, H.; Olanj, N.

2016-01-01

In the spirit of performing a complete phenomenological investigation of the merits of Kimber-Martin-Ryskin (KMR) and Martin-Ryskin-Watt (MRW) unintegrated parton distribution functions (UPDF), we have computed the longitudinal structure function of the proton, FL (x ,Q2), from the so-called dipole approximation, using the LO and the NLO-UPDF, prepared in the respective frameworks. The preparation process utilizes the PDF of Martin et al., MSTW2008-LO and MSTW2008-NLO, as the inputs. Afterwards, the numerical results are undergone a series of comparisons against the exact kt-factorization and the kt-approximate results, derived from the work of Golec-Biernat and Stasto, against each other and the experimental data from ZEUS and H1 Collaborations at HERA. Interestingly, our results show a much better agreement with the exact kt-factorization, compared to the kt-approximate outcome. In addition, our results are completely consistent with those prepared from embedding the KMR and MRW UPDF directly into the kt-factorization framework. One may point out that the FL, prepared from the KMR UPDF shows a better agreement with the exact kt-factorization. This is despite the fact that the MRW formalism employs a better theoretical description of the DGLAP evolution equation and has an NLO expansion. Such unexpected consequence appears, due to the different implementation of the angular ordering constraint in the KMR approach, which automatically includes the resummation of ln ⁡ (1 / x), BFKL logarithms, in the LO-DGLAP evolution equation.

PQBP1 Is a Proximal Sensor of the cGAS-Dependent Innate Response to HIV-1.

PubMed

Yoh, Sunnie M; Schneider, Monika; Seifried, Janna; Soonthornvacharin, Stephen; Akleh, Rana E; Olivieri, Kevin C; De Jesus, Paul D; Ruan, Chunhai; de Castro, Elisa; Ruiz, Pedro A; Germanaud, David; des Portes, Vincent; García-Sastre, Adolfo; König, Renate; Chanda, Sumit K

2015-06-04

Dendritic cells (DCs) play a critical role in the immune response to viral infection through the facilitation of cell-intrinsic antiviral activity and the activation of adaptive immunity. HIV-1 infection of DCs triggers an IRF3-dependent innate immune response, which requires the activity of cyclic GAMP synthase (cGAS). We report the results of a targeted RNAi screen utilizing primary human monocyte-derived DCs (MDDCs) to identify immune regulators that directly interface with HIV-1-encoded features to initiate this innate response. Polyglutamine binding protein 1 (PQBP1) emerged as a strong candidate through this analysis. We found that PQBP1 directly binds to reverse-transcribed HIV-1 DNA and interacts with cGAS to initiate an IRF3-dependent innate response. MDDCs derived from Renpenning syndrome patients, who harbor mutations in the PQBP1 locus, possess a severely attenuated innate immune response to HIV-1 challenge, underscoring the role of PQBP1 as a proximal innate sensor of a HIV-1 infection. Copyright © 2015 Elsevier Inc. All rights reserved.

HPV Prevalence in Multiple Anatomical Sites among Men Who Have Sex with Men in Peru

PubMed Central

Blas, Magaly M.; Brown, Brandon; Menacho, Luis; Alva, Isaac E.; Silva-Santisteban, Alfonso; Carcamo, Cesar

2015-01-01

Background Human Papilloma Virus (HPV) infection is the most common sexually transmitted viral infection worldwide. HPV is highly prevalent in sexually active men who have sex with men (MSM) and has been associated with anal cancer, penile cancer, and oropharyngeal cancer. Methods From March to September 2011, we conducted a cross-sectional study of HPV prevalence among MSM above age 18 years. Participants were recruited using respondent driven sampling at Clinica Cayetano Heredia. All participants provided anal, genital, and oral samples for HPV DNA testing, and blood for HIV and HPV antibody testing. Results A total of 200 MSM were recruited in the study. The mean age was 34 years (range 18–59 years, SD = 9.4) and101 participants were HIV negative (99 HIV positive). HPV 6/11/16/18 or quadrivalent HPV vaccine (HPV4) genotype seroprevalence among HIV negative and positive MSM was 64.3% (55%-75.9%) and 93.8% (87.6%-99.2%) respectively (p<0.001). HIV positivity was associated with a higher prevalence of HPV4 and HPV 16/18 DNA at external genital sites and the anal canal. HPV4 DNA prevalence at external genital sites among HIV negative and positive MSM was 14.9% and 28.7% (p = 0.02) respectively, at anal canal was 50.9% and 79.0% (p = 0.001), and at the oral cavity was 9.9% and 8.5% (p = 0.6). Conclusions HPV4 seroprevalence was high in our study among both HIV positives and negatives, with HPV DNA prevalence much lower, and the anal canal being the anatomical site with the highest HPV DNA prevalence. HPV prevention interventions are needed among MSM at high-risk for HIV infection. PMID:26437318

HPV Prevalence in Multiple Anatomical Sites among Men Who Have Sex with Men in Peru.

PubMed

Blas, Magaly M; Brown, Brandon; Menacho, Luis; Alva, Isaac E; Silva-Santisteban, Alfonso; Carcamo, Cesar

2015-01-01

Human Papilloma Virus (HPV) infection is the most common sexually transmitted viral infection worldwide. HPV is highly prevalent in sexually active men who have sex with men (MSM) and has been associated with anal cancer, penile cancer, and oropharyngeal cancer. From March to September 2011, we conducted a cross-sectional study of HPV prevalence among MSM above age 18 years. Participants were recruited using respondent driven sampling at Clinica Cayetano Heredia. All participants provided anal, genital, and oral samples for HPV DNA testing, and blood for HIV and HPV antibody testing. A total of 200 MSM were recruited in the study. The mean age was 34 years (range 18-59 years, SD = 9.4) and101 participants were HIV negative (99 HIV positive). HPV 6/11/16/18 or quadrivalent HPV vaccine (HPV4) genotype seroprevalence among HIV negative and positive MSM was 64.3% (55%-75.9%) and 93.8% (87.6%-99.2%) respectively (p<0.001). HIV positivity was associated with a higher prevalence of HPV4 and HPV 16/18 DNA at external genital sites and the anal canal. HPV4 DNA prevalence at external genital sites among HIV negative and positive MSM was 14.9% and 28.7% (p = 0.02) respectively, at anal canal was 50.9% and 79.0% (p = 0.001), and at the oral cavity was 9.9% and 8.5% (p = 0.6). HPV4 seroprevalence was high in our study among both HIV positives and negatives, with HPV DNA prevalence much lower, and the anal canal being the anatomical site with the highest HPV DNA prevalence. HPV prevention interventions are needed among MSM at high-risk for HIV infection.

A Pilot Study of the Immunologic, Virologic, and Pathologic Consequences of Intra-anal 5% Imiquimod in HIV-1-Infected Men With High-Grade Squamous Intraepithelial Lesions.

PubMed

Cranston, Ross D; Baker, Jonathan R; Siegel, Aaron; Brand, Rhonda M; Janocko, Laura; McGowan, Ian

2018-03-01

Imiquimod can be used to treat internal anal high-grade squamous intraepithelial lesions. In HIV-1-infected individuals there is a theoretical concern for increased HIV replication in anorectal tissue secondary to imiquimod-induced mucosal inflammation. The purpose of this study was to assess local virologic, immunologic, and pathologic effects of imiquimod treatment in HIV-infected individuals. This was a pilot study at a single academic center. The study was conducted at the University of Pittsburgh Anal Dysplasia Clinic. HIV-1-infected individuals with biopsy-confirmed internal anal high-grade squamous intraepithelial lesions were included. Imiquimod cream was prescribed for intra-anal use 3 times per week for 9 weeks. Anal human papillomavirus typing, anal and rectal tissue HIV-1 RNA and DNA quantification, cytokine gene expression, and anal histology were measured. Nine evaluable participants (1 participant was lost to follow-up) were all white men with a median age of 46 years (interquartile range = 12 y) and a median CD4 T-cell count of 480 cells per cubic millimeter (interquartile range = 835). All were taking antiretroviral therapy, and 7 of 9 had HIV-1 RNA <50 copies per milliliter. The median dose of imiquimod used was 27.0 (interquartile range = 3.5), and there was a median of 11 days (interquartile range = 10 d) from last dose to assessment. There was no progression to cancer, no significant change in the number of human papillomavirus types detected, and no significant change in quantifiable cytokines/HIV-1 RNA or DNA levels in anal or rectal tissue. Seven (35%) of 20 high-grade lesions resolved to low-grade squamous intraepithelial lesions. The study was limited by the small number of participants and variable time to final assessment. Intra-anal imiquimod showed no evidence of immune activation or increase in HIV-1 viral replication in anal and rectal tissue and confirmed efficacy for intra-anal high-grade squamous intraepithelial lesion treatment morbidity. See Video Abstract at http://links.lww.com/DCR/A498.

HIV-1 vaccines

PubMed Central

Excler, Jean-Louis; Robb, Merlin L; Kim, Jerome H

2014-01-01

The development of a safe and effective preventive HIV-1 vaccine remains a public health priority. Despite scientific difficulties and disappointing results, HIV-1 vaccine clinical development has, for the first time, established proof-of-concept efficacy against HIV-1 acquisition and identified vaccine-associated immune correlates of risk. The correlate of risk analysis showed that IgG antibodies against the gp120 V2 loop correlated with decreased risk of HIV infection, while Env-specific IgA directly correlated with increased risk. The development of vaccine strategies such as improved envelope proteins formulated with potent adjuvants and DNA and vectors expressing mosaics, or conserved sequences, capable of eliciting greater breadth and depth of potentially relevant immune responses including neutralizing and non-neutralizing antibodies, CD4+ and CD8+ cell-mediated immune responses, mucosal immune responses, and immunological memory, is now proceeding quickly. Additional human efficacy trials combined with other prevention modalities along with sustained funding and international collaboration remain key to bring an HIV-1 vaccine to licensure. PMID:24637946

Quantification of integrated HIV DNA by repetitive-sampling Alu-HIV PCR on the basis of poisson statistics.

PubMed

De Spiegelaere, Ward; Malatinkova, Eva; Lynch, Lindsay; Van Nieuwerburgh, Filip; Messiaen, Peter; O'Doherty, Una; Vandekerckhove, Linos

2014-06-01

Quantification of integrated proviral HIV DNA by repetitive-sampling Alu-HIV PCR is a candidate virological tool to monitor the HIV reservoir in patients. However, the experimental procedures and data analysis of the assay are complex and hinder its widespread use. Here, we provide an improved and simplified data analysis method by adopting binomial and Poisson statistics. A modified analysis method on the basis of Poisson statistics was used to analyze the binomial data of positive and negative reactions from a 42-replicate Alu-HIV PCR by use of dilutions of an integration standard and on samples of 57 HIV-infected patients. Results were compared with the quantitative output of the previously described Alu-HIV PCR method. Poisson-based quantification of the Alu-HIV PCR was linearly correlated with the standard dilution series, indicating that absolute quantification with the Poisson method is a valid alternative for data analysis of repetitive-sampling Alu-HIV PCR data. Quantitative outputs of patient samples assessed by the Poisson method correlated with the previously described Alu-HIV PCR analysis, indicating that this method is a valid alternative for quantifying integrated HIV DNA. Poisson-based analysis of the Alu-HIV PCR data enables absolute quantification without the need of a standard dilution curve. Implementation of the CI estimation permits improved qualitative analysis of the data and provides a statistical basis for the required minimal number of technical replicates. © 2014 The American Association for Clinical Chemistry.

HIV Integration at Certain Sites in Host DNA is Linked to the Expansion and Persistence of Infected Cells | Center for Cancer Research

Cancer.gov

When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with HIV are currently treated with combined antiretroviral therapy (cART), which prevents viral replication in the majority of treated patients. When cART is initiated, most HIV-infected cells die in one or two days, and more of the infected cells die over a period of weeks to months. However there are some long-lived infected cells that do not die, which prevents patients from being cured.

HIV Integration at Certain Sites in Host DNA Is Linked to the Expansion and Persistence of Infected Cells | Poster

Cancer.gov

Editor’s note: This article was originally published on the Center for Cancer Research website. When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with HIV are currently treated with combined antiretroviral therapy (cART), which prevents viral replication in the majority of treated patients. When cART is initiated, most HIV-infected cells die in one or two days, and more of the infected cells die over a period of weeks to months. However there are some long-lived infected cells that do not die, which prevents patients from being cured.

Design and use of mouse control DNA for DNA biomarker extraction and PCR detection from urine: application for transrenal Mycobacterium tuberculosis DNA detection

PubMed Central

Bordelon, Hali; Ricks, Keersten M.; Pask, Megan E.; Russ, Patricia K.; Solinas, Francesca; Baglia, Mark L.; Short, Philip A.; Nel, Andrew; Blackburn, Jonathan; Dheda, Keertan; Zamudio, Carlos; Cáceres, Tatiana; Wright, David W.; Haselton, Frederick R.; Pettit, April C.

2017-01-01

Urine samples are increasingly used for diagnosing infections including Escherichia coli, Ebola virus, and Zika virus. However, extraction and concentration of nucleic acid biomarkers from urine is necessary for many molecular detection strategies such as polymerase chain reaction (PCR). Since urine samples typically have large volumes with dilute biomarker concentrations making them prone to false negatives, another impediment for urine-based diagnostics is the establishment of appropriate controls particularly to rule out false negatives. In this study, a mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) DNA target was added to retrospectively collected urine samples from tuberculosis (TB)-infected and TB-uninfected patients to indicate extraction of intact DNA and removal of PCR inhibitors from urine samples. We tested this design on surrogate urine samples, retrospective 1 milliliter (mL) urine samples from patients in Lima, Peru and retrospective 5 mL urine samples from patients in Cape Town, South Africa. Extraction/PCR control DNA was detectable in 97% of clinical samples with no statistically significant differences among groups. Despite the inclusion of this control, there was no difference in the amount of TB IS6110 Tr-DNA detected between TB-infected and TB-uninfected groups except for samples from known HIV-infected patients. We found a increase in TB IS6110 Tr-DNA between TB/HIV co-infected patients compared to TB-uninfected/HIV-infected patients (N=18, p=0.037). The inclusion of an extraction/PCR control DNA to indicate successful DNA extraction and removal of PCR inhibitors should be easily adaptable as a sample preparation control for other acellular sample types. PMID:28285168

Design and use of mouse control DNA for DNA biomarker extraction and PCR detection from urine: Application for transrenal Mycobacterium tuberculosis DNA detection.

PubMed

Bordelon, Hali; Ricks, Keersten M; Pask, Megan E; Russ, Patricia K; Solinas, Francesca; Baglia, Mark L; Short, Philip A; Nel, Andrew; Blackburn, Jonathan; Dheda, Keertan; Zamudio, Carlos; Cáceres, Tatiana; Wright, David W; Haselton, Frederick R; Pettit, April C

2017-05-01

Urine samples are increasingly used for diagnosing infections including Escherichia coli, Ebola virus, and Zika virus. However, extraction and concentration of nucleic acid biomarkers from urine is necessary for many molecular detection strategies such as polymerase chain reaction (PCR). Since urine samples typically have large volumes with dilute biomarker concentrations making them prone to false negatives, another impediment for urine-based diagnostics is the establishment of appropriate controls particularly to rule out false negatives. In this study, a mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) DNA target was added to retrospectively collected urine samples from tuberculosis (TB)-infected and TB-uninfected patients to indicate extraction of intact DNA and removal of PCR inhibitors from urine samples. We tested this design on surrogate urine samples, retrospective 1milliliter (mL) urine samples from patients in Lima, Peru and retrospective 5mL urine samples from patients in Cape Town, South Africa. Extraction/PCR control DNA was detectable in 97% of clinical samples with no statistically significant differences among groups. Despite the inclusion of this control, there was no difference in the amount of TB IS6110 Tr-DNA detected between TB-infected and TB-uninfected groups except for samples from known HIV-infected patients. We found an increase in TB IS6110 Tr-DNA between TB/HIV co-infected patients compared to TB-uninfected/HIV-infected patients (N=18, p=0.037). The inclusion of an extraction/PCR control DNA to indicate successful DNA extraction and removal of PCR inhibitors should be easily adaptable as a sample preparation control for other acellular sample types. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of HIV infection-related DNA methylation sites and advanced epigenetic aging in HIV-positive, treatment-naive U.S. veterans.

PubMed

Nelson, Kristin N; Hui, Qin; Rimland, David; Xu, Ke; Freiberg, Matthew S; Justice, Amy C; Marconi, Vincent C; Sun, Yan V

2017-02-20

HIV-positive individuals are at higher risk than healthy persons for aging-related diseases, including myocardial infarction and non-AIDS defining cancers. Recent evidence suggests that HIV infection may modulate changes in the host cell epigenome, and these changes represent a potential mechanism through which HIV infection accelerates aging. We assessed the difference in DNA methylation (DNAm) age, an aging marker involving multiple age-related cytosine-guanine dinucleotide (CpG) sites, among antiretroviral treatment (ART)-naive HIV-positive and HIV-negative individuals in a cohort of veterans from the Veterans Aging Cohort Study. Peripheral blood samples were collected from 19 ART-naive, HIV-positive, and 19 HIV-negative male participants, matched by age and race. Blood samples were collected from HIV-positive participants 7-11 years after ART initiation. We compared DNAm age between HIV-positive and HIV-negative groups at baseline and between HIV-positive patients at baseline and follow-up. We also performed an epigenome-wide analysis to identify CpG methylation sites associated with HIV infection. DNAm age in HIV-positive individuals is, on average, 11.2 years higher than HIV study participants at baseline, and two of 10 HIV-positive individuals showed an increase in DNAm age after ART initiation. Epigenome-wide association studies showed an association of HIV infection with one site, in gene VPS37B, which approached statistical significance in our cohort (P = 3.30 × 10, Bonferroni-corrected threshold = 1.22 × 10) and was replicated in a second, larger cohort. ART treatment-naive HIV-positive individuals have significantly older DNAm age compared to HIV-negative individuals in the Veterans Aging Cohort Study cohort. Longitudinal changes in DNAm age are highly variable across individuals after initiation of antiretroviral therapy.

Structure, interaction and real-time monitoring of the enzymatic reaction of wild-type APOBEC3G.

PubMed

Furukawa, Ayako; Nagata, Takashi; Matsugami, Akimasa; Habu, Yuichirou; Sugiyama, Ryuichi; Hayashi, Fumiaki; Kobayashi, Naohiro; Yokoyama, Shigeyuki; Takaku, Hiroshi; Katahira, Masato

2009-02-18

Human APOBEC3G exhibits anti-human immunodeficiency virus-1 (HIV-1) activity by deaminating cytidines of the minus strand of HIV-1. Here, we report a solution structure of the C-terminal deaminase domain of wild-type APOBEC3G. The interaction with DNA was examined. Many differences in the interaction were found between the wild type and recently studied mutant APOBEC3Gs. The position of the substrate cytidine, together with that of a DNA chain, in the complex, was deduced. Interestingly, the deamination reaction of APOBEC3G was successfully monitored using NMR signals in real time. Real-time monitoring has revealed that the third cytidine of the d(CCCA) segment is deaminated at an early stage and that then the second one is deaminated at a late stage, the first one not being deaminated at all. This indicates that the deamination is carried out in a strict 3' --> 5' order. Virus infectivity factor (Vif) of HIV-1 counteracts the anti-HIV-1 activity of APOBEC3G. The structure of the N-terminal domain of APOBEC3G, with which Vif interacts, was constructed with homology modelling. The structure implies the mechanism of species-specific sensitivity of APOBEC3G to Vif action.

Structure, interaction and real-time monitoring of the enzymatic reaction of wild-type APOBEC3G

PubMed Central

Furukawa, Ayako; Nagata, Takashi; Matsugami, Akimasa; Habu, Yuichirou; Sugiyama, Ryuichi; Hayashi, Fumiaki; Kobayashi, Naohiro; Yokoyama, Shigeyuki; Takaku, Hiroshi; Katahira, Masato

2009-01-01

Human APOBEC3G exhibits anti-human immunodeficiency virus-1 (HIV-1) activity by deaminating cytidines of the minus strand of HIV-1. Here, we report a solution structure of the C-terminal deaminase domain of wild-type APOBEC3G. The interaction with DNA was examined. Many differences in the interaction were found between the wild type and recently studied mutant APOBEC3Gs. The position of the substrate cytidine, together with that of a DNA chain, in the complex, was deduced. Interestingly, the deamination reaction of APOBEC3G was successfully monitored using NMR signals in real time. Real-time monitoring has revealed that the third cytidine of the d(CCCA) segment is deaminated at an early stage and that then the second one is deaminated at a late stage, the first one not being deaminated at all. This indicates that the deamination is carried out in a strict 3′ → 5′ order. Virus infectivity factor (Vif) of HIV-1 counteracts the anti-HIV-1 activity of APOBEC3G. The structure of the N-terminal domain of APOBEC3G, with which Vif interacts, was constructed with homology modelling. The structure implies the mechanism of species-specific sensitivity of APOBEC3G to Vif action. PMID:19153609

Expanded breadth of the T-cell response to mosaic HIV-1 envelope DNA vaccination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korber, Bette; Fischer, William; Wallstrom, Timothy

2009-01-01

An effective AIDS vaccine must control highly diverse circulating strains of HIV-1. Among HIV -I gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV -I Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential Tcell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining (ICS) in inbred mice with a standardized panel of highlymore » conserved 15-mer PTE peptides. I, 2 and 3 mosaic sets were developed that increased theoretical epitope coverage. The breadth and magnitude ofT-cell immunity stimulated by these vaccines were compared to natural strain Env's; additional comparisons were performed on mutant Env's, including gpl60 or gpl45 with or without V regions and gp41 deletions. Among them, the 2 or 3 mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the 3 mosaic set elicited responses to an average of 8 peptide pools compared to 2 pools for a set of3 natural Env's. Synthetic mosaic HIV -I antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T -cell-based HIV -1 vaccines.« less

Impairment of Human Immunodeficiency Virus Type-1 Integrase SUMOylation Correlates with an Early Replication Defect*

PubMed Central

Zamborlini, Alessia; Coiffic, Audrey; Beauclair, Guillaume; Delelis, Olivier; Paris, Joris; Koh, Yashuiro; Magne, Fabian; Giron, Marie-Lou; Tobaly-Tapiero, Joelle; Deprez, Eric; Emiliani, Stephane; Engelman, Alan; de Thé, Hugues; Saïb, Ali

2011-01-01

HIV-1 integrase (IN) orchestrates the integration of the reverse transcribed viral cDNA into the host cell genome and participates also in other steps of HIV-1 replication. Cellular and viral factors assist IN in performing its multiple functions, and post-translational modifications contribute to modulate its activities. Here, we show that HIV-1 IN is modified by SUMO proteins and that phylogenetically conserved SUMOylation consensus motifs represent major SUMO acceptor sites. Viruses harboring SUMOylation site IN mutants displayed a replication defect that was mapped during the early stages of infection, before integration but after reverse transcription. Because SUMOylation-defective IN mutants retained WT catalytic activity, we hypothesize that SUMOylation might regulate the affinity of IN for co-factors, contributing to efficient HIV-1 replication. PMID:21454548

Patterns of herpes simplex virus shedding over 1 month and the impact of acyclovir and HIV in HSV-2-seropositive women in Tanzania

PubMed Central

Weiss, Helen A; LeGoff, Jerome; Changalucha, John; Clayton, Tim C; Ross, David A; Belec, Laurent; Hayes, Richard J; Watson-Jones, Deborah

2011-01-01

Objectives Few studies have examined the frequency and duration of genital herpes simplex virus (HSV) shedding in sub-Saharan Africa. This study describes HSV shedding patterns among a sample of HSV-2-seropositive women enrolled in a placebo-controlled trial of HSV suppressive therapy (acyclovir 400 mg twice a day) in Tanzania. Methods Trial participants were invited to participate in a substudy involving 12 clinic visits over 4 weeks. At each visit, cervical, vaginal and external skin swabs were taken and analysed for HSV DNA using inhouse real-time PCR. Results HSV shedding was mainly subclinical (90%; 57/63 shedding days in the placebo arm). The most frequent shedding site was the external skin, but HSV DNA was detected from all three sites on 42% (27/63) of shedding days. In HIV-negative women, HSV DNA was detected on 3% (9/275) of days in the acyclovir versus 11% (33/309) in the placebo arm, while in HIV-positive women, detection was on 14% (23/160) versus 19% (30/155) of days, respectively. Conclusions HSV shedding was common, varying greatly by individual. Shedding rates were similar to studies in African and non-African settings. Among HIV-negative women, shedding rates were lower in the acyclovir arm; however, acyclovir did not substantially impact on HSV shedding in HIV-positive women. PMID:21653932

Brief Report: Effect of CMV and HIV Transcription on CD57 and PD-1 T-Cell Expression During Suppressive ART

PubMed Central

Massanella, Marta; Smith, Davey M.; Spina, Celsa A.; Schrier, Rachel; Daar, Eric S.; Dube, Michael P.; Morris, Sheldon R.; Gianella, Sara

2016-01-01

Abstract: HIV-infected men who have sex with men are nearly universally coinfected with cytomegalovirus (CMV). In this study of 45 HIV-infected men who have sex with men virologically suppressed on ART, we found that presence of seminal CMV DNA shedding and higher levels of systemic cellular HIV RNA transcription were both independently associated with increased PD-1 expression on circulating CD4+ T cells, but not with higher levels of senescent (CD57+) T cells. In addition, greater HIV RNA transcription was associated with lower CD57 expression on CD8 T cells. Although causality cannot be inferred from this retrospective study, these results suggest that asymptomatic CMV replication and residual cellular HIV transcription may contribute to persistent immune dysregulation during suppressive ART. PMID:26818740

Antiretroviral Agents Effectively Block HIV Replication after Cell-to-Cell Transfer

PubMed Central

Permanyer, Marc; Ballana, Ester; Ruiz, Alba; Badia, Roger; Riveira-Munoz, Eva; Gonzalo, Encarna; Clotet, Bonaventura

2012-01-01

Cell-to-cell transmission of HIV has been proposed as a mechanism contributing to virus escape to the action of antiretrovirals and a mode of HIV persistence during antiretroviral therapy. Here, cocultures of infected HIV-1 cells with primary CD4+ T cells or lymphoid cells were used to evaluate virus transmission and the effect of known antiretrovirals. Transfer of HIV antigen from infected to uninfected cells was resistant to the reverse transcriptase inhibitors (RTIs) zidovudine (AZT) and tenofovir, but was blocked by the attachment inhibitor IgGb12. However, quantitative measurement of viral DNA production demonstrated that all anti-HIV agents blocked virus replication with similar potency to cell-free virus infections. Cell-free and cell-associated infections were equally sensitive to inhibition of viral replication when HIV-1 long terminal repeat (LTR)-driven green fluorescent protein (GFP) expression in target cells was measured. However, detection of GFP by flow cytometry may incorrectly estimate the efficacy of antiretrovirals in cell-associated virus transmission, due to replication-independent Tat-mediated LTR transactivation as a consequence of cell-to-cell events that did not occur in short-term (48-h) cell-free virus infections. In conclusion, common markers of virus replication may not accurately correlate and measure infectivity or drug efficacy in cell-to-cell virus transmission. When accurately quantified, active drugs blocked proviral DNA and virus replication in cell-to-cell transmission, recapitulating the efficacy of antiretrovirals in cell-free virus infections and in vivo. PMID:22696642

Immunotherapy with an HIV-DNA Vaccine in Children and Adults

PubMed Central

Palma, Paolo; Gudmundsdotter, Lindvi; Finocchi, Andrea; Eriksson, Lars E.; Mora, Nadia; Santilli, Veronica; Aquilani, Angela; Manno, Emma C.; Zangari, Paola; Romiti, Maria Luisa; Montesano, Carla; Grifoni, Alba; Brave, Andreas; Ljungberg, Karl; Blomberg, Pontus; Bernardi, Stefania; Sandström, Eric; Hejdeman, Bo; Rossi, Paolo; Wahren, Britta

2014-01-01

Therapeutic HIV immunization is intended to induce new HIV-specific cellular immune responses and to reduce viral load, possibly permitting extended periods without antiretroviral drugs. A multigene, multi-subtype A, B, C HIV-DNA vaccine (HIVIS) has been used in clinical trials in both children and adults with the aim of improving and broadening the infected individuals’ immune responses. Despite the different country locations, different regimens and the necessary variations in assays performed, this is, to our knowledge, the first attempt to compare children’s and adults’ responses to a particular HIV vaccine. Ten vertically HIV-infected children aged 4–16 years were immunized during antiretroviral therapy (ART). Another ten children were blindly recruited as controls. Both groups continued their antiretroviral treatment during and after vaccinations. Twelve chronically HIV-infected adults were vaccinated, followed by repeated structured therapy interruptions (STI) of their antiretroviral treatment. The adult group included four controls, receiving placebo vaccinations. The HIV-DNA vaccine was generally well tolerated, and no serious adverse events were registered in any group. In the HIV-infected children, an increased specific immune response to Gag and RT proteins was detected by antigen-specific lymphoproliferation. Moreover, the frequency of HIV-specific CD8+ T-cell lymphocytes releasing perforin was significantly higher in the vaccinees than the controls. In the HIV-infected adults, increased CD8+ T-cell responses to Gag, RT and viral protease peptides were detected. No augmentation of HIV-specific lymphoproliferative responses were detected in adults after vaccination. In conclusion, the HIV-DNA vaccine can elicit new HIV-specific cellular immune responses, particularly to Gag antigens, in both HIV-infected children and adults. Vaccinated children mounted transient new HIV-specific immune responses, including both CD4+ T-cell lymphoproliferation and late CD8+ T-cell responses. In the adult cohort, primarily CD8+ T-cell responses related to MHC class I alleles were noted. However, no clinical benefits with respect to viral load reduction were ascribable to the vaccinations alone. No severe adverse effects related to the vaccine were found in either cohort, and no virological failures or drug resistances were detected. PMID:26344746

Optimal vitamin D plasma levels are associated with lower bacterial DNA translocation in HIV/hepatitis c virus coinfected patients.

PubMed

García-Álvarez, Mónica; Berenguer, Juan; Jiménez-Sousa, Maria Ángeles; Vázquez-Morón, Sonia; Carrero, Ana; Gutiérrez-Rivas, Mónica; Aldámiz-Echevarría, Teresa; López, Juan Carlos; García-Broncano, Pilar; Resino, Salvador

2016-04-24

Vitamin D has been linked to the immune response modulation and the integrity of the intestinal mucosal barrier. Therefore, vitamin D might be involved in bacterial translocation related to HIV infection. Our major aim was to analyze the association between plasma levels of 25-hydroxy-vitamin D [25(OH)D] and bacterial 16S ribosomal DNA (bactDNA) in 120 HIV/hepatitis c virus (HCV) coinfected patients. Cross-sectional study. Plasma 25(OH)D levels were quantified by enzyme immunoassay. The vitamin D status was defined as deficient (<25 nmol/l), insufficient (25-74 nmol/l), and optimal (≥75 nmol/l) plasma levels. Plasma bactDNA levels were measured by quantitative real-time PCR. For bactDNA levels the cutoffs used were as follows: low [p75th). Eighteen (15%) patients had 25(OH)D deficiency, 93 (77.5%) had insufficiency and nine (7.5%) had 25(OH)D optimal values. The bactDNA levels were lower in patients with 25(OH)D at least 75 nmol/l [37 copies/μl] than in patients with 25(OH)D insufficiency [84.2 copies/μl; P = 0.042]. Conversely, low bactDNA levels (

Effect of cytomegalovirus and Epstein-Barr virus replication on intestinal mucosal gene expression and microbiome composition of HIV-infected and uninfected individuals.

PubMed

Gianella, Sara; Chaillon, Antoine; Mutlu, Ece A; Engen, Phillip A; Voigt, Robin M; Keshavarzian, Ali; Losurdo, John; Chakradeo, Prachi; Lada, Steven M; Nakazawa, Masato; Landay, Alan L

2017-09-24

HIV-infection is associated with dramatic changes in the intestinal mucosa. The impact of other viral pathogens is unclear. One hundred and eight (108) biopsies from left and right colon (n = 79) and terminal ileum (n = 29) were collected from 19 HIV-infected and 22 HIV-uninfected participants. Levels of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNA were measured by droplet digital PCR. Mucosal gene expression was measured via multiplex-assay. Microbiome analysis was performed using bacterial 16S-rDNA-pyrosequencing. The effect of CMV and EBV replication on the microbiome composition and mRNA-expression of selected cytokines (IL-6, IFN-γ, IL-1β, CCL2, IL-8, and IFN-β1) was evaluated. Overall, CMV and EBV were detected in at least one intestinal site in 60.5 and 78.9% of participants, respectively. HIV-infected individuals demonstrated less detectable CMV (P = 0.04); CMV was more frequently detected in terminal ileum than colon (P = 0.04). Detectable EBV was more frequent among HIV-infected (P = 0.05) without differences by intestinal site. The number of operational taxonomic units did not differ by CMV or EBV detection status. Among HIV-infected participants, higher CMV was only associated with lower relative abundance of Actinobacteria in the ileum (P = 0.03). Presence of CMV was associated with upregulated expression of all selected cytokines in the ileum (all P = 0.02) and higher expression of IL-8 and IFN-β1 in the colon (all P < 0.05) of HIV-uninfected participants, but not among HIV-infected. EBV had no effect on cytokine expression or microbiome composition whatsoever. These results illustrate a complex interplay among HIV-infection, intestinal CMV replication, and mucosal gut environment, and highlight a possible modulatory effect of CMV on the microbial and immune homeostasis.

Prime, Shock, and Kill: Priming CD4 T Cells from HIV Patients with a BCL-2 Antagonist before HIV Reactivation Reduces HIV Reservoir Size

PubMed Central

Cummins, Nathan W.; Sainski, Amy M.; Dai, Haiming; Natesampillai, Sekar; Pang, Yuan-Ping; Bren, Gary D.; de Araujo Correia, Maria Cristina Miranda; Sampath, Rahul; Rizza, Stacey A.; O'Brien, Daniel; Yao, Joseph D.

2016-01-01

ABSTRACT Understanding how some HIV-infected cells resist the cytotoxicity of HIV replication is crucial to enabling HIV cure efforts. HIV killing of CD4 T cells that replicate HIV can involve HIV protease-mediated cleavage of procaspase 8 to generate a fragment (Casp8p41) that directly binds and activates the mitochondrial proapoptotic protein BAK. Here, we demonstrate that Casp8p41 also binds with nanomolar affinity to the antiapoptotic protein Bcl-2, which sequesters Casp8p41 and prevents apoptosis. Further, we show that central memory CD4 T cells (TCM) from HIV-infected individuals have heightened expression of BCL-2 relative to procaspase 8, possibly explaining the persistence of HIV-infected TCM despite generation of Casp8p41. Consistent with this hypothesis, the selective BCL-2 antagonist venetoclax induced minimal killing of uninfected CD4 T cells but markedly increased the death of CD4 T cells and diminished cell-associated HIV DNA when CD4 T cells from antiretroviral therapy (ART)-suppressed HIV patients were induced with αCD3/αCD28 to reactivate HIV ex vivo. Thus, priming CD4 T cells from ART suppressed HIV patients with a BCL-2 antagonist, followed by HIV reactivation, achieves reductions in cell-associated HIV DNA, whereas HIV reactivation alone does not. IMPORTANCE HIV infection is incurable due to a long-lived reservoir of HIV+ memory CD4 T cells, and no clinically relevant interventions have been identified that reduce the number of these HIV DNA-containing cells. Since postintegration HIV replication can result in HIV protease generation of Casp8p41, which activates BAK, causing infected CD4 T cell death, we sought to determine whether this occurs in memory CD4 T cells. Here, we demonstrate that memory CD4 T cells can generate Casp8p41 and yet are intrinsically resistant to death induced by diverse stimuli, including Casp8p41. Furthermore, BCL-2 expression is relatively increased in these cells and directly binds and inhibits Casp8p41's proapoptotic effects. Antagonizing BCL-2 with venetoclax derepresses this antagonism, resulting in death, preferentially in HIV DNA containing cells, since only these cells generate Casp8p41. Thus, BCL-2 antagonism is a clinically relevant intervention with the potential to reduce HIV reservoir size in patients. PMID:26842479

Cyclophilin B enhances HIV-1 Infection

PubMed Central

DeBoer, Jason; Madson, Christian J.; Belshan, Michael

2016-01-01

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. PMID:26774171

Cyclophilin B enhances HIV-1 infection.

PubMed

DeBoer, Jason; Madson, Christian J; Belshan, Michael

2016-02-01

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. Copyright © 2015 Elsevier Inc. All rights reserved.

Human immunodeficiency virus type 1 Tat does not transactivate mature trans-acting responsive region RNA species in the nucleus or cytoplasm of primate cells.

PubMed Central

Chin, D J; Selby, M J; Peterlin, B M

1991-01-01

Human immunodeficiency virus (HIV)-encoded transactivator Tat is essential for viral gene expression and replication. By interacting with a nascent RNA stem-loop called the trans-acting responsive region (TAR). Tat increases rates of initiation and/or elongation of HIV transcription. Several reports have also suggested that Tat has additional effects on mature HIV RNA species including modification of primary transcripts in the nucleus and their increased translation in the cytoplasm. These posttranscriptional effects are most pronounced in the Xenopus oocyte. To investigate directly whether Tat has similar effects on viral transcripts in cells that are permissive for HIV replication, we cotransfected and microinjected human and monkey cells with Tat and TAR in the form of DNA or RNA. Whereas Tat transactivated TAR DNA targets, it did not transactivate TAR RNA targets in the nucleus of microinjected cells or in the cytoplasm of transfected cells. We conclude that in cells permissive for viral replication, Tat exerts its effect primarily at the level of HIV transcription. Images PMID:1900539

A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter.

PubMed

Isalan, M; Klug, A; Choo, Y

2001-07-01

DNA-binding domains with predetermined sequence specificity are engineered by selection of zinc finger modules using phage display, allowing the construction of customized transcription factors. Despite remarkable progress in this field, the available protein-engineering methods are deficient in many respects, thus hampering the applicability of the technique. Here we present a rapid and convenient method that can be used to design zinc finger proteins against a variety of DNA-binding sites. This is based on a pair of pre-made zinc finger phage-display libraries, which are used in parallel to select two DNA-binding domains each of which recognizes given 5 base pair sequences, and whose products are recombined to produce a single protein that recognizes a composite (9 base pair) site of predefined sequence. Engineering using this system can be completed in less than two weeks and yields proteins that bind sequence-specifically to DNA with Kd values in the nanomolar range. To illustrate the technique, we have selected seven different proteins to bind various regions of the human immunodeficiency virus 1 (HIV-1) promoter.

Synthesis of an integrated cockpit management system

NASA Technical Reports Server (NTRS)

Dasaro, J. A.; Elliott, C. T.

1982-01-01

The process used in the synthesis of an integrated cockpit management system was discussed. Areas covered included flight displays, subsystem management, checklists, and procedures (both normal and emergency). The process of evolving from the unintegrated conventional system to the integrated system is examined and a brief description of the results presented.

Mitochondrial Oxidative Phosphorylation Protein Levels in Peripheral Blood Mononuclear Cells Correlate with Levels in Subcutaneous Adipose Tissue within Samples Differing by HIV and Lipoatrophy Status

PubMed Central

Gerschenson, Mariana; Chow, Dominic; Libutti, Daniel E.; Willis, John H.; Murray, James; Capaldi, Roderick A.; Marusich, Michael

2008-01-01

Abstract Depletion of mitochondrial DNA (mtDNA) and mtDNA-encoded respiratory chain proteins in subcutaneous (SC) fat from patients with HIV lipoatrophy have clearly demonstrated the role of mitochondrial dysfunction in this syndrome. Research in HIV lipoatrophy, however, has been severely hampered by the lack of a suitable surrogate marker in blood or other easily obtained clinical specimens as fat biopsies are invasive and mtDNA levels in peripheral blood mononuclear cells (PBMC) do not consistently correlate with the disease process. We used a simple, rapid, quantitative 2-site dipstick immunoassay to measure OXPHOS enzymes Complex I (CI) and Complex IV (CIV), and rtPCR to measure mtDNA in 26 matched SC fat and PBMC specimens previously banked from individuals on potent antiretroviral (ARV) therapy with HIV lipoatrophy, on similar ARV therapy without lipoatrophy, and in HIV seronegative controls. Significant correlations were found between the respective PBMC and fat levels for both CI (r = 0.442, p = 0.024) and for CIV (r = 0.507, p = 0.008). Both CI and CIV protein levels were also significantly reduced in both PBMCs and fat in lipoatrophic subjects compared to HIV seronegative controls (p ≤ 0.05), while a comparative reduction in mtDNA levels in lipoatrophic subjects was observed only in fat. We conclude that CI and CIV levels in PBMCs correlate to their respective levels in fat and may have utility as surrogate markers of mitochondrial dysfunction in lipoatrophy. PMID:18844460

Lipopolysaccharide is a potent monocyte/macrophage-specific stimulator of human immunodeficiency virus type 1 expression

PubMed Central

1990-01-01

Lipopolysaccharide (LPS) potently stimulates human immunodeficiency virus type 1-long terminal repeat (HIV-1-LTR) CAT constructs transfected into monocyte/macrophage-like cell lines but not a T cell line. This effect appears to be mediated through the induction of nuclear factor kappa B (NF-kappa B). Electrophoretic mobility shift assays demonstrate that LPS induces a DNA binding activity indistinguishable from NF-kappa B in U937 and THP-1 cells. LPS is also shown to dramatically increase HIV-1 production from a chronically infected monocyte/macrophage-like cloned cell line, U1, which produces very low levels of HIV-1 at baseline. The stimulation of viral production from this cell line occurs only if these cells are treated with granulocyte/macrophage colony-stimulating factor (GM-CSF) before treatment with LPS. This stimulation of HIV-1 production is correlated with an increase in the level of HIV-1 RNA and and activation of NF- kappa B. LPS is not able to induce HIV-1 production in a cloned T cell line. The effect of LPS on HIV-1 replication occurs at picogram per milliliter concentrations and may be clinically significant in understanding the variability of the natural history of HIV-1 infection. PMID:2193097

Experiences of social harm and changes in sexual practices among volunteers who had completed a phase I/II HIV vaccine trial employing HIV-1 DNA priming and HIV-1 MVA boosting in Dar es Salaam, Tanzania.

PubMed

Tarimo, Edith A M; Munseri, Patricia; Aboud, Said; Bakari, Muhammad; Mhalu, Fred; Sandstrom, Eric

2014-01-01

Volunteers in phase I/II HIV vaccine trials are assumed to be at low risk of acquiring HIV infection and are expected to have normal lives in the community. However, during participation in the trials, volunteers may encounter social harm and changes in their sexual behaviours. The current study aimed to study persistence of social harm and changes in sexual practices over time among phase I/II HIV vaccine immunogenicity (HIVIS03) trial volunteers in Dar es Salaam, Tanzania. A descriptive prospective cohort study was conducted among 33 out of 60 volunteers of HIVIS03 trial in Dar es Salaam, Tanzania, who had received three HIV-1 DNA injections boosted with two HIV-1 MVA doses. A structured interview was administered to collect data. Analysis was carried out using SPSS and McNemars' chi-square (χ2) was used to test the association within-subjects. Participants reported experiencing negative comments from their colleagues about the trial; but such comments were less severe during the second follow up visits (χ2 = 8.72; P<0.001). Most of the comments were associated with discrimination (χ2 = 26.72; P<0.001), stigma (χ2 = 6.06; P<0.05), and mistrust towards the HIV vaccine trial (χ2 = 4.9; P<0.05). Having a regular sexual partner other than spouse or cohabitant declined over the two follow-up periods (χ2 = 4.45; P<0.05). Participants in the phase I/II HIV vaccine trial were likely to face negative comments from relatives and colleagues after the end of the trial, but those comments decreased over time. In this study, the inherent sexual practice of having extra sexual partners other than spouse declined over time. Therefore, prolonged counselling and support appears important to minimize risky sexual behaviour among volunteers after participation in HIV Vaccine trials.

Host Methyltransferases and Demethylases: Potential New Epigenetic Targets for HIV Cure Strategies and Beyond.

PubMed

Boehm, Daniela; Ott, Melanie

2017-11-01

A successful HIV cure strategy may require reversing HIV latency to purge hidden viral reservoirs or enhancing HIV latency to permanently silence HIV transcription. Epigenetic modifying agents show promise as antilatency therapeutics in vitro and ex vivo, but also affect other steps in the viral life cycle. In this review, we summarize what we know about cellular DNA and protein methyltransferases (PMTs) as well as demethylases involved in HIV infection. We describe the biology and function of DNA methyltransferases, and their controversial role in HIV infection. We further explain the biology of PMTs and their effects on lysine and arginine methylation of histone and nonhistone proteins. We end with a focus on protein demethylases, their unique modes of action and their emerging influence on HIV infection. An outlook on the use of methylation-modifying agents in investigational HIV cure strategies is provided.

Infant peripheral blood repetitive element hypomethylation associated with antiretroviral therapy in utero.

PubMed

Marsit, Carmen J; Brummel, Sean S; Kacanek, Deborah; Seage, George R; Spector, Stephen A; Armstrong, David A; Lester, Barry M; Rich, Kenneth

2015-01-01

The use of combination antiretroviral therapy (cART) to prevent HIV mother-to-child transmission during pregnancy and delivery is generally considered safe. However, vigilant assessment of potential risks of these agents remains warranted. Epigenetic changes including DNA methylation are considered potential mechanisms linking the in utero environment with long-term health outcomes. Few studies have examined the epigenetic effects of prenatal exposure to pharmaceutical agents, including antiretroviral therapies, on children. In this study, we examined the methylation status of the LINE-1 and ALU-Yb8 repetitive elements as markers of global DNA methylation alteration in peripheral blood mononuclear cells obtained from newborns participating in the Pediatric HIV/AIDS Cohort Study SMARTT cohort of HIV-exposed, cART-exposed uninfected infants compared to a historical cohort of HIV-exposed, antiretroviral-unexposed infants from the Women and Infants Transmission Study Cohort. In linear regression models controlling for potential confounders, we found the adjusted mean difference of AluYb8 methylation of the cART-exposed compared to the -unexposed was -0.568 (95% CI: -1.023, -0.149) and for LINE-1 methylation was -1.359 (95% CI: -1.860, -0.857). Among those exposed to cART, subjects treated with atazanavir (ATV), compared to those on other treatments, had less AluYb8 methylation (-0.524, 95% CI: -0.025, -1.024). Overall, these results suggest a small but statistically significant reduction in the methylation of these repetitive elements in an HIV-exposed, cART-exposed cohort compared to an HIV-exposed, cART-unexposed historic cohort. The potential long-term implications of these differences are worthy of further examination.

Single-diffractive production of dijets within the kt-factorization approach

NASA Astrophysics Data System (ADS)

Łuszczak, Marta; Maciuła, Rafał; Szczurek, Antoni; Babiarz, Izabela

2017-09-01

We discuss single-diffractive production of dijets. The cross section is calculated within the resolved Pomeron picture, for the first time in the kt-factorization approach, neglecting transverse momentum of the Pomeron. We use Kimber-Martin-Ryskin unintegrated parton (gluon, quark, antiquark) distributions in both the proton as well as in the Pomeron or subleading Reggeon. The unintegrated parton distributions are calculated based on conventional mmht2014nlo parton distribution functions in the proton and H1 Collaboration diffractive parton distribution functions used previously in the analysis of diffractive structure function and dijets at HERA. For comparison, we present results of calculations performed within the collinear-factorization approach. Our results remain those obtained in the next-to-leading-order approach. The calculation is (must be) supplemented by the so-called gap survival factor, which may, in general, depend on kinematical variables. We try to describe the existing data from Tevatron and make detailed predictions for possible LHC measurements. Several differential distributions are calculated. The E¯T, η ¯ and xp ¯ distributions are compared with the Tevatron data. A reasonable agreement is obtained for the first two distributions. The last one requires introducing a gap survival factor which depends on kinematical variables. We discuss how the phenomenological dependence on one kinematical variable may influence dependence on other variables such as E¯T and η ¯. Several distributions for the LHC are shown.

Molecular mechanisms of retroviral integration site selection

PubMed Central

Kvaratskhelia, Mamuka; Sharma, Amit; Larue, Ross C.; Serrao, Erik; Engelman, Alan

2014-01-01

Retroviral replication proceeds through an obligate integrated DNA provirus, making retroviral vectors attractive vehicles for human gene-therapy. Though most of the host cell genome is available for integration, the process of integration site selection is not random. Retroviruses differ in their choice of chromatin-associated features and also prefer particular nucleotide sequences at the point of insertion. Lentiviruses including HIV-1 preferentially integrate within the bodies of active genes, whereas the prototypical gammaretrovirus Moloney murine leukemia virus (MoMLV) favors strong enhancers and active gene promoter regions. Integration is catalyzed by the viral integrase protein, and recent research has demonstrated that HIV-1 and MoMLV targeting preferences are in large part guided by integrase-interacting host factors (LEDGF/p75 for HIV-1 and BET proteins for MoMLV) that tether viral intasomes to chromatin. In each case, the selectivity of epigenetic marks on histones recognized by the protein tether helps to determine the integration distribution. In contrast, nucleotide preferences at integration sites seem to be governed by the ability for the integrase protein to locally bend the DNA duplex for pairwise insertion of the viral DNA ends. We discuss approaches to alter integration site selection that could potentially improve the safety of retroviral vectors in the clinic. PMID:25147212

Herpes viruses and HIV-1 drug resistance mutations influence the virologic and immunologic milieu of the male genital tract.

PubMed

Gianella, Sara; Morris, Sheldon R; Anderson, Christy; Spina, Celsa A; Vargas, Milenka V; Young, Jason A; Richman, Douglas D; Little, Susan J; Smith, Davey M

2013-01-02

To further understand the role that chronic viral infections of the male genital tract play on HIV-1 dynamics and replication. Retrospective, observational study including 236 paired semen and blood samples collected from 115 recently HIV-1 infected antiretroviral naive men who have sex with men. In this study, we evaluated the association of seminal HIV-1 shedding to coinfections with seven herpes viruses, blood plasma HIV-1 RNA levels, CD4 T-cell counts, presence of transmitted drug resistance mutations (DRMs) in HIV-1 pol, participants' age and stage of HIV-infection using multivariate generalized estimating equation methods. Associations between herpes virus shedding, seminal HIV-1 levels, number and immune activation of seminal T-cells was also investigated (Mann-Whitney). Seminal herpes virus shedding was observed in 75.7% of individuals. Blood HIV-1 RNA levels (P < 0.01) and seminal cytomegalovirus (CMV) and human herpes virus (HHV)-8 levels (P < 0.05) were independent predictors of detectable seminal HIV-1 RNA; higher seminal HIV-1 levels were associated with CMV and Epstein-Barr virus (EBV) seminal shedding, and absence of DRM (P < 0.05). CMV and EBV seminal shedding was associated with higher number of seminal T-lymphocytes, but only presence of seminal CMV DNA was associated with increased immune activation of T-lymphocytes in semen and blood. Despite high median CD4 T-cells numbers, we found a high frequency of herpes viruses seminal shedding in our cohort. Shedding of CMV, EBV and HHV-8 and absence of DRM were associated with increased frequency of HIV-1 shedding and/or higher levels of HIV-1 RNA in semen, which are likely important cofactors for HIV-1 transmission.

Actinomycin D binding mode reveals the basis for its potent HIV-1 and cancer activity

NASA Astrophysics Data System (ADS)

Paramanathan, Thayaparan; Vladescu, Ioana D.; McCauley, Micah J.; Rouzina, Ioulia; Williams, Mark C.

2011-03-01

Actinomycin D (ActD) is one of the most studied antibiotics, which has been used as an anti-cancer agent and also shown to inhibit HIV reverse transcription. Initial studies with ActD established that it intercalates double stranded DNA (dsDNA). However, recent studies have shown that ActD binds with even higher affinity to single stranded DNA (ssDNA). In our studies we use optical tweezers to stretch and hold single dsDNA molecule at constant force in the presence of varying ActD concentrations until the binding reaches equilibrium. The change in dsDNA length upon ActD binding measured as a function of time yields the rate of binding in addition to the equilibrium lengthening of DNA. The results suggest extremely slow kinetics, on the order of several minutes and 0.52 +/- 0.06 μ M binding affinity. Holding DNA at constant force while stretching and relaxing suggests that ActD binds to two single strands that are close to each other rather than to pure dsDNA or ssDNA. This suggests that biological activity of ActD that contributes towards the inhibition of cellular replication is due to its ability to bind at DNA bubbles during RNA transcription, thereby stalling the transcription process.

Two Case Studies in the Scientific Method: Antisense Experiments and HIV Vaccination Studies.

ERIC Educational Resources Information Center

Guilfoile, Patrick

1999-01-01

Presents two recent cases that can be used in the classroom to illustrate the application of scientific methods in biological research: (1) the use of a complementary RNA or DNA molecule to block the production or translation of an mRNA molecule; and (2) the development of HIV trial vaccines. Contains 20 references. (WRM)

Structural Insights into HIV Reverse Transcriptase Mutations Q151M and Q151M Complex That Confer Multinucleoside Drug Resistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Das, Kalyan; Martinez, Sergio E.; Arnold, Eddy

HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations that confer resistance to nucleoside RT inhibitors (NRTIs) emerge during clinical use. Q151M and four associated mutations, A62V, V75I, F77L, and F116Y, were detected in patients failing therapies with dideoxynucleosides (didanosine [ddI], zalcitabine [ddC]) and/or zidovudine (AZT). The cluster of the five mutations is referred to as the Q151M complex (Q151Mc), and an RT or virus containing Q151Mc exhibits resistance to multiple NRTIs. To understand the structural basis for Q151M and Q151Mc resistance, we systematically determined the crystal structures of the wild-type RT/double-stranded DNA (dsDNA)/dATP (complex I), wild-type RT/dsDNA/ddATPmore » (complex II), Q151M RT/dsDNA/dATP (complex III), Q151Mc RT/dsDNA/dATP (complex IV), and Q151Mc RT/dsDNA/ddATP (complex V) ternary complexes. The structures revealed that the deoxyribose rings of dATP and ddATP have 3'-endo and 3'-exo conformations, respectively. The single mutation Q151M introduces conformational perturbation at the deoxynucleoside triphosphate (dNTP)-binding pocket, and the mutated pocket may exist in multiple conformations. The compensatory set of mutations in Q151Mc, particularly F116Y, restricts the side chain flexibility of M151 and helps restore the DNA polymerization efficiency of the enzyme. The altered dNTP-binding pocket in Q151Mc RT has the Q151-R72 hydrogen bond removed and has a switched conformation for the key conserved residue R72 compared to that in wild-type RT. On the basis of a modeled structure of hepatitis B virus (HBV) polymerase, the residues R72, Y116, M151, and M184 in Q151Mc HIV-1 RT are conserved in wild-type HBV polymerase as residues R41, Y89, M171, and M204, respectively; functionally, both Q151Mc HIV-1 and wild-type HBV are resistant to dideoxynucleoside analogs.« less

Brief Report: HIV/HBV Coinfection is a Significant Risk Factor for Liver Fibrosis in Tanzanian HIV-Infected Adults.

PubMed

Hawkins, Claudia; Christian, Beatrice; Fabian, Emanuel; Macha, Irene; Gawile, Cecilia; Mpangala, Shida; Ulenga, Nzovu; Thio, Chloe L; Ammerman, Lauren R; Mugusi, Ferdinand; Fawzi, Wafaie; Green, Richard; Murphy, Robert

2017-11-01

In sub-Saharan Africa, the burden of liver disease associated with chronic hepatitis B virus (HBV) and HIV is unknown. We characterized liver disease using aspartate aminotransferase-to-platelet ratio index (APRI) and FIB-4 in patients with HIV, HBV, and HIV/HBV coinfection in Tanzania. Using a cross-sectional design, we compared the prevalence of liver fibrosis in treatment-naive HIV monoinfected, HBV monoinfected, and HIV/HBV-coinfected adults enrolled at Management and Development for Health (MDH)-supported HIV treatment clinics in Dar es Salaam, Tanzania. Risk factors associated with significant fibrosis (APRI >0.5 and FIB-4 >1.45) were examined. Two hundred sixty-seven HIV-infected, 165 HBV-infected, and 63 HIV/HBV-coinfected patients were analyzed [44% men, median age 37 (interquartile range 14), body mass index 23 (7)]. APRI and FIB-4 were strongly correlated (r = 0.78, P < 0.001, R = 0.61). Overall median APRI scores were low {HIV/HBV [0.36 (interquartile range 0.4)], HIV [0.23 (0.17)], HBV [0.29 (0.15)] (P < 0.01)}. In multivariate analyses, HIV/HBV coinfection was associated with APRI >0.5 [HIV/HBV vs. HIV: odds ratio (OR) 3.78 (95% confidence interval: 1.91 to 7.50)], [HIV/HBV vs. HBV: OR 2.61 (1.26 to 5.44)]. HIV RNA per 1 log10 copies/mL increase [OR 1.53 (95% confidence interval: 1.04 to 2.26)] and HBV DNA per 1 log10 copies/mL increase [OR 1.36 (1.15, 1.62)] were independently associated with APRI >0.5 in HIV-infected and HBV-infected patients, respectively. HIV/HBV coinfection is an important risk factor for significant fibrosis. Higher levels of circulating HIV and HBV virus may play a direct role in liver fibrogenesis. Prompt diagnosis and aggressive monitoring of liver disease in HIV/HBV coinfection is warranted.

Characterization of Equine Infectious Anemia Virus Integration in the Horse Genome.

PubMed

Liu, Qiang; Wang, Xue-Feng; Ma, Jian; He, Xi-Jun; Wang, Xiao-Jun; Zhou, Jian-Hua

2015-06-19

Human immunodeficiency virus (HIV)-1 has a unique integration profile in the human genome relative to murine and avian retroviruses. Equine infectious anemia virus (EIAV) is another well-studied lentivirus that can also be used as a promising retro-transfection vector, but its integration into its native host has not been characterized. In this study, we mapped 477 integration sites of the EIAV strain EIAVFDDV13 in fetal equine dermal (FED) cells during in vitro infection. Published integration sites of EIAV and HIV-1 in the human genome were also analyzed as references. Our results demonstrated that EIAVFDDV13 tended to integrate into genes and AT-rich regions, and it avoided integrating into transcription start sites (TSS), which is consistent with EIAV and HIV-1 integration in the human genome. Notably, the integration of EIAVFDDV13 favored long interspersed elements (LINEs) and DNA transposons in the horse genome, whereas the integration of HIV-1 favored short interspersed elements (SINEs) in the human genome. The chromosomal environment near LINEs or DNA transposons potentially influences viral transcription and may be related to the unique EIAV latency states in equids. The data on EIAV integration in its natural host will facilitate studies on lentiviral infection and lentivirus-based therapeutic vectors.

Characterization of Equine Infectious Anemia Virus Integration in the Horse Genome

PubMed Central

Liu, Qiang; Wang, Xue-Feng; Ma, Jian; He, Xi-Jun; Wang, Xiao-Jun; Zhou, Jian-Hua

2015-01-01

Human immunodeficiency virus (HIV)-1 has a unique integration profile in the human genome relative to murine and avian retroviruses. Equine infectious anemia virus (EIAV) is another well-studied lentivirus that can also be used as a promising retro-transfection vector, but its integration into its native host has not been characterized. In this study, we mapped 477 integration sites of the EIAV strain EIAVFDDV13 in fetal equine dermal (FED) cells during in vitro infection. Published integration sites of EIAV and HIV-1 in the human genome were also analyzed as references. Our results demonstrated that EIAVFDDV13 tended to integrate into genes and AT-rich regions, and it avoided integrating into transcription start sites (TSS), which is consistent with EIAV and HIV-1 integration in the human genome. Notably, the integration of EIAVFDDV13 favored long interspersed elements (LINEs) and DNA transposons in the horse genome, whereas the integration of HIV-1 favored short interspersed elements (SINEs) in the human genome. The chromosomal environment near LINEs or DNA transposons potentially influences viral transcription and may be related to the unique EIAV latency states in equids. The data on EIAV integration in its natural host will facilitate studies on lentiviral infection and lentivirus-based therapeutic vectors. PMID:26102582

The conserved N-terminal basic residues and zinc-finger motifs of HIV-1 nucleocapsid restrict the viral cDNA synthesis during virus formation and maturation

PubMed Central

Didierlaurent, Ludovic; Houzet, Laurent; Morichaud, Zakia; Darlix, Jean-Luc; Mougel, Marylène

2008-01-01

Reverse transcription of the genomic RNA by reverse transcriptase occurs soon after HIV-1 infection of target cells. The viral nucleocapsid (NC) protein chaperones this process via its nucleic acid annealing activities and its interactions with the reverse transcriptase enzyme. To function, NC needs its two conserved zinc fingers and flanking basic residues. We recently reported a new role for NC, whereby it negatively controls reverse transcription in the course of virus formation. Indeed, deleting its zinc fingers causes reverse transcription activation in virus producer cells. To investigate this new NC function, we used viruses with subtle mutations in the conserved zinc fingers and its flanking domains. We monitored by quantitative PCR the HIV-1 DNA content in producer cells and in produced virions. Results showed that the two intact zinc-finger structures are required for the temporal control of reverse transcription by NC throughout the virus replication cycle. The N-terminal basic residues also contributed to this new role of NC, while Pro-31 residue between the zinc fingers and Lys-59 in the C-terminal region did not. These findings further highlight the importance of NC as a major target for anti-HIV-1 drugs. PMID:18641038

Immunization with Clinical HIV-1 Env Proteins Induces Broad Antibody Dependent Cellular Cytotoxicity-Mediating Antibodies in a Rabbit Vaccination Model.

PubMed

Karlsson, Ingrid; Borggren, Marie; Jensen, Sanne Skov; Heyndrickx, Leo; Stewart-Jones, Guillaume; Scarlatti, Gabriella; Fomsgaard, Anders

2017-11-17

The induction of both neutralizing antibodies and non-neutralizing antibodies with effector functions, for example, antibody-dependent cellular cytotoxicity (ADCC), is desired in the search for effective vaccines against HIV-1. In the pursuit of novel immunogens capable of inducing an efficient antibody response, rabbits were immunized with selected antigens using different prime-boost strategies. We immunized 35 different groups of rabbits with Env antigens from clinical HIV-1 subtypes A and B, including immunization with DNA alone, protein alone, and DNA prime with protein boost. The rabbit sera were screened for ADCC activity using a GranToxiLux-based assay with human peripheral blood mononuclear cells as effector cells and CEM.NKR CCR5 cells coated with HIV-1 envelope as target cells. The groups with the highest ADCC activity were further characterized for cross-reactivity between HIV-1 subtypes. The immunogen inducing the most potent and broadest ADCC response was a trimeric gp140. The ADCC activity was highest against the HIV-1 subtype corresponding to the immunogen. The ADCC activity did not necessarily reflect neutralizing activity in the pseudovirus-TZMbl assay, but there was an overall correlation between the two antiviral activities. We present a rabbit vaccination model and an assay suitable for screening HIV-1 vaccine candidates for the induction of ADCC-mediating antibodies in addition to neutralizing antibodies. The antigens and/or immunization strategies capable of inducing antibodies with ADCC activity did not necessarily induce neutralizing activity and vice versa. Nevertheless, we identified vaccine candidates that were able to concurrently induce both types of responses and that had ADCC activity that was cross-reactive between different subtypes. When searching for an effective vaccine candidate, it is important to evaluate the antibody response using a model and an assay measuring the desired function.

Functional Coupling between HIV-1 Integrase and the SWI/SNF Chromatin Remodeling Complex for Efficient in vitro Integration into Stable Nucleosomes

PubMed Central

Lesbats, Paul; Botbol, Yair; Chevereau, Guillaume; Vaillant, Cédric; Calmels, Christina; Arneodo, Alain; Andreola, Marie-Line; Lavigne, Marc; Parissi, Vincent

2011-01-01

Establishment of stable HIV-1 infection requires the efficient integration of the retroviral genome into the host DNA. The molecular mechanism underlying the control of this process by the chromatin structure has not yet been elucidated. We show here that stably associated nucleosomes strongly inhibit in vitro two viral-end integration by decreasing the accessibility of DNA to integrase. Remodeling of the chromatinized template by the SWI/SNF complex, whose INI1 major component interacts with IN, restores and redirects the full-site integration into the stable nucleosome region. These effects are not observed after remodeling by other human remodeling factors such as SNF2H or BRG1 lacking the integrase binding protein INI1. This suggests that the restoration process depends on the direct interaction between IN and the whole SWI/SNF complex, supporting a functional coupling between the remodeling and integration complexes. Furthermore, in silico comparison between more than 40,000 non-redundant cellular integration sites selected from literature and nucleosome occupancy predictions also supports that HIV-1 integration is promoted in the genomic region of weaker intrinsic nucleosome density in the infected cell. Our data indicate that some chromatin structures can be refractory for integration and that coupling between nucleosome remodeling and HIV-1 integration is required to overcome this natural barrier. PMID:21347347

Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.

PubMed

Boyd, Anders; Lacombe, Karine; Lavocat, Fabien; Maylin, Sarah; Miailhes, Patrick; Lascoux-Combe, Caroline; Delaugerre, Constance; Girard, Pierre-Marie; Zoulim, Fabien

2016-10-01

In the presence of highly-potent antivirals, persistence of hepatitis B virus (HBV) is most well-characterized by covalently-closed circular DNA (cccDNA) and total intrahepatic DNA (IH-DNA). We sought to determine how antiviral therapy could affect their levels during human immunodeficiency virus (HIV)-HBV co-infection. Sixty co-infected patients from a well-defined cohort with ⩾1 liver biopsy were studied. HBV cccDNA and total IH-DNA were extracted from biopsies and quantified by real-time PCR. Factors associated with intrahepatic viral load were determined using mixed-effect linear regression and half-life viral kinetics during reconstructed follow-up using non-linear exponential decay models. At biopsy, 35 (58.3%) patients were hepatitis B "e" antigen (HBeAg)-positive and 33 (55.0%) had detectable plasma HBV-DNA (median=4.58log10IU/ml, IQR=2.95-7.43). Overall, median cccDNA was -0.95log10copies/cell (IQR=-1.70, -0.17) and total IH-DNA was 0.27log10copies/cell (IQR=-0.39, 2.00). In multivariable analysis, significantly lower levels of cccDNA and total IH-DNA were observed in patients with HBeAg-negative serology, nadir CD4(+) cell counts >250/mm(3), and longer cumulative TDF-duration, but not lamivudine- or adefovir-duration. In post-hoc analysis using reconstructed TDF-duration (median 29.6months, IQR=15.0-36.1, n=31), average half-life of cccDNA was estimated at 9.2months (HBeAg-positive=8.6, HBeAg-negative=26.2) and total IH DNA at 5.8months (HBeAg-positive=1.3, HBeAg-negative=13.6). Intrahepatic viral loads remained detectable for all patients, even with prolonged TDF-exposure. In co-infection, TDF-use is associated with lower levels of HBV replication intermediates and cccDNA. Slow decay of intrahepatic viral loads underscores that TDF is unable to completely block intracellular viral DNA synthesis, which possibly accounts for continuous replenishment of the cccDNA pool. Chronic hepatitis B virus (HBV) is a persistent infection, while the only real way of knowing the extent of this persistence is through measuring levels of virus in the liver. In this study, we examine levels of HBV in the liver among patients with both HBV and human immunodeficiency virus, or HIV, infection. It would appear that the currently available medication, namely "tenofovir", works well to decrease virus levels in the liver, but it remains at low levels despite long periods of treatment. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Suppression of HIV-1 Infection by APOBEC3 Proteins in Primary Human CD4+ T Cells Is Associated with Inhibition of Processive Reverse Transcription as Well as Excessive Cytidine Deamination

PubMed Central

Gillick, Kieran; Pollpeter, Darja; Phalora, Prabhjeet; Kim, Eun-Young; Wolinsky, Steven M.

2013-01-01

The Vif protein of human immunodeficiency virus type 1 (HIV-1) promotes viral replication by downregulation of the cell-encoded, antiviral APOBEC3 proteins. These proteins exert their suppressive effects through the inhibition of viral reverse transcription as well as the induction of cytidine deamination within nascent viral cDNA. Importantly, these two effects have not been characterized in detail in human CD4+ T cells, leading to controversies over their possible contributions to viral inhibition in the natural cell targets of HIV-1 replication. Here we use wild-type and Vif-deficient viruses derived from the CD4+ T cells of multiple donors to examine the consequences of APOBEC3 protein function at natural levels of expression. We demonstrate that APOBEC3 proteins impart a profound deficiency to reverse transcription from the initial stages of cDNA synthesis, as well as excessive cytidine deamination (hypermutation) of the DNAs that are synthesized. Experiments using viruses from transfected cells and a novel method for mapping the 3′ termini of cDNAs indicate that the inhibition of reverse transcription is not limited to a few specific sites, arguing that APOBEC3 proteins impede enzymatic processivity. Detailed analyses of mutation spectra in viral cDNA strongly imply that one particular APOBEC3 protein, APOBEC3G, provides the bulk of the antiviral phenotype in CD4+ T cells, with the effects of APOBEC3F and APOBEC3D being less significant. Taken together, we conclude that the dual mechanisms of action of APOBEC3 proteins combine to deliver more effective restriction of HIV-1 than either function would by itself. PMID:23152537

Incorporation of excess wild-type and mutant tRNA(3Lys) into human immunodeficiency virus type 1.

PubMed Central

Huang, Y; Mak, J; Cao, Q; Li, Z; Wainberg, M A; Kleiman, L

1994-01-01

Human immunodeficiency virus (HIV) particles produced in COS-7 cells transfected with HIV type 1 (HIV-1) proviral DNA contain 8 molecules of tRNA(3Lys) per 2 molecules of genomic RNA and 12 molecules of tRNA1,2Lys per 2 molecules of genomic RNA. When COS-7 cells are transfected with a plasmid containing both HIV-1 proviral DNA and a human tRNA3Lys gene, there is a large increase in the amount of cytoplasmic tRNA3Lys per microgram of total cellular RNA, and the tRNA3Lys content in the virus increases from 8 to 17 molecules per 2 molecules of genomic RNA. However, the total number of tRNALys molecules per 2 molecules of genomic RNA remains constant at 20; i.e., the viral tRNA1,2Lys content decreases from 12 to 3 molecules per 2 molecules of genomic RNA. All detectable tRNA3Lys is aminoacylated in the cytoplasm of infected cells and deacylated in the virus. When COS-7 cells are transfected with a plasmid containing both HIV-1 proviral DNA and a mutant amber suppressor tRNA3Lys gene (in which the anticodon is changed from TTT to CTA), there is also a large increase in the relative concentration of cytoplasmic tRNA3Lys, and the tRNA3Lys content in the virus increases from 8 to 15 molecules per 2 molecules of genomic RNA, with a decrease in viral tRNA1,2Lys from 12 to 5 molecules per 2 molecules of genomic RNA. Thus, the total number of molecules of tRNALys in the virion remains at 20. The alteration of the anticodon has little effect on the viral packaging of this mutant tRNA in spite of the fact that it no longer contains the modified base mcm 5s2U at position 34, and its ability to be aminoacylated is significantly impaired compared with that of wild-type tRNA3Lys. Viral particles which have incorporated either excess wild-type tRNA3Lys or mutant suppressor tRNA3Lys show no differences in viral infectivity compared with wild-type HIV-1. Images PMID:7966556

Epidemiological trends of HIV-1 infection in blood donors from Catalonia, Spain (2005-2014).

PubMed

Bes, Marta; Piron, Maria; Casamitjana, Natàlia; Gregori, Josep; Esteban, Juan Ignacio; Ribera, Esteban; Quer, Josep; Puig, Lluís; Sauleda, Sílvia

2017-09-01

Human immunodeficiency virus 1 (HIV-1) subtype B is predominant in Spain. However, the recent arrival of immigrant populations has increased the prevalence of non-B subtypes and circulating recombinant forms. The objective of this study was to determine the prevalence of HIV-1 subtypes and transmitted drug-resistance mutations in blood donors from the Catalonian region (northeastern Spain). HIV-1-positive blood donors identified in Catalonia from 2005 to 2014 were included. Demographic variables and risk factors for HIV-1 acquisition were recorded. HIV-1 subtyping was carried out by HIV-1 DNA polymerase region sequencing, and phylogenetic analyses were performed using the neighbor-joining method. During the study period, 2.8 million blood donations were screened, and 214 HIV-1-positive donors were identified, yielding an overall prevalence of 7.7 per 100,000 donations (89% men; mean age, 34 ± 10 years). Most HIV-1-positive donors were native to Spain (81%), and 61% were regular blood donors. When risk factors were known, 62% reportedly were men who had sex with men. HIV-1 subtyping was possible in 176 HIV-1-positive individuals: 143 (81%) had HIV-1 subtype B, and 33 (19%) had non-B subtypes. Most HIV-1 non-B subtypes were circulating recombinant forms (n = 20; 61%). Factors associated with HIV-1 subtype B were male sex (p = 0.007) and men who had sex with men (p < 0.001). The overall prevalence of transmitted drug-resistance mutations was 14%. Non-B subtypes, circulating recombinant forms, and transmitted drug-resistance mutation sequences circulate among HIV-1-positive blood donors in Catalonia. Continuous local epidemiological surveillance is required to implement optimal prevention strategies for controlling transfusion-transmitted HIV and to improve health policies regarding HIV infection. © 2017 AABB.

Flexible DNA binding of the BTB/POZ-domain protein FBI-1.

PubMed

Pessler, Frank; Hernandez, Nouria

2003-08-01

POZ-domain transcription factors are characterized by the presence of a protein-protein interaction domain called the POZ or BTB domain at their N terminus and zinc fingers at their C terminus. Despite the large number of POZ-domain transcription factors that have been identified to date and the significant insights that have been gained into their cellular functions, relatively little is known about their DNA binding properties. FBI-1 is a BTB/POZ-domain protein that has been shown to modulate HIV-1 Tat trans-activation and to repress transcription of some cellular genes. We have used various viral and cellular FBI-1 binding sites to characterize the interaction of a POZ-domain protein with DNA in detail. We find that FBI-1 binds to inverted sequence repeats downstream of the HIV-1 transcription start site. Remarkably, it binds efficiently to probes carrying these repeats in various orientations and spacings with no particular rotational alignment, indicating that its interaction with DNA is highly flexible. Indeed, FBI-1 binding sites in the adenovirus 2 major late promoter, the c-fos gene, and the c-myc P1 and P2 promoters reveal variously spaced direct, inverted, and everted sequence repeats with the consensus sequence G(A/G)GGG(T/C)(C/T)(T/C)(C/T) for each repeat.

Analytical Framework for Identifying and Differentiating Recent Hitchhiking and Severe Bottleneck Effects from Multi-Locus DNA Sequence Data

DOE PAGES

Sargsyan, Ori

2012-05-25

Hitchhiking and severe bottleneck effects have impact on the dynamics of genetic diversity of a population by inducing homogenization at a single locus and at the genome-wide scale, respectively. As a result, identification and differentiation of the signatures of such events from DNA sequence data at a single locus is challenging. This study develops an analytical framework for identifying and differentiating recent homogenization events at multiple neutral loci in low recombination regions. The dynamics of genetic diversity at a locus after a recent homogenization event is modeled according to the infinite-sites mutation model and the Wright-Fisher model of reproduction withmore » constant population size. In this setting, I derive analytical expressions for the distribution, mean, and variance of the number of polymorphic sites in a random sample of DNA sequences from a locus affected by a recent homogenization event. Based on this framework, three likelihood-ratio based tests are presented for identifying and differentiating recent homogenization events at multiple loci. Lastly, I apply the framework to two data sets. First, I consider human DNA sequences from four non-coding loci on different chromosomes for inferring evolutionary history of modern human populations. The results suggest, in particular, that recent homogenization events at the loci are identifiable when the effective human population size is 50000 or greater in contrast to 10000, and the estimates of the recent homogenization events are agree with the “Out of Africa” hypothesis. Second, I use HIV DNA sequences from HIV-1-infected patients to infer the times of HIV seroconversions. The estimates are contrasted with other estimates derived as the mid-time point between the last HIV-negative and first HIV-positive screening tests. Finally, the results show that significant discrepancies can exist between the estimates.« less

Chimeric peptide-mediated siRNA transduction to inhibit HIV-1 infection.

PubMed

Bivalkar-Mehla, Shalmali; Mehla, Rajeev; Chauhan, Ashok

2017-04-01

Persistent human immunodeficiency virus 1 (HIV-1) infection provokes immune activation and depletes CD4 +  lymphocytes, leading to acquired immunodeficiency syndrome. Uninterrupted administration of combination antiretroviral therapy (cART) in HIV-infected patients suppresses viral replication to below the detectable level and partially restores the immune system. However, cART-unresponsive residual HIV-1 infection and elusive transcriptionally silent but reactivatable viral reservoirs maintain a permanent viral DNA blue print. The virus rebounds within a few weeks after interruption of suppressive therapy. Adjunct gene therapy to control viral replication by ribonucleic acid interference (RNAi) is a post-transcriptional gene silencing strategy that could suppress residual HIV-1 burden and overcome viral resistance. Small interfering ribonucleic acids (siRNAs) are efficient transcriptional inhibitors, but need delivery systems to reach inside target cells. We investigated the potential of chimeric peptide (FP-PTD) to deliver specific siRNAs to HIV-1-susceptible and permissive cells. Chimeric FP-PTD peptide was designed with an RNA binding domain (PTD) to bind siRNA and a cell fusion peptide domain (FP) to enter cells. FP-PTD-siRNA complex entered and inhibited HIV-1 replication in susceptible cells, and could be a candidate for in vivo testing.

Epidemiological and clinical characteristics of hepatitis B virus in HIV-infected patients in Guangdong, China.

PubMed

Huang, S M; Cai, W P; Hu, F Y; Lan, Y; Liao, B L; Chen, Y P; Tang, X P

2016-09-01

This study investigated the epidemiological and clinical characteristics of hepatitis B virus (HBV) in HIV-infected adults at the time of antiretroviral therapy (ART) initiation in Guangdong province, China. A total of 2793 HIV-infected adults were enrolled between January 2004 and September 2011. Demographic data and laboratory parameters were collected, HBV-DNA levels were measured, and HBV genotypes were identified before ART initiation. The prevalence of hepatitis B surface antigen (HBsAg) in HIV-infected patients was 13.2%. A total of 266 HIV/HBV co-infected patients and 1469 HIV mono-infected patients were recruited. The median alanine aminotransferase and aspartate aminotransferase levels of HIV/HBV co-infected patients were higher than HIV mono-infected patients (32 U/L vs. 22 U/L, p < 0.001 and 35 U/L vs. 24 U/L, p < 0.001, respectively), whereas the median CD4 cell count of HIV/HBV co-infected patients was lower than HIV mono-infected patients (59 cells/mm(3) vs. 141 cells/mm(3), p < 0.001). The level of CD4 cell count was lower in hepatitis B e-antigen (HBeAg)-positive co-infected patients than HBeAg-negative patients (36 cells/mm(3) vs. 69 cells/mm(3), p = 0.014). A similar result was found in high level of HBV-DNA and low level of HBV-DNA groups (33 cells/mm(3) vs. 89 cells/mm(3), p < 0.001). HBV genotypes were classified as genotypes B and C. Patients infected with genotypes B and C differed significantly in terms of proportion of those who were HBeAg-positive (40.5% vs. 62.2%, p = 0.014). This study indicates a high prevalence of HBsAg in HIV-infected adults in Guangdong. The level of CD4 cell count in HIV/HBV co-infected patients was much lower than HIV mono-infected patients, especially in patients who were HBeAg-positive and had a high level of HBV-DNA. The predominant HBV genotype in HIV/HBV co-infected patients is genotype B. © The Author(s) 2015.

Malnutrition in HIV-Infected Children Is an Indicator of Severe Disease with an Impaired Response to Antiretroviral Therapy.

PubMed

Muenchhoff, Maximilian; Healy, Michael; Singh, Ravesh; Roider, Julia; Groll, Andreas; Kindra, Chirjeev; Sibaya, Thobekile; Moonsamy, Angeline; McGregor, Callum; Phan, Michelle Q; Palma, Alejandro; Kloverpris, Henrik; Leslie, Alasdair; Bobat, Raziya; LaRussa, Philip; Ndung'u, Thumbi; Goulder, Philip; Sobieszczyk, Magdalena E; Archary, Mohendran

2018-01-01

This observational study aimed to describe immunopathogenesis and treatment outcomes in children with and without severe acute malnutrition (SAM) and HIV-infection. We studied markers of microbial translocation (16sDNA), intestinal damage (iFABP), monocyte activation (sCD14), T-cell activation (CD38, HLA-DR) and immune exhaustion (PD1) in 32 HIV-infected children with and 41 HIV-infected children without SAM prior to initiation of antiretroviral therapy (ART) and cross-sectionally compared these children to 15 HIV-uninfected children with and 19 HIV-uninfected children without SAM. We then prospectively measured these markers and correlated them to treatment outcomes in the HIV-infected children at 48 weeks following initiation of ART. Plasma levels of 16sDNA, iFABP and sCD14 were measured by quantitative real time PCR, ELISA and Luminex, respectively. T cell phenotype markers were measured by flow cytometry. Multiple regression analysis was performed using generalized linear models (GLMs) and the least absolute shrinkage and selection operator (LASSO) approach for variable selection. Microbial translocation, T cell activation and exhaustion were increased in HIV-uninfected children with SAM compared to HIV-uninfected children without SAM. In HIV-infected children microbial translocation, immune activation, and exhaustion was strongly increased but did not differ by SAM-status. SAM was associated with increased mortality rates early after ART initiation. Malnutrition, age, microbial translocation, monocyte, and CD8 T cell activation were independently associated with decreased rates of CD4% immune recovery after 48 weeks of ART. SAM is associated with increased microbial translocation, immune activation, and immune exhaustion in HIV-uninfected children and with worse prognosis and impaired immune recovery in HIV-infected children on ART.

Malnutrition in HIV-Infected Children Is an Indicator of Severe Disease with an Impaired Response to Antiretroviral Therapy

PubMed Central

Healy, Michael; Singh, Ravesh; Roider, Julia; Groll, Andreas; Kindra, Chirjeev; Sibaya, Thobekile; Moonsamy, Angeline; McGregor, Callum; Phan, Michelle Q.; Palma, Alejandro; Kloverpris, Henrik; Leslie, Alasdair; Bobat, Raziya; LaRussa, Philip; Ndung'u, Thumbi; Goulder, Philip; Sobieszczyk, Magdalena E.; Archary, Mohendran

2018-01-01

Abstract This observational study aimed to describe immunopathogenesis and treatment outcomes in children with and without severe acute malnutrition (SAM) and HIV-infection. We studied markers of microbial translocation (16sDNA), intestinal damage (iFABP), monocyte activation (sCD14), T-cell activation (CD38, HLA-DR) and immune exhaustion (PD1) in 32 HIV-infected children with and 41 HIV-infected children without SAM prior to initiation of antiretroviral therapy (ART) and cross-sectionally compared these children to 15 HIV-uninfected children with and 19 HIV-uninfected children without SAM. We then prospectively measured these markers and correlated them to treatment outcomes in the HIV-infected children at 48 weeks following initiation of ART. Plasma levels of 16sDNA, iFABP and sCD14 were measured by quantitative real time PCR, ELISA and Luminex, respectively. T cell phenotype markers were measured by flow cytometry. Multiple regression analysis was performed using generalized linear models (GLMs) and the least absolute shrinkage and selection operator (LASSO) approach for variable selection. Microbial translocation, T cell activation and exhaustion were increased in HIV-uninfected children with SAM compared to HIV-uninfected children without SAM. In HIV-infected children microbial translocation, immune activation, and exhaustion was strongly increased but did not differ by SAM-status. SAM was associated with increased mortality rates early after ART initiation. Malnutrition, age, microbial translocation, monocyte, and CD8 T cell activation were independently associated with decreased rates of CD4% immune recovery after 48 weeks of ART. SAM is associated with increased microbial translocation, immune activation, and immune exhaustion in HIV-uninfected children and with worse prognosis and impaired immune recovery in HIV-infected children on ART. PMID:28670966

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin, Zhiqi; Shi, Ke; Banerjee, Surajit

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase–DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNAmore » remained elusive. In this paper, we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein–DNA and protein–protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Finally, our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.« less

Evolutionary genomics and HIV restriction factors.

PubMed

Pyndiah, Nitisha; Telenti, Amalio; Rausell, Antonio

2015-03-01

To provide updated insights into innate antiviral immunity and highlight prototypical evolutionary features of well characterized HIV restriction factors. Recently, a new HIV restriction factor, Myxovirus resistance 2, has been discovered and the region/residue responsible for its activity identified using an evolutionary approach. Furthermore, IFI16, an innate immunity protein known to sense several viruses, has been shown to contribute to the defense to HIV-1 by causing cell death upon sensing HIV-1 DNA. Restriction factors against HIV show characteristic signatures of positive selection. Different patterns of accelerated sequence evolution can distinguish antiviral strategies--offense or defence--as well as the level of specificity of the antiviral properties. Sequence analysis of primate orthologs of restriction factors serves to localize functional domains and sites responsible for antiviral action. We use recent discoveries to illustrate how evolutionary genomic analyses help identify new antiviral genes and their mechanisms of action.

The early effects of stavudine compared with tenofovir on adipocyte gene expression, mitochondrial DNA copy number and metabolic parameters in South African HIV-infected patients: a randomized trial.

PubMed

Menezes, C N; Duarte, R; Dickens, C; Dix-Peek, T; Van Amsterdam, D; John, M-A; Ive, P; Maskew, M; Macphail, P; Fox, M P; Raal, F; Sanne, I; Crowther, N J

2013-04-01

Stavudine is being phased out because of its mitochondrial toxicity and tenofovir (TDF) is recommended as part of first-line highly active antiretroviral therapy (HAART) in South Africa. A prospective, open-label, randomized controlled trial comparing standard- and low-dose stavudine with TDF was performed to assess early differences in adipocyte mtDNA copy number, gene expression and metabolic parameters in Black South African HIV-infected patients. Sixty patients were randomized 1:1:1 to either standard-dose (30-40 mg) or low-dose (20-30 mg) stavudine or TDF (300 mg) each combined with lamivudine and efavirenz. Subcutaneous fat biopsies were obtained at weeks 0 and 4. Adipocyte mtDNA copies/cell and gene expression were measured using quantitative polymerase chain reaction (qPCR). Markers of inflammation and lipid and glucose metabolism were also assessed. A 29% and 32% decrease in the mean mtDNA copies/cell was noted in the standard-dose (P < 0.05) and low-dose stavudine (P < 0.005) arms, respectively, when compared with TDF at 4 weeks. Nuclear respiratory factor-1 (NRF1) and mitochondrial cytochrome B (MTCYB) gene expression levels were affected by stavudine, with a significantly (P < 0.05) greater fall in expression observed with the standard, but not the low dose compared with TDF. No significant differences were observed in markers of inflammation and lipid and glucose metabolism. These results demonstrate early mitochondrial depletion among Black South African patients receiving low and standard doses of stavudine, with preservation of gene expression levels, except for NRF1 and MTCYB, when compared with patients on TDF. © 2012 British HIV Association.

Productive HIV-1 Infection of Human Cervical Tissue Ex Vivo is Associated with the Secretory Phase of the Menstrual Cycle

PubMed Central

Saba, Elisa; Origoni, Massimo; Taccagni, Gianluca; Ferrari, Davide; Doglioni, Claudio; Nava, Alice; Lisco, Andrea; Grivel, Jean-Charles; Margolis, Leonid; Poli, Guido

2013-01-01

Cervical tissue explants (CTE) from 22 HIV-1 seronegative women were exposed to R5 HIV-1 ex vivo. Eight CTE were productively infected in terms of HIV-1 p24Gag release in culture supernatants whereas 14 were not. Nonetheless, both accumulation of HIV-1gag DNA and of p24Gag+ CD4+ T cells and macrophages occurred in both productive and, at lower levels, in nonproductive CTE. Nonproductive CTE differed from productive CTE for higher secretion of CCL3 and CCL5. A post-hoc analysis revealed that all productive CTE were established from women in their secretory phase of the menstrual cycle, whereas nonproductive CTE derived from women either in their secretory (28%) or proliferative (36%) menstrual cycle phases or with an atrophic endometrium (36%). Thus, our results support the epidemiological observation that sexual HIV-1 transmission from males to women as well as from women to men is more efficient during their secretory phase of the menstrual cycle. PMID:23385427

Performance of an Early Infant Diagnostic Test, AmpliSens DNA-HIV-FRT, Using Dried Blood Spots Collected from Children Born to Human Immunodeficiency Virus-Infected Mothers in Ukraine.

PubMed

Chang, Joy; Tarasova, Tetyana; Shanmugam, Vedapuri; Azarskova, Marianna; Nguyen, Shon; Hurlston, Mackenzie; Sabatier, Jennifer; Zhang, Guoqing; Osmanov, Saladin; Ellenberger, Dennis; Yang, Chunfu; Vitek, Charles; Liulchuk, Maria; Nizova, Natalya

2015-12-01

An accurate accessible test for early infant diagnosis (EID) is crucial for identifying HIV-infected infants and linking them to treatment. To improve EID services in Ukraine, dried blood spot (DBS) samples obtained from 237 HIV-exposed children (≤18 months of age) in six regions in Ukraine in 2012 to 2013 were tested with the AmpliSens DNA-HIV-FRT assay, the Roche COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) HIV-1 Qual test, and the Abbott RealTime HIV-1 Qualitative assay. In comparison with the paired whole-blood results generated from AmpliSens testing at the oblast HIV reference laboratories in Ukraine, the sensitivity was 0.99 (95% confidence interval [CI], 0.95 to 1.00) for the AmpliSens and Roche CAP/CTM Qual assays and 0.96 (95% CI, 0.90 to 0.98) for the Abbott Qualitative assay. The specificity was 1.00 (95% CI, 0.97 to 1.00) for the AmpliSens and Abbott Qualitative assays and 0.99 (95% CI, 0.96 to 1.00) for the Roche CAP/CTM Qual assay. McNemar analysis indicated that the proportions of positive results for the tests were not significantly different (P > 0.05). Cohen's kappa (0.97 to 0.99) indicated almost perfect agreement among the three tests. These results indicated that the AmpliSens DBS and whole-blood tests performed equally well and were comparable to the two commercially available EID tests. More importantly, the performance characteristics of the AmpliSens DBS test meets the World Health Organization EID test requirements; implementing AmpliSens DBS testing might improve EID services in resource-limited settings. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Unusual and unique distribution of anal high-risk human papillomavirus (HR-HPV) among men who have sex with men living in the Central African Republic

PubMed Central

Camengo, Serge Police; Veyer, David; Matta, Mathieu; Robin, Leman; Longo, Jean De Dieu; Grésenguet, Gérard; Péré, Hélène; Meye, Jean-François; Belec, Laurent

2018-01-01

Background High-risk (HR) human papillomavirus (HPV) infection remains a great concern in relation to African men who have sex with men (MSM), especially those infected with HIV. The prevalence of HR-HPV and associated risk factors was estimated in a cross-sectional observational study covering MSM living in Bangui, Central African Republic. Methods MSM receiving care at the Centre National de Référence des Infections Sexuellement Transmissibles et de la Thérapie Antirétrovirale, Bangui, were included. HIV serostatus and socio-demographic and behavioral characteristics were collected. HPV DNA was detected and genotyped on anal swabs using Anyplex™ II HPV28 test (Seegene, South Korea), and HSV DNA by in-house real-time PCR. Logistic regression analyses were used to determine risk factors associated with HPV outcomes. Results 42 MSM (mean age, 23.2 years; range, 14–39) including 69.1% HIV-1-positive and 30.9% HIV-negative were prospectively enrolled. The prevalence of anal HPV was 69.1%, including 82.7% of HR-HPV which were multiple in 52.0%. The most prevalent genotypes were HPV-35, HPV-58, HPV-59 and HPV-31. While, HPV-16 and HPV-18 were present in a minority of samples. Multiple HR-HPV infection was more frequent in HIV-positive MSM (41.4%) with 2.7 genotypes per anal samples than in HIV-negative (7.7%) with 1.5 genotypes per anal samples. HPV types included in the prophylactic Gardasil-9® vaccine were detected in 68.9% of specimens and HPV-58 was the most frequently detected. MSM infected by HPV-16 and HPV-18 were all infected by HIV-1. Few anal swabs (11.9%) contained HSV-2 DNA without relationship with HPV detection. Condomless receptive anal intercourse was the main risk factor to being infected with any type of HPV and condomless insertive anal intercourse was significantly less associated with HPV contamination than receptive anal intercourse (Odd ratio = 0.02). Conclusion MSM in Bangui are at-risk of HIV and HR-HPV anal infections. The unusual distribution of HPV-35 as predominant HPV suggests possible geographic specificities in the molecular epidemiology of HR-HPV in sub-Saharan Africa. Scaling up prevention strategies against HPV infection and related cancers adapted for MSM in Africa should be prioritized. Innovative interventions should be conceived for the MSM population living in Bangui. PMID:29795661

Nuclear factor kappa B: a potential target for anti-HIV chemotherapy.

PubMed

Pande, V; Ramos, M J

2003-08-01

The Nuclear Factor Kappa B (NF-kappaB) is a lymphoid-specific transcription factor, which is sequestered in the cytoplasm by the protein IkappaB. NF-kappaB plays a major role in the regulation of HIV-1 gene expression. Upon activation, NF-kappaB is released from IkappaB, moves to the nucleus, and binds to its sites on the HIV long terminal repeat to start transcription of integrated HIV genome. The present review focuses on the NF-kappaB as a potential target for the development of chemotherapy against HIV-1. Beginning from the viral-binding to reverse transcription, integration, and gene expression, to the virion maturation, the life cycle of HIV presents drug-targets at all the stages. As a result, many drugs have been developed and have entered clinical trials. Some of the most important of these are reverse transcriptase and protease inhibitors, which have been used mostly in clinical studies in the form of combined therapy. But, this combined therapy has presented the problem of resistance, due to mutations in the virus. However, targeting NF-kappaB for the suppression of virus does not present the problem of resistance, as NF-kappaB is a normal part of the human T-4 cell, and is not subject to mutations, as is the virus. An overview of the NF-kappaB system and its role in HIV-1 is presented, followed by a critical review of its current and potential synthetic inhibitors. The drugs studied against NF-kappaB fall mainly into three categories: (1) Antioxidants, against oxidative stress conditions, which aid in NF-kappaB activation, (2) IkappaB phosphorylation and degradation inhibitors (the phosphorylation and degradation of IkappaB is necessary to make NF-kappaB free and move to the nucleus), and (3) NF-kappaB DNA binding inhibitors. The antioxidants include N-Acetyl-L-cysteine (NAC), alpha-Lipoic acid, glutathione monoester, pyrrolidine dithiocarbamate, and tepoxalin, of which NAC is the best studied. The IkappaB phosphorylation and degradation inhibitors, which have been studied in the context of HIV-1 include the salicylates (sodium salicylate, and acetylsalicylic acid (aspirin)). Finally, the NF-kappaB DNA binding inhibitors, which have received attention only recently, are reviewed. These include the most potential, aurine tricarboxylic acid (ATA), a chelating agent, which has been found to inhibit NF-kappaB DNA binding at a low concentration of 30 micro M. The probable mechanism of action of these drugs is discussed alongwith relevant suggestions and conclusions.

Electrochemical detection of sequence-specific DNA based on formation of G-quadruplex-hemin through continuous hybridization chain reaction.

PubMed

Sun, Xiaofan; Chen, Haohan; Wang, Shuling; Zhang, Yiping; Tian, Yaping; Zhou, Nandi

2018-08-27

A high-sensitive detection of sequence-specific DNA was established based on the formation of G-quadruplex-hemin complex through continuous hybridization chain reaction (HCR). Taking HIV DNA sequence as an example, a capture probe complementary to part of HIV DNA was firstly self-assembled onto the surface of Au electrode. Then a specially designed assistant probe with both terminals complementary to the target DNA and a G-quadruplex-forming sequence in the center was introduced into the detection solution. In the presence of both the target DNA and the assistant probe, the target DNA can be captured on the electrode surface and then a continuous HCR can be conducted due to the mutual recognition of the target DNA and the assistant probe, leading to the formation of a large number of G-quadruplex on the electrode surface. With the help of hemin, a pronounced electrochemical signal can be observed in differential pulse voltammetry (DPV), due to the formation of G-quadruplex-hemin complex. The peak current is linearly related with the logarithm of the concentration of the target DNA in the range from 10 fM to 10 pM. The electrochemical sensor has high selectivity to clearly discriminate single-base mismatched and three-base mismatched sequences from the original HIV DNA sequence. Moreover, the established DNA sensor was challenged by detection of HIV DNA in human serum samples, which showed the low detection limit of 6.3 fM. Thus it has great application prospect in the field of clinical diagnosis and environmental monitoring. Copyright © 2018 Elsevier B.V. All rights reserved.

Six-year pilot study on nucleic acid testing for blood donations in China.

PubMed

Ye, Xianlin; Yang, Baocheng; Zhu, Weigang; Zheng, Xin; Du, Peng; Zeng, Jingfeng; Li, Chengyao

2013-10-01

A six-year pilot study on nucleic acid testing for HBV, HCV and HIV-1 has been undertaken on sero-negative plasmas in mini-pool and individual donation testing at Shenzhen Blood Center. Of 307,740 sero-negative blood samples, 95 of 102 HBV DNA yields were confirmed positive, 80/95 (84.2%) were classified as occult HBV infection (OBI) and 15 (15.8%) as window period cases. Amongst OBIs, 45% carried anti-HBc only, 41.3% anti-HBc and anti-HBs and 13.7% anti-HBs only. HBV DNA yield was 1:3239. One HCV WP and one HIV-1 infected donations were detected. High residual risk was found in current blood donations screening in China. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development of a Novel Anti-HIV-1 Agent from within: Effect of Chimeric Vpr-Containing Protease Cleavage Site Residues on Virus Replication

NASA Astrophysics Data System (ADS)

Serio, D.; Rizvi, T. A.; Cartas, M.; Kalyanaraman, V. S.; Weber, I. T.; Koprowski, H.; Srinivasan, A.

1997-04-01

Effective antiviral agents will be of great value in controlling virus replication and delaying the onset of HIV-1-related disease symptoms. Current therapy involves the use of antiviral agents that target the enzymatic functions of the virus, resulting in the emergence of resistant viruses to these agents, thus lowering their effectiveness. To overcome this problem, we have considered the idea of developing novel agents from within HIV-1 as inhibitors of virus replication. The specificity of the Vpr protein for the HIV-1 virus particle makes it an attractive molecule for the development of antiviral agents targeting the events associated with virus maturation. We have generated chimeric Vpr proteins containing HIV-1-specific sequences added to the C terminus of Vpr. These sequences correspond to nine cleavage sites of the Gag and Gag-Pol precursors of HIV-1. The chimeric Vpr constructs were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity using single- and multiple-round replication assays. The virus particles generated exhibited a variable replication pattern depending on the protease cleavage site used as a fusion partner. Interestingly, the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2, 24/2, completely abolished virus infectivity. These results show that chimeric proteins generated from within HIV-1 have the ability to suppress HIV-1 replication and make ideal agents for gene therapy or intracellular immunization to treat HIV-1 infection.

Sexually-Transmitted/Founder HIV-1 Cannot Be Directly Predicted from Plasma or PBMC-Derived Viral Quasispecies in the Transmitting Partner

PubMed Central

Frange, Pierre; Meyer, Laurence; Jung, Matthieu; Goujard, Cecile; Zucman, David; Abel, Sylvie; Hochedez, Patrick; Gousset, Marine; Gascuel, Olivier; Rouzioux, Christine; Chaix, Marie-Laure

2013-01-01

Objective Characterization of HIV-1 sequences in newly infected individuals is important for elucidating the mechanisms of viral sexual transmission. We report the identification of transmitted/founder viruses in eight pairs of HIV-1 sexually-infected patients enrolled at the time of primary infection (“recipients”) and their transmitting partners (“donors”). Methods Using a single genome-amplification approach, we compared quasispecies in donors and recipients on the basis of 316 and 376 C2V5 env sequences amplified from plasma viral RNA and PBMC-associated DNA, respectively. Results Both DNA and RNA sequences indicated very homogeneous viral populations in all recipients, suggesting transmission of a single variant, even in cases of recent sexually transmitted infections (STIs) in donors (n = 2) or recipients (n = 3). In all pairs, the transmitted/founder virus was derived from an infrequent variant population within the blood of the donor. The donor variant sequences most closely related to the recipient sequences were found in plasma samples in 3/8 cases and/or in PBMC samples in 6/8 cases. Although donors were exclusively (n = 4) or predominantly (n = 4) infected by CCR5-tropic (R5) strains, two recipients were infected with highly homogeneous CXCR4/dual-mixed-tropic (X4/DM) viral populations, identified in both DNA and RNA. The proportion of X4/DM quasispecies in donors was higher in cases of X4/DM than R5 HIV transmission (16.7–22.0% versus 0–2.6%), suggesting that X4/DM transmission may be associated with a threshold population of X4/DM circulating quasispecies in donors. Conclusions These suggest that a severe genetic bottleneck occurs during subtype B HIV-1 heterosexual and homosexual transmission. Sexually-transmitted/founder virus cannot be directly predicted by analysis of the donor’s quasispecies in plasma and/or PBMC. Additional studies are required to fully understand the traits that confer the capacity to transmit and establish infection, and determine the role of concomitant STIs in mitigating the genetic bottleneck in mucosal HIV transmission. PMID:23874894

Infusing Physical Activities into the Classroom: Effects on Preschool Children's Geography Learning

ERIC Educational Resources Information Center

Mavilidi, Myrto-Foteini; Okely, Anthony D.; Chandler, Paul; Paas, Fred

2016-01-01

In this intervention study, we investigated the effects of physical activities that were integrated into a geography task on preschool children's learning performance and enjoyment. Eight childcare centers with 87 four-to-five-year-old children were randomly assigned across an integrated physical activity condition, an unintegrated physical…

Notes on Unintegration and Disintegration from Historical and Developmental Perspectives

ERIC Educational Resources Information Center

Urwin, Cathy

2006-01-01

This paper contextualises Bick's contribution historically, particularly in relation to Winnicott's work which challenged the Kleinian tradition to provide a developmental account of psychotic phenomena that would give a role to environmental factors. A difference in emphasis between Bick's early and later account of the functions of the skin in…

HIV Integration at Certain Sites in Host DNA is Linked to the Expansion and Persistence of Infected Cells | Center for Cancer Research

Cancer.gov

When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with

Comparison of Newly Assembled Full Length HIV-1 Integrase With Prototype Foamy Virus Integrase: Structure-Function Prospective.

PubMed

Dayer, Mohammad Reza

2016-05-01

Drug design against human immunodeficiency virus type 1 (HIV-1) integrase through its mechanistic study is of great interest in the area in biological research. The main obstacle in this area is the absence of the full-length crystal structure for HIV-1 integrase to be used as a model. A complete structure, similar to HIV-1 of a prototype foamy virus integrase in complex with DNA, including all conservative residues, is available and has been extensively used in recent investigations. The aim of this study was to determine whether the above model is precisely representative of HIV-1 integrase. This would critically determine the success of any designed drug using the model in deactivation of integrase and AIDS treatment. Primarily, a new structure for HIV-1 was constructed, using a crystal structure of prototype foamy virus as the starting structure. The constructed structure of HIV-1 integrase was simultaneously simulated with a prototype foamy virus integrase on a separate occasion. Our results indicate that the HIV-1 system behaves differently from the prototype foamy virus in terms of folding, hydration, hydrophobicity of binding site and stability. Based on our findings, we can conclude that HIV-1 integrase is vastly different from the prototype foamy virus integrase and does not resemble it, and the modeling output of the prototype foamy virus simulations could not be simply generalized to HIV-1 integrase. Therefore, our HIV-1 model seems to be more representative and more useful for future research.

Clinical validation of a novel diagnostic HIV-2 total nucleic acid qualitative assay using the Abbott m2000 platform: Implications for complementary HIV-2 nucleic acid testing for the CDC 4th generation HIV diagnostic testing algorithm.

PubMed

Chang, Ming; Wong, Audrey J S; Raugi, Dana N; Smith, Robert A; Seilie, Annette M; Ortega, Jose P; Bogusz, Kyle M; Sall, Fatima; Ba, Selly; Seydi, Moussa; Gottlieb, Geoffrey S; Coombs, Robert W

2017-01-01

The 2014 CDC 4th generation HIV screening algorithm includes an orthogonal immunoassay to confirm and discriminate HIV-1 and HIV-2 antibodies. Additional nucleic acid testing (NAT) is recommended to resolve indeterminate or undifferentiated HIV seroreactivity. HIV-2 NAT requires a second-line assay to detect HIV-2 total nucleic acid (TNA) in patients' blood cells, as a third of untreated patients have undetectable plasma HIV-2 RNA. To validate a qualitative HIV-2 TNA assay using peripheral blood mononuclear cells (PBMC) from HIV-2-infected Senegalese study participants. We evaluated the assay precision, sensitivity, specificity, and diagnostic performance of an HIV-2 TNA assay. Matched plasma and PBMC samples were collected from 25 HIV-1, 30 HIV-2, 8 HIV-1/-2 dual-seropositive and 25 HIV seronegative individuals. Diagnostic performance was evaluated by comparing the outcome of the TNA assay to the results obtained by the 4th generation HIV screening and confirmatory immunoassays. All PBMC from 30 HIV-2 seropositive participants tested positive for HIV-2 TNA including 23 patients with undetectable plasma RNA. Of the 30 matched plasma specimens, one was HIV non-reactive. Samples from 50 non-HIV-2 infected individuals were confirmed as non-reactive for HIV-2 Ab and negative for HIV-2 TNA. The agreement between HIV-2 TNA and the combined immunoassay results was 98.8% (79/80). Furthermore, HIV-2 TNA was detected in 7 of 8 PBMC specimens from HIV-1/HIV-2 dual-seropositive participants. Our TNA assay detected HIV-2 DNA/RNA in PBMC from serologically HIV-2 reactive, HIV indeterminate or HIV undifferentiated individuals with undetectable plasma RNA, and is suitable for confirming HIV-2 infection in the HIV testing algorithm. Copyright © 2016 Elsevier B.V. All rights reserved.

A single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity

PubMed Central

Wahba, Alexander S.; Esmaeili, Abbasali; Damha, Masad J.; Hudson, Robert H. E.

2010-01-01

6-Phenylpyrrolocytidine (PhpC), a structurally conservative and highly fluorescent cytidine analog, was incorporated into oligoribonucleotides. The PhpC-containing RNA formed native-like duplex structures with complementary DNA or RNA. The PhpC-modification was found to act as a sensitive reporter group being non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single PhpC insert was an excellent substrate for HIV-1 RT Ribonuclease H and rapidly reported cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The PhpC-based assay for RNase H was superior to the traditional molecular beacon approach in terms of responsiveness, rapidity and ease (single label versus dual). Furthermore, the PhpC-based assay is amenable to high-throughput microplate assay format and may form the basis for a new screen for inhibitors of HIV-RT RNase H. PMID:19933258

First report of human parvovirus 4 detection in Iran.

PubMed

Asiyabi, Sanaz; Nejati, Ahmad; Shoja, Zabihollah; Shahmahmoodi, Shohreh; Jalilvand, Somayeh; Farahmand, Mohammad; Gorzin, Ali-Akbar; Najafi, Alireza; Haji Mollahoseini, Mostafa; Marashi, Sayed Mahdi

2016-08-01

Parvovirus 4 (PARV4) is an emerging and intriguing virus that currently received many attentions. High prevalence of PARV4 infection in high-risk groups such as HIV infected patients highlights the potential clinical outcomes that this virus might have. Molecular techniques were used to determine both the presence and the genotype of circulating PARV4 on previously collected serum samples from 133 HIV infected patients and 120 healthy blood donors. Nested PCR was applied to assess the presence of PARV4 DNA genome in both groups. PARV4 DNA was detected in 35.3% of HIV infected patients compared to 16.6% healthy donors. To genetically characterize the PARV4 genotype in these groups, positive samples were randomly selected and subjected for sequencing and phylogenetic analysis. All PARV4 sequences were found to be genotype 1 and clustered with the reference sequences of PARV4 genotype 1. J. Med. Virol. 88:1314-1318, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Therapeutic DNA vaccination of vertically HIV-infected children: report of the first pediatric randomised trial (PEDVAC).

PubMed

Palma, Paolo; Romiti, Maria Luisa; Montesano, Carla; Santilli, Veronica; Mora, Nadia; Aquilani, Angela; Dispinseri, Stefania; Tchidjou, Hyppolite K; Montano, Marco; Eriksson, Lars E; Baldassari, Stefania; Bernardi, Stefania; Scarlatti, Gabriella; Wahren, Britta; Rossi, Paolo

2013-01-01

Twenty vertically HIV-infected children, 6-16 years of age, with stable viral load control and CD4+ values above 400 cells/mm(3). Ten subjects continued their ongoing antiretroviral treatment (ART, Group A) and 10 were immunized with a HIV-DNA vaccine in addition to their previous therapy (ART and vaccine, Group B). The genetic vaccine represented HIV-1 subtypes A, B and C, encoded Env, Rev, Gag and RT and had no additional adjuvant. Immunizations took place at weeks 0, 4 and 12, with a boosting dose at week 36. Monitoring was performed until week 60 and extended to week 96. Safety data showed good tolerance of the vaccine. Adherence to ART remained high and persistent during the study and did not differ significantly between controls and vaccinees. Neither group experienced either virological failure or a decline of CD4+ counts from baseline. Higher HIV-specific cellular immune responses were noted transiently to Gag but not to other components of the vaccine. Lymphoproliferative responses to a virion antigen HIV-1 MN were higher in the vaccinees than in the controls (p = 0.047), whereas differences in reactivity to clade-specific Gag p24, RT or Env did not reach significance. Compared to baseline, the percentage of HIV-specific CD8+ lymphocytes releasing perforin in the Group B was higher after the vaccination schedule had been completed (p = 0.031). No increased CD8+ perforin levels were observed in control Group A. The present study demonstrates the feasibility, safety and moderate immunogenicity of genetic vaccination in vertically HIV-infected children, paving the way for amplified immunotherapeutic approaches in the pediatric population. clinicaltrialsregister.eu _2007-002359-18IT.

Therapeutic DNA Vaccination of Vertically HIV-Infected Children: Report of the First Pediatric Randomised Trial (PEDVAC)

PubMed Central

Palma, Paolo; Romiti, Maria Luisa; Montesano, Carla; Santilli, Veronica; Mora, Nadia; Aquilani, Angela; Dispinseri, Stefania; Tchidjou, Hyppolite K.; Montano, Marco; Eriksson, Lars E.; Baldassari, Stefania; Bernardi, Stefania; Scarlatti, Gabriella

2013-01-01

Subjects Twenty vertically HIV-infected children, 6–16 years of age, with stable viral load control and CD4+ values above 400 cells/mm3. Intervention Ten subjects continued their ongoing antiretroviral treatment (ART, Group A) and 10 were immunized with a HIV-DNA vaccine in addition to their previous therapy (ART and vaccine, Group B). The genetic vaccine represented HIV-1 subtypes A, B and C, encoded Env, Rev, Gag and RT and had no additional adjuvant. Immunizations took place at weeks 0, 4 and 12, with a boosting dose at week 36. Monitoring was performed until week 60 and extended to week 96. Results Safety data showed good tolerance of the vaccine. Adherence to ART remained high and persistent during the study and did not differ significantly between controls and vaccinees. Neither group experienced either virological failure or a decline of CD4+ counts from baseline. Higher HIV-specific cellular immune responses were noted transiently to Gag but not to other components of the vaccine. Lymphoproliferative responses to a virion antigen HIV-1 MN were higher in the vaccinees than in the controls (p = 0.047), whereas differences in reactivity to clade-specific Gag p24, RT or Env did not reach significance. Compared to baseline, the percentage of HIV-specific CD8+ lymphocytes releasing perforin in the Group B was higher after the vaccination schedule had been completed (p = 0.031). No increased CD8+ perforin levels were observed in control Group A. Conclusions The present study demonstrates the feasibility, safety and moderate immunogenicity of genetic vaccination in vertically HIV-infected children, paving the way for amplified immunotherapeutic approaches in the pediatric population. Trial registration clinicaltrialsregister.eu _2007-002359-18 IT PMID:24312194

Identification of HIV-1 genitourinary tract compartmentalization by analyzing the env gene sequences in urine.

PubMed

Blasi, Maria; Carpenter, J Harris; Balakumaran, Bala; Cara, Andrea; Gao, Feng; Klotman, Mary E

2015-08-24

HIV-1 persists indefinitely in memory CD4 T cells and other long-lived cellular reservoirs despite antiretroviral therapy. Our group had previously demonstrated that HIV-1 can establish a productive infection in renal epithelial cells and that the kidney represents a separate compartment for HIV-1 replication. Here, to better understand the viruses in this unique site, we genetically characterized and compared the viruses in blood and urine specimens from 24 HIV-1 infected patients with detectable viremia. Blood and urine samples were obtained from 35 HIV-1 positive patients. Single-genome amplification was performed on HIV-1 env RNA and DNA isolated from urine supernatants and urine-derived cell pellets, respectively, as well as from plasma and peripheral blood mononuclear cell from the same individuals. Neighbor-joining trees were constructed under the Kimura 2-parameter model. We amplified and sequenced the full-length HIV-1 envelope (env) gene from 12 of the 24 individuals, indicating that 50% of the viremic HIV-1-positive patients had viral RNA in their urine. Phylogenetic analysis of the env sequences from four individuals with more than 15 urine-derived env sequences showed that the majority of the sequences from urine formed distinct cluster(s) independent of those peripheral blood mononuclear cell and plasma-derived sequences, consistent with viral compartmentalization in the urine. Our results suggest the presence of a distinct HIV compartment in the genitourinary tract.

Identification of HIV-1 Genitourinary Tract Compartmentalization by Analyzing the env Gene Sequences in Urine

PubMed Central

BLASI, Maria; CARPENTER, J. Harris; BALAKUMARAN, Bala; CARA, Andrea; GAO, Feng; KLOTMAN, Mary E.

2015-01-01

Objective HIV-1 persists indefinitely in memory CD4+ T cells and other long-lived cellular reservoirs despite antiretroviral therapy (ART). Our group had previously demonstrated that HIV-1 can establish a productive infection in renal epithelial cells and that the kidney represents a separate compartment for HIV-1 replication. Here, to better understand the viruses in this unique site, we genetically characterized and compared the viruses in blood and urine specimens from twenty-four HIV-1 infected subjects with detectable viremia. Design and Methods Blood and urine samples were obtained from 35 HIV-1 positive subjects. Single-genome amplification was performed on HIV-1 env RNA and DNA isolated from urine supernatants and urine derived cell pellets respectively, as well as from plasma and PBMC from the same individuals. Neighbor-joining trees were constructed under the Kimura 2-parameter mode. Results We amplified and sequenced the full-length HIV-1 envelope (env) gene from twelve of the twenty-four individuals, indicating that fifty percent (50%) of the viremic HIV-1 positive patients had viral RNA in their urine. Phylogenetic analysis of the env sequences from four subjects with more than fifteen urine-derived env sequences showed that the majority of the sequences from urine formed distinct cluster(s) independent of those PBMC and plasma-derived sequences, consistent with viral compartmentalization in the urine. Conclusions Our results suggest the presence of a distinct HIV compartment in the genitourinary tract. PMID:26372275

HIV genetic information and clonal growth

Cancer.gov

Based on an analysis of blood cells from five HIV-infected individuals, NCI researchers have identified more than 2,400 HIV DNA insertion sites. Analysis of these sites showed that there is extensive clonal expansion (growth) of HIV infected cells.

Simian Immunodeficiency Virus Vif and Human APOBEC3B Interactions Resemble Those between HIV-1 Vif and Human APOBEC3G.

PubMed

Wang, Jiayi; Shaban, Nadine M; Land, Allison M; Brown, William L; Harris, Reuben S

2018-06-15

Several members of the APOBEC3 DNA cytosine deaminase family can potently inhibit Vif-deficient human immunodeficiency virus type 1 (HIV-1) by catalyzing cytosine deamination in viral cDNA and impeding reverse transcription. HIV-1 counteracts restriction with the virally encoded Vif protein, which targets relevant APOBEC3 proteins for proteasomal degradation. HIV-1 Vif is optimized for degrading the restrictive human APOBEC3 repertoire, and, in general, lentiviral Vif proteins specifically target the restricting APOBEC3 enzymes of each host species. However, simian immunodeficiency virus SIV mac239 Vif elicits a curiously wide range of APOBEC3 degradation capabilities that include degradation of several human APOBEC3s and even human APOBEC3B, a non-HIV-1-restricting APOBEC3 enzyme. To better understand the molecular determinants of the interaction between SIV mac239 Vif and human APOBEC3B, we analyzed an extensive series of mutants. We found that SIV mac239 Vif interacts with the N-terminal domain of human APOBEC3B and, interestingly, that this occurs within a structural region homologous to the HIV-1 Vif interaction surface of human APOBEC3G. An alanine scan of SIV mac239 Vif revealed several residues required for human APOBEC3B degradation activity. These residues overlap HIV-1 Vif surface residues that interact with human APOBEC3G and are distinct from those that engage APOBEC3F or APOBEC3H. Overall, these studies indicate that the molecular determinants of the functional interaction between human APOBEC3B and SIV mac239 Vif resemble those between human APOBEC3G and HIV-1 Vif. These studies contribute to the growing knowledge of the APOBEC-Vif interaction and may help guide future efforts to disrupt this interaction as an antiviral therapy or exploit the interaction as a novel strategy to inhibit APOBEC3B-dependent tumor evolution. IMPORTANCE Primate APOBEC3 proteins provide innate immunity against retroviruses such as HIV and SIV. HIV-1, the primary cause of AIDS, utilizes its Vif protein to specifically counteract restrictive human APOBEC3 enzymes. SIV mac239 Vif exhibits a much wider range of anti-APOBEC3 activities that includes several rhesus macaque enzymes and extends to multiple proteins in the human APOBEC3 repertoire, including APOBEC3B. Understanding the molecular determinants of the interaction between SIV mac239 Vif and human APOBEC3B adds to existing knowledge on the APOBEC3-Vif interaction and has potential to shed light on what processes may have shaped Vif functionality over evolutionary time. An intimate understanding of this interaction may also lead to a novel cancer therapy because, for instance, creating a derivative of SIV mac239 Vif that specifically targets human APOBEC3B could be used to suppress tumor genomic DNA mutagenesis by this enzyme, slow ongoing tumor evolution, and help prevent poor clinical outcomes. Copyright © 2018 American Society for Microbiology.

Hepatitis B virus prevalence, risk factors and genotype distribution in HIV infected patients from West Java, Indonesia.

PubMed

Fibriani, Azzania; Wisaksana, Rudi; Alisjahbana, Bachti; Indrati, Agnes; Schutten, Martin; van Crevel, Reinout; van der Ven, Andre; Boucher, Charles A B

2014-04-01

Indonesia currently faces both an increasing HIV incidence and a high hepatitis B virus (HBV) burden. The objective of our study is to examine the prevalence, risk factors, and genotypic distribution of HBV infection among HIV infected patients in West Java, Indonesia. A cross sectional study was conducted among a cohort of HIV infected patients in 2008. Demographic and disease related variables were compared between HBV negative and positive patients. Logistic regression was applied to determine risk factors for HBV co-infection. HBV and HIV genotyping was performed in co-infected patients. Of 636 HIV-infected patients, the rate of HBV co-infection was 7%. The proportion of males was higher in HBV/HIV co-infected patients than in HIV mono-infected patients (93% vs. 72%, P=0.001). A history of injecting drug use (IDU), but not tattooing, was associated with HBV co-infection [P=0.035 OR 2.41 (95% CI 1.06-5.47)]. In the HIV and HBV treatment naive patients, CD4 cells counts <50cells/mm(3), HIV-RNA plasma ≥10,000copies/ml and AST level above normal were more often found in patients with high HBV-DNA levels (≥20,000IU/ml) as compared to those with low HBV DNA (<20.000IU/ml) (P<0.05). As in the general population, B3 was the dominant subtype in HBV co-infected patients. The prevalence of active HBV infection and the genotype distribution among HIV infected individuals is similar to the overall population in Java. However, an increased prevalence was observed in men with a history of IDU, underlining the need for routine HBV screening and monitoring. Copyright © 2014 Elsevier B.V. All rights reserved.

Options in human papillomavirus (HPV) detection for cervical cancer screening: comparison between full genotyping and a rapid qualitative HPV-DNA assay in Ghana.

PubMed

Obiri-Yeboah, Dorcas; Adu-Sarkodie, Yaw; Djigma, Florencia; Akakpo, Kafui; Aniakwa-Bonsu, Ebenezer; Amoako-Sakyi, Daniel; Jacques, Simpore; Mayaud, Philippe

2017-01-01

Modern cervical cancer screening increasingly relies on the use of molecular techniques detecting high-risk oncogenic human papillomavirus (hr-HPV). A major challenge for developing countries like Ghana has been the unavailability and costs of HPV DNA-based testing. This study compares the performance of care HPV, a semi-rapid and affordable qualitative detection assay for 14 hr-HPV genotypes, with HPV genotyping, for the detection of cytological cervical squamous intraepithelial lesions (SIL). A study comparing between frequency matched HIV-1 seropositive and HIV-seronegative women was conducted in the Cape Coast Teaching Hospital, Ghana. A systematic sampling method was used to select women attending clinics in the hospital. Cervical samples were tested for HPV by care HPV and Anyplex-II HPV28 genotyping assay, and by conventional cytology. A total of 175 paired results (94 from HIV-1 seropositive and 81 from HIV-seronegative women) were analyzed based on the ability of both tests to detect the 14 hr-HPV types included in the care HPV assay. The inter-assay concordance was 94.3% (95%CI: 89.7-97.2%, kappa = 0.88), similar by HIV serostatus. The care HPV assay was equally sensitive among HIV-1 seropositive and seronegative women (97.3% vs. 95.7%, p  = 0.50) and slightly more specific among HIV-seronegative women (85.0% vs. 93.1%, p  = 0.10). care HPV had good sensitivity (87.5%) but low specificity (52.1%) for the detection of low SIL or greater lesions, but its performance was superior to genotyping (87.5 and 38.8%, respectively). Reproducibility of care HPV, tested on 97 samples by the same individual was 82.5% (95%CI: 73.4-89.4%). The performance characteristics of care HPV compared to genotyping suggest that this simpler and cheaper HPV detection assay could offer a suitable alternative for HPV screening in Ghana.

HIV DNA Is Frequently Present within Pathologic Tissues Evaluated at Autopsy from Combined Antiretroviral Therapy-Treated Patients with Undetectable Viral Loads

PubMed Central

Lamers, Susanna L.; Rose, Rebecca; Maidji, Ekaterina; Agsalda-Garcia, Melissa; Nolan, David J.; Fogel, Gary B.; Salemi, Marco; Garcia, Debra L.; Bracci, Paige; Yong, William; Commins, Deborah; Said, Jonathan; Khanlou, Negar; Hinkin, Charles H.; Sueiras, Miguel Valdes; Mathisen, Glenn; Donovan, Suzanne; Shiramizu, Bruce; Stoddart, Cheryl A.; Singer, Elyse J.

2016-01-01

ABSTRACT HIV infection treatment strategies have historically defined effectiveness through measuring patient plasma HIV RNA. While combined antiretroviral therapy (cART) can reduce plasma viral load (pVL) to undetectable levels, the degree that HIV is eliminated from other anatomical sites remains unclear. We investigated the HIV DNA levels in 229 varied autopsy tissues from 20 HIV-positive (HIV+) cART-treated study participants with low or undetectable plasma VL and cerebrospinal fluid (CSF) VL prior to death who were enrolled in the National Neurological AIDS Bank (NNAB) longitudinal study and autopsy cohort. Extensive medical histories were obtained for each participant. Autopsy specimens, including at least six brain and nonbrain tissues per participant, were reviewed by study pathologists. HIV DNA, measured in tissues by quantitative and droplet digital PCR, was identified in 48/87 brain tissues and 82/142 nonbrain tissues at levels >200 HIV copies/million cell equivalents. No participant was found to be completely free of tissue HIV. Parallel sequencing studies from some tissues recovered intact HIV DNA and RNA. Abnormal histological findings were identified in all participants, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. All brain tissues demonstrated some degree of pathology. Ninety-five percent of participants had some degree of atherosclerosis, and 75% of participants died with cancer. This study assists in characterizing the anatomical locations of HIV, in particular, macrophage-rich tissues, such as the central nervous system (CNS) and testis. Additional studies are needed to determine if the HIV recovered from tissues promotes the pathogenesis of inflammatory diseases, such as HIV-associated neurocognitive disorders, cancer, and atherosclerosis. IMPORTANCE It is well-known that combined antiretroviral therapy (cART) can reduce plasma HIV to undetectable levels; however, cART cannot completely clear HIV infection. An ongoing question is, “Where is HIV hiding?” A well-studied HIV reservoir is “resting” T cells, which can be isolated from blood products and succumb to cART once activated. Less-studied reservoirs are anatomical tissue samples, which have unknown cART penetration, contain a comparably diverse spectrum of potentially HIV-infected immune cells, and are important since <2% of body lymphocytes actually reside in blood. We examined 229 varied autopsy specimens from 20 HIV+ participants who died while on cART and identified that >50% of tissues were HIV infected. Additionally, we identified considerable pathology in participants' tissues, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. This study substantiates that tissue-associated HIV is present despite cART and can inform future studies into HIV persistence. PMID:27466426

HIV DNA Is Frequently Present within Pathologic Tissues Evaluated at Autopsy from Combined Antiretroviral Therapy-Treated Patients with Undetectable Viral Loads.

PubMed

Lamers, Susanna L; Rose, Rebecca; Maidji, Ekaterina; Agsalda-Garcia, Melissa; Nolan, David J; Fogel, Gary B; Salemi, Marco; Garcia, Debra L; Bracci, Paige; Yong, William; Commins, Deborah; Said, Jonathan; Khanlou, Negar; Hinkin, Charles H; Sueiras, Miguel Valdes; Mathisen, Glenn; Donovan, Suzanne; Shiramizu, Bruce; Stoddart, Cheryl A; McGrath, Michael S; Singer, Elyse J

2016-10-15

HIV infection treatment strategies have historically defined effectiveness through measuring patient plasma HIV RNA. While combined antiretroviral therapy (cART) can reduce plasma viral load (pVL) to undetectable levels, the degree that HIV is eliminated from other anatomical sites remains unclear. We investigated the HIV DNA levels in 229 varied autopsy tissues from 20 HIV-positive (HIV(+)) cART-treated study participants with low or undetectable plasma VL and cerebrospinal fluid (CSF) VL prior to death who were enrolled in the National Neurological AIDS Bank (NNAB) longitudinal study and autopsy cohort. Extensive medical histories were obtained for each participant. Autopsy specimens, including at least six brain and nonbrain tissues per participant, were reviewed by study pathologists. HIV DNA, measured in tissues by quantitative and droplet digital PCR, was identified in 48/87 brain tissues and 82/142 nonbrain tissues at levels >200 HIV copies/million cell equivalents. No participant was found to be completely free of tissue HIV. Parallel sequencing studies from some tissues recovered intact HIV DNA and RNA. Abnormal histological findings were identified in all participants, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. All brain tissues demonstrated some degree of pathology. Ninety-five percent of participants had some degree of atherosclerosis, and 75% of participants died with cancer. This study assists in characterizing the anatomical locations of HIV, in particular, macrophage-rich tissues, such as the central nervous system (CNS) and testis. Additional studies are needed to determine if the HIV recovered from tissues promotes the pathogenesis of inflammatory diseases, such as HIV-associated neurocognitive disorders, cancer, and atherosclerosis. It is well-known that combined antiretroviral therapy (cART) can reduce plasma HIV to undetectable levels; however, cART cannot completely clear HIV infection. An ongoing question is, "Where is HIV hiding?" A well-studied HIV reservoir is "resting" T cells, which can be isolated from blood products and succumb to cART once activated. Less-studied reservoirs are anatomical tissue samples, which have unknown cART penetration, contain a comparably diverse spectrum of potentially HIV-infected immune cells, and are important since <2% of body lymphocytes actually reside in blood. We examined 229 varied autopsy specimens from 20 HIV(+) participants who died while on cART and identified that >50% of tissues were HIV infected. Additionally, we identified considerable pathology in participants' tissues, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. This study substantiates that tissue-associated HIV is present despite cART and can inform future studies into HIV persistence. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

HIV-1-associated PKA acts as a cofactor for genome reverse transcription

PubMed Central

2013-01-01

Background Host cell proteins, including cellular kinases, are embarked into intact HIV-1 particles. We have previously shown that the Cα catalytic subunit of cAMP-dependent protein kinase is packaged within HIV-1 virions as an enzymatically active form able to phosphorylate a synthetic substrate in vitro (Cartier et al. J. Biol. Chem. 278:35211 (2003)). The present study was conceived to investigate the contribution of HIV-1-associated PKA to the retroviral life cycle. Results NL4.3 viruses were produced from cells cultured in the presence of PKA inhibitors H89 (H89-NL4.3) or Myr-PKI (PKI-NL4.3) and analyzed for viral replication. Despite being mature and normally assembled, and containing expected levels of genomic RNA and RT enzymatic activity, such viruses showed poor infectivity. Indeed, infection generated reduced amounts of strong-strop minus strand DNA, while incoming RNA levels in target cells were unaffected. Decreased cDNA synthesis was also evidenced in intact H89-NL4.3 and PKI-NL4.3 cell free particles using endogenous reverse transcription (ERT) experiments. Moreover, similar defects were reproduced when wild type NL4.3 particles preincubated with PKA inhibitors were subjected to ERT reactions. Conclusions Altogether, our results indicate that HIV-1-associated PKA is required for early reverse transcription of the retroviral genome both in cell free intact viruses and in target cells. Accordingly, virus-associated PKA behaves as a cofactor of an intraviral process required for optimal reverse transcription and for early post-entry events. PMID:24344931

Select Host Restriction Factors Are Associated with HIV Persistence During Antiretroviral Therapy

PubMed Central

ABDEL-MOHSEN, Mohamed; WANG, Charlene; STRAIN, Matthew C.; LADA, Steven M.; DENG, Xutao; COCKERHAM, Leslie R.; PILCHER, Christopher D.; HECHT, Frederick M.; LIEGLER, Teri; RICHMAN, Douglas D.; DEEKS, Steven G.; PILLAI, Satish K.

2015-01-01

Objective The eradication of HIV necessitates elimination of the HIV latent reservoir. Identifying host determinants governing latency and reservoir size in the setting of antiretroviral therapy (ART) is an important step in developing strategies to cure HIV infection. We sought to determine the impact of cell-intrinsic immunity on the HIV latent reservoir. Design We investigated the relevance of a comprehensive panel of established anti-HIV-1 host restriction factors to multiple established virologic and immunologic measures of viral persistence in HIV-1-infected, ART-suppressed individuals. Methods We measured the mRNA expression of 42 anti-HIV-1 host restriction factors, levels of cell-associated HIV-1 RNA, levels of total pol and 2-LTR circle HIV-1 DNA, and immunophenotypes of CD4+ T cells in 72 HIV-1-infected subjects on suppressive ART (23 subjects initiated ART <1 year post-infection, and 49 subjects initiated ART >1 year post-infection). Correlations were analyzed using non-parametric tests. Results The enhanced expression of a few select host restriction factors, p21, schlafen 11, and PAF1, was strongly associated with reduced CD4+ T cell-associated HIV RNA during ART (p<0.001). In addition, our data suggested that ART perturbs the regulatory relationship between CD4+ T cell activation and restriction factor expression. Lastly, cell-intrinsic immune responses were significantly enhanced in subjects who initiated ART during early versus chronic infection, and may contribute to the reduced reservoir size observed in these individuals. Conclusions Intrinsic immune responses modulate HIV persistence during suppressive ART, and may be manipulated to enhance the efficacy of ART and promote viral eradication through reversal of latency in vivo. PMID:25602681

HIV1 V3 loop hypermutability is enhanced by the guanine usage bias in the part of env gene coding for it.

PubMed

Khrustalev, Vladislav Victorovich

2009-01-01

Guanine is the most mutable nucleotide in HIV genes because of frequently occurring G to A transitions, which are caused by cytosine deamination in viral DNA minus strands catalyzed by APOBEC enzymes. Distribution of guanine between three codon positions should influence the probability for G to A mutation to be nonsynonymous (to occur in first or second codon position). We discovered that nucleotide sequences of env genes coding for third variable regions (V3 loops) of gp120 from HIV1 and HIV2 have different kinds of guanine usage biases. In the HIV1 reference strain and 100 additionally analyzed HIV1 strains the guanine usage bias in V3 loop coding regions (2G>1G>3G) should lead to elevated nonsynonymous G to A transitions occurrence rates. In the HIV2 reference strain and 100 other HIV2 strains guanine usage bias in V3 loop coding regions (3G>2G>1G) should protect V3 loops from hypermutability. According to the HIV1 and HIV2 V3 alignment, insertion of the sequence enriched with 2G (21 codons in length) occurred during the evolution of HIV1 predecessor, while insertion of the different sequence enriched with 3G (19 codons in length) occurred during the evolution of HIV2 predecessor. The higher is the level of 3G in the V3 coding region, the lower should be the immune escaping mutation occurrence rates. This hypothesis was tested in this study by comparing the guanine usage in V3 loop coding regions from HIV1 fast and slow progressors. All calculations have been performed by our algorithms "VVK In length", "VVK Dinucleotides" and "VVK Consensus" (www.barkovsky.hotmail.ru).

Using Cellular Proteins to Reveal Mechanisms of HIV Infection | Center for Cancer Research

Cancer.gov

A vital step in HIV infection is the insertion of viral DNA into the genome of the host cell. In order for the insertion to occur, viral nucleic acid must be transported through the membrane that separates the main cellular compartment (the cytoplasm) from the nucleus, where the host DNA is located. Scientists are actively studying the mechanism used to transport viral DNA

Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART.

PubMed

Wiegand, Ann; Spindler, Jonathan; Hong, Feiyu F; Shao, Wei; Cyktor, Joshua C; Cillo, Anthony R; Halvas, Elias K; Coffin, John M; Mellors, John W; Kearney, Mary F

2017-05-02

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2-18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.

Cyclophilin B enhances HIV-1 infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

DeBoer, Jason; Madson, Christian J.; Belshan, Michael, E-mail: michaelbelshan@creighton.edu

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence,more » putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. - Highlights: • CypB has been identified in several proteomic studies of HIV-1 infection. • CypB expression is upregulated in activated and infected T-cells. • Over-expression of CypB enhances HIV nuclear import and infection. • The N-terminus of CypB is necessary for these effects.« less

Smoking and anal high-risk human papillomavirus DNA loads in HIV-positive men who have sex with men.

PubMed

Wieland, Ulrike; Hellmich, Martin; Wetendorf, Janna; Potthoff, Anja; Höfler, Daniela; Swoboda, Jochen; Fuchs, Wolfgang; Brockmeyer, Norbert; Pfister, Herbert; Kreuter, Alexander

2015-10-01

HIV-positive men who have sex with men (MSM) have an increased risk for anal human papillomavirus (HPV) infection, anal high-grade intraepithelial lesions (HSIL), and anal cancer. Smoking is associated with abnormal anal cytology and with an increased risk for anal cancer. We collected 3736 intraanal swabs from 803 HIV-positive MSM who participated in an anal cancer screening program between October 2003 and August 2014. HPV prevalence, anal cytology and HPV DNA load of high-risk (HR) HPV-types 16, 18, 31 and 33 of non-smokers and smokers were compared. HPV-typing was performed by alpha-HPV genus-specific PCR and hybridization with 38 type-specific probes using a multiplex genotyping assay. In samples positive for HPV16, 18, 31, or 33, HPV DNA loads were determined by type-specific real-time PCRs and expressed as HPV DNA copies per betaglobin gene copy. At baseline, HR-HPV DNA (80.5 vs. 89.0%, p=0.001), HPV16 DNA (41.6 vs. 52.3%, p=0.003), HPV18 DNA (15.5 vs. 26.0%, p<0.001), anal dysplasia (LSIL+HSIL; 51.5 vs. 58.4%, p=0.045) and HSIL (17.2 vs. 22.7%, p=0.048) were detected more frequently in smokers compared to non-smokers. Throughout the study period 32.7% of non-smokers and 39.9% of smokers developed HSIL (p=0.011), and three smokers developed anal cancer. Considering swabs from the entire study period (median HPV load value per patient per cytology grade), smokers with normal anal cytology had significantly higher HPV16 loads (median 0.29 vs. 0.87, n=201, p=0.007) and cumulative high-risk-HPV loads (median 0.53 vs. 1.08, n=297, p=0.004) than non-smokers. Since elevated HR-HPV DNA loads are associated with an increased risk for HPV-induced anogenital cancers, HPV-infected HIV-positive MSM should be counseled to refrain from smoking. Additionally, for smokers, shorter anal cancer screening intervals than for non-smokers may be appropriate. Copyright © 2015 Elsevier GmbH. All rights reserved.

Clinical Outcomes of Virologically-Suppressed Patients with Pre-existing HIV-1 Drug Resistance Mutations Switching to Rilpivirine/Emtricitabine/Tenofovir Disoproxil Fumarate in the SPIRIT Study.

PubMed

Porter, Danielle P; Toma, Jonathan; Tan, Yuping; Solberg, Owen; Cai, Suqin; Kulkarni, Rima; Andreatta, Kristen; Lie, Yolanda; Chuck, Susan K; Palella, Frank; Miller, Michael D; White, Kirsten L

2016-02-01

Antiretroviral regimen switching may be considered for HIV-1-infected, virologically-suppressed patients to enable treatment simplification or improve tolerability, but should be guided by knowledge of pre-existing drug resistance. The current study examined the impact of pre-existing drug resistance mutations on virologic outcomes among virologically-suppressed patients switching to Rilpivirine (RPV)/emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF). SPIRIT was a phase 3b study evaluating the safety and efficacy of switching to RPV/FTC/TDF in virologically-suppressed HIV-1-infected patients. Pre-existing drug resistance at baseline was determined by proviral DNA genotyping for 51 RPV/FTC/TDF-treated patients with known mutations by historical RNA genotype and matched controls and compared with clinical outcome at Week 48. Drug resistance mutations in protease or reverse transcriptase were detected in 62.7% of patients by historical RNA genotype and in 68.6% by proviral DNA genotyping at baseline. Proviral DNA sequencing detected 89% of occurrences of NRTI and NNRTI resistance-associated mutations reported by historical genotype. Mutations potentially affecting RPV activity, including E138A/G/K/Q, Y181C, and H221Y, were detected in isolates from 11 patients by one or both assays. None of the patients with single mutants had virologic failure through Week 48. One patient with pre-existing Y181Y/C and M184I by proviral DNA genotyping experienced virologic failure. Nineteen patients with K103N present by historical genotype were confirmed by proviral DNA sequencing and 18/19 remained virologically-suppressed. Virologic success rates were high among virologically-suppressed patients with pre-existing NRTI and NNRTI resistance-associated mutations who switched to RPV/FTC/TDF in the SPIRIT study. While plasma RNA genotyping remains preferred, proviral DNA genotyping may provide additional value in virologically-suppressed patients for whom historical resistance data are unavailable.

Inhibition of HIV replication by pokeweed antiviral protein targeted to CD4+ cells by monoclonal antibodies

NASA Astrophysics Data System (ADS)

Zarling, Joyce M.; Moran, Patricia A.; Haffar, Omar; Sias, Joan; Richman, Douglas D.; Spina, Celsa A.; Myers, Dorothea E.; Kuebelbeck, Virginia; Ledbetter, Jeffrey A.; Uckun, Fatih M.

1990-09-01

FUNCTIONAL impairment and selective depletion of CD4+ T cells, the hallmark of AIDS, are at least partly caused by human immunodeficiency virus (HIV-1) type 1 binding to the CD4 molecule and infecting CD4+ cells1,2. It may, therefore, be of therapeutic value to target an antiviral agent to CD4+ cells to prevent infection and to inhibit HIV-1 production in patients' CD4+ cells which contain proviral DNA3,4. We report here that HIV-1 replication in normal primary CD4+ T cells can be inhibited by pokeweed antiviral protein, a plant protein of relative molecular mass 30,000 (ref. 5), which inhibits replication of certain plant RNA viruses6-8, and of herpes simplex virus, poliovirus and influenza virus9-11. Targeting pokeweed antiviral protein to CD4+ T cells by conjugating it to monoclonal antibodies reactive with CDS, CD7 or CD4 expressed on CD4+ cells, increased its anti-HIV potency up to 1,000-fold. HIV-1 replication is inhibited at picomolar concentrations of conjugates of pokeweed antiviral protein and monoclonal antibodies, which do not inhibit proliferation of normal CD4+ T cells or CD4-dependent responses. These conjugates inhibit HIV-1 protein synthesis and also strongly inhibit HIV-1 production in activated CD4+ T cells from infected patients.

Human Immunodeficiency Virus Integration Protein Expressed in Escherichia Coli Possesses Selective DNA Cleaving Activity

NASA Astrophysics Data System (ADS)

Sherman, Paula A.; Fyfe, James A.

1990-07-01

The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2+ over Mg2+. Reaction selectivity was further demonstrated by the lack of cleavage of an HIV U5 substrate on the complementary (minus) strand, an analogous substrate that mimics the U3 terminus of an avian retrovirus, and an HIV U5 substrate in which the conserved CA dinucleotide was replaced with a TA dinucleotide. Such an integration protein-mediated cleavage reaction is expected to occur as part of the integration event in the retroviral life cycle, in which a double-stranded DNA copy of the viral RNA genome is inserted into the host cell DNA.

HIV-1 induction-maintenance at the lymph node level: the "Apollo-97" Study.

PubMed

Lafeuillade, A; Poggi, C; Chadapaud, S; Hittinger, G; Chouraqui, M; Delbeke, E

2001-10-01

To assess the effects of five-drug combination therapy on HIV-1 load in lymph nodes and subsequent maintenance with four and three drugs. Ten pharmacotherapeutically naive patients received a combination of zidovudine, lamivudine, didanosine, ritonavir, and saquinavir for 24 weeks, then zidovudine, lamivudine, didanosine, and saquinavir for the next 24 weeks, and finally zidovudine, lamivudine, and saquinavir for the last 24 weeks. HIV-1 RNA in lymph nodes was measured using quantitative polymerase chain reaction (PCR) at baseline, after 12, 24, 48, and 78 weeks. Plasma HIV-1 RNA, proviral DNA in peripheral blood mononuclear cells (PBMCs), circulating lymphocyte subsets, and protease inhibitor levels in blood were also regularly measured. Genotypic resistance was assessed in the different compartments in 2 patients who were failed by therapy. HIV-1 RNA decreased in lymph nodes in 9 patients and was stable in 1 despite initial control of plasma replication <20 copies/ml in each patient. Lymph node levels rebounded in 1 patient at week 72 as a result of lack of adherence and remained stable in the 8 others despite maintenance regimens. This represents a mean drop of -3.17 log in lymph nodes for the 8 patients maintaining undetectable viremia at 72 weeks. In the patient with stable lymph node viral RNA, selection of the M184V mutation was demonstrated at this level before detection in plasma and low blood saquinavir levels were found throughout the study. Continuous improvements in immune parameters were observed in all cases, although PBMC proviral DNA levels either showed a continuous decrease or stabilized to a plateau. More complex regimens do not perform better in lymph nodes than classic triple therapy. The persistence of HIV-1 RNA in lymph nodes could be related with cellular resistance mechanisms rather than an insufficient potency of the regimens.

Curcumin-Loaded Apotransferrin Nanoparticles Provide Efficient Cellular Uptake and Effectively Inhibit HIV-1 Replication In Vitro

PubMed Central

Gandapu, Upendhar; Chaitanya, R. K.; Kishore, Golla; Reddy, Raju C.; Kondapi, Anand K.

2011-01-01

Background Curcumin (diferuloylmethane) shows significant activity across a wide spectrum of conditions, but its usefulness is rather limited because of its low bioavailability. Use of nanoparticle formulations to enhance curcumin bioavailability is an emerging area of research. Methodology/Principal Findings In the present study, curcumin-loaded apotransferrin nanoparticles (nano-curcumin) prepared by sol-oil chemistry and were characterized by electron and atomic force microscopy. Confocal studies and fluorimetric analysis revealed that these particles enter T cells through transferrin-mediated endocytosis. Nano-curcumin releases significant quantities of drug gradually over a fairly long period, ∼50% of curcumin still remaining at 6 h of time. In contrast, intracellular soluble curcumin (sol-curcumin) reaches a maximum at 2 h followed by its complete elimination by 4 h. While sol-curcumin (GI50 = 15.6 µM) is twice more toxic than nano-curcumin (GI50 = 32.5 µM), nano-curcumin (IC50<1.75 µM) shows a higher anti-HIV activity compared to sol-curcumin (IC50 = 5.1 µM). Studies in vitro showed that nano-curcumin prominently inhibited the HIV-1 induced expression of Topo II α, IL-1β and COX-2, an effect not seen with sol-curcumin. Nano-curcumin did not affect the expression of Topoisomerase II β and TNF α. This point out that nano-curcumin affects the HIV-1 induced inflammatory responses through pathways downstream or independent of TNF α. Furthermore, nano-curcumin completely blocks the synthesis of viral cDNA in the gag region suggesting that the nano-curcumin mediated inhibition of HIV-1 replication is targeted to viral cDNA synthesis. Conclusion Curcumin-loaded apotransferrin nanoparticles are highly efficacious inhibitors of HIV-1 replication in vitro and promise a high potential for clinical usefulness. PMID:21887247