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Sample records for increases hiv dna

  1. HIV DNA Reservoir Increases Risk for Cognitive Disorders in cART-Naïve Patients

    PubMed Central

    Valcour, Victor G.; Ananworanich, Jintanat; Agsalda, Melissa; Sailasuta, Napapon; Chalermchai, Thep; Schuetz, Alexandra; Shikuma, Cecilia; Liang, Chin-Yuan; Jirajariyavej, Supunee; Sithinamsuwan, Pasiri; Tipsuk, Somporn; Clifford, David B.; Paul, Robert; Fletcher, James L. K.; Marovich, Mary A.; Slike, Bonnie M.; DeGruttola, Victor; Shiramizu, Bruce

    2013-01-01

    Objectives Cognitive impairment remains frequent in HIV, despite combination antiretroviral therapy (cART). Leading theories implicate peripheral monocyte HIV DNA reservoirs as a mechanism for spread of the virus to the brain. These reservoirs remain present despite cART. The objective of this study was to determine if the level of HIV DNA in CD14+ enriched monocytes predicted cognitive impairment and brain injury. Methods We enrolled 61 cART-naïve HIV-infected Thais in a prospective study and measured HIV DNA in CD14+ enriched monocyte samples in a blinded fashion. We determined HAND diagnoses by consensus panel and all participants underwent magnetic resonance spectroscopy (MRS) to measure markers of brain injury. Immune activation was measured via cytokines in cerebrospinal fluid (CSF). Results The mean (SD) age was 35 (6.9) years, CD4 T-lymphocyte count was 236 (139) and log10 plasma HIV RNA was 4.8 (0.73). Twenty-eight of 61 met HAND criteria. The log10 CD14+ HIV DNA was associated with HAND in unadjusted and adjusted models (p = 0.001). There was a 14.5 increased odds ratio for HAND per 1 log-value of HIV DNA (10-fold increase in copy number). Plasma CD14+ HIV DNA was associated with plasma and CSF neopterin (p = 0.023) and with MRS markers of neuronal injury (lower N-acetyl aspartate) and glial dysfunction (higher myoinositol) in multiple brain regions. Interpretation Reservoir burden of HIV DNA in monocyte-enriched (CD14+) peripheral blood cells increases risk for HAND in treatment-naïve HIV+ subjects and is directly associated with CSF immune activation and both brain injury and glial dysfunction by MRS. PMID:23936155

  2. HIV-1 p24(gag) derived conserved element DNA vaccine increases the breadth of immune response in mice.

    PubMed

    Kulkarni, Viraj; Rosati, Margherita; Valentin, Antonio; Ganneru, Brunda; Singh, Ashish K; Yan, Jian; Rolland, Morgane; Alicea, Candido; Beach, Rachel Kelly; Zhang, Gen-Mu; Le Gall, Sylvie; Broderick, Kate E; Sardesai, Niranjan Y; Heckerman, David; Mothe, Beatriz; Brander, Christian; Weiner, David B; Mullins, James I; Pavlakis, George N; Felber, Barbara K

    2013-01-01

    Viral diversity is considered a major impediment to the development of an effective HIV-1 vaccine. Despite this diversity, certain protein segments are nearly invariant across the known HIV-1 Group M sequences. We developed immunogens based on the highly conserved elements from the p24(gag) region according to two principles: the immunogen must (i) include strictly conserved elements of the virus that cannot mutate readily, and (ii) exclude both HIV regions capable of mutating without limiting virus viability, and also immunodominant epitopes located in variable regions. We engineered two HIV-1 p24(gag) DNA immunogens that express 7 highly Conserved Elements (CE) of 12-24 amino acids in length and differ by only 1 amino acid in each CE ('toggle site'), together covering >99% of the HIV-1 Group M sequences. Altering intracellular trafficking of the immunogens changed protein localization, stability, and also the nature of elicited immune responses. Immunization of C57BL/6 mice with p55(gag) DNA induced poor, CD4(+) mediated cellular responses, to only 2 of the 7 CE; in contrast, vaccination with p24CE DNA induced cross-clade reactive, robust T cell responses to 4 of the 7 CE. The responses were multifunctional and composed of both CD4(+) and CD8(+) T cells with mature cytotoxic phenotype. These findings provide a method to increase immune response to universally conserved Gag epitopes, using the p24CE immunogen. p24CE DNA vaccination induced humoral immune responses similar in magnitude to those induced by p55(gag), which recognize the virus encoded p24(gag) protein. The inclusion of DNA immunogens composed of conserved elements is a promising vaccine strategy to induce broader immunity by CD4(+) and CD8(+) T cells to additional regions of Gag compared to vaccination with p55(gag) DNA, achieving maximal cross-clade reactive cellular and humoral responses. PMID:23555935

  3. Increased presence of Epstein-Barr virus DNA in ocular fluid samples from HIV negative immunocompromised patients with uveitis

    PubMed Central

    Ongkosuwito, J.; Van der Lelij, A.; Bruinenberg, M.; Doorn, M. W.; Feron, E.; Hoyng, C.; de Keizer, R. J W; Klok, A.; Kijlstra, A.

    1998-01-01

    AIMS—To investigate whether routine testing for Epstein-Barr virus (EBV) is necessary in the examination of a patient with uveitis.
METHODS—Intraocular EBV DNA was determined in 183 ocular fluid samples taken from patients with AIDS and uveitis, HIV negative immunocompromised uveitis, acute retinal necrosis, toxoplasma chorioretinitis, intraocular lymphoma, anterior uveitis, and miscellaneous uveitis of unknown cause. In 82 samples from this group of patients paired serum/ocular fluid analysis was performed to detect local antibody production against EBV. Controls (n=46) included ocular fluid samples taken during surgery for diabetic retinopathy, macular pucker, or cataract.
RESULTS—Serum antibody titres to EBV capsid antigen proved to be significantly increased in HIV negative immunocompromised patients with uveitis (p<0.01) compared with controls. Local antibody production revealed only three positive cases out of 82 patients tested, two results were borderline positive and one patient had uveitis caused by VZV. EBV DNA was detected in three out of 46 control ocular fluid samples. In the different uveitis groups EBV DNA was noted, but was not significantly higher than in the controls, except in six out of 11 HIV negative immunocompromised patients (p=0.0008). In four out of these six cases another infectious agent (VZV, HSV, CMV, or Toxoplasma gondii) had previously been identified as the cause of the uveitis.
CONCLUSIONS—When comparing various groups of uveitis patients, EBV DNA was found more often in HIV negative immunocompromised patients with uveitis. Testing for EBV does not have to be included in the routine management of patients with uveitis, since indications for an important role of this virus were not found in the pathogenesis of intraocular inflammation.

 Keywords: Epstein-Barr virus; intraocular fluid; polymerase chain reaction; uveitis PMID:9602620

  4. Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques

    PubMed Central

    Kulkarni, Viraj; Valentin, Antonio; Rosati, Margherita; Alicea, Candido; Singh, Ashish K.; Jalah, Rashmi; Broderick, Kate E.; Sardesai, Niranjan Y.; Le Gall, Sylvie; Mothe, Beatriz; Brander, Christian; Rolland, Morgane; Mullins, James I.; Pavlakis, George N.; Felber, Barbara K.

    2014-01-01

    HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24gag elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55gag increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist. PMID:24465991

  5. Altered response hierarchy and increased T-cell breadth upon HIV-1 conserved element DNA vaccination in macaques.

    PubMed

    Kulkarni, Viraj; Valentin, Antonio; Rosati, Margherita; Alicea, Candido; Singh, Ashish K; Jalah, Rashmi; Broderick, Kate E; Sardesai, Niranjan Y; Le Gall, Sylvie; Mothe, Beatriz; Brander, Christian; Rolland, Morgane; Mullins, James I; Pavlakis, George N; Felber, Barbara K

    2014-01-01

    HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24(gag) elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55(gag) increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist. PMID:24465991

  6. 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. PMID:26162319

  7. China update: HIV increasing.

    PubMed

    Gil, V E

    1993-01-01

    The HIV/AIDS case rate in China increased 117% over the period 1990-1992, from 446 to 957 HIV infections. While the majority of cases in 1990 were localized in Yunnan among minority farmers and manual laborers, infection is now found in 19 provinces, counties, and urban areas over a wider spectrum of society. The concentration of cases among IV-drug users has decreased from 83.4% to 72.6%. HIV monitoring and prevention stations have been in place in the country since 1986. The government also encouraged special zones and semiautonomous cities as well as the World Health Organization to take steps to monitor and prevent the spread of HIV. While these effort have served to augment the degree of sex education previously provided, low budgets, bureaucracy, and ambivalence have impeded control efforts. Only 1.25 million of the 1.16 billion population has been serosampled over 8 years and 100,000 fewer serosamples were taken in 1992 compared to in 1991. Neither the general population nor health workers have sufficient knowledge about HIV/AIDS to prevent its continued spread. While gay men sampled in Beijing were better informed about transmission means and risk groups, over two thirds believed they were not at risk if they avoided having sex with foreigners. Recent economic reform measures allowing large movements of population from rural to urban areas, increased disposable income available for prostitutes, and greater exposure to alternative sexual norms and behaviors through the media and music further increase the risk of HIV transmission, especially among the younger generation. To counter these risks, an AIDS hotline for information and referrals has been established in Beijing which openly reaches out to the homosexual community and fields 8-12 calls/day. Training programs for doctors, counselors, professors, and social workers have been attended by people from more than 30 provinces and regions. In addition, modest research into sexual behavior is also being

  8. Blockade of immunoregulatory Fc-signalling by HIV peptides: oligopeptides from HIV gp120 and gp41 bind the Fc portion of IgG and increase the in vitro anti-ssDNA response.

    PubMed Central

    Rahimpour, R; Anderson, C C; Sinclair, N R

    1993-01-01

    Concomitant ligation of antigen receptors with Fc-receptors negatively signals B cells. Antibodies to the Fc portion of IgG prevent this negative Fc-signalling, provided that these antibodies do not emit Fc signals. Prevention of Fc signals leads to augmented antibody responses to self and foreign antigens, and reduces the requirement for T cells by 10- to 100-fold in T cell-dependent antibody responses. In ELISA assays, peptides from conserved portions of the glycoproteins, HIV-1 gp120 or gp41 from HIV-1 and HIV-2 bind to the Fc portion of IgG, but do not bind the F(ab')2 portion of IgG. HIV-derived peptides, which bind to the Fc portion of IgG, augment the antibody-forming cell response to single-stranded (ss)DNA. The spontaneous response to ssDNA using spleen cells from young mice, and the response in the presence of exogenous DNA using spleen cells from old mice, are augmented to the greatest extent. These results demonstrate that HIV peptides bind to the Fc portion of IgG and augment immune responses to DNA; they suggest the possibility that blockade of the Fc portion of IgG antibodies is associated with a reduction in Fc-mediated regulation of anti-self responses. Blockade of regulatory Fc-signalling may account for increased circulating immunoglobulins and autoantibodies in clinical AIDS. PMID:8403512

  9. Increased Generation of HIV-1 gp120-Reactive CD8+ T Cells by a DNA Vaccine Construct Encoding the Chemokine CCL3

    PubMed Central

    Øynebråten, Inger; Hinkula, Jorma; Fredriksen, Agnete B.; Bogen, Bjarne

    2014-01-01

    DNA vaccines based on subunits from pathogens have several advantages over other vaccine strategies. DNA vaccines can easily be modified, they show good safety profiles, are stable and inexpensive to produce, and the immune response can be focused to the antigen of interest. However, the immunogenicity of DNA vaccines which is generally quite low needs to be improved. Electroporation and co-delivery of genetically encoded immune adjuvants are two strategies aiming at increasing the efficacy of DNA vaccines. Here, we have examined whether targeting to antigen-presenting cells (APC) could increase the immune response to surface envelope glycoprotein (Env) gp120 from Human Immunodeficiency Virus type 1 (HIV-1). To target APC, we utilized a homodimeric vaccine format denoted vaccibody, which enables covalent fusion of gp120 to molecules that can target APC. Two molecules were tested for their efficiency as targeting units: the antibody-derived single chain Fragment variable (scFv) specific for the major histocompatilibility complex (MHC) class II I-E molecules, and the CC chemokine ligand 3 (CCL3). The vaccines were delivered as DNA into muscle of mice with or without electroporation. Targeting of gp120 to MHC class II molecules induced antibodies that neutralized HIV-1 and that persisted for more than a year after one single immunization with electroporation. Targeting by CCL3 significantly increased the number of HIV-1 gp120-reactive CD8+ T cells compared to non-targeted vaccines and gp120 delivered alone in the absence of electroporation. The data suggest that chemokines are promising molecular adjuvants because small amounts can attract immune cells and promote immune responses without advanced equipment such as electroporation. PMID:25122197

  10. DNA Immunization for HIV Vaccine Development

    PubMed Central

    Chen, Yuxin; Wang, Shixia; Lu, Shan

    2014-01-01

    DNA vaccination has been studied in the last 20 years for HIV vaccine research. Significant experience has been accumulated in vector design, antigen optimization, delivery approaches and the use of DNA immunization as part of a prime-boost HIV vaccination strategy. Key historical data and future outlook are presented. With better understanding on the potential of DNA immunization and recent progress in HIV vaccine research, it is anticipated that DNA immunization will play a more significant role in the future of HIV vaccine development. PMID:26344472

  11. Circulating HIV DNA Correlates With Neurocognitive Impairment in Older HIV-infected Adults on Suppressive ART

    PubMed Central

    Oliveira, Michelli Faria de; Murrel, Ben; Pérez-Santiago, Josué; Vargas, Milenka; Ellis, Ronald J.; Letendre, Scott; Grant, Igor; Smith, Davey M.; Woods, Steven Paul; Gianella, Sara

    2015-01-01

    Older HIV-infected adults have a higher risk of neurocognitive impairment, but the underlying mechanisms are poorly understood. Here, we investigated the associations between levels of HIV DNA in peripheral blood, soluble markers of inflammation and cellular trafficking in blood and cerebrospinal fluid (CSF) and neurocognitive functioning among 18 younger (22–40 years) and 26 older (50–71 years) HIV-infected subjects, who were administered a comprehensive neurocognitive battery. Older HIV-infected individuals presented higher levels of inflammation in CSF and blood compared to younger individuals, but no difference was observed in HIV DNA levels. Among older participants, higher HIV DNA levels were significantly associated with more severe neurocognitive impairment (p = 0.005), particularly in the Executive Functions domain (p = 0.004). No association was observed between HIV DNA and neurocognition among younger individuals. Despite significantly increased inflammation observed in the older group, none of the inflammatory markers were associated with neurocognitive impairment among older HIV+ individuals (p > 0.05). Our study supports the involvement of peripheral HIV DNA reservoir in the pathogenesis of neurocognitive disorder during suppressive ART. Correlates of neurocognitive impairment might differ between younger and older adults, suggesting that future treatment and prevention strategies for HIV-associated neurocognitive disorders likely need to be tailored based on age. PMID:26603568

  12. Mutagenicity and pausing of HIV reverse transcriptase during HIV plus-strand DNA synthesis.

    PubMed Central

    Ji, J; Hoffmann, J S; Loeb, L

    1994-01-01

    The unusually high frequency of misincorporation by HIV-1 reverse transcriptase (HIV RT) is likely to be the major factor in the rapid accumulation of viral mutations in AIDS, especially in the env gene. To investigate the ability of HIV RT to copy the env gene, we subcloned an HIV env gene fragment into a single-stranded DNA vector and measured the progression of synthesis by HIV RT. We observed that HIV RT, but not RT from avian myeloblastosis virus, DNA polymerase-alpha or T7 DNA polymerase, pauses specifically at poly-deoxyadenosine stretches within the env gene. The frequency of bypassing the polyadenosine stretches by HIV RT is enhanced by increasing the ratio of enzyme to template. We measured the fidelity of DNA synthesis within a segment of the hypervariable region 1 of the env gene (V-1) containing a poly-deoxyadenosine sequence by repetitively copying the DNA by HIV RT, and then cloning and sequencing the copied fragments. We found that 27% of the errors identified in V-1 sequence were frameshift mutations opposite the poly-adenosine tract, a site where strong pausing was observed. Pausing of HIV RT at the polyadenosine tract could be enhanced by either distamycin A or netropsin, (A-T)-rich minor groove binding peptides. Moreover, netropsin increases the frequency of frameshift mutations in experiments in which HIV RT catalyzes gap filling synthesis within the lacZ gene in double-stranded circular M13mp2 DNA. These combined results suggest that the enhanced mutation frequency may be due to increased pausing at netropsin-modified polyadenosine tracts. Therefore, netropsin and related A-T binding chemicals may selectively enhance frameshift mutagenesis induced by HIV RT and yield predominantly non-viable virus. Images PMID:7510388

  13. Highly precise measurement of HIV DNA by droplet digital PCR.

    PubMed

    Strain, Matthew C; Lada, Steven M; Luong, Tiffany; Rought, Steffney E; Gianella, Sara; Terry, Valeri H; Spina, Celsa A; Woelk, Christopher H; Richman, Douglas D

    2013-01-01

    Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR (long terminal repeat) circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it. PMID:23573183

  14. Highly Precise Measurement of HIV DNA by Droplet Digital PCR

    PubMed Central

    Strain, Matthew C.; Lada, Steven M.; Luong, Tiffany; Rought, Steffney E.; Gianella, Sara; Terry, Valeri H.; Spina, Celsa A.; Woelk, Christopher H.; Richman, Douglas D.

    2013-01-01

    Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR (long terminal repeat) circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it. PMID:23573183

  15. HIV-1 Integrase-DNA Recognition Mechanisms

    PubMed Central

    Kessl, Jacques J.; McKee, Christopher J.; Eidahl, Jocelyn O.; Shkriabai, Nikolozi; Katz, Ari; Kvaratskhelia, Mamuka

    2009-01-01

    Integration of a reverse transcribed DNA copy of the HIV viral genome into the host chromosome is essential for virus replication. This process is catalyzed by the virally encoded protein integrase. The catalytic activities, which involve DNA cutting and joining steps, have been recapitulated in vitro using recombinant integrase and synthetic DNA substrates. Biochemical and biophysical studies of these model reactions have been pivotal in advancing our understanding of mechanistic details for how IN interacts with viral and target DNAs, and are the focus of the present review. PMID:21994566

  16. 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

  17. Quantification of Total and 2-LTR (Long terminal repeat) HIV DNA, HIV RNA and Herpesvirus DNA in PBMCs

    PubMed Central

    Massanella, Marta; Gianella, Sara; Lada, Steven M.; Richman, Douglas D.; Strain, Matthew C.

    2016-01-01

    Almost all individuals infected with human immunodeficiency virus (HIV) are also infected with cytomegalovirus (CMV) and Epstein Barr virus (EBV). The aims of our studies have included characterizing and measuring the latent HIV reservoir and understanding the association between asymptomatic replication of CMV (and other herpesvirus, including EBV) and this HIV reservoir (Gianella et al., 2014). This protocol was designed to simultaneously co-extract DNA and RNA from the same peripheral blood mononuclear cell (PBMC) aliquot and quantify HIV, CMV and EBV DNA, as well as HIV RNA using droplet digital PCR (ddPCR). For collection and processing of male genital secretions and quantification of HIV RNA and DNA from seven human herpesviruses from seminal plasma, refer to protocol “Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma” (Vargas-Meneses et al., 2015).

  18. DNA confinement drives uncoating of the HIV Virus

    NASA Astrophysics Data System (ADS)

    Rouzina, I.; Bruinsma, R.

    2014-09-01

    The enzyme reverse transcriptase converts single-stranded RNA molecules into double-stranded DNA molecules inside mature HIV viral capsids. We present a model for the uncoating of the HIV virus where the capsid uncoating process is driven by the confinement force exerted on the capsid wall porduced to the double-stranded DNA generated by reverse transcriptase.

  19. Increased HIV-1 activity in anal high-grade squamous intraepithelial lesions compared with unaffected anal mucosa in men who have sex with men.

    PubMed

    Pollakis, Georgios; Richel, Olivier; Vis, Joost D; Prins, Jan M; Paxton, William A; de Vries, Henry J C

    2014-06-01

    We studied 3 patients with focal intra-anal tissue high-grade squamous intraepithelial lesions (HSILs). All had increased human immunodeficiency virus type 1 (HIV-1) RNA and DNA in lesions compared with that in healthy mucosa. HIV-1 RNA and HIV-1 episomal DNA were indicative of ongoing viral replication, more so in anal HSILs. PMID:24604897

  20. HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins.

    PubMed

    Hrecka, Kasia; Hao, Caili; Shun, Ming-Chieh; Kaur, Sarabpreet; Swanson, Selene K; Florens, Laurence; Washburn, Michael P; Skowronski, Jacek

    2016-07-01

    HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4(DCAF1) E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4(DCAF1) E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4(DCAF1) E3. Thus, separate functions of HIV-1 Vpr usurp CRL4(DCAF1) E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host. PMID:27335459

  1. 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.

  2. HIV-1-Specific Antibody Response and Function after DNA Prime and Recombinant Adenovirus 5 Boost HIV Vaccine in HIV-Infected Subjects.

    PubMed

    Gach, Johannes S; Gorlani, Andrea; Dotsey, Emmanuel Y; Becerra, Juan C; Anderson, Chase T M; Berzins, Baiba; Felgner, Philip L; Forthal, Donald N; Deeks, Steven G; Wilkin, Timothy J; Casazza, Joseph P; Koup, Richard A; Katlama, Christine; Autran, Brigitte; Murphy, Robert L; Achenbach, Chad J

    2016-01-01

    Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5) boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART). Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB) and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC). We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir. PMID:27500639

  3. HIV-1-Specific Antibody Response and Function after DNA Prime and Recombinant Adenovirus 5 Boost HIV Vaccine in HIV-Infected Subjects

    PubMed Central

    Gach, Johannes S.; Gorlani, Andrea; Dotsey, Emmanuel Y.; Becerra, Juan C.; Anderson, Chase T. M.; Berzins, Baiba; Felgner, Philip L.; Forthal, Donald N.; Deeks, Steven G.; Wilkin, Timothy J.; Casazza, Joseph P.; Koup, Richard A.; Katlama, Christine; Autran, Brigitte; Murphy, Robert L.; Achenbach, Chad J.

    2016-01-01

    Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5) boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART). Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB) and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC). We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir. PMID:27500639

  4. Increased DNA methylation in the suicide brain.

    PubMed

    Haghighi, Fatemeh; Xin, Yurong; Chanrion, Benjamin; O'Donnell, Anne H; Ge, Yongchao; Dwork, Andrew J; Arango, Victoria; Mann, J John

    2014-09-01

    Clinical studies find that childhood adversity and stressful life events in adulthood increase the risk for major depression and for suicide. The predispositions to either major depression or suicide are thought to depend on genetic risk factors or epigenetic effects. We investigated DNA methylation signatures postmortem in brains of suicides with diagnosis of major depressive disorder. DNA methylation levels were determined at single C-phosphate-G (CpG) resolution sites within ventral prefrontal cortex of 53 suicides and nonpsychiatric controls, aged 16 to 89 years. We found that DNA methylation increases throughout the lifespan. Suicides showed an 8-fold greater number of methylated CpG sites relative to controls (P < 2.2 x 10(-16)), with greater DNA methylation changes over and above the increased methylation observed in normal aging. This increased DNA methylation may be a significant contributor to the neuropathology and psychopathology underlying the risk of suicide in depression. PMID:25364291

  5. Autograft HIV-DNA Load Predicts HIV-1 Peripheral Reservoir After Stem Cell Transplantation for AIDS-Related Lymphoma Patients

    PubMed Central

    Bortolin, Maria Teresa; Pratesi, Chiara; Tedeschi, Rosamaria; Basaglia, Giancarlo; Abbruzzese, Luciano; Mazzucato, Mario; Spina, Michele; Vaccher, Emanuela; Tirelli, Umberto; Rupolo, Maurizio; Michieli, Mariagrazia; Di Mascio, Michele; De Paoli, Paolo

    2015-01-01

    Abstract Autologous stem cell transplantation (ASCT) is a widely used procedure for AIDS-related lymphomas, and it represents an opportunity to evaluate strategies curing HIV-1 infection. The association of autograft HIV-DNA load with peripheral blood HIV-1 reservoir before ASCT and its contribution in predicting HIV-1 reservoir size and stability during combination antiretroviral therapy (cART) after transplantation are unknown. Aiming to obtain information suggesting new functional cure strategies by ASCT, we retrospectively evaluated HIV-DNA load in autograft and in peripheral blood before and after transplantation in 13 cART-treated HIV-1 relapse/refractoring lymphoma patients. Among them seven discontinued cART after autograft infusion. HIV-DNA was evaluated by a sensitive quantitative real-time polymerase chain reaction (PCR). After debulking chemotherapy/mobilization, the autograft HIV-1 reservoir was higher than and not associated with the peripheral HIV-1 reservoir at baseline [median 215 HIV-DNA copies/106 autograft mononuclear cells, range 13–706 vs. 82 HIV-DNA copies/106 peripheral blood mononuclear cells (PBMCs), range 13–479, p=0.03]. After high dose chemotherapy and autograft infusion, HIV-DNA levels reached a plateau between month 6 and 12 of follow-up. No association was found between peripheral HIV-DNA levels at baseline and after infusion in both cART interrupting and not interrupting patients. Only in the last subgroup, a stable significant linear association between autograft and peripheral blood HIV-1 reservoir emerged from month 1 (R2=0.84, p=0.01) to month 12 follow-up (R2=0.99, p=0.0005). In summary, autograft HIV-1 reservoir size could be influenced by the mobilization phase and predicts posttransplant peripheral HIV-1 reservoir size in patients on continuous cART. These findings could promote new research on strategies reducing the HIV-1 reservoir by using the ASCT procedure. PMID:25581618

  6. Human Macrophages Support Persistent Transcription From Unintegrated HIV-1 DNA

    PubMed Central

    Kelly, Jeremy; Beddall, Margaret H.; Yu, Dongyang; Iyer, Subashini R.; Marsh, Jon W.; Wu, Yuntao

    2008-01-01

    Retroviruses require integration of their RNA genomes for both stability and productive viral replication. In HIV infection of non-dividing, resting CD4 T cells, where integration is greatly impeded, the reverse transcribed HIV DNA has limited biological activity and a short half-life. In metabolically active and proliferating T cells unintegrated DNA rapidly diminishes with cell division. HIV also infects the non-dividing, but metabolically active macrophage population. In an in vitro examination of HIV infection of macrophages, we find that unintegrated viral DNA not only has an unusual stability, but also maintains biological activity. The unintegrated linear DNA, 1-LTR, and 2-LTR circles are stable for at least 30 days. Additionally there is persistent viral gene transcription, which is selective and skewed towards viral early genes such as nef and tat with highly diminished rev and vif. One viral early gene product Nef was measurably synthesized. We also find that independent of integration, the HIV infection process in macrophages leads to generation of numerous chemokines. PMID:18054979

  7. Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering.

    PubMed

    Novitsky, Vlad; Zahralban-Steele, Melissa; McLane, Mary Fran; Moyo, Sikhulile; van Widenfelt, Erik; Gaseitsiwe, Simani; Makhema, Joseph; Essex, M

    2015-08-01

    The goal of the study was to improve the methodology of HIV genotyping for analysis of HIV drug resistance and HIV clustering. Using the protocol of Gall et al. (A. Gall, B. Ferns, C. Morris, S. Watson, M. Cotten, M. Robinson, N. Berry, D. Pillay, and P. Kellam, J Clin Microbiol 50:3838-3844, 2012, doi:10.1128/JCM.01516-12), we developed a robust methodology for amplification of two large fragments of viral genome covering about 80% of the unique HIV-1 genome sequence. Importantly, this method can be applied to both viral RNA and proviral DNA amplification templates, allowing genotyping in HIV-infected subjects with suppressed viral loads (e.g., subjects on antiretroviral therapy [ART]). The two amplicons cover critical regions across the HIV-1 genome (including pol and env), allowing analysis of mutations associated with resistance to protease inhibitors, reverse transcriptase inhibitors (nucleoside reverse transcriptase inhibitors [NRTIs] and nonnucleoside reverse transcriptase inhibitors [NNRTIs]), integrase strand transfer inhibitors, and virus entry inhibitors. The two amplicons generated span 7,124 bp, providing substantial sequence length and numbers of informative sites for comprehensive phylogenic analysis and greater refinement of viral linkage analyses in HIV prevention studies. The long-range HIV genotyping from proviral DNA was successful in about 90% of 212 targeted blood specimens collected in a cohort where the majority of patients had suppressed viral loads, including 65% of patients with undetectable levels of HIV-1 RNA loads. The generated amplicons could be sequenced by different methods, such as population Sanger sequencing, single-genome sequencing, or next-generation ultradeep sequencing. The developed method is cost-effective-the cost of the long-range HIV genotyping is under $140 per subject (by Sanger sequencing)-and has the potential to enable the scale up of public health HIV prevention interventions. PMID:26041893

  8. 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. PMID:25332688

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

    PubMed Central

    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. PMID:25332688

  10. Estimation of HIV-1 DNA Level Interfering with Reliability of HIV-1 RNA Quantification Performed on Dried Blood Spots Collected from Successfully Treated Patients.

    PubMed

    Zida, Sylvie; Tuaillon, Edouard; Barro, Makoura; Kwimatouo Lekpa Franchard, Arnaud; Kagoné, Thérèse; Nacro, Boubacar; Ouedraogo, Abdoul Salam; Bolloré, Karine; Sanosyan, Armen; Plantier, Jean-Christophe; Meda, Nicolas; Sangaré, Lassana; Rouzioux, Christine; Rouet, François; Kania, Dramane

    2016-06-01

    The impact of HIV-1 DNA coamplification during HIV-1 RNA quantification on dried blood spots (DBS) was explored. False-positive HIV RNA detection (22/62, 35%) was associated with high HIV-1 DNA levels. Specificity of HIV-1 RNA assays on DBS should be evaluated following manufacturer protocols on samples with HIV-1 DNA levels of ≥1,000 copies/10(6) peripheral blood mononuclear cells. PMID:27008874

  11. Cheminformatics methods for novel nanopore analysis of HIV DNA termini

    PubMed Central

    Winters-Hilt, Stephen; Landry, Matthew; Akeson, Mark; Tanase, Maria; Amin, Iftekhar; Coombs, Amy; Morales, Eric; Millet, John; Baribault, Carl; Sendamangalam, Srikanth

    2006-01-01

    Background Channel current feature extraction methods, using Hidden Markov Models (HMMs) have been designed for tracking individual-molecule conformational changes. This information is derived from observation of changes in ionic channel current blockade "signal" upon that molecule's interaction with (and occlusion of) a single nanometer-scale channel in a "nanopore detector". In effect, a nanopore detector transduces single molecule events into channel current blockades. HMM analysis tools described are used to help systematically explore DNA dinucleotide flexibility, with particular focus on HIV's highly conserved (and highly flexible/reactive) viral DNA termini. One of the most critical stages in HIV's attack is the binding between viral DNA and the retroviral integrase, which is influenced by the dynamic-coupling induced high flexibility of a CA/TG dinucleotide positioned precisely two base-pairs from the blunt terminus of the duplex viral DNA. This suggests the study of a family of such CA/TG dinucleotide molecules via nanopore measurement and cheminformatics analysis. Results HMMs are used for level identification on the current blockades, HMM/EM with boosted variance emissions are used for level projection pre-processing, and time-domain FSAs are used to parse the level-projected waveform for kinetic information. The observed state kinetics of the DNA hairpins containing the CA/TG dinucleotide provides clear evidence for HIV's selection of a peculiarly flexible/interactive DNA terminus. PMID:17118144

  12. Multiple factors affect immunogenicity of DNA plasmid HIV vaccines in human clinical trials

    PubMed Central

    Jin, Xia; Morgan, Cecilia; Yu, Xuesong; DeRosa, Stephen; Tomaras, Georgia D.; Montefiori, David C.; Kublin, James; Corey, Larry; Keefer, Michael C.

    2015-01-01

    Plasmid DNA vaccines have been licensed for use in domesticated animals because of their excellent immunogenicity, but none have yet been licensed for use in humans. Here we report a retrospective analysis of 1218 healthy human volunteers enrolled in 10 phase I clinical trials in which DNA plasmids encoding HIV antigens were administered. Elicited T-cell immune responses were quantified by validated intracellular cytokine staining (ICS) stimulated with HIV peptide pools. HIV-specific binding and neutralizing antibody activities were also analyzed using validated assays. Results showed that, in the absence of adjuvants and boosting with alternative vaccines, DNA vaccines elicited CD8+ and CD4+ T-cell responses in an average of 13.3% (95% CI: 9.8% to 17.8%) and 37.7% (95% CI: 31.9% to 43.8%) of vaccine recipients, respectively. Three vaccinations (versus 2) improved the proportion of subjects with antigen-specific CD8+ responses (p=0.02), as did increased DNA dosage (p=0.007). Furthermore, female gender and participants having a lower Body Mass Index were independently associated with higher CD4+ T-cell response rate (p=0.001 and p=0.008, respectively). These vaccines elicited minimal neutralizing and binding antibody responses. These findings of the immunogenicity of HIV DNA vaccines in humans can provide guidance for future clinical trials. PMID:25820067

  13. HIV-1 increases TLR responses in human primary astrocytes

    PubMed Central

    Serramía, M Jesús; Muñoz-Fernández, M Ángeles; Álvarez, Susana

    2015-01-01

    Astrocytes are the major glial cell within the central nervous system and have a number of important physiological properties related to brain homeostasis. They provide trophic support to neurons and are immune cells with key roles during states-of-inflammation. The potential for production of proinflammatory cytokines and its consequences has been studied in the context of HIV-1 infection of normal human astrocytes (NHA). NHA express TLR3, TLR4, and TLR5. TLR3 ligation induced the strongest proinflammatory polarizing response, characterized by generation of high levels of TNF-α, IL-6, and IL-8. HIV-1 increased the transient production of key inflammatory mediators, and exposure to LPS of HIV-1-infected cells increased significantly the cytokine secretion. We confirmed that it is necessary viral gene expression from the moment of pretreatment with antiretrovirals inhibited totally HIV-1-induced TLR response. The higher response to LPS from HIV-1-infected cells did not correlate with TLR4 or MyD88 increased expression. LPS responsiveness of infected cells parallels MHC class II expression, but not CD14. HIV-1-infected NHA present increased sensitivity to the proinflammatory effects of LPS. If this phenomenon occurs in vivo, it will contribute to the immunopathogenesis of this disease and may ultimately offer novel targets for immunomodulatory therapy. PMID:26671458

  14. Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

    PubMed Central

    Gaskill, Peter J.; Yano, Hideaki H.; Kalpana, Ganjam V.; Javitch, Jonathan A.; Berman, Joan W.

    2014-01-01

    Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. PMID:25268786

  15. The increase in global HIV epidemics in MSM.

    PubMed

    Beyrer, Chris; Sullivan, Patrick; Sanchez, Jorge; Baral, Stefan D; Collins, Chris; Wirtz, Andrea L; Altman, Dennis; Trapence, Gift; Mayer, Kenneth

    2013-11-13

    Epidemics of HIV in MSM continue to expand in most low, middle, and upper income countries in 2013 and rates of new infection have been consistently high among young MSM. Current prevention and treatment strategies are insufficient for this next wave of HIV spread. We conducted a series of comprehensive reviews of HIV prevalence and incidence, risks for HIV, prevention and care, stigma and discrimination, and policy and advocacy options. The high per act transmission probability of receptive anal intercourse, sex role versatility among MSM, network level effects, and social and structural determinants play central roles in disproportionate disease burdens. HIV can be transmitted through large MSM networks at great speed. Molecular epidemiologic data show marked clustering of HIV in MSM networks, and high proportions of infections due to transmission from recent infections. Prevention strategies that lower biological risks, including those using antiretrovirals, offer promise for epidemic control, but are limited by structural factors including, discrimination, criminalization, and barriers to healthcare. Subepidemics, including among racial and ethnic minority MSM in the United States and UK, are particularly severe and will require culturally tailored efforts. For the promise of new and combined bio-behavioral interventions to be realized, clinically competent healthcare is necessary and community leadership, engagement, and empowerment are likely to be key. Addressing the expanding epidemics of HIV in MSM will require continued research, increased resources, political will, policy change, structural reform, community engagement, and strategic planning and programming, but it can and must be done. PMID:23842129

  16. Y chromosome and HIV DNA Detection in Vaginal Swabs as Biomarkers of Semen and HIV Exposure in Women

    PubMed Central

    Penrose, Kerri J.; Richardson, Barbra A.; Besson, Guillaume; Dezzutti, Charlene S.; Herold, Betsy C.; Abdool Karim, Salim S.; Mellors, John; Parikh, Urvi M.

    2014-01-01

    Background The inability to quantify sexual exposure to HIV limits the power of HIV prevention trials of vaccines, microbicides and pre-exposure prophylaxis (PrEP) in women. We investigated detection of HIV-1 and Y chromosomal (Yc) DNA in vaginal swabs from 83 participants in the HPTN 035 microbicide trial as biomarkers of HIV exposure and unprotected sexual activity. Methods 143 vaginal swabs from 85 women were evaluated for the presence of Y chromosomal DNA (Quantifiler Duo DNA quantification kit, Applied Biosystems) and total HIV-1 DNA (single copy in-house qPCR assay). Y DNA detection was paired with self-reported behavioral data with regard to recent coitus (≤ 1 week prior to collection) and condom usage (100% vs. <100% compliance). Results Yc DNA was detected in 62/143 (43%) swabs. For the 126 visits at which both behavioral data and swabs were collected, Yc DNA was significantly more frequent in women reporting <100% condom usage (OR 10.69; 95% confidence interval: 2.27 – 50.32; p=0.003). Notably, 27 of 83 (33%) swabs from women reporting 100% condom usage were positive for Yc DNA. HIV DNA was only detected in swabs collected post-seroconversion. Conclusions The use of Yc DNA in HIV prevention trials could reliably identify sub-groups of women who have unprotected sexual activity and could provide valuable exposure-based estimates of efficacy. PMID:25299415

  17. Cytomegalovirus DNA in Semen and Blood Is Associated With Higher Levels of Proviral HIV DNA

    PubMed Central

    Gianella, Sara; Anderson, Christy M.; Vargas, Milenka V.; Richman, Douglas D.; Little, Susan J.; Morris, Sheldon R.; Smith, Davey M.

    2013-01-01

    Over three-fourths of human immunodeficiency virus (HIV)–infected men who have sex with men (MSM) have at least one herpesvirus detected in their semen, and cytomegalovirus (CMV) is the most prevalent. The presence of CMV is associated with higher T-cell immune activation and with HIV disease progression in treated and untreated individuals. In this study of 113 antiretroviral (ART)–naive HIV-infected MSM, we found that CMV replication in blood and semen was associated with higher levels of HIV DNA in peripheral blood mononuclear cells. These observations suggest that interventions aimed to reduce CMV replication and, thus, systemic immune activation could decrease the size of the latent HIV reservoir. PMID:23275608

  18. HIV-1 Tat depresses DNA-PK{sub CS} expression and DNA repair, and sensitizes cells to ionizing radiation

    SciTech Connect

    Sun Yi; Huang Yuechen; Xu Qinzhi; Wang Huiping; Bai Bei; Sui Jianli; Zhou Pingkun . E-mail: zhoupk@nic.bmi.ac.cn

    2006-07-01

    Purpose There is accumulating evidence that cancer patients with human immmunodeficiency virus-1/acquired immunodeficency syndrome (HIV-1/AIDS) have more severe tissue reactions and often develop cutaneous toxic effects when subjected to radiotherapy. Here we explored the effects of the HIV-1 Tat protein on cellular responses to ionizing radiation. Methods and Materials Two Tat-expressing cell lines, TT2 and TE671-Tat, were derived from human rhabdomyosarcoma cells by transfecting with the HIV-1 tat gene. Radiosensitivity was determined using colony-forming ability. Gene expression was assessed by cDNA microarray and immunohybridization. The Comet assay and {gamma}-H2AX foci were use to detect DNA double-strand breaks (DSBs) and repair. Radiation-induced cell cycle changes were detected by flow cytometry. Results The radiosensitivity of TT2 and TE671-Tat cells was significantly increased as compared with parental TE671 cells or the control TE671-pCI cells. Tat also increased proliferation activity. The comet assay and {gamma}H2AX foci detection revealed a decreased capacity to repair radiation-induced DNA DSBs in Tat-expressing cells. Microarray assay demonstrated that the DNA repair gene DNA-PKcs, and cell cycle-related genes Cdc20, Cdc25C, KIF2C and CTS1 were downregulated in Tat-expressing cells. Depression of DNA-PKcs in Tat-expressing cells was further confirmed by RT-PCR and immuno-hybridization analysis. Tat-expressing cells exhibited a prolonged S phase arrest after 4 Gy {gamma}-irradiation, and a noticeable delay in the initiation and elimination of radiation-induced G{sub 2}/M arrest as compared with parental cells. In addition, the G{sub 2}/M arrest was incomplete in TT2 cells. Moreover, HIV-1 Tat resulted in a constitutive overexpression of cyclin B1 protein. Conclusion HIV-1 Tat protein sensitizes cells to ionizing radiation via depressing DNA repair and dysregulating cell cycle checkpoints. These observations provide new insight into the increased

  19. Total HIV-1 DNA, a Marker of Viral Reservoir Dynamics with Clinical Implications.

    PubMed

    Avettand-Fènoël, Véronique; Hocqueloux, Laurent; Ghosn, Jade; Cheret, Antoine; Frange, Pierre; Melard, Adeline; Viard, Jean-Paul; Rouzioux, Christine

    2016-10-01

    HIV-1 DNA persists in infected cells despite combined antiretroviral therapy (cART), forming viral reservoirs. Recent trials of strategies targeting latent HIV reservoirs have rekindled hopes of curing HIV infection, and reliable markers are thus needed to evaluate viral reservoirs. Total HIV DNA quantification is simple, standardized, sensitive, and reproducible. Total HIV DNA load influences the course of the infection and is therefore clinically relevant. In particular, it is predictive of progression to AIDS and death, independently of HIV RNA load and the CD4 cell count. Baseline total HIV DNA load is predictive of the response to cART. It declines during cART but remains quantifiable, at a level that reflects both the history of infection (HIV RNA zenith, CD4 cell count nadir) and treatment efficacy (residual viremia, cumulative viremia, immune restoration, immune cell activation). Total HIV DNA load in blood is also predictive of the presence and severity of some HIV-1-associated end-organ disorders. It can be useful to guide individual treatment, notably, therapeutic de-escalation. Although it does not distinguish between replication-competent and -defective latent viruses, the total HIV DNA load in blood, tissues, and cells provides insights into HIV pathogenesis, probably because all viral forms participate in host cell activation and HIV pathogenesis. Total HIV DNA is thus a biomarker of HIV reservoirs, which can be defined as all infected cells and tissues containing all forms of HIV persistence that participate in pathogenesis. This participation may occur through the production of new virions, creating new cycles of infection and disseminating infected cells; maintenance or amplification of reservoirs by homeostatic cell proliferation; and viral transcription and synthesis of viral proteins without new virion production. These proteins can induce immune activation, thus participating in the vicious circle of HIV pathogenesis. PMID:27559075

  20. Increase of HIV-1 subtype A in Central African Republic.

    PubMed

    Müller-Trutwin, M C; Chaix, M L; Letourneur, F; Bégaud, E; Beaumont, D; Deslandres, A; You, B; Morvan, J; Mathiot, C; Barré-Sinoussi, F; Saragosti, S

    1999-06-01

    The concomitant presence of five distinct HIV-1 subtypes and of unclassified HIV-1 was reported in Bangui, Central African Republic (C.A.R.) between 1990 and 1991. This previous study was conducted in individuals belonging to the C.A.R. Armed Forces (FACA) Cohort and in patients from the University Hospital of Baugui. To follow the HIV-1 subtype distribution in Bangui over time, we conducted a cross-sectional surveillance of HIV-1 subtypes between 1987 and 1997 in three groups of individuals in Bangui: 47 men belonging to the FACA Cohort, 38 patients from the CNHUB hospital, and 51 individuals consulting the sexually transmitted diseases (STD) clinic. One hundred and ten HIV-1 were subtyped by heteroduplex mobility assay (HMA) and/or sequencing of env regions encompassing the V3 domain. By comparing the HIV-1 distribution in two time periods (1987-1991 and 1991-1996) in the FACA cohort, we observed a significant increase of subtype A from 43.7% to 83.9%. This subtype distribution does not seem specific to the FACA cohort, in that subtype A accounted for 46.7% of the HIV-1 infections in CNHUB patients in the first time period studied and for 69.6% in the second time period. In STD patients, subtype A infections were predominant in 1995 (72.7%) and 1997 (89.7%). Subtype E viruses could be identified in the second time period, but represented only between 6.5% and 21.8% of the infections in the three groups of individuals studied. Other subtypes (B, C, H) and non-classified HIV-1 in C2-V3 were detected with only a 3.2% to 9.1% frequency for each in the second time period. Phylogenetic analysis excluded infection by a single source for the individuals included in the study. Our data demonstrate an increase in the proportion of HIV-1 subtype A infections in Bangui that raises the question of a preferential transmissibility of specific HIV-1 variants. PMID:10360809

  1. Autograft HIV-DNA load predicts HIV-1 peripheral reservoir after stem cell transplantation for AIDS-related lymphoma patients.

    PubMed

    Zanussi, Stefania; Bortolin, Maria Teresa; Pratesi, Chiara; Tedeschi, Rosamaria; Basaglia, Giancarlo; Abbruzzese, Luciano; Mazzucato, Mario; Spina, Michele; Vaccher, Emanuela; Tirelli, Umberto; Rupolo, Maurizio; Michieli, Mariagrazia; Di Mascio, Michele; De Paoli, Paolo

    2015-01-01

    Autologous stem cell transplantation (ASCT) is a widely used procedure for AIDS-related lymphomas, and it represents an opportunity to evaluate strategies curing HIV-1 infection. The association of autograft HIV-DNA load with peripheral blood HIV-1 reservoir before ASCT and its contribution in predicting HIV-1 reservoir size and stability during combination antiretroviral therapy (cART) after transplantation are unknown. Aiming to obtain information suggesting new functional cure strategies by ASCT, we retrospectively evaluated HIV-DNA load in autograft and in peripheral blood before and after transplantation in 13 cART-treated HIV-1 relapse/refractoring lymphoma patients. Among them seven discontinued cART after autograft infusion. HIV-DNA was evaluated by a sensitive quantitative real-time polymerase chain reaction (PCR). After debulking chemotherapy/mobilization, the autograft HIV-1 reservoir was higher than and not associated with the peripheral HIV-1 reservoir at baseline [median 215 HIV-DNA copies/10(6) autograft mononuclear cells, range 13-706 vs. 82 HIV-DNA copies/10(6) peripheral blood mononuclear cells (PBMCs), range 13-479, p = 0.03]. After high dose chemotherapy and autograft infusion, HIV-DNA levels reached a plateau between month 6 and 12 of follow-up. No association was found between peripheral HIV-DNA levels at baseline and after infusion in both cART interrupting and not interrupting patients. Only in the last subgroup, a stable significant linear association between autograft and peripheral blood HIV-1 reservoir emerged from month 1 (R(2) = 0.84, p = 0.01) to month 12 follow-up (R(2) = 0.99, p = 0.0005). In summary, autograft HIV-1 reservoir size could be influenced by the mobilization phase and predicts posttransplant peripheral HIV-1 reservoir size in patients on continuous cART. These findings could promote new research on strategies reducing the HIV-1 reservoir by using the ASCT procedure. PMID:25581618

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

    PubMed

    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; Koelsch, Kersten K; Kelleher, Anthony D; Phillips, Rodney E; Frater, John

    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. PMID:25217531

  3. Nucleic Acid Chaperone Activity of HIV-1 NC Proteins Investigated by Single Molecule DNA Stretching

    NASA Astrophysics Data System (ADS)

    Williams, Mark C.; Gorelick, Robert J.; Musier-Forsyth, Karin; Bloomfield, Victor A.

    2002-03-01

    HIV-1 Nucleocapsid Protein (NC) is a nucleic acid chaperone protein that is responsible for facilitating numerous nucleic acid rearrangements throughout the reverse transcription cycle of HIV-1. To understand the mechanism of NC’s chaperone function, we carried out single molecule DNA stretching studies in the presence of NC and mutant forms of NC. Using an optical tweezers instrument, we stretch single DNA molecules from the double-stranded helical state to the single-stranded (coil) state. Based on the observed cooperativity of DNA force-induced melting, we find that the fraction of melted base pairs at room temperature is increased dramatically in the presence of NC. Thus, upon NC binding, increased thermal fluctuations cause continuous melting and reannealing of base pairs so that DNA strands are able to rapidly sample configurations in order to find the lowest energy state. While NC destabilizes the double-stranded form of DNA, a mutant form of NC that lacks the zinc finger structures does not. DNA stretching experiments carried out in the presence of NC variants containing more subtle changes in the zinc finger structures were conducted to elucidate the contribution of each individual finger to NC’s chaperone activity, and these results will be reported.

  4. Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants.

    PubMed

    McManus, Margaret; Mick, Eric; Hudson, Richard; Mofenson, Lynne M; Sullivan, John L; Somasundaran, Mohan; Luzuriaga, Katherine

    2016-01-01

    The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log10 copies per million PBMC, p<0.001; ET: -1.04±0.11 log10 DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log10 DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log10 DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible. PMID:27104621

  5. Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants

    PubMed Central

    McManus, Margaret; Mick, Eric; Hudson, Richard; Mofenson, Lynne M.; Sullivan, John L.; Somasundaran, Mohan; Luzuriaga, Katherine

    2016-01-01

    The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log10 copies per million PBMC, p<0.001; ET: -1.04±0.11 log10 DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log10 DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log10 DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible. PMID:27104621

  6. Impact of the DNA extraction method on 2-LTR DNA circle recovery from HIV-1 infected cells

    PubMed Central

    Badralmaa, Yunden; Natarajan, Ven

    2013-01-01

    Detection of episomal 2-LTR DNA circles is used as a marker for the ongoing virus replication in patients infected with HIV-1, and efficient extraction of episomal DNA is critical for accurate estimation of the 2-LTR circles. The impact of different methods of DNA extraction on the recovery of 2-LTR circles was compared using mitochondrial DNA extracted as an internal control. The bacterial plasmid DNA isolation method extracted less than 10% of cellular DNA, 40% of mitochondrial DNA and 12-20 % of the input 2-LTR DNA. The total DNA isolation method recovered about 70% of mitochondrial DNA and 45% of the input 2-LTR DNA. The total nucleic acid isolation method recovered 90% of mitochondrial DNA and 60% of the input 2-LTR DNA. Similar results were obtained when the DNA was extracted from HIV-1 infected cells. Plasmid DNA isolation could not distinguish between 12 and 25 copies of 2-LTR DNA per million cells, whereas the total nucleic acid isolation showed a consistent and statistically significant difference between 12 and 25 copies. In conclusion, the total nucleic acid isolation method is more efficient than the plasmid DNA isolation method in recovering mitochondrial DNA and 2-LTR DNA circles from HIV-1 infected cells. PMID:23773807

  7. Expanded breadth of the T-cell response to mosaic HIV-1 envelope DNA vaccination

    SciTech Connect

    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 highly 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.

  8. 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

  9. Safety and Immunogenicity Study of Multiclade HIV-1 Adenoviral Vector Vaccine Alone or as Boost following a Multiclade HIV-1 DNA Vaccine in Africa

    PubMed Central

    Allen, Susan; Than, Soe; Adams, Elizabeth M.; Graham, Barney S.; Koup, Richard A.; Bailer, Robert T.; Smith, Carol; Dally, Len; Tarragona-Fiol, Tony; Bergin, Philip J.; Hayes, Peter; Ho, Martin; Loughran, Kelley; Komaroff, Wendy; Stevens, Gwynneth; Thomson, Helen; Boaz, Mark J.; Cox, Josephine H.; Schmidt, Claudia; Gilmour, Jill; Nabel, Gary J.; Fast, Patricia

    2010-01-01

    Background We conducted a double-blind, randomized, placebo-controlled Phase I study of a recombinant replication-defective adenovirus type 5 (rAd5) vector expressing HIV-1 Gag and Pol from subtype B and Env from subtypes A, B and C, given alone or as boost following a DNA plasmid vaccine expressing the same HIV-1 proteins plus Nef, in 114 healthy HIV-uninfected African adults. Methodology/Principal Findings Volunteers were randomized to 4 groups receiving the rAd5 vaccine intramuscularly at dosage levels of 1×1010 or 1×1011 particle units (PU) either alone or as boost following 3 injections of the DNA vaccine given at 4 mg/dose intramuscularly by needle-free injection using Biojector® 2000. Safety and immunogenicity were evaluated for 12 months. Both vaccines were well-tolerated. Overall, 62% and 86% of vaccine recipients in the rAd5 alone and DNA prime - rAd5 boost groups, respectively, responded to the HIV-1 proteins by an interferon-gamma (IFN-γ) ELISPOT. The frequency of immune responses was independent of rAd5 dosage levels. The highest frequency of responses after rAd5 alone was detected at 6 weeks; after DNA prime - rAd5 boost, at 6 months (end of study). At baseline, neutralizing antibodies against Ad5 were present in 81% of volunteers; the distribution was similar across the 4 groups. Pre-existing immunity to Ad5 did not appear to have a significant impact on reactogenicity or immune response rates to HIV antigens by IFN-γ ELISPOT. Binding antibodies against Env were detected in up to 100% recipients of DNA prime - rAd5 boost. One volunteer acquired HIV infection after the study ended, two years after receipt of rAd5 alone. Conclusions/Significance The HIV-1 rAd5 vaccine, either alone or as a boost following HIV-1 DNA vaccine, was well-tolerated and immunogenic in African adults. DNA priming increased the frequency and magnitude of cellular and humoral immune responses, but there was no effect of rAd5 dosage on immunogenicity endpoints. Trial

  10. Osteopontin enhances HIV replication and is increased in the brain and cerebrospinal fluid of HIV-infected individuals

    PubMed Central

    Islam, Tanzeem; Adams, Robert; Nerle, Sujata; Kamara, Masiray; Eger, Caitlin; Marder, Karen; Cohen, Bruce; Schifitto, Giovanni; McArthur, Justin C.; Sacktor, Ned; Pardo, Carlos A.

    2012-01-01

    Despite effective and widely available suppressive anti-HIV therapy, the prevalence of mild neurocognitive dysfunction continues to increase. HIV-associated neurocognitive disorder (HAND) is a multifactorial disease with sustained central nervous system inflammation and immune activation as prominent features. Inflammatory macrophages, HIV-infected and uninfected, play a central role in the development of HIV dementia. There is a critical need to identify biomarkers and to better understand the molecular mechanisms leading to cognitive dysfunction in HAND. In this regard, we identified through a subtractive hybridization strategy osteopontin (OPN, SPP1, gene) an inflammatory marker, as an upregulated gene in HIV-infected primary human monocyte-derived macrophages. Knockdown of OPN in primary macrophages resulted in a threefold decrease in HIV-1 replication. Ectopic expression of OPN in the TZM-bl cell line significantly enhanced HIV infectivity and replication. A significant increase in the degradation of the NF-κB inhibitor, IκBα and an increase in the nuclear-to-cytoplasmic ratio of NF-κB were found in HIV-infected cells expressing OPN compared to controls. Moreover, mutation of the NF-κB binding domain in the HIV-LTR abrogated enhanced promoter activity stimulated by OPN. Interestingly, compared to cerebrospinal fluid from normal and multiple sclerosis controls, OPN levels were significantly higher in HIV-infected individuals both with and without neurocognitive disorder. OPN levels were highest in HIV-infected individuals with moderate to severe cognitive impairment. Moreover, OPN was significantly elevated in brain tissue from HIV-infected individuals with cognitive disorder versus those without impairment. Collectively, these data suggest that OPN stimulates HIV-1 replication and that high levels of OPN are present in the CNS compartment of HIV-infected individuals, reflecting ongoing inflammatory processes at this site despite anti-HIV therapy. PMID

  11. Increased Risk of HIV-1 Transmission in Pregnancy: A Prospective Study among African HIV-1 Serodiscordant Couples

    PubMed Central

    MUGO, Nelly R.; HEFFRON, Renee; DONNELL, Deborah; WALD, Anna; WERE, Edwin O.; REES, Helen; CELUM, Connie; KIARIE, James N.; COHEN, Craig R.; KAYINTEKORE, Kayitesi; BAETEN, Jared M.

    2011-01-01

    Background Physiologic and behavioral changes during pregnancy may alter HIV-1 susceptibility and infectiousness. Prospective studies exploring pregnancy and HIV-1 acquisition risk in women have found inconsistent results. No study has explored the effect of pregnancy on HIV-1 transmission risk from HIV-1 infected women to male partners. Methods In a prospective study of African HIV-1 serodiscordant couples, we evaluated the relationship between pregnancy and the risk of 1) HIV-1 acquisition among women and 2) HIV-1 transmission from women to men. Results 3321 HIV-1 serodiscordant couples were enrolled, 1085 (32.7%) with HIV-1 susceptible female partners and 2236 (67.3%) with susceptible male partners. HIV-1 incidence in women was 7.35 versus 3.01 per 100 person-years during pregnant and non-pregnant periods (hazard ratio [HR] 2.34, 95% confidence interval [CI] 1.33–4.09). This effect was attenuated and not statistically significant after adjusting for sexual behavior and other confounding factors (adjusted HR 1.71, 95% CI 0.93–3.12). HIV-1 incidence in male partners of infected women was 3.46 versus 1.58 per 100 person-years when their partners were pregnant versus not pregnant (HR 2.31, 95% CI 1.22–4.39). This effect was not attenuated in adjusted analysis (adjusted HR 2.47, 95% CI 1.26–4.85). Conclusions HIV-1 risk increased two-fold during pregnancy. Elevated risk of HIV-1 acquisition in pregnant women appeared in part to be explained by behavioral and other factors. This is the first study to show pregnancy increased the risk of female-to-male HIV-1 transmission, which may reflect biological changes of pregnancy that could increase HIV-1 infectiousness. PMID:21785321

  12. Activities of Human Immunodeficiency Virus (HIV) Integration Protein In vitro: Specific Cleavage and Integration of HIV DNA

    NASA Astrophysics Data System (ADS)

    Bushman, Frederic D.; Craigie, Robert

    1991-02-01

    Growth of human immunodeficiency virus (HIV) after infection requires the integration of a DNA copy of the viral RNA genome into a chromosome of the host. Here we present a simple in vitro system that carries out the integration reaction and the use of this system to probe the mechanism of integration. The only HIV protein necessary is the integration (IN) protein, which has been overexpressed in insect cells and then partially purified. DNA substrates are supplied as oligonucleotides that match the termini of the linear DNA product of reverse transcription. In the presence of HIV IN protein, oligonucleotide substrates are cleaved to generate the recessed 3' ends that are the precursor for integration, and the cleaved molecules are efficiently inserted into a DNA target. Analysis of reaction products reveals that HIV IN protein joins 3' ends of the viral DNA to 5' ends of cuts made by IN protein in the DNA target. We have also used this assay to characterize the sequences at the ends of the viral DNA involved in integration. The assay provides a simple screen for testing candidate inhibitors of HIV IN protein; some such inhibitors might have useful antiviral activity.

  13. Antiretroviral genotypic resistance in plasma RNA and whole blood DNA in HIV-1 infected patients failing HAART.

    PubMed

    Saracino, Annalisa; Gianotti, Nicola; Marangi, Marianna; Cibelli, Donatella C; Galli, Andrea; Punzi, Grazia; Monno, Laura; Lazzarin, Adriano; Angarano, Gioacchino

    2008-10-01

    The extent to which HIV-1 proviral DNA mutations cause clinically relevant antiretroviral resistance is still controversial. Paired plasma HIV-1 RNA and whole blood DNA were compared in patients failing HAART to investigate if the additional knowledge of archived mutations could improve the selection of potentially active drugs. Seventy-three HIV-1-infected patients with first/second HAART failure were studied before starting a new regimen based on RNA genotyping. Follow-up data after a 12-week therapy were available. DNA genotyping was retrospectively performed on stored whole blood samples and mutational profiles were compared to those from RNA. The mean number of IAS pol mutations was significantly higher in RNA (4.45 +/- 2.76) than in DNA (2.88 +/- 2.47) (P < 0.001). DNA genotyping provided a 6% increase in detection of resistance-associated mutations. Among 64/73 patients showing discordant DNA/RNA profiles, 54 (84%) also differed for predicted active drugs. 16/73 (22%) patients had >or=1 mutation revealed by DNA genotyping alone, probably affecting therapy success in 2/16. However, neither RNA/DNA discordance nor detection of isolated DNA mutations were statistically associated with outcome. In conclusion, plasma RNA remains the elective choice for HIV genotyping in patients with therapy failure, even if the detection of proviral resistance-associated mutations, not simultaneously found in RNA, is a frequent event. Therefore, in some cases DNA plus RNA genotyping might assist in choosing more accurately subsequent antiretroviral regimens. PMID:18712823

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

    PubMed

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

    2016-03-01

    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

  15. 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

  16. Comparative Analysis of Cell-Associated HIV DNA Levels in Cerebrospinal Fluid and Peripheral Blood by Droplet Digital PCR

    PubMed Central

    de Oliveira, Michelli Faria; Gianella, Sara; Letendre, Scott; Scheffler, Konrad; Kosakovsky Pond, Sergei L.; Smith, Davey M.; Strain, Matt; Ellis, Ronald J.

    2015-01-01

    Background Measurement of HIV DNA-bearing cells in cerebrospinal fluid (CSF) is challenging because few cells are present. We present a novel application of the sensitive droplet digital (dd)PCR in this context. Methods We analyzed CSF cell pellets and paired peripheral blood mononuclear cells (PBMC) from 28 subjects, 19 of whom had undetectable HIV RNA (<48 copies/mL) in both compartments. We extracted DNA from PBMC using silica-based columns and used direct lysis on CSF cells. HIV DNA and the host housekeeping gene (RPP30) were measured in CSF and PBMC by (dd)PCR. We compared HIV DNA levels in virally-suppressed and-unsuppressed subgroups and calculated correlations between HIV DNA and RNA levels in both compartments using non-parametric tests. Results HIV DNA was detected in 18/28 (64%) CSF cell pellets, including 10/19 (53%) samples with undetectable HIV RNA. HIV DNA levels in CSF cell pellets were not correlated with RPP30 (p = 0.3), but correlated positively with HIV RNA in CSF (p = 0.04) and HIV DNA in PBMC (p = 0.03). Cellular HIV DNA in CSF was detected in comparable levels in HIV RNA-suppressed and unsuppressed subjects (p = 0.14). In contrast, HIV DNA levels in PBMC were significantly lower in HIV RNA-suppressed than in unsuppressed subjects (p = 0.014). Among subjects with detectable HIV DNA in both compartments, HIV DNA levels in CSF were significantly higher than in PBMC (p<0.001). Conclusions Despite low mononuclear cell numbers in CSF, HIV DNA was detected in most virally suppressed individuals. In contrast to PBMC, suppressive ART was not associated with lower HIV DNA levels in CSF cells, compared to no ART, perhaps due to poorer ART penetration, slower decay of HIV DNA, or enrichment of HIV DNA-bearing mononuclear cells into the CSF, compared to blood. Future studies should determine what fraction of HIV DNA is replication-competent in CSF leukocytes, compared to PBMC. PMID:26431315

  17. Boosting with Subtype C CN54rgp140 Protein Adjuvanted with Glucopyranosyl Lipid Adjuvant after Priming with HIV-DNA and HIV-MVA Is Safe and Enhances Immune Responses: A Phase I Trial

    PubMed Central

    Joseph, Sarah; Geldmacher, Christof; Munseri, Patricia J.; Aboud, Said; Missanga, Marco; Mann, Philipp; Wahren, Britta; Ferrari, Guido; Polonis, Victoria R.; Robb, Merlin L.; Weber, Jonathan; Tatoud, Roger; Maboko, Leonard; Hoelscher, Michael; Lyamuya, Eligius F.; Biberfeld, Gunnel; Sandström, Eric; Kroidl, Arne; Bakari, Muhammad; Nilsson, Charlotta; McCormack, Sheena

    2016-01-01

    -MVA prior to the two Env protein immunizations as compared to unprimed vaccinees (p = 0.07). Conclusion We report excellent tolerability, enhanced binding antibody responses and Env-specific cell-mediated immune responses but no ADCC antibody increase after two immunizations with a subtype C rgp140 protein adjuvanted in GLA-AF in healthy volunteers previously immunized with HIV-DNA and HIV-MVA. Trial Registration International Clinical Trials Registry PACTR2010050002122368 PMID:27192151

  18. HIV-1 Structural Proteins Serve as PAMPs for TLR2 Heterodimers Significantly Increasing Infection and Innate Immune Activation

    PubMed Central

    Henrick, Bethany M.; Yao, Xiao-Dan; Rosenthal, Kenneth Lee

    2015-01-01

    Immune activation is critical to HIV infection and pathogenesis; however, our understanding of HIV innate immune activation remains incomplete. Recently we demonstrated that soluble TLR2 (sTLR2) physically inhibited HIV-induced NFκB activation and inflammation, as well as HIV-1 infection. In light of these findings, we hypothesized that HIV-1 structural proteins may serve as pathogen-associated molecular patterns (PAMPs) for cellular TLR2 heterodimers. These studies made use of primary human T cells and TZMbl cells stably transformed to express TLR2 (TZMbl-2). Our results demonstrated that cells expressing TLR2 showed significantly increased proviral DNA compared to cells lacking TLR2, and mechanistically this may be due to a TLR2-mediated increased CCR5 expression. Importantly, we show that HIV-1 structural proteins, p17, p24, and gp41, act as viral PAMPs signaling through TLR2 and its heterodimers leading to significantly increased immune activation via the NFκB signaling pathway. Using co-immunoprecipitation and a dot blot method, we demonstrated direct protein interactions between these viral PAMPs and TLR2, while only p17 and gp41 bound to TLR1. Specifically, TLR2/1 heterodimer recognized p17 and gp41, while p24 lead to immune activation through TLR2/6. These results were confirmed using TLR2/1 siRNA knock down assays which ablated p17 and gp41-induced cellular activation and through studies of HEK293 cells expressing selected TLRs. Interestingly, our results show in the absence of TLR6, p24 bound to TLR2 and blocked p17 and gp41-induced activation, thus providing a novel mechanism by which HIV-1 can manipulate innate sensing. Taken together, our results identified, for the first time, novel HIV-1 PAMPs that play a role in TLR2-mediated cellular activation and increased proviral DNA. These findings have important implications for our fundamental understanding of HIV-1 immune activation and pathogenesis, as well as HIV-1 vaccine development. PMID:26347747

  19. 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

  20. Cholera toxin B subunit acts as a potent systemic adjuvant for HIV-1 DNA vaccination intramuscularly in mice

    PubMed Central

    Hou, Jue; Liu, Ying; Hsi, Jenny; Wang, Hongzhi; Tao, Ran; Shao, Yiming

    2014-01-01

    Cholera toxin B subunit (CTB) was investigated as a classical mucosal adjuvant that can increase vaccine immunogenicity. In this study, we found out the in vitro efficacy of cholera toxin B subunit (CTB) in activating mice bone marrow-derived dendritic cells (BMDCs) through Toll-like receptor signaling pathways. In vitro RNA and transcriptional level profiling arrays revealed that CTB guides high levels of Th1 and Th2 type cytokines, inflammatory cytokines, and chemokines. Based on the robustness of these profiling results, we examined the induction of HIV Env-specific immunity by CTB co-inoculated with HIV Env DNA vaccine intramuscularly in vivo. CTB enhanced HIV-Env specific cellular immune responses in Env-specific IFN-γ ELISPOT, compared with DNA vaccine alone. Moreover, CTB induced high levels of Env specific humoral response and promoted antibody maturation after the third round of vaccination. This combination immunization strategy induced a Th2-type bias response which is indicative of a high ratio of IgG1/IgG2a. This study reports that CTB as a classical mucosal adjuvant could enhance HIV-1 DNA-based vaccine immunogenicity intramuscularly; therefore, these findings suggest that CTB could serve as an effective candidate adjuvant for DNA vaccination. PMID:24633335

  1. Mitochondrial DNA variation and HIV-associated sensory neuropathy in CHARTER.

    PubMed

    Holzinger, Emily R; Hulgan, Todd; Ellis, Ronald J; Samuels, David C; Ritchie, Marylyn D; Haas, David W; Kallianpur, Asha R; Bloss, Cinnamon S; Clifford, David B; Collier, Ann C; Gelman, Benjamin B; Marra, Christina M; McArthur, Justin C; McCutchan, J Allen; Morgello, Susan; Simpson, David M; Franklin, Donald R; Rosario, Debralee; Selph, Doug; Letendre, Scott; Grant, Igor

    2012-12-01

    HIV-associated sensory neuropathy remains an important complication of combination antiretroviral therapy and HIV infection. Mitochondrial DNA haplogroups and single nucleotide polymorphisms (SNPs) have previously been associated with symptomatic neuropathy in clinical trial participants. We examined associations between mitochondrial DNA variation and HIV-associated sensory neuropathy in CNS HIV Antiretroviral Therapy Effects Research (CHARTER). CHARTER is a USA-based longitudinal observational study of HIV-infected adults who underwent a structured interview and standardized examination. HIV-associated sensory neuropathy was determined by trained examiners as ≥1 sign (diminished vibratory and sharp-dull discrimination or ankle reflexes) bilaterally. Mitochondrial DNA sequencing was performed and haplogroups were assigned by published algorithms. Multivariable logistic regression of associations between mitochondrial DNA SNPs, haplogroups, and HIV-associated sensory neuropathy were performed. In analyses of associations of each mitochondrial DNA SNP with HIV-associated sensory neuropathy, the two most significant SNPs were at positions A12810G [odds ratio (95 % confidence interval) = 0.27 (0.11-0.65); p = 0.004] and T489C [odds ratio (95 % confidence interval) = 0.41 (0.21-0.80); p = 0.009]. These synonymous changes are known to define African haplogroup L1c and European haplogroup J, respectively. Both haplogroups were associated with decreased prevalence of HIV-associated sensory neuropathy compared with all other haplogroups [odds ratio (95 % confidence interval) = 0.29 (0.12-0.71); p = 0.007 and odds ratio (95 % confidence interval) = 0.42 (0.18-1.0); p = 0.05, respectively]. In conclusion, in this cohort of mostly combination antiretroviral therapy-treated subjects, two common mitochondrial DNA SNPs and their corresponding haplogroups were associated with a markedly decreased prevalence of HIV-associated sensory neuropathy

  2. X4 and R5 HIV-1 have distinct post-entry requirements for uracil DNA glycosylase during infection of primary cells.

    PubMed

    Jones, Kate L; Roche, Michael; Gantier, Michael P; Begum, Nasim A; Honjo, Tasuku; Caradonna, Salvatore; Williams, Bryan R G; Mak, Johnson

    2010-06-11

    It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is approximately 10-20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations. PMID:20371602

  3. X4 and R5 HIV-1 Have Distinct Post-entry Requirements for Uracil DNA Glycosylase during Infection of Primary Cells

    PubMed Central

    Jones, Kate L.; Roche, Michael; Gantier, Michael P.; Begum, Nasim A.; Honjo, Tasuku; Caradonna, Salvatore; Williams, Bryan R. G.; Mak, Johnson

    2010-01-01

    It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is ∼10–20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations. PMID:20371602

  4. HIV DNA and cognition in a Thai longitudinal HAART initiation cohort

    PubMed Central

    Valcour, V G.; Shiramizu, B T.; Sithinamsuwan, P; Nidhinandana, S; Ratto-Kim, S; Ananworanich, J; Siangphoe, U; Kim, J H.; de Souza, M; Degruttola, V; Paul, R H.; Shikuma, C M.

    2009-01-01

    Objectives: The extent to which highly active antiretroviral therapy (HAART) era cognitive disorders are due to active processes, incomplete clearance of reservoirs, or comorbidities is controversial. This study aimed to determine if immunologic and virologic factors influence cognition after first-time HAART in Thai individuals with HIV-associated dementia (HAD) and Thai individuals without HAD (non-HAD). Methods: Variables were captured longitudinally to determine factors predictive of degree of cognitive recovery after first-time HAART. Neuropsychological data were compared to those of 230 HIV-negative Thai controls. Results: HIV RNA and CD4 lymphocyte counts were not predictive of HAD cross-sectionally or degree of cognitive improvement longitudinally. In contrast, baseline and longitudinal HIV DNA isolated from monocytes correlated to cognitive performance irrespective of plasma HIV RNA and CD4 lymphocyte counts pre-HAART (p < 0.001) and at 48 weeks post HAART (p < 0.001). Levels exceeding 3.5 log10 copies HIV DNA/106 monocyte at baseline distinguished all HAD and non-HAD cases (p < 0.001). At 48 weeks, monocyte HIV DNA was below the level of detection of our assay (10 copies/106 cells) in 15/15 non-HAD compared to only 4/12 HAD cases, despite undetectable plasma HIV RNA in 26/27 cases. Baseline monocyte HIV DNA predicted 48-week cognitive performance on a composite score, independently of concurrent monocyte HIV DNA and CD4 count (p < 0.001). Conclusions: Monocyte HIV DNA level correlates to cognitive performance before highly active antiretroviral therapy (HAART) and 48 weeks after HAART in this cohort and baseline monocyte HIV DNA may predict 48-week cognitive performance. These findings raise the possibility that short-term incomplete cognitive recovery with HAART may represent an active process related to this peripheral reservoir. GLOSSARY ARV = antiretroviral; CI = confidence interval; CRF = circulating recombinant form; GDS = global deficit score

  5. Inhibition of reverse transcriptase activity increases stability of the HIV-1 core.

    PubMed

    Yang, Yang; Fricke, Thomas; Diaz-Griffero, Felipe

    2013-01-01

    Previous studies showed that HIV-1 reverse transcription occurs during or before uncoating, linking mechanistically reverse transcription with uncoating. Here we show that inhibition of reverse transcriptase (RT) during HIV-1 infection by pharmacologic or genetic means increased the stability of the HIV-1 core during infection. Interestingly, HIV-1 particles with increased core stability were resistant to the core-destabilizing effects of rhesus TRIM5α (TRIM5α(rh)). Collectively, this work implies that the surface of the HIV-1 core is dynamic and changes upon the ongoing processes within the core. PMID:23077298

  6. 9G4 Autoreactivity Is Increased in HIV-Infected Patients and Correlates with HIV Broadly Neutralizing Serum Activity

    PubMed Central

    Kobie, James J.; Alcena, Danielle C.; Zheng, Bo; Bryk, Peter; Mattiacio, Jonelle L.; Brewer, Matthew; LaBranche, Celia; Young, Faith M.; Dewhurst, Stephen; Montefiori, David C.; Rosenberg, Alexander F.; Feng, Changyong; Jin, Xia; Keefer, Michael C.; Sanz, Ignacio

    2012-01-01

    The induction of a broadly neutralizing antibody (BNAb) response against HIV-1 would be a desirable feature of a protective vaccine. Vaccine strategies thus far have failed to elicit broadly neutralizing antibody responses; however a minority of HIV-infected patients do develop circulating BNAbs, from which several potent broadly neutralizing monoclonal antibodies (mAbs) have been isolated. The findings that several BNmAbs exhibit autoreactivity and that autoreactive serum antibodies are observed in some HIV patients have advanced the possibility that enforcement of self-tolerance may contribute to the rarity of BNAbs. To examine the possible breakdown of tolerance in HIV patients, we utilized the 9G4 anti-idiotype antibody system, enabling resolution of both autoreactive VH4-34 gene-expressing B cells and serum antibodies. Compared with healthy controls, HIV patients had significantly elevated 9G4+ serum IgG antibody concentrations and frequencies of 9G4+ B cells, a finding characteristic of systemic lupus erythematosus (SLE) patients, both of which positively correlated with HIV viral load. Compared to the global 9G4−IgD− memory B cell population, the 9G4+IgD− memory fraction in HIV patients was dominated by isotype switched IgG+ B cells, but had a more prominent bias toward “IgM only" memory. HIV envelope reactivity was observed both in the 9G4+ serum antibody and 9G4+ B cell population. 9G4+ IgG serum antibody levels positively correlated (r = 0.403, p = 0.0019) with the serum HIV BNAbs. Interestingly, other serum autoantibodies commonly found in SLE (anti-dsDNA, ANA, anti-CL) did not correlate with serum HIV BNAbs. 9G4-associated autoreactivity is preferentially expanded in chronic HIV infection as compared to other SLE autoreactivities. Therefore, the 9G4 system provides an effective tool to examine autoreactivity in HIV patients. Our results suggest that the development of HIV BNAbs is not merely a consequence of a general breakdown in

  7. Single-Molecule FRET Studies of HIV TAR–DNA Hairpin Unfolding Dynamics

    PubMed Central

    2015-01-01

    We directly measure the dynamics of the HIV trans-activation response (TAR)–DNA hairpin with multiple loops using single-molecule Förster resonance energy transfer (smFRET) methods. Multiple FRET states are identified that correspond to intermediate melting states of the hairpin. The stability of each intermediate state is calculated from the smFRET data. The results indicate that hairpin unfolding obeys a “fraying and peeling” mechanism, and evidence for the collapse of the ends of the hairpin during folding is observed. These results suggest a possible biological function for hairpin loops serving as additional fraying centers to increase unfolding rates in otherwise stable systems. The experimental and analytical approaches developed in this article provide useful tools for studying the mechanism of multistate DNA hairpin dynamics and of other general systems with multiple parallel pathways of chemical reactions. PMID:25254491

  8. Mitochondrial DNA variation and virologic and immunological HIV outcomes in African Americans

    PubMed Central

    Aissani, Brahim; Shrestha, Sadeep; Wiener, Howard W.; Tang, Jianming; Kaslow, Richard A.; Wilson, Craig M.

    2016-01-01

    Objective To evaluate the impact of mitochondrial DNA (mtDNA) haplogroups on virologic and immunological outcomes of HIV infection. Design HAART-naive African American adolescent participants to the Reaching for Excellence in Adolescent Care and Health study. Methods The mtDNA haplogroups were inferred from sequenced mtDNA hypervariable regions HV1 and HV2 and their predictive value on HIV outcomes were evaluated in linear mixed models, controlled for human leukocyte antigen (HLA)-B27, HLA-B57 and HLA-B35-Px alleles and other covariates. Results We report data showing that the mtDNA L2 lineage, a group composed of L2a, L2b and L2e mtDNA haplogroups in the studied population, is significantly associated (beta=−0.08; Bonferroni-adjusted P=0.004) with decline of CD4+ T cells (median loss of 8 ± 1 cells per month) in HAART-naive HIV-infected individuals of African American descent (n=133). No significant association (P<0.05) with set-point viral load was observed with any of the tested mtDNA haplogroups. The present data concur with previous findings in the AIDS Clinical Trials Group study 384, implicating the L2 lineage with slower CD4+ T-cell recovery after antiretroviral therapy in African Americans. Conclusions Whereas the L2 lineage showed an association with unfavorable immunological outcomes of HIV infection, its phylogenetic divergence from J and U5a, two lineages associated with accelerated HIV progression in European Americans, raises the possibility that interactions with common nucleus-encoded variants drive HIV progression. Disentangling the effects of mitochondrial and nuclear gene variants on the outcomes of HIV infection is an important step to be taken toward a better understanding of HIV/AIDS pathogenesis and pharmacogenomics. PMID:24932613

  9. Increased Frequency of α-Synuclein in the Substantia Nigra in HIV Infection

    PubMed Central

    Khanlou, Negar; Moore, David J.; Chana, Gursharan; Cherner, Mariana; Lazzaretto, Deborah; Dawes, Sharron; Grant, Igor; Masliah, Eliezer; Everall, Ian P.

    2014-01-01

    The frequency of neurodegenerative markers among long surviving HIV infected individuals is unknown, therefore, the present study investigated the frequency of α-synuclein, β-amyloid and HIV-associated brain pathology in the brains of older HIV infected individuals. We examined the substantia nigra of 73 clinically well-characterized HIV infected individuals aged 50 to 76 years from the National NeuroAIDS Tissue Consortium. We also examined the frontal and temporal cortical regions of a subset of 36 individuals. The brain regions were examined for the presence of α-synuclein, β-amyloid and HIV-associated brain pathology. Neuritic α-synuclein expression was found in 16% (12/73) of the substantia nigra of the HIV+ cases and none of the older control cases (0/18). β-amyloid deposits were prevalent and found in nearly all of the HIV+ cases (35/36). Despite these increases of degenerative pathology, HIV-associated brain pathology was present in only 10% of cases. Among older HIV+ adults HIV-associated brain pathology does not appear elevated; however, the frequency of both α-synuclein and β-amyloid is higher than that found in older healthy persons. The increased prevalence of α-synuclein and β-amyloid in the brains of older HIV-infected individuals may predict an increased risk of developing neurodegenerative disease. PMID:19115126

  10. 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

  11. Nano-Delivery Vehicles/Adjuvants for DNA Vaccination Against HIV.

    PubMed

    Dong, Yaqiong; Yang, Jun; Zhang, Jinchao; Zhang, Xin

    2016-03-01

    More than 75 million people has been infected HIV and it is responsible for nearly 36 million deaths on a global scale. As one of the deadliest infectious diseases, HIV is becoming the urgent issue of the global epidemic to tackle. In order to settle this problem from the source, some effective prevention strategies should be developed to control the pandemic of HIV. Vaccines, especially DNA vaccines, could be the optimal way to control the spread of HIV due to the unparalleled superiority that DNA vaccines could generate long-term humoral and cellular immune responses which could provide protective immunity for HIV. But the naked DNA could hardly enter into cells and is easily degraded by DNases and lysosomes, so designing effective delivery system is a promising strategy. Since delivery system could be constructed to promote efficient delivery of DNA into mammalian cells, protect them from degradation, and also could be established to be a target system to arrive at certain position of expectation. The current review discusses the potential of various nano-delivery vehicles/adjuvants such as polymer, lipid, liposome, peptide and inorganic material in improving efficiency of diverse modalities available for HIV DNA vaccines. PMID:27455611

  12. Dose-dependent inhibition of Gag cellular immunity by Env in SIV/HIV DNA vaccinated macaques

    PubMed Central

    Valentin, Antonio; Li, Jinyao; Rosati, Margherita; Kulkarni, Viraj; Patel, Vainav; Jalah, Rashmi; Alicea, Candido; Reed, Steven; Sardesai, Niranjan; Berkower, Ira; Pavlakis, George N; Felber, Barbara K

    2015-01-01

    The induction of a balanced immune response targeting the major structural proteins, Gag and Env of HIV, is important for the development of an efficacious vaccine. The use of DNA plasmids expressing different antigens offers the opportunity to test in a controlled manner the influence of different vaccine components on the magnitude and distribution of the vaccine-induced cellular and humoral immune responses. Here, we show that increasing amounts of env DNA results in greatly enhanced Env antibody titers without significantly affecting the levels of anti-Env cellular immune responses. Co-immunization with Env protein further increased antibody levels, indicating that vaccination with DNA only is not sufficient for eliciting maximal humoral responses against Env. In contrast, under high env:gag DNA plasmid ratio, the development of Gag cellular responses was significantly reduced by either SIV or HIV Env, whereas Gag humoral responses were not affected. Our data indicate that a balanced ratio of the 2 key HIV/SIV vaccine components, Gag and Env, is important to avoid immunological interference and to achieve both maximal humoral responses against Env to prevent virus acquisition and maximal cytotoxic T cell responses against Gag to prevent virus spread. PMID:26125521

  13. Methylome-wide Analysis of Chronic HIV Infection Reveals Five-Year Increase in Biological Age and Epigenetic Targeting of HLA.

    PubMed

    Gross, Andrew M; Jaeger, Philipp A; Kreisberg, Jason F; Licon, Katherine; Jepsen, Kristen L; Khosroheidari, Mahdieh; Morsey, Brenda M; Swindells, Susan; Shen, Hui; Ng, Cherie T; Flagg, Ken; Chen, Daniel; Zhang, Kang; Fox, Howard S; Ideker, Trey

    2016-04-21

    HIV-infected individuals are living longer on antiretroviral therapy, but many patients display signs that in some ways resemble premature aging. To investigate and quantify the impact of chronic HIV infection on aging, we report a global analysis of the whole-blood DNA methylomes of 137 HIV+ individuals under sustained therapy along with 44 matched HIV- individuals. First, we develop and validate epigenetic models of aging that are independent of blood cell composition. Using these models, we find that both chronic and recent HIV infection lead to an average aging advancement of 4.9 years, increasing expected mortality risk by 19%. In addition, sustained infection results in global deregulation of the methylome across >80,000 CpGs and specific hypomethylation of the region encoding the human leukocyte antigen locus (HLA). We find that decreased HLA methylation is predictive of lower CD4 / CD8 T cell ratio, linking molecular aging, epigenetic regulation, and disease progression. PMID:27105112

  14. Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

    PubMed Central

    Weil, Amy F.; Ghosh, Devlina; Zhou, Yan; Seiple, Lauren; McMahon, Moira A.; Spivak, Adam M.; Siliciano, Robert F.; Stivers, James T.

    2013-01-01

    HIV-1 reverse transcriptase discriminates poorly between dUTP and dTTP, and accordingly, viral DNA products become heavily uracilated when viruses infect host cells that contain high ratios of dUTP:dTTP. Uracilation of invading retroviral DNA is thought to be an innate immunity barrier to retroviral infection, but the mechanistic features of this immune pathway and the cellular fate of uracilated retroviral DNA products is not known. Here we developed a model system in which the cellular dUTP:dTTP ratio can be pharmacologically increased to favor dUTP incorporation, allowing dissection of this innate immunity pathway. When the virus-infected cells contained elevated dUTP levels, reverse transcription was found to proceed unperturbed, but integration and viral protein expression were largely blocked. Furthermore, successfully integrated proviruses lacked detectable uracil, suggesting that only nonuracilated viral DNA products were integration competent. Integration of the uracilated proviruses was restored using an isogenic cell line that had no detectable human uracil DNA glycosylase (hUNG2) activity, establishing that hUNG2 is a host restriction factor in cells that contain high dUTP. Biochemical studies in primary cells established that this immune pathway is not operative in CD4+ T cells, because these cells have high dUTPase activity (low dUTP), and only modest levels of hUNG activity. Although monocyte-derived macrophages have high dUTP levels, these cells have low hUNG activity, which may diminish the effectiveness of this restriction pathway. These findings establish the essential elements of this pathway and reconcile diverse observations in the literature. PMID:23341616

  15. Encoded novel forms of HSP70 or a cytolytic protein increase DNA vaccine potency

    PubMed Central

    Garrod, Tamsin; Grubor-Bauk, Branka; Yu, Stanley; Gargett, Tessa; Gowans, Eric J

    2014-01-01

    In humans, DNA vaccines have failed to demonstrate the equivalent levels of immunogenicity that were shown in smaller animals. Previous studies have encoded adjuvants, predominantly cytokines, within these vaccines in an attempt to increase antigen-specific immune responses. However, these strategies have lacked breadth of innate immune activation and have led to disappointing results in clinical trials. Damage associated molecular patterns (DAMPs) have been identified as pattern recognition receptor (PRR) agonists. DAMPs can bind to a wide range of PRRs on dendritic cells (DCs) and thus our studies have aimed to utilize this characteristic to act as an adjuvant in a DNA vaccine approach. Specifically, HSP70 has been identified as a DAMP, but has been limited by its lack of accessibility to PRRs in and on DCs. Here, we discuss the promising results achieved with the inclusion of membrane-bound or secreted HSP70 into a DNA vaccine encoding HIV gag as the model immunogen. PMID:25483501

  16. Project VOGUE: A partnership for increasing HIV knowledge and HIV vaccine trial awareness among House Ball leaders in Western New York

    PubMed Central

    Alio, Amina P.; Fields, Sheldon D.; Humes, Damon L.; Bunce, Catherine A.; Wallace, Stephaun E.; Lewis, Cindi; Elder, Heather; Wakefield, Steven; Keefer, Michael C.

    2014-01-01

    Men who sleep with men (MSM) and transgender individuals of color, the largest demographic in the House Ball community (HBC) are amongst the group at highest risk for HIV infection in the United States. The HBC have limited access to culturally appropriate HIV education. This study aimed to develop a partnership with HBC leaders to uncover strategies for increasing HIV prevention knowledge, including participation in HIV vaccine trials. To this end a research institution-community-HBC partnership was established. In-depth qualitative and quantitative data were collected from the 14 HBC leaders in western New York, revealing that knowledge of HIV and related vaccine trials was limited. Barriers to increasing HIV knowledge included fear of peer judgment, having inaccurate information about HIV, and lack of education. Among the HBC, community partnerships will further aid in the development of future HIV prevention programs and increase individuals’ willingness to participate in future HIV vaccine trials. PMID:25642120

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

    PubMed

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

    2016-02-12

    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

  18. Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR

    PubMed Central

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; Vandekerckhove, Linos; De Spiegelaere, Ward

    2014-01-01

    Introduction In HIV-infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples. Materials and Methods We compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR). Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59). Samples for the plasmid DNA isolation method were spiked with an internal control plasmid. Results Genomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974) and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs), (R2=0.671). Furthermore, Bland–Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10). Conclusions 2-LTR circles

  19. HIV Drug Resistance Mutations in Proviral DNA from a Community Treatment Program

    PubMed Central

    Derache, Anne; Shin, Hyoung-Shik; Balamane, Maya; White, Elizabeth; Israelski, Dennis; Klausner, Jeffrey D.; Freeman, Alexandra H.; Katzenstein, David

    2015-01-01

    Background Drug resistance mutations archived in resting memory CD4+ cells may persist despite suppression of HIV RNA to <50 copies/ml. We sequenced pol gene from proviral DNA among viremic and suppressed patients to identify drug resistance mutations. Methods The Peninsula AIDS Research Cohort study enrolled and followed over 2 years 120 HIV infected patients from San Mateo and San Francisco Counties. HIV-1 pol genotyping by bulk sequencing was performed on 38 DNA and RNA from viremic patients and DNA only among 82 suppressed patients at baseline. Antiretroviral susceptibility was predicted by HIVDB.stanford.edu. Results Among 120 subjects, 81% were on antiretroviral therapy and had been treated for a median time of 7 years. Thirty-two viremic patients showed concordant RNA and DNA genotypes (84%); the discordant profiles were mainly observed in patients with low-level viremia. Among suppressed patients, 21 had drug resistance mutations in proviral DNA (26%) with potential resistance to one, two or three ARV classes in 16, 4 and 1 samples respectively. Conclusions The high level of genotype concordance between DNA and RNA in viremic patients suggested that DNA genotyping might be used to assess drug resistance in resource-limited settings, and further investigation of extracted DNA from dried blood spots is needed. Drug resistance mutations in proviral DNA in 26% of subjects with less than 50 copies/ml pose a risk for the transmission of drug resistant virus with virologic failure, treatment interruption or decreased adherence. PMID:25635815

  20. DNA Triplex-Based Complexes Display Anti-HIV-1-Cell Fusion Activity.

    PubMed

    Xu, Liang; Zhang, Tao; Xu, Xiaoyu; Chong, Huihui; Lai, Wenqing; Jiang, Xifeng; Wang, Chao; He, Yuxian; Liu, Keliang

    2015-08-01

    DNA triplexes with hydrophobic modifications were designed and evaluated for their activity as inhibitors of the cell fusion of human immunodeficiency virus type 1 (HIV-1). Triplex inhibitors displayed low micromolar activities in the cell-cell fusion assay and nanomolar activities in the anti-HIV-1 pseudovirus test. Helix structure and the presence of sufficient numbers of hydrophobic regions were essential for the antifusion activity. Results from native polyacrylamide gel electrophoresis and a fluorescent resonance energy transfer-based inhibitory assay indicated that these triplexes may interact with the primary pocket at the glycoprotein 41 (gp41) N-heptad repeat, thereby inhibiting formation of the HIV-1 gp41 6-helical bundle. Triplex-based complexes may represent a novel category of HIV-1 inhibitors in anti-HIV-1 drug discovery. PMID:26192705

  1. Increased Hot Flash Severity and Related Interference in Perimenopausal HIV-infected Women

    PubMed Central

    Looby, Sara E.; Shifren, Jan; Corless, Inge; Rope, Alison; Pedersen, Maria C.; Joffe, Hadine; Grinspoon, Steven

    2013-01-01

    Objective As women with HIV are living longer, more are entering the perimenopause. Prior studies suggest that HIV-infected women are more likely to have hot flashes than non-HIV-infected women. However, little is known regarding the severity and degree of interference that hot flashes have on daily function, mood, and quality of life in this population. Methods Perimenopausal HIV-infected and non-HIV-infected women matched by age, race, and menstrual patterns completed the Menopause Rating Scale (MRS) to assess hot flash severity and the Hot Flash Related Daily Interference Scale (HFRDIS). MRS and HFRDIS scores and subscores were compared between groups. Results 33 HIV-infected and 33 non-HIV-infected women similar in age (47 (45,48) median (interquartile range) vs. 47 (46,49) yrs; race (64% vs. 52% non-white; P=0.32), and menstrual patterns (# periods in past year; 5 (4,9) vs. 6 (4,10); P=0.53) were studied. Perimenopausal HIV-infected women reported greater hot flash severity (HIV; 2 (1,3) vs. non-HIV: 1 (0,3); P=0.03), and hot flash-related interference (HFRDIS total score; 37 (10,60) vs. 6 (0,20); P=0.001). Conclusions Perimenopausal HIV-infected women experience greater hot flash severity and related interference compared to non-HIV-infected perimenopausal women. Increased distress secondary to hot flashes may reduce quality of life and negatively impact important health-promoting behaviors including adherence to antiretroviral therapy in HIV-infected women. PMID:23820600

  2. Does HIV increase the risk of spousal violence in sub-Saharan Africa?

    PubMed

    Chin, Yoo-Mi

    2013-09-01

    Although a positive association is found between HIV prevalence and intimate partner violence, a causal interpretation is hard to establish due to the endogeneity of HIV prevalence. Using the distance from the origin of the virus as an instrument, I find that an exogenous increase in HIV prevalence in a cluster has a sizable positive effect on the risk of physical and sexual violence against women within marriage. The results of this study confirm a gender-specific negative externality of the disease and encourage policy efforts to incorporate services for violence against women into existing HIV programs. PMID:24012689

  3. 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

    2013-01-01

    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. PMID:23549916

  4. Rapid Detection of HIV-1 Proviral DNA for Early Infant Diagnosis Using Recombinase Polymerase Amplification

    PubMed Central

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

    2013-01-01

    ABSTRACT 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. PMID:23549916

  5. Synthetic Consensus HIV-1 DNA Induces Potent Cellular Immune Responses and Synthesis of Granzyme B, Perforin in HIV Infected Individuals

    PubMed Central

    Morrow, Matthew P; Tebas, Pablo; Yan, Jian; Ramirez, Lorenzo; Slager, Anna; Kraynyak, Kim; Diehl, Malissa; Shah, Divya; Khan, Amir; Lee, Jessica; Boyer, Jean; Kim, J Joseph; Sardesai, Niranjan Y; Weiner, David B; Bagarazzi, Mark L

    2015-01-01

    This study evaluated the safety and immunogenicity of PENNVAX-B in 12 HIV infected individuals. PENNVAX-B is a combination of three optimized synthetic plasmids encoding for multiclade HIV Gag and Pol and a consensus CladeB Env delivered by electroporation. HIV infected individuals whose virus was effectively suppressed using highly active antiretroviral therapy (HAART) received PENNVAX-B DNA followed by electroporation with CELLECTRA-5P at study weeks 0, 4, 8, and 16. Local administration site and systemic reactions to PENNVAX-B were recorded after each treatment along with any adverse events. Pain of the treatment procedure was assessed using a Visual Analog Scale. Whole PBMCs were isolated for use in IFN ELISpot and Flow Cytometric assays. PENNVAX-B was generally safe and well tolerated. Overall, the four dose regimen was not associated with any serious adverse events or severe local or systemic reactions. A rise in antigen-specific SFU was detected in the INFγ ELISpot assay in all 12 participants. T cells from 8/12 participants loaded with both granzyme B and perforin in response to HIV antigen, an immune finding characteristic of long-term nonprogressors (LTNPs) and elite controllers (ECs). Thus administration of PENNVAX-B may prove useful adjunctive therapy to ART for treatment and control of HIV infection. PMID:25531694

  6. Synthetic consensus HIV-1 DNA induces potent cellular immune responses and synthesis of granzyme B, perforin in HIV infected individuals.

    PubMed

    Morrow, Matthew P; Tebas, Pablo; Yan, Jian; Ramirez, Lorenzo; Slager, Anna; Kraynyak, Kim; Diehl, Malissa; Shah, Divya; Khan, Amir; Lee, Jessica; Boyer, Jean; Kim, J Joseph; Sardesai, Niranjan Y; Weiner, David B; Bagarazzi, Mark L

    2015-03-01

    This study evaluated the safety and immunogenicity of PENNVAX-B in 12 HIV infected individuals. PENNVAX-B is a combination of three optimized synthetic plasmids encoding for multiclade HIV Gag and Pol and a consensus CladeB Env delivered by electroporation. HIV infected individuals whose virus was effectively suppressed using highly active antiretroviral therapy (HAART) received PENNVAX-B DNA followed by electroporation with CELLECTRA-5P at study weeks 0, 4, 8, and 16. Local administration site and systemic reactions to PENNVAX-B were recorded after each treatment along with any adverse events. Pain of the treatment procedure was assessed using a Visual Analog Scale. Whole PBMCs were isolated for use in IFN ELISpot and Flow Cytometric assays. PENNVAX-B was generally safe and well tolerated. Overall, the four dose regimen was not associated with any serious adverse events or severe local or systemic reactions. A rise in antigen-specific SFU was detected in the INFγ ELISpot assay in all 12 participants. T cells from 8/12 participants loaded with both granzyme B and perforin in response to HIV antigen, an immune finding characteristic of long-term nonprogressors (LTNPs) and elite controllers (ECs). Thus administration of PENNVAX-B may prove useful adjunctive therapy to ART for treatment and control of HIV infection. PMID:25531694

  7. Excision of HIV-1 DNA by gene editing: a proof-of-concept in vivo study.

    PubMed

    Kaminski, R; Bella, R; Yin, C; Otte, J; Ferrante, P; Gendelman, H E; Li, H; Booze, R; Gordon, J; Hu, W; Khalili, K

    2016-08-01

    A CRISPR/Cas9 gene editing strategy has been remarkable in excising segments of integrated HIV-1 DNA sequences from the genome of latently infected human cell lines and by introducing InDel mutations, suppressing HIV-1 replication in patient-derived CD4+ T-cells, ex vivo. Here, we employed a short version of the Cas9 endonuclease, saCas9, together with a multiplex of guide RNAs (gRNAs) for targeting the viral DNA sequences within the 5'-LTR and the Gag gene for removing critically important segments of the viral DNA in transgenic mice and rats encompassing the HIV-1 genome. Tail-vein injection of transgenic mice with a recombinant Adeno-associated virus 9 (rAAV9) vector expressing saCas9 and the gRNAs, rAAV:saCas9/gRNA, resulted in the cleavage of integrated HIV-1 DNA and excision of a 978 bp DNA fragment spanning between the LTR and Gag gene in the spleen, liver, heart, lung and kidney as well as in the circulating lymphocytes. Retro-orbital inoculation of rAAV9:saCas9/gRNA in transgenic rats eliminated a targeted segment of viral DNA and substantially decreased the level of viral gene expression in circulating blood lymphocytes. The results from the proof-of-concept studies, for the first time, demonstrate the in vivo eradication of HIV-1 DNA by CRISPR/Cas9 on delivery by an rAAV9 vector in a range of cells and tissues that harbor integrated copies of viral DNA. PMID:27194423

  8. Oxidized LDL levels are increased in HIV infection and may drive monocyte activation

    PubMed Central

    Zidar, David A.; Juchnowski, Steven; Ferrari, Brian; Clagett, Brian; Pilch-Cooper, Heather A.; Rose, Shawn; Rodriguez, Benigno; McComsey, Grace A.; Sieg, Scott F.; Mehta, Nehal N.; Lederman, Michael M.; Funderburg, Nicholas T.

    2015-01-01

    Background Human immunodeficiency virus (HIV) infection is associated with increased cardiovascular risk, and this risk correlates with markers of monocyte activation. We have shown that HIV is associated with a prothrombotic monocyte phenotype, which can be partially mitigated by statin therapy. We therefore explored the relationship between oxidized LDL particles and monocyte activation. Methods We performed phenotypic analysis of monocytes using flow cytometry on fresh whole blood in 54 patients with HIV and 24 controls without HIV. Plasma levels of oxLDL, soluble CD14, IL-6, soluble CD163 were measured by ELISA. In vitro experiments were performed using flow cytometry. Results Plasma levels of oxLDL were significantly increased in HIV-infection compared to controls (60.1 units vs 32.1 units, p<0.001). Monocyte expression of the oxLDL receptors, CD36 and Toll-like receptor 4, were also increased in HIV. OxLDL levels correlated with markers of monocyte activation, including soluble CD14, TF expression on inflammatory monocytes, and CD36. In vitro, stimulation with oxLDL, but not to LDL, resulted in expansion of inflammatory monocytes and increased monocyte expression of TF, recapitulating the monocyte profile we find in HIV disease. Conclusions OxLDL may contribute to monocyte activation and further study in the context of HIV disease is warranted. PMID:25647528

  9. HIV

    PubMed Central

    Chawla, Sumit; Sahoo, Soumya Swaroop; Jain, Rambilas; Khanna, Pardeep; Mehta, Bharti; Singh, Inderjeet

    2014-01-01

    Getting to zero: zero new HIV infections, zero deaths from AIDS-related illness, zero discrimination is the theme of World AIDS Day 2012. Given the spread of the epidemic today, getting to zero may sound difficult, but significant progress is underway. The total annual loss for the entire country due to HIV is 7% of GDP, which exceeds India’s annual health expenditure in 2004. The additional loss due to loss of labor income and increased medical expenditure as measured by the external transfers, account for 5% of the country’s health expenditure and 0.23% of GDP. Given that the HIV incidence rate is only 0.27% in India, these losses are quite staggering. Despite the remarkable achievements in development of anti-retroviral therapies against HIV and the recent advances in new prevention technologies, the rate of new HIV infections continue to outpace efforts on HIV prevention and control. Thus, the development of a safe and effective vaccine for prevention and control of AIDS remains a global public health priority and the greatest opportunity to eventually end the AIDS pandemic. PMID:24056755

  10. Neisseria gonorrhoeae-induced human defensins 5 and 6 increase HIV infectivity: role in enhanced transmission.

    PubMed

    Klotman, Mary E; Rapista, Aprille; Teleshova, Natalia; Micsenyi, Amanda; Jarvis, Gary A; Lu, Wuyuan; Porter, Edith; Chang, Theresa L

    2008-05-01

    Sexually transmitted infections (STIs) increase the likelihood of HIV transmission. Defensins are part of the innate mucosal immune response to STIs and therefore we investigated their role in HIV infection. We found that human defensins 5 and 6 (HD5 and HD6) promoted HIV infection, and this effect was primarily during viral entry. Enhancement was seen with primary viral isolates in primary CD4(+) T cells and the effect was more pronounced with R5 virus compared with X4 virus. HD5 and HD6 promoted HIV reporter viruses pseudotyped with vesicular stomatitis virus and murine leukemia virus envelopes, indicating that defensin-mediated enhancement was not dependent on CD4 and coreceptors. Enhancement of HIV by HD5 and HD6 was influenced by the structure of the peptides, as loss of the intramolecular cysteine bonds was associated with loss of the HIV-enhancing effect. Pro-HD5, the precursor and intracellular form of HD5, also exhibited HIV-enhancing effect. Using a cervicovaginal tissue culture system, we found that expression of HD5 and HD6 was induced in response to Neisseria gonorrhoeae (GC, for gonococcus) infection and that conditioned medium from GC-exposed cervicovaginal epithelial cells with elevated levels of HD5 also enhanced HIV infection. Introduction of small interfering RNAs for HD5 or HD6 abolished the HIV-enhancing effect mediated by GC. Thus, the induction of these defensins in the mucosa in the setting of GC infection could facilitate HIV infection. Furthermore, this study demonstrates the complexity of defensins as innate immune mediators in HIV transmission and warrants further investigation of the mechanism by which defensins modulate HIV infection. PMID:18424739

  11. Plasma Levels of Bacterial DNA Correlate with Immune Activation and the Magnitude of Immune Restoration in Persons with Antiretroviral-Treated HIV Infection

    PubMed Central

    Jiang, Wei; Lederman, Michael M.; Hunt, Peter; Sieg, Scott F.; Haley, Kathryn; Rodriguez, Benigno; Landay, Alan; Martin, Jeffrey; Sinclair, Elizabeth; Asher, Ava I.; Deeks, Steven G.; Douek, Daniel C.; Brenchley, Jason M.

    2009-01-01

    The significance of elevated plasma levels of bacterial lipopolysaccharide (LPS) in persons with chronic HIV infection remains undefined. We measured LPS levels by use of limulus lysate assay, and DNA sequences encoding bacterial ribosomal 16S RNA (16S rDNA) were assessed by quantitative polymerase chain reactions in plasma samples obtained from 242 donors. Plasma levels of 16S rDNA were significantly higher in human immunodeficiency virus (HIV)–infected subjects than in uninfected subjects, and they correlated with LPS levels. Higher levels of 16S rDNA were associated with higher levels of T cell activation and with lower levels of CD4 T cell restoration during antiretroviral therapy. Antiretroviral therapy reduces but does not fully normalize plasma levels of bacterial 16S rDNA, an index of microbial translocation from the gastrointestinal tract. High levels of 16S rDNA during therapy are strongly associated with reduced increases in the CD4+ T lymphocyte count, irrespective of plasma HIV RNA levels. These findings are consistent with the importance of microbial translocation in immunodeficiency and T cell homeostasis in chronic HIV infection. PMID:19265479

  12. HIV infection among heterosexuals at increased risk--United States, 2010.

    PubMed

    2013-03-15

    In 2009, an estimated 27% of human immunodeficiency virus (HIV) infections in the United States were attributed to heterosexual contact. During 2006-2007, as part of the data collection for the National HIV Behavioral Surveillance System (NHBS), CDC surveyed heterosexuals who lived in urban areas with a high prevalence of acquired immunodeficiency syndrome (AIDS) and found an overall HIV prevalence of 2.0% and a prevalence of 2.3% among persons with annual household incomes at or below the poverty level and 2.8% among persons with less than a high school education. This report summarizes HIV testing results from the second cycle of NHBS, conducted in 2010, which focused on heterosexual persons with low socioeconomic status (SES) living in areas with high AIDS case rates. The results indicated that HIV prevalence was 2.3% overall and 1.1% among participants who did not report a previous positive HIV test result. Overall, 25.8% of participants had never been tested for HIV until the NHBS survey. Given the high HIV prevalence in this sample, additional research should be conducted to identify culturally appropriate interventions that overcome barriers to HIV testing and increase linkage to care for heterosexuals with low SES in urban areas with high prevalence of AIDS. PMID:23486383

  13. Lability of DNA polymerase alpha correlated with decreased DNA synthesis and increased age in human cells

    SciTech Connect

    Busbee, D.; Sylvia, V.; Stec, J.; Cernosek, Z.; Norman, J.

    1987-12-01

    DNA excision repair and mitogen-initiated blastogenesis in human cells declined in efficiency as an apparent function of decreased DNA polymerase alpha specific activity with increased age of the cell donor. DNA polymerase alpha isolated from fetal cells contained a single, high-specific-activity enzyme form that could not be further activated and that was stable with regard to enzyme activity and affinity for DNA template-primer. DNA polymerase alpha isolated from adult-derived cells contained both low-specific-activity and high-specific-activity forms. The low-activity enzyme form, which showed low affinity of binding to DNA template-primer, was activated by treatment with phosphatidylinositol, /sup 32/P-ATP, and phosphatidylinositol kinase, resulting in a /sup 32/P-labeled enzyme that exhibited high affinity of binding to DNA template-primer. The activated enzyme was unstable, exhibiting a loss of /sup 32/P-label correlated with the loss of both specific activity and high affinity of binding to DNA template-primer. The data suggest that DNA polymerase alpha isolated from adult-derived human cells has low-activity and high-activity forms. Decreased specific activity of DNA polymerase alpha correlated with increased age of the donor appears to be a function of loss of an enzyme activator molecule resulting in diminished ability of the enzyme to bind DNA template-primer.

  14. Increased HIV testing among hospitalized patients who declined testing in the emergency department.

    PubMed

    Felsen, Uriel R; Cunningham, Chinazo O; Zingman, Barry S

    2016-05-01

    Health-care systems have serial encounters with many of the same patients across care settings; however, few studies have examined the role of reoffering HIV testing after a patient declines. We assessed whether an intervention to increase HIV testing among hospitalized patients was associated with increased testing among those who declined a test while in the Emergency Department (ED). We studied 8-week periods pre- and post-implementation of an electronic medical record (EMR)-based intervention to increase HIV testing among hospitalized patients. We included all patients 21-64 years old who had no prior HIV test, declined HIV testing in the ED, and were subsequently hospitalized. We used logistic regression to test for an association between time of hospital admission (pre- vs. post-intervention) and whether an HIV test was performed prior to discharge. Pre- and post-implementation, 220 and 218 patients who declined HIV testing in the ED were hospitalized, respectively. There were no significant demographic or clinical differences among patients pre- and post-implementation. Pre- and post-implementation, the median proportion of patients tested weekly was 6.7% (IQR 6.5%, 10.0%) and 41.4% (IQR 33.3%, 41.9%), respectively (aOR 6.2: 95%CI: 3.6, 10.6). HIV testing increased among hospitalized patients who declined a test in the ED after implementation of an EMR-based intervention. Almost half of the patients who declined testing in the ED ultimately underwent testing after it was reoffered during hospitalization, suggesting that the decision to undergo HIV testing is a dynamic process. Leveraging EMR resources may be an effective tool for expanding HIV testing, and testing should be reoffered to patients who previously declined. PMID:26654431

  15. 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.

  16. 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.

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

    PubMed

    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. PMID:26738439

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

    PubMed Central

    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. PMID:26738439

  19. 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. PMID:24873435

  20. DNA bar coding and pyrosequencing to identify rare HIV drug resistance mutations.

    PubMed

    Hoffmann, Christian; Minkah, Nana; Leipzig, Jeremy; Wang, Gary; Arens, Max Q; Tebas, Pablo; Bushman, Frederic D

    2007-01-01

    Treatment of HIV-infected individuals with antiretroviral agents selects for drug-resistant mutants, resulting in frequent treatment failures. Although the major antiretroviral resistance mutations are routinely characterized by DNA sequencing, treatment failures are still common, probably in part because undetected rare resistance mutations facilitate viral escape. Here we combined DNA bar coding and massively parallel pyrosequencing to quantify rare drug resistance mutations. Using DNA bar coding, we were able to analyze seven viral populations in parallel, overall characterizing 118 093 sequence reads of average length 103 bp. Analysis of a control HIV mixture showed that resistance mutations present as 5% of the population could be readily detected without false positive calls. In three samples of multidrug-resistant HIV populations from patients, all the drug-resistant mutations called by conventional analysis were identified, as well as four additional low abundance drug resistance mutations, some of which would be expected to influence the response to antiretroviral therapy. Methods for sensitive characterization of HIV resistance alleles have been reported, but only the pyrosequencing method allows all the positions at risk for drug resistance mutations to be interrogated deeply for many HIV populations in a single experiment. PMID:17576693

  1. 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. PMID:16045743

  2. Cellular HIV-1 DNA quantitation in patients during simplification therapy with protease inhibitor-sparing regimens.

    PubMed

    Sarmati, Loredana; Parisi, Saverio Giuseppe; Nicastri, Emanuele; d'Ettorre, Gabriella; Andreoni, Carolina; Dori, Luca; Gatti, Francesca; Montano, Marco; Buonomini, Anna Rita; Boldrin, Caterina; Palù, Giorgio; Vullo, Vincenzo; Andreoni, Massimo

    2007-07-01

    Simplified regimens containing protease-inhibitors (PI)-sparing combinations were used in patients with virological suppression after prolonged highly active antiretroviral therapy. This study evaluated the total HIV-1 DNA quantitation as a predictor of long-term success for PI-sparing simplified therapy. Sixty-two patients were enrolled in a prospective non-randomized cohort. All patients have been receiving a triple-therapy regimen, two nucleoside reverse transcriptase inhibitors (NRTIs) plus one PI, for at least 9 months and were characterized by undetectable plasma HIV-1 RNA levels (<50 cp/ml) for at least 6 months. Patients were changed to a simplified PI-sparing regimen to overcome PI-associated adverse effects. HIV-DNA levels in peripheral blood mononuclear cells (PBMCs) were evaluated at baseline and at the end of follow-up. Patients with proviral DNA levels below the median value (226 copies/10(6) PBMCs) had a significant higher CD4 cell count at nadir (P = 0.003) and at enrolment (P = 0.001) with respect to patients with HIV-DNA levels above the median value. At month 18, 53 out of 62 (85%) patients on simplified regimen showed virological success, 4 (6.4%) patients experienced virological failure and 5 (8%) patients showed viral blip. At logistic regression analysis, HIV-DNA levels below 226 copies/10(6) PBMCs at baseline were associated independently to a reduced risk of virological failure or viral blip during simplified therapy (OR 0.002, 95% CI 0.001-0.46, P = 0.025). The substitution of PI with NRTI or non-NRTIs may represent an effective treatment option. Indeed, treatment failure or viral blip were experienced by 6% and 8% of the patients on simplified therapy, respectively. In addition, sustained suppression of the plasma viral load was significantly correlated with low levels of proviral DNA before treatment simplification. PMID:17516532

  3. Inducible Escherichia coli fermentation for increased plasmid DNA production.

    PubMed

    Carnes, Aaron E; Hodgson, Clague P; Williams, James A

    2006-11-01

    Bacterial plasmids are the vectors of choice for DNA vaccines and short-term gene therapeutics. Growing plasmid DNA by microbial (Escherichia coli) fermentation is usually combined with alkaline lysis/chromatography methods of purification. To date, typical plasmid fermentation media and processes result in yields of 100-250 mg of plasmid DNA/l of culture medium, using standard high-copy pUC origin-containing plasmids. In order to address this initial and yield-limiting upstream step, we identified novel fermentation control parameters for fed-batch fermentation. The resulting fermentation strategies significantly increased specific plasmid yield with respect to cell mass while enhancing plasmid integrity and maintaining supercoiled DNA content. Fed-batch fermentation yield exceeding 1000 mg of plasmid DNA/l was obtained after reduction of plasmid-mediated metabolic burden during growth, and yields up to 1500 mg of plasmid DNA/l have been achieved with optimized plasmid backbones. Interestingly, by inducing high plasmid levels after sufficient biomass accumulation at low temperature and restricted growth, cells were able to tolerate significantly higher plasmid quantities than cells grown by conventional processes. This 5-10-fold increase in plasmid yield dramatically decreases plasmid manufacturing costs and improves the effectiveness of downstream purification by reducing the fraction of impurities. PMID:16819941

  4. Increasing HIV/AIDS awareness among African-American women: an exploratory study.

    PubMed

    Brown, Ludella; Tabi, Marian M

    2013-07-01

    This exploratory study was conducted to assess the effect of an HIV/AIDS prevention program on producing positive changing attitudes among African-American women in Southeast Georgia. This study used a faith-based approach. Data were collected from 23 respondents recruited from a local African-American church. HIV training was conducted over four 1-hour sessions using web-based interactive videos and lectures on HIV/AIDS. Constructs from the Social Cognitive Theory comprised the framework upon which the women received HIV/AIDS prevention training. Participants completed a 25-item pre- and post-intervention questionnaire to measure any changes that occurred in their attitudes regarding HIV/AIDS. Results showed a statistically significant difference in mean scores of individual knowledge and attitudes about HIV. The difference in mean scores for the remaining items was found to be statistically insignificant. The overall change in attitudes was also statistically significant, t = 2.27, df = 22, p < 0.05, which provided further evidence that when peers educate their communities on HIV/AIDS, it makes a significant difference in changing their attitudes about this disease. Although findings were positive, further data is needed to substantiate and validate the use of community peers to increase knowledge and awareness about HIV/AIDS among the African-American population. PMID:24218873

  5. Motif-Optimized Subtype A HIV Envelope-based DNA Vaccines Rapidly Elicit Neutralizing Antibodies When Delivered Sequentially

    PubMed Central

    Pissani, Franco; Malherbe, Delphine C.; Robins, Harlan; DeFilippis, Victor R.; Park, Byung; Sellhorn, George; Stamatatos, Leonidas; Overbaugh, Julie; Haigwood, Nancy L.

    2012-01-01

    HIV-1 infection results in the development of a diverging quasispecies unique to each infected individual. Envelope (Env)-specific neutralizing antibodies (NAbs) typically develop over months to years after infection and initially are limited to the infecting virus. In some subjects, antibody responses develop that neutralize heterologous isolates (HNAbs), a phenomenon termed broadening of the NAb response. Studies of co-crystalized antibodies and proteins have facilitated the identification of some targets of broadly neutralizing monoclonal antibodies (NmAbs) capable of neutralizing many or most heterologous viruses; however, the ontogeny of these antibodies in vivo remains elusive. We hypothesize that Env protein escape variants stimulate broad NAb development in vivo and could generate such NAbs when used as immunogens. Here we test this hypothesis in rabbits using HIV Env vaccines featuring: (1) use of individual quasispecies env variants derived from an HIV-1 subtype A-infected subject exhibiting high levels of NAbs within the first year of infection that increased and broadened with time; (2) motif optimization of envs to enhance in vivo expression of DNA formulated as vaccines; and (3) a combined DNA plus protein boosting regimen. Vaccines consisted of multiple env variants delivered sequentially and a simpler regimen that utilized only the least and most divergent clones. The simpler regimen was as effective as the more complex approach in generating modest HNAbs and was more efficient when modified, motif-optimized DNA was used in combination with trimeric gp140 protein. This is a rationally designed strategy that facilitates future vaccine design by addressing the difficult problem of generating HNAbs to HIV by empirically testing the immunogenicity of naturally occurring quasispecies env variants. PMID:22749601

  6. Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation

    PubMed Central

    Mouquet, Hugo; Scheid, Johannes F.; Zoller, Markus J.; Krogsgaard, Michelle; Ott, Rene G.; Shukair, Shetha; Artyomov, Maxim N.; Pietzsch, John; Connors, Mark; Pereyra, Florencia; Walker, Bruce D.; Ho, David D.; Wilson, Patrick C.; Seaman, Michael S.; Eisen, Herman N.; Chakraborty, Arup K.; Hope, Thomas J.; Ravetch, Jeffrey V.; Wardemann, Hedda; Nussenzweig, Michel C.

    2013-01-01

    During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV disfavours homotypic bivalent antibody binding1–3. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development4, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients5 with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV. PMID:20882016

  7. Binding of DNA with Abf2p Increases Efficiency of DNA Uptake by Isolated Mitochondria.

    PubMed

    Samoilova, E O; Krasheninnikov, I A; Vinogradova, E N; Kamenski, P A; Levitskii, S A

    2016-07-01

    Mutations in mitochondrial DNA often lead to severe hereditary diseases that are virtually resistant to symptomatic treatment. During the recent decades, many efforts were made to develop gene therapy approaches for treatment of such diseases using nucleic acid delivery into the organelles. The possibility of DNA import into mitochondria has been shown, but this process has low efficiency. In the present work, we demonstrate that the efficiency of DNA import can be significantly increased by preforming its complex with a mitochondria-targeted protein nonspecifically binding with DNA. As a model protein, we used the yeast protein Abf2p. In addition, we measured the length of the DNA site for binding this protein and the dissociation constant of the corresponding DNA-protein complex. Our data can serve as a basis for development of novel, highly efficient approaches for suppressing mutations in the mitochondrial genome. PMID:27449618

  8. Increase in the frequency of hepadnavirus DNA integrations by oxidative DNA damage and inhibition of DNA repair.

    PubMed Central

    Petersen, J; Dandri, M; Bürkle, A; Zhang, L; Rogler, C E

    1997-01-01

    Persistent hepadnavirus infection leads to oxidative stress and DNA damage through increased production of toxic oxygen radicals. In addition, hepadnaviral DNA integrations into chromosomal DNA can promote the process of hepatocarcinogenesis (M. Feitelson, Clin. Microbiol. Rev. 5:275-301, 1992). While previous studies have identified preferred integration sites in hepadnaviral genomes and suggested integration mechanisms (M. A. Buendia, Adv. Cancer Res. 59:167-226, 1992; C. E. Rogler, Curr. Top. Microbiol. Immunol. 168:103-141, 1991; C. Shih et al., J. Virol. 61:3491-3498, 1987), very little is known about the effects of agents which damage chromosomal DNA on the frequency of hepadnaviral DNA integrations. Using a recently developed subcloning approach to detect stable new integrations of duck hepatitis B virus (DHBV) (S. S. Gong, A. D. Jensen, and C. E. Rogler, J. Virol. 70:2000-2007, 1996), we tested the effects of increased chromosomal DNA damage induced by H2O2, or of the disturbance in DNA repair due to the inhibition of poly(ADP-ribose) polymerase (PARP), on the frequency of DHBV DNA integrations. Subclones of LMH-D21-6 cells, which replicate DHBV, were grown in the presence of various H2O2 concentrations and exhibited up to a threefold increase in viral DNA integration frequency in a dose-dependent manner. Moreover, inhibition of PARP, which plays a role in cellular responses to DNA breakage, by 3-aminobenzamide (3-AB) resulted in a sevenfold increase in the total number of new DHBV DNA integrations into host chromosomal DNA. Removal of either H2O2 or 3-AB from the culture medium in a subsequent cycle of subcloning was accompanied by a reversion back towards the original lower frequency of stable DHBV DNA integrations for LMH-D21-6 cells. These data support the hypothesis that DNA damage sites can serve as sites for hepadnaviral DNA integration, and that increasing the number of DNA damage sites dramatically increases viral integration frequency. PMID

  9. Increasing global participation in genetics research through DNA barcoding.

    PubMed

    Adamowicz, Sarah J; Steinke, Dirk

    2015-12-01

    DNA barcoding--the sequencing of short, standardized DNA regions for specimen identification and species discovery--has promised to facilitate rapid access to biodiversity knowledge by diverse users. Here, we advance our opinion that increased global participation in genetics research is beneficial, both to scientists and for science, and explore the premise that DNA barcoding can help to democratize participation in genetics research. We examine publication patterns (2003-2014) in the DNA barcoding literature and compare trends with those in the broader, related domain of genomics. While genomics is the older and much larger field, the number of nations contributing to the published literature is similar between disciplines. Meanwhile, DNA barcoding exhibits a higher pace of growth in the number of publications as well as greater evenness among nations in their proportional contribution to total authorships. This exploration revealed DNA barcoding to be a highly international discipline, with growing participation by researchers in especially biodiverse nations. We briefly consider several of the challenges that may hinder further participation in genetics research, including access to training and molecular facilities as well as policy relating to the movement of genetic resources. PMID:26642251

  10. Trichosanthin, a potent HIV-1 inhibitor, can cleave supercoiled DNA in vitro.

    PubMed Central

    Li, M X; Yeung, H W; Pan, L P; Chan, S I

    1991-01-01

    Trichosanthin, an abortifacient, immunosuppressive and anti-tumor protein purified from the traditional Chinese herb medicine Tian Hua Fen, is a potent inhibitor against HIV-1 replication. Under normal enzymatic digestion conditions, trichosanthin cleaves the supercoiled double-stranded DNA to produce nicked circular and linear DNA. Trichosanthin has no effect on linear double-stranded DNA. Neither does it convert relaxed circular duplex DNA into a supercoiled form in the presence of ATP. Thus trichosanthin is not a DNA gyrase. However, trichosanthin can cleave the relaxed circular DNA into a linear form, indicating that both the circular as well as the supercoiled forms are essential for trichosanthin recognition. In addition, trichosanthin contains one calcium metal ion per protein molecule, which presumably is related to its endonucleolytic activity. Images PMID:1659689

  11. Novel bimodular DNA aptamers with guanosine quadruplexes inhibit phylogenetically diverse HIV-1 reverse transcriptases

    PubMed Central

    Michalowski, Daniel; Chitima-Matsiga, Rebecca; Held, Daniel M.; Burke, Donald H.

    2008-01-01

    DNA aptamers RT5, RT6 and RT47 form a group of related sequences that inhibit HIV-1 reverse transcriptase (RT). The essential inhibitory structure is identified here as bimodular, with a 5′ stem–loop module physically connected to a 3′-guanosine quadruplex module. The stem–loop tolerates considerable sequence plasticity. Connections between the guanosine triplets in the quadruplex could be simplified to a single nucleotide or a nonnucleic acid linker, such as hexaethylene glycol. All 12 quadruplex guanosines are required in an aptamer retaining most of the original loop sequence from RT6; only 11 are required for aptamer R1T (single T residue in intra-quadruplex loops). Circular dichroism (CD) spectroscopy gave ellipticity minima and maxima at 240 nm and 264 nm, indicating a parallel arrangement of the quadruplex strands. The simplified aptamers displayed increased overall stability. An aptamer carrying the original intra-quadruplex loops from RT6 inhibited RT in K+ buffers but not in Na+ buffers and displayed significant CD spectral broadening in Na+ buffers, while R1T inhibited RT in both buffers and displayed less broadening in Na+ buffers. The bimodular ssDNA aptamers inhibited RT from diverse primate lentiviruses with low nM IC50 values. These data provide insight into the requirements for broad-spectrum RT inhibition by nucleic acid aptamers. PMID:18996899

  12. Increased DNA fragmentation and ultrastructural changes in fibromyalgic muscle fibres

    PubMed Central

    Sprott, H; Salemi, S; Gay, R; Bradley, L; Alarcon, G; Oh, S; Michel, B; Gay, S

    2004-01-01

    Objective: To determine whether there is evidence of increased DNA fragmentation and ultrastructural changes in muscle tissue of patients with fibromyalgia (FM) compared with healthy controls. Methods: Muscle tissues from 10 community residents with FM and 10 age and sex matched healthy controls were examined "blindly" for the presence of DNA fragmentation by two different methods: terminal deoxynucleotidyl transferase (TdT) staining (TUNEL) and the FragEL-Klenow DNA fragmentation detection kit. Ultrastructural analysis of tissue was performed by electron microscopy. Results: DNA fragmentation was detected by both methods in 55.4 (SEM 2.5)% of the nuclei in muscle tissue of patients with FM compared with 16.1 (4.1)% (p<0.001) of the nuclei in healthy controls. Contrary to expectation, no typical features of apoptosis could be detected by electron microscopy. The myofibres and actin filaments were disorganised and lipofuscin bodies were seen; glycogen and lipid accumulation were also found. The number of mitochondria was significantly lower in patients with FM than in controls and seemed to be morphologically altered. Conclusion: The ultrastructural changes described suggest that patients with FM are characterised by abnormalities in muscle tissue that include increased DNA fragmentation and changes in the number and size of mitochondria. These cellular changes are not signs of apoptosis. Persistent focal contractions in muscle may contribute to ultrastructural tissue abnormalities as well as to the induction and/or chronicity of nociceptive transmission from muscle to the central nervous system. PMID:14962957

  13. 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. PMID:26319939

  14. Increases in HIV Testing among Men Who Have Sex with Men — National HIV Behavioral Surveillance System, 20 U.S. Metropolitan Statistical Areas, 2008 and 2011

    PubMed Central

    Cooley, Laura A.; Oster, Alexandra M.; Rose, Charles E.; Wejnert, Cyprian; Le, Binh C.; Paz-Bailey, Gabriela

    2014-01-01

    In 2011, 62% of estimated new HIV diagnoses in the United States were attributed to male-to-male sexual contact (men who have sex with men, MSM); 39% of these MSM were black or African American. HIV testing, recommended at least annually by CDC for sexually active MSM, is an essential first step in HIV care and treatment for HIV-positive individuals. A variety of HIV testing initiatives, designed to reach populations disproportionately affected by HIV, have been developed at both national and local levels. We assessed changes in HIV testing behavior among MSM participating in the National HIV Behavioral Surveillance System in 2008 and 2011. We compared the percentages tested in the previous 12 months in 2008 and 2011, overall and by race/ethnicity and age group. In unadjusted analyses, recent HIV testing increased from 63% in 2008 to 67% in 2011 overall (P<0.001), from 63% to 71% among black MSM (P<0.001), and from 63% to 75% among MSM of other/multiple races (P<0.001); testing did not increase significantly for white or Hispanic/Latino MSM. Multivariable model results indicated an overall increase in recent HIV testing (adjusted prevalence ratio [aPR] = 1.07, P<0.001). Increases were largest for black MSM (aPR = 1.12, P<0.001) and MSM of other/multiple races (aPR = 1.20, P<0.001). Among MSM aged 18–19 years, recent HIV testing was shown to increase significantly among black MSM (aPR = 1.20, P = 0.007), but not among MSM of other racial/ethnic groups. Increases in recent HIV testing among populations most affected by HIV are encouraging, but despite these increases, improved testing coverage is needed to meet CDC recommendations. PMID:25180514

  15. Persistent HIV-related stigma in rural Uganda during a period of increasing HIV incidence despite treatment expansion

    PubMed Central

    Chan, Brian T.; Weiser, Sheri D.; Boum, Yap; Siedner, Mark J.; Mocello, A. Rain; Haberer, Jessica E.; Hunt, Peter W.; Martin, Jeffrey N.; Mayer, Kenneth H.; Bangsberg, David R.; Tsai, Alexander C.

    2014-01-01

    Objective Program implementers have argued that the increasing availability of anti-retroviral therapy (ART) will reduce the stigma of HIV. We analyzed data from Uganda to assess how HIV-related stigma has changed during a period of ART expansion. Design Serial cross-sectional surveys. Methods We analyzed data from the Uganda AIDS Rural Treatment Outcomes (UARTO) study during 2007-2012 to estimate trends in internalized stigma among people living with HIV (PLHIV) at the time of treatment initiation. We analyzed data from the Uganda Demographic and Health Surveys (DHS) from 2006 and 2011 to estimate trends in stigmatizing attitudes and anticipated stigma in the general population. We fitted regression models adjusted for socio-demographic characteristics, with year of data collection as the primary explanatory variable. Results We estimated an upward trend in internalized stigma among PLHIV presenting for treatment initiation (adjusted b=0.18; 95% CI, 0.06 to 0.30). In the general population, the odds of reporting anticipated stigma were greater in 2011 compared to 2006 (adjusted OR=1.80; 95% CI, 1.51 to 2.13), despite an apparent decline in stigmatizing attitudes (adjusted OR=0.62; 95% CI, 0.52 to 0.74). Conclusions Internalized stigma has increased over time among PLHIV in the setting of worsening anticipated stigma in the general population. Further study is needed to better understand the reasons for increasing HIV-related stigma in Uganda and its impact on HIV prevention efforts. PMID:25268886

  16. Increases in HIV diagnoses at the U.S.-Mexico border, 2003-2006.

    PubMed

    Espinoza, Lorena; Hall, H Irene; Hu, Xiaohong

    2009-10-01

    The population at the U.S.-Mexico border has experienced growth, more than double the U.S. national average. Movements of populations in this region have contributed to increased incidence of certain infectious diseases. We used information on persons diagnosed with HIV during 2003 to 2006 and aged 13 years or older (n = 4,279) reported to the Centers for Disease Control and Prevention for 45 U.S. border counties. We estimated the annual percent change and rates with Poisson regression. Overall, 47% of persons diagnosed with HIV in the border region were Hispanic; 39% nonHispanic white; and 10% nonHispanic black. During 2003 to 2006, HIV diagnoses increased 7.8% per year. Increases were observed among males, particularly among men who have sex with men. Among females, HIV diagnoses remained stable but decreased among females in nonborder regions. The number of HIV diagnoses at the border has increased. To decrease incidence of HIV disease it is necessary to develop prevention and education programs specific to this region. PMID:19824832

  17. Quantification of viral DNA during HIV-1 infection: A review of relevant clinical uses and laboratory methods.

    PubMed

    Alidjinou, E K; Bocket, L; Hober, D

    2015-02-01

    Effective antiretroviral therapy usually leads to undetectable HIV-1 RNA in the plasma. However, the virus persists in some cells of infected patients as various DNA forms, both integrated and unintegrated. This reservoir represents the greatest challenge to the complete cure of HIV-1 infection and its characteristics highly impact the course of the disease. The quantification of HIV-1 DNA in blood samples constitutes currently the most practical approach to measure this residual infection. Real-time quantitative PCR (qPCR) is the most common method used for HIV-DNA quantification and many strategies have been developed to measure the different forms of HIV-1 DNA. In the literature, several "in-house" PCR methods have been used and there is a need for standardization to have comparable results. In addition, qPCR is limited for the precise quantification of low levels by background noise. Among new assays in development, digital PCR was shown to allow an accurate quantification of HIV-1 DNA. Total HIV-1 DNA is most commonly measured in clinical routine. The absolute quantification of proviruses and unintegrated forms is more often used for research purposes. PMID:25201144

  18. Secondary Structure and Secondary Structure Dynamics of DNA Hairpins Complexed with HIV-1 NC Protein

    PubMed Central

    Cosa, Gonzalo; Harbron, Elizabeth J.; Zeng, Yining; Liu, Hsiao-Wei; O'Connor, Donald B.; Eta-Hosokawa, Chie; Musier-Forsyth, Karin; Barbara, Paul F.

    2004-01-01

    Reverse transcription of the HIV-1 RNA genome involves several complex nucleic acid rearrangement steps that are catalyzed by the HIV-1 nucleocapsid protein (NC), including for example, the annealing of the transactivation response (TAR) region of the viral RNA to the complementary region (TAR DNA) in minus-strand strong-stop DNA. We report herein single-molecule fluorescence resonance energy transfer measurements on single immobilized TAR DNA hairpins and hairpin mutants complexed with NC (i.e., TAR DNA/NC). Using this approach we have explored the conformational distribution and dynamics of the hairpins in the presence and absence of NC protein. The data demonstrate that NC shifts the equilibrium secondary structure of TAR DNA hairpins from a fully “closed” conformation to essentially one specific “partially open” conformation. In this specific conformation, the two terminal stems are “open” or unwound and the other stems are closed. This partially open conformation is arguably a key TAR DNA intermediate in the NC-induced annealing mechanism of TAR DNA. PMID:15454467

  19. Increasing HIV-1 molecular complexity among men who have sex with men in Bangkok.

    PubMed

    Leelawiwat, Wanna; Rutvisuttinunt, Wiriya; Arroyo, Miguel; Mueanpai, Famui; Kongpechsatit, Oranuch; Chonwattana, Wannee; Chaikummao, Supaporn; de Souza, Mark; vanGriensven, Frits; McNicholl, Janet M; Curlin, Marcel E

    2015-04-01

    In Thailand, new HIV-1 infections are largely concentrated in certain risk groups such as men who have sex with men (MSM), where annual incidence may be as high as 12% per year. The paucity of information on the molecular epidemiology of HIV-1 in Thai MSM limits progress in understanding the epidemic and developing new prevention methods. We evaluated HIV-1 subtypes in seroincident and seroprevalent HIV-1-infected men enrolled in the Bangkok MSM Cohort Study (BMCS) between 2006 and 2011. We characterized HIV-1 subtype in 231 seroprevalent and 194 seroincident subjects using the multihybridization assay (MHA). Apparent dual infections, recombinant strains, and isolates found to be nontypeable by MHA were further characterized by targeted genomic sequencing. Most subjects were infected with HIV-1 CRF01_AE (82%), followed by infections with recombinants (11%, primarily CRF01_AE/B recombinants), subtype B (5%), and dual infections (2%). More than 11 distinct chimeric patterns were observed among CRF01B_AE/B recombinants, most involving recombination within integrase. A significant increase in the proportion of nontypeable strains was observed among seroincident MSM between 2006 and 2011. CRF01_AE and subtype B were the most and least common infecting strains, respectively. The predominance of CRF01_AE among HIV-1 infections in Thai MSM participating in the BMCS parallels trends observed in Thai heterosexuals and injecting drug users. The presence of complex recombinants and a significant rise in nontypeable strains suggest ongoing changes in the genetic makeup of the HIV-1 epidemic in Thailand, which may pose challenges for HIV-1 prevention efforts and vaccine development. PMID:25366819

  20. Immunogenicity of a novel engineered HIV-1 clade C synthetic consensus-based envelope DNA vaccine.

    PubMed

    Yan, Jian; Corbitt, Natasha; Pankhong, Panyupa; Shin, Thomas; Khan, Amir; Sardesai, Niranjan Y; Weiner, David B

    2011-09-22

    DNA vaccines require significant engineering in order to generate strong CTL responses in both non-human primates and humans. In this study, we designed a clade C env gene (EY3E1-C) to decrease the genetic distances of virus isolates within clade C and focus the induced T cell responses to conserved clade C epitopes. After generating a consensus sequence by analyzing full-length clade C env early transmitter sequences, several modifications were performed to increase the expression of the EY3E1-C, including codon/RNA optimization, addition of Kozak sequence and addition of an IgE leader sequence. We also shortened the V1 and V2 loops to approximate early transmitter isolate sequences and the cytoplasmic tail was truncated to prevent envelope recycling. When studied as a DNA vaccine in Balb/c mice, compared to a primary codon-optimized clade C envelope DNA vaccine (p96ZM651gp140-CD5), this novel construct is up to three times more potent in driving CTL responses. Importantly this construct not only induces stronger cross-reactive cellular responses within clade C, it also induces stronger immune responses against clade B and group M envelope peptide pools than p96ZM651gp140-CD5. Epitope mapping demonstrated that EY3E1-C was able to induce clade C envelope-specific immune responses against 15 peptide pools, clade B envelope-specific immune responses against 19 peptide pools and group M envelope-specific immune responses against 16 peptide pools out of 29, respectively, indicating that a significant increase in the breadth of induced immune responses. The analysis of antibody responses suggested that vaccination of pEY3E1-C could induce a clade C envelope-specific antibody response. The cellular immune responses of pEY3E1-C could be further enhanced when the DNA was delivered by using electroporation (EP). Thus, the synthetic engineered consensus EY3E1-C gene is capable of eliciting stronger and broader CTL responses than primary clade C envelopes. This finding

  1. Soluble multi-trimeric TNF superfamily ligand adjuvants enhance immune responses to a HIV-1 Gag DNA vaccine

    PubMed Central

    Kanagavelu, Saravana K.; Snarsky, Victoria; Termini, James M.; Gupta, Sachin; Barzee, Suzanne; Wright, Jacqueline A.; Khan, Wasif N.; Kornbluth, Richard S.; Stone, Geoffrey W.

    2011-01-01

    Background DNA vaccines remain an important component of HIV vaccination strategies, typically as part of a prime/boost vaccination strategy with viral vector or protein boost. A number of DNA prime/viral vector boost vaccines are currently being evaluated for both preclinical studies and in Phase I and Phase II clinical trials. These vaccines would benefit from molecular adjuvants that increase correlates of immunity during the DNA prime. While HIV vaccine immune correlates are still not well defined, there are a number of immune assays that have been shown to correlate with protection from viral challenge including CD8+ T cell avidity, antigen-specific proliferation, and polyfunctional cytokine secretion. Methodology and Principal Findings Recombinant DNA vaccine adjuvants composed of a fusion between Surfactant Protein D (SP-D) and either CD40 Ligand (CD40L) or GITR Ligand (GITRL) were previously shown to enhance HIV-1 Gag DNA vaccines. Here we show that similar fusion constructs composed of the TNF superfamily ligands (TNFSFL) 4-1BBL, OX40L, RANKL, LIGHT, CD70, and BAFF can also enhanced immune responses to a HIV-1 Gag DNA vaccine. BALB/c mice were vaccinated intramuscularly with plasmids expressing secreted Gag and SP-D-TNFSFL fusions. Initially, mice were analyzed 2 weeks or 7 weeks following vaccination to evaluate the relative efficacy of each SP-D-TNFSFL construct. All SP-D-TNFSFL constructs enhanced at least one Gag-specific immune response compared to the parent vaccine. Importantly, the constructs SP-D-4-1BBL, SP-D-OX40L, and SP-D-LIGHT enhanced CD8+ T cell avidity and CD8+/CD4+ T cell proliferation 7 weeks post vaccination. These avidity and proliferation data suggest that 4-1BBL, OX40L, and LIGHT fusion constructs may be particularly effective as vaccine adjuvants. Constructs SP-D-OX40L, SP-D-LIGHT, and SP-D-BAFF enhanced Gag-specific IL-2 secretion in memory T cells, suggesting these adjuvants can increase the number of self-renewing Gag-specific CD8

  2. Inhibition of HIV-1 gene expression by novel macrophage-tropic DNA enzymes targeted to cleave HIV-1 TAT/Rev RNA.

    PubMed Central

    Unwalla, H; Banerjea, A C

    2001-01-01

    Many regions of the HIV-1 genome have been targeted in earlier studies by RNA-cleaving DNA enzymes possessing the 10-23 catalytic motif, and efficient inhibition of HIV-1 gene expression was reported. All these studies employed charged synthetic lipids to introduce the catalytic DNA into the mammalian cells, which severely limits its practical application and usefulness in vivo. Taking advantage of the ability of G residues to interact directly with the scavenger receptors on the macrophages, we synthesized a DNA enzyme 5970 that contained 10 G residues at the 3' end. With the aim of improving the intracellular stability of the DNA enzyme 5970, we added two short stretches of stem-loop structures that were 12 bases long on either side of the DNA enzyme 5970. DNA enzyme 5970 without the poly-G tracts cleaved the synthetic RNA of HIV-1 TAT/Rev, two important regulatory proteins of HIV, very efficiently in a sequence-specific manner. Addition of 10 G residues at the 3' end of the DNA enzyme affected the cleavage efficiency only marginally whereas the same DNA enzyme with stem-loop structures on either end was significantly less efficient. The DNA enzyme with the poly-G tract at its 3' end was taken up specifically by a human macrophage-specific cell line directly in the absence of Lipofectin and was also able to inhibit HIV-1 gene expression in a transient-expression system as well as when challenged with the virus. The potential applications of these novel macrophage-tropic DNA enzymes are discussed. PMID:11415445

  3. A multigene HIV type 1 subtype C modified vaccinia Ankara (MVA) vaccine efficiently boosts immune responses to a DNA vaccine in mice.

    PubMed

    Shephard, Enid; Burgers, Wendy A; Van Harmelen, Joanne H; Monroe, James E; Greenhalgh, Trish; Williamson, Carolyn; Williamson, Anna-Lise

    2008-02-01

    Heterologous prime-boost vaccine strategies have generated high frequencies of antigen-specific T cells in preclinical and clinical trials of candidate HIV vaccines. We have developed a DNA (SAAVI DNA-C) and MVA (SAAVI MVA-C) vaccine based on HIV-1 subtype C for testing in clinical trials. Both vaccines contain five subtype C genes: gag, reverse transcriptase, tat, and nef, expressed as a polyprotein, and a truncated env (gp150). The individual vaccines induced CD8(+) and CD4(+) T cells specific for the vaccine-expressed antigens in BALB/c mice. Combining the vaccines in a DNA prime and MVA boost regimen increased the cumulative peptide response compared to the DNA vaccine alone 10-fold, to over 6000 SFU/10(6) splenocytes in the IFN-gamma ELISPOT assay. Th1 cytokine IFN-gamma and TNF-alpha levels from HIV-specific CD8(+) and CD4(+) T cells increased 20- and 8-fold, respectively, with a SAAVI MVA-C boost. Effector and effector memory RT- and Env-specific memory CD8(+) T cell subsets were boosted after MVA immunization, and over time the cells returned to an intermediate memory phenotype similar to that prior to the boost. Immunization of guinea pigs with the DNA-MVA combination induced high titers of antibodies to gp120, although neutralizing activity was weak or absent. The demonstration that these vaccines induce potent cellular immune responses merits their testing in clinical trials. PMID:18240963

  4. Changing spatial patterns and increasing rurality of HIV prevalence in the Democratic Republic of the Congo between 2007 and 2013.

    PubMed

    Carrel, Margaret; Janko, Mark; Mwandagalirwa, Melchior Kashamuka; Morgan, Camille; Fwamba, Franck; Muwonga, Jérémie; Tshefu, Antoinette K; Meshnick, Steven; Emch, Michael

    2016-05-01

    The Democratic Republic of the Congo (DRC) has one of the lowest HIV prevalence in sub-Saharan Africa, estimated at 1.1% [0.9-1.3] of adults aged 15-49 in 2013 (UNAIDS). Within the 2 million km(2) country, however, there exists spatial variation in HIV prevalence, with the highest HIV prevalence observed in the large cities of Kinshasa and Lubumbashi. Globally, HIV is an increasingly rural disease, diffusing outwards from urban centers of high HIV prevalence to places where HIV was previously absent or present at very low levels. Utilizing data collected during Demographic and Health Surveillance (DHS) in 2007 and 2013 in the DRC, we sought to update the map of HIV prevalence in the DRC as well as to explore whether HIV in the DRC is an increasingly rural disease or remains confined to urban areas. Bayesian kriging and regression indicate that HIV prevalence in rural areas of the DRC is higher in 2013 than in 2007 and that increased distance to an urban area is no longer protective against HIV as it was in 2007. These findings suggest that HIV education, testing and prevention efforts need to diffuse from urban to rural areas just as HIV is doing. PMID:26974234

  5. Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma

    PubMed Central

    Vargas-Meneses, Milenka V.; Massanella, Marta; Ignacio, Caroline C.; Gianella, Sara

    2016-01-01

    Multiple viruses can co-infect the genital tract, modifying the immunologic and virologic milieu and possibly playing a role in viral transmission and pathogenesis. The aim of our studies has been to understand the complex relationships between HIV-1 RNA, and multiple human herpesviruses known to frequently replicate in the genital tract of HIV-infected men (i.e. cytomegalovirus [CMV], Epstein Bar virus [EBV], herpes simplex virus [HSV] types 1 and 2, and human herpesviruses [HHV] 6, 7 and 8) (Gianella et al., 2013a; Gianella et al., 2013b; Gianella et al., 2013c; Gianella et al., 2014). This protocol was designed to collect and process male genital secretion (GS), and to isolate and further quantify HIV RNA and DNA of seven HHV from seminal plasma using quantitative real time PCR technology. PMID:27376109

  6. Autoantibodies against peripheral blood cells appear early in HIV infection and their prevalence increases with disease progression.

    PubMed Central

    Klaassen, R J; Mulder, J W; Vlekke, A B; Eeftinck Schattenkerk, J K; Weigel, H M; Lange, J M; von dem Borne, A E

    1990-01-01

    The presence of platelet- and neutrophil-bound immunoglobulin (PBIg and NBIg) in thrombocytopenic or neutropenic HIV-infected individuals has led to the concept that in HIV infection thrombocytopenia and neutropenia are mediated by autoimmunity. However, PBIg and NBIg were also demonstrated in non-cytopenic HIV-infected individuals. We determined the prevalence of autoantibodies against neutrophils and platelets by immunofluorescence in randomly chosen persons in different stages of asymptomatic and symptomatic HIV infection. Platelet and neutrophil autoantibodies already appeared in the asymptomatic stage and their prevalence further increased in the symptomatic stages. No correlation was found between the presence of either platelet or neutrophil antibodies and the occurrence of circulating immune complexes in the blood or the serum immunoglobulin level. There was no significant difference in neutrophil counts in HIV-infected persons with or without neutrophil autoantibodies. In addition, no significant difference in neutrophil count was found between HIV-infected and non-HIV-infected persons. HIV-infected individuals with platelet autoantibodies tended to have a lower platelet count than HIV-infected individuals without these antibodies. However, the platelet count in HIV-infected individuals without platelet antibodies was significantly lower than in the non-HIV infected persons. Thus, autoantibodies against platelets and neutrophils occur early in HIV infection and their prevalence is correlated with disease progression. Their presence is associated with cytopenia only in a limited number of persons. Non-immune mechanisms also mediate thrombocytopenia in HIV infection. PMID:1974174

  7. Increased HIV Testing among Men Who Have Sex with Men from 2008 to 2012, Nanjing, China

    PubMed Central

    Yan, Hongjing; Li, Jianjun; Raymond, H. Fisher; Huan, Xiping; Guan, Wenhui; Hu, Haiyang; Yang, Haitao; McFarland, Willi; Wei, Chongyi

    2016-01-01

    Background HIV testing is the first point of HIV treatment entry for HIV-infected individuals and an avenue to engage persons at risk in prevention. In China, where the prevalence of HIV among men who have sex with men (MSM) has been rising over the last decade, uptake of HIV testing has been low. Methods We examined changes in HIV testing in the preceding 12 months through two cross-sectional surveys conducted among MSM in Nanjing, Jiangsu province, China in 2008 and 2012. Respondent-driven sampling (RDS) was used to recruit participants. Questionnaire interviews and venous blood were collected to measure HIV testing, risk behaviors, and prevalence of HIV, syphilis, and HSV-2. Results A total of 430 and 589 MSM were surveyed in 2008 and 2012, respectively, with comparable samples in each round with respect to demographic characteristics. HIV testing in the past 12 months increased significantly from 20.1% (95% CI 13.3–26.8) in 2008 to 46.0% (95% CI 39.3–51.4, p < 0.001) in 2012. HIV prevalence was stable, at 6.6% (95% CI 2.5–11.3) in 2008 and 10.1% (95% CI 6.6–13.9, p = 0.240) in 2012, as was syphilis (14.3% in 2008 vs. 9.9% in 2012, p = 0.240). HSV-2 prevalence (18.6% in 2008 vs. 10.2% in 2012, p = 0.040) and self-reported STI in the last year (24.3% in 2008 vs. 14.3% in 2012, p = 0.020) significantly decreased. Changes in reported sexual behaviors were mixed and the profiles of who did and did not test varied between 2008 and 2012. Conclusions HIV testing uptake more than doubled among MSM in Nanjing from 2008 to 2012 –a period of massive promotion and scale up of testing programs for MSM. However, additional efforts are still needed to further increase the proportion of men being not only tested but also undergoing repeat testing if they engage in continued risk taking behavior. PMID:27111540

  8. NLRX1 Sequesters STING to Negatively Regulate the Interferon Response, Thereby Facilitating the Replication of HIV-1 and DNA Viruses.

    PubMed

    Guo, Haitao; König, Renate; Deng, Meng; Riess, Maximilian; Mo, Jinyao; Zhang, Lu; Petrucelli, Alex; Yoh, Sunnie M; Barefoot, Brice; Samo, Melissa; Sempowski, Gregory D; Zhang, Aiping; Colberg-Poley, Anamaris M; Feng, Hui; Lemon, Stanley M; Liu, Yong; Zhang, Yanping; Wen, Haitao; Zhang, Zhigang; Damania, Blossom; Tsao, Li-Chung; Wang, Qi; Su, Lishan; Duncan, Joseph A; Chanda, Sumit K; Ting, Jenny P-Y

    2016-04-13

    Understanding the negative regulators of antiviral immune responses will be critical for advancing immune-modulated antiviral strategies. NLRX1, an NLR protein that negatively regulates innate immunity, was previously identified in an unbiased siRNA screen as required for HIV infection. We find that NLRX1 depletion results in impaired nuclear import of HIV-1 DNA in human monocytic cells. Additionally, NLRX1 was observed to reduce type-I interferon (IFN-I) and cytokines in response to HIV-1 reverse-transcribed DNA. NLRX1 sequesters the DNA-sensing adaptor STING from interaction with TANK-binding kinase 1 (TBK1), which is a requisite for IFN-1 induction in response to DNA. NLRX1-deficient cells generate an amplified STING-dependent host response to cytosolic DNA, c-di-GMP, cGAMP, HIV-1, and DNA viruses. Accordingly, Nlrx1(-/-) mice infected with DNA viruses exhibit enhanced innate immunity and reduced viral load. Thus, NLRX1 is a negative regulator of the host innate immune response to HIV-1 and DNA viruses. PMID:27078069

  9. Liver ultrastructural morphology and mitochondrial DNA levels in HIV/hepatitis C virus coinfection: no evidence of mitochondrial damage with highly active antiretroviral therapy.

    PubMed

    Matsukura, Motoi; Chu, Fanny F S; Au, May; Lu, Helen; Chen, Jennifer; Rietkerk, Sonja; Barrios, Rolando; Farley, John D; Montaner, Julio S; Montessori, Valentina C; Walker, David C; Côté, Hélène C F

    2008-06-19

    Liver mitochondrial toxicity is a concern, particularly in HIV/hepatitis C virus (HCV) coinfection. Liver biopsies from HIV/HCV co-infected patients, 14 ON-highly active antiretroviral therapy (HAART) and nine OFF-HAART, were assessed by electron microscopy quantitative morphometric analyses. Hepatocytes tended to be larger ON-HAART than OFF-HAART (P = 0.05), but mitochondrial volume, cristae density, lipid volume, mitochondrial DNA and RNA levels were similar. We found no evidence of increased mitochondrial toxicity in individuals currently on HAART, suggesting that concomitant HAART should not delay HCV therapy. PMID:18525271

  10. Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR.

    PubMed

    Henrich, Timothy J; Gallien, Sebastien; Li, Jonathan Z; Pereyra, Florencia; Kuritzkes, Daniel R

    2012-12-01

    Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia were also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets. PMID:22974526

  11. Human papillomavirus DNA and mRNA prevalence and association with cervical cytological abnormalities in the Irish HIV population.

    PubMed

    Loy, Aisling; McInerney, Jamie; Pilkington, Loretto; Keegan, Helen; Delamere, Sandra; Martin, Cara M; Sheils, Orla; O'Leary, John J; Mulcahy, Fiona

    2015-10-01

    The complex interplay between HIV and human papillomavirus and its link to cervical dysplasia is poorly understood. This is the first study to assess the prevalence of oncogenic human papillomavirus mRNA in HIV-positive women, its relationship to HIV and its potential use in the triage of cervical cancer screening in HIV-positive women. In this cross-sectional study, we included 321 HIV-positive women. In all, 28.7% had abnormal cervical cytology, 51.1% were human papillomavirus DNA-positive and 21.8% tested positive for human papillomavirus mRNA. Women with a CD4 count of <200 × 10(6)/L were more likely to test positive for human papillomavirus DNA and mRNA. Virally suppressed women were less likely to be human papillomavirus DNA-positive; however, the same did not hold true for human papillomavirus mRNA. We found the human papillomavirus mRNA screening to be more specific when screening for low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion than human papillomavirus DNA at 84.53% compared to 57.36%. However, the sensitivity was less at 51.59% versus 91.07% for human papillomavirus DNA. It may be possible in the future to use human papillomavirus mRNA/DNA testing within a triage algorithm for the screening and management of cervical cancer in the HIV-positive patient. PMID:25258395

  12. Low-Level Detection and Quantitation of Cellular HIV-1 DNA and 2-LTR Circles Using Droplet Digital PCR

    PubMed Central

    Henrich, Timothy J.; Gallien, Sebastien; Li, Jonathan Z.; Pereyra, Florencia; Kuritzkes, Daniel R.

    2012-01-01

    Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia was also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets. PMID:22974526

  13. Safety and Comparative Immunogenicity of an HIV-1 DNA Vaccine in Combination with Plasmid Interleukin 12 and Impact of Intramuscular Electroporation for Delivery

    PubMed Central

    Kalams, Spyros A.; Parker, Scott D.; Elizaga, Marnie; Metch, Barbara; Edupuganti, Srilatha; Hural, John; De Rosa, Stephen; Carter, Donald K.; Rybczyk, Kyle; Frank, Ian; Fuchs, Jonathan; Koblin, Beryl; Kim, Denny H.; Joseph, Patrice; Keefer, Michael C.; Baden, Lindsey R.; Eldridge, John; Boyer, Jean; Sherwat, Adam; Cardinali, Massimo; Allen, Mary; Pensiero, Michael; Butler, Chris; Khan, Amir S.; Yan, Jian; Sardesai, Niranjan Y.; Kublin, James G.; Weiner, David B.

    2013-01-01

    Background. DNA vaccines have been very poorly immunogenic in humans but have been an effective priming modality in prime-boost regimens. Methods to increase the immunogenicity of DNA vaccines are needed. Methods. HIV Vaccine Trials Network (HVTN) studies 070 and 080 were multicenter, randomized, clinical trials. The human immunodeficiency virus type 1 (HIV-1) PENNVAX®-B DNA vaccine (PV) is a mixture of 3 expression plasmids encoding HIV-1 Clade B Env, Gag, and Pol. The interleukin 12 (IL-12) DNA plasmid expresses human IL-12 proteins p35 and p40. Study subjects were healthy HIV-1–uninfected adults 18–50 years old. Four intramuscular vaccinations were given in HVTN 070, and 3 intramuscular vaccinations were followed by electroporation in HVTN 080. Cellular immune responses were measured by intracellular cytokine staining after stimulation with HIV-1 peptide pools. Results. Vaccination was safe and well tolerated. Administration of PV plus IL-12 with electroporation had a significant dose-sparing effect and provided immunogenicity superior to that observed in the trial without electroporation, despite fewer vaccinations. A total of 71.4% of individuals vaccinated with PV plus IL-12 plasmid with electroporation developed either a CD4+ or CD8+ T-cell response after the second vaccination, and 88.9% developed a CD4+ or CD8+ T-cell response after the third vaccination. Conclusions. Use of electroporation after PV administration provided superior immunogenicity than delivery without electroporation. This study illustrates the power of combined DNA approaches to generate impressive immune responses in humans. PMID:23840043

  14. HIV-1 nucleocapsid and ESCRT-component Tsg101 interplay prevents HIV from turning into a DNA-containing virus.

    PubMed

    Chamontin, Célia; Rassam, Patrice; Ferrer, Mireia; Racine, Pierre-Jean; Neyret, Aymeric; Lainé, Sébastien; Milhiet, Pierre-Emmanuel; Mougel, Marylène

    2015-01-01

    HIV-1, the agent of the AIDS pandemic, is an RNA virus that reverse transcribes its RNA genome (gRNA) into DNA, shortly after its entry into cells. Within cells, retroviral assembly requires thousands of structural Gag proteins and two copies of gRNA as well as cellular factors, which converge to the plasma membrane in a finely regulated timeline. In this process, the nucleocapsid domain of Gag (GagNC) ensures gRNA selection and packaging into virions. Subsequent budding and virus release require the recruitment of the cellular ESCRT machinery. Interestingly, mutating GagNC results into the release of DNA-containing viruses, by promo-ting reverse transcription (RTion) prior to virus release, through an unknown mechanism. Therefore, we explored the biogenesis of these DNA-containing particles, combining live-cell total internal-reflection fluorescent microscopy, electron microscopy, trans-complementation assays and biochemical characterization of viral particles. Our results reveal that DNA virus production is the consequence of budding defects associated with Gag aggregation at the plasma membrane and deficiency in the recruitment of Tsg101, a key ESCRT-I component. Indeed, targeting Tsg101 to virus assembly sites restores budding, restricts RTion and favors RNA packaging into viruses. Altogether, our results highlight the role of GagNC in the spatiotemporal control of RTion, via an ESCRT-I-dependent mechanism. PMID:25488808

  15. A trend towards increasing viral load in newly diagnosed HIV-infected inpatients in southeast China.

    PubMed

    Chen, Y; Wang, Z; Huang, A; Yuan, J; Wei, D; Ye, H

    2016-06-01

    Peripheral blood viral load is an important indicator of viral production and clearance. Previous studies have suggested that viral load might predict the rate of decrease in CD4+ cell count and progression to AIDS and death. Here, we conducted a retrospective analysis of the trends in HIV-1 viral load in southeast China. Among inpatients newly diagnosed with HIV infection, we found that viral load has increased over the past decade from 4·20 log10 copies/ml in 2002 to 6·61 log10 copies/ml in 2014, with a mean increase of 0·19 log10 copies/ml each year. However, the CD4+ cell count was stable and insensitive to changes in viral load. Thus, increasing viral load appears to be an emerging trend in newly diagnosed HIV-infected inpatients. PMID:26732896

  16. RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study.

    PubMed

    Bernacchi, Serena; Henriet, Simon; Dumas, Philippe; Paillart, Jean-Christophe; Marquet, Roland

    2007-09-01

    The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors. PMID:17609216

  17. Which gay men would increase their frequency of HIV testing with home self-testing?

    PubMed

    Bavinton, Benjamin R; Brown, Graham; Hurley, Michael; Bradley, Jack; Keen, Phillip; Conway, Damian P; Guy, Rebecca; Grulich, Andrew E; Prestage, Garrett

    2013-07-01

    Many Australian gay men do not get tested for HIV at the recommended frequency. Barriers to HIV testing may be reduced by the availability of home HIV self-testing (HHST). An online cross-sectional questionnaire was conducted with 2,306 Australian gay men during 2009. Multivariate logistic regression identified factors associated with being likely to increase testing frequency if HHST was available, among previously-tested and never-tested men. Among 2,018 non-HIV-positive men, 83.9% had been tested. Two-thirds indicated they would test more often if HHST was available irrespective of previous testing history. In multivariate analysis, independent predictors of increased testing frequency with HHST included preferences for more convenient testing, not having to see a doctor when testing and wanting immediate results among all men, as well as not being from an Anglo-Australian background and recent unprotected anal sex with casual partners among previously-tested men only. The majority of gay men report that being able to test themselves at home would increase their frequency of HIV testing. PMID:23525790

  18. Mechanism of polyoxometalate-mediated inactivation of DNA polymerases: an analysis with HIV-1 reverse transcriptase indicates specificity for the DNA-binding cleft.

    PubMed Central

    Sarafianos, S G; Kortz, U; Pope, M T; Modak, M J

    1996-01-01

    The anti-DNA polymerase activity of a structural family of polyoxometalates has been determined. Two representative compounds of this family, possessing a saddle-like structure [(O3POPO3)4W12O36]16- (polyoxometalate I) and [(O3PCH2PO3)4W12O36]16- (polyoxometalate II) were found to inhibit all the DNA polymerases tested, with IC50 values ranging from 2 to 10 microM. A comparative study with HIV-1 reverse transcriptase (RT) and Klenow polymerase as representative DNA polymerases indicated that protection from inactivation was achieved by inclusion of DNA but not by deoxynucleotide triphosphates (dNTPs). Kinetic analysis revealed that the mode of HIV-1 RT inhibition is competitive with respect to DNA, and non-competitive with respect to dNTP binding. Cross-linking experiments confirmed that the inhibitors interfere with the DNA-binding function of HIV-1 reverse transcriptase. Interestingly, a number of drug-resistant mutants of HIV-1 RT exhibit a sensitivity to polyoxometalate comparable to the wild-type HIV-1 RT, suggesting that these polyoxometalates interact at a novel site. Because different polymerases contain DNA-binding clefts of various dimensions, it should be possible to modify polyoxometalates or to add a link to an enzyme-specific drug so that more effective inhibitors could be developed. Using a computer model of HIV-1 RT we performed docking studies in a binary complex (enzyme-polyoxometalate I) to propose tentatively a possible interacting site in HIV-1 RT consistent with the available biochemical results as well as with the geometric and charge constraints of the two molecules. PMID:8912703

  19. Integrin α4β7 expression increases HIV susceptibility in activated cervical CD4+ T cells via an HIV attachment-independent mechanism

    PubMed Central

    Ding, Jian; Tasker, Carley; Lespinasse, Pierre; Dai, Jihong; Fitzgerald-Bocarsly, Patricia; Lu, Wuyuan; Heller, Debra; Chang, Theresa L.

    2015-01-01

    Background CD4+ T cells, the principal target in acute SIV and HIV infection, are crucial for the establishment and dissemination of HIV infection in mucosal tissues. Studies indicate that α4β7 CD4+ T cells are preferentially infected by HIV in vitro and during acute SIV infection. The integrin α4β7 is thought to promote HIV capture by target cells; however, the role of integrin α4β7 in HIV transmission remains controversial. In this study, we characterized immune phenotypes of human cervical T cells and examined HIV preference in integrin α4β7+ CD4+ T cells. In vitro all-trans retinoic acid differentiated peripheral CD4+ T cells (at-RA differentiated cells) were included as a comparison. Results In both peripheral and cervical cells, the majority of HIV p24+ cells were activated CD4+ T cells expressing integrin α4β7. Among infected at-RA differentiated cells, the frequency of CCR5 expression was higher in HIV p24+ cells than in HIV p24- cells; no such difference was observed in cervical cells. Neither the cyclic hexapeptide CWLDVC nor a monoclonal antibody against integrin α4β7 blocked HIV attachment or gp120 binding to target cells regardless of the presence of CD4, indicating that integrin α4β7 did not facilitate HIV capture by target cells. Conclusion Integrin α4β7 expression increases HIV susceptibility, but the mechanism is not through promoting HIV binding to target cells. PMID:26167616

  20. Mucosal DNA vaccination with highly attenuated Shigella is superior to attenuated Salmonella and comparable to intramuscular DNA vaccination for T cells against HIV.

    PubMed

    Vecino, William H; Morin, Paul M; Agha, Rabia; Jacobs, William R; Fennelly, Glenn J

    2002-07-01

    An immunization strategy using attenuated bacteria to deliver DNA vaccine plasmids to mucosal sites may induce protective T cell responses against sexual HIV transmission. In a murine intranasal (i.n.) immunization model, we demonstrate that transiently persistent Deltaasd Shigella flexneri strain 15D harboring DNA vaccines induces HIV- and SIV-specific gamma interferon (IFN-gamma) producing CD8+ T cells among splenocytes more efficiently than either a longer persisting DeltaaroD Salmonella typhimurium strain SL7207 or transiently persistent S. typhi strain Ty21a harboring DNA vaccines. Also, the frequency of antigen-specific gamma interferon (IFN-gamma) producing cells induced by Shigella 15D harboring a DNA vaccine were comparable to that induced by intramuscular (i.m.) immunization with purified DNA vaccine. Moreover, the magnitude of mucosal and systemic antigen-specific IgA and IgG responses after immunization were dependent upon the route (i.m. vs. i.n.) of inoculation, with i.n. Shigella 15D DNA vaccines generating higher levels of HIV-specific IgA in vaginal washings than i.m. purified DNA vaccine. Deltaasd S. flexneri is a promising vector for mucosal DNA vaccine immunization against HIV. PMID:12036602

  1. Optimizing HIV/AIDS resources in Armenia: increasing ART investment and examining HIV programmes for seasonal migrant labourers

    PubMed Central

    Kelly, Sherrie L; Shattock, Andrew J; Kerr, Cliff C; Stuart, Robyn M; Papoyan, Arshak; Grigoryan, Trdat; Hovhannisyan, Ruben; Grigoryan, Samvel; Benedikt, Clemens; Wilson, David P

    2016-01-01

    Introduction HIV prevalence is declining in key populations in Armenia including in people who inject drugs (PWID), men who have sex with men, prison inmates, and female sex workers (FSWs); however, prevalence is increasing among Armenians who seasonally migrate to work in countries with higher HIV prevalence, primarily to the Russian Federation. Methods We conducted a modelling study using the Optima model to assess the optimal resource allocation to meet targets from the 2013 to 2016 national strategic plan to minimize HIV incidence and AIDS-related deaths by 2020. Demographic, epidemiological, behavioural, and programme cost data from 2000 through 2014 were used to inform the model. The levels of coverage that could be attained among targeted populations with different investments, as well as their expected outcomes, were determined. In the absence of evidence of the efficacy of HIV programmes targeted at seasonal labour migrants, we conducted a sensitivity analysis to determine the cost-effective funding threshold for the seasonal labour migrant programme. Results The optimization analysis revealed that shifts in funding allocations could further minimize incidence and deaths by 2020 within the available resource envelope. The largest emphasis should be on antiretroviral therapy (ART), with the optimal investment to increase treatment coverage by 40%. Optimal investments also involve increases in opiate substitution therapy and FSW programmes, as well as maintenance of other prevention programmes for PWID and prevention of mother-to-child transmission. Additional funding for these increases should come from budgets for general population programmes. This is projected to avert 17% of new infections and 29% of AIDS-related deaths by 2020 compared to a baseline scenario of maintaining 2013 spending. Our sensitivity analysis demonstrated that, at current spending, coverage of annual testing among migrants of at least 43% should be achieved to warrant continuation

  2. A polymerase chain reaction method for the amplification of full-length envelope genes of HIV-1 from DNA samples containing single molecules of HIV-1 provirus.

    PubMed

    McClure, P; Curran, R; Boneham, S; Ball, J K

    2000-07-01

    Polymerase chain reaction (PCR) amplification of full-length envelope genes from the human immunodeficiency virus type 1 (HIV-1) directly from uncultured clinical samples is difficult. This paper describes a comparative assessment of the performance of three thermostable polymerases in an HIV-1 full-length envelope gene PCR. The PCR method utilising Expand HiFi polymerase was successful when using DNA samples extracted from a variety of sources including blood, semen and various tissues. This method generated high and specific yields of product from samples containing as little as one copy of HIV-1 proviral DNA. The resulting PCR products were suitable for a variety of downstream analytical methods including DNA sequence analysis. PMID:10921844

  3. Ethical reflections on pharmacogenetics and DNA banking in a cohort of HIV-infected patients

    PubMed Central

    de Montgolfier, Sandrine; Moutel, Grégoire; Duchange, Nathalie; Theodorou, Ioannis; Hervé, Christian; Leport, Catherine

    2002-01-01

    The aim of this study was to analyze ethical questions concerning the storage of human biological samples to be used in genetic analyses and pharmacogenetic research based on a French experience of DNA banking in a cohort of HIV-infected patients receiving protease inhibitor treatment (APROCO). We describe the ethical issues raised during the establishment of a DNA bank, including questions dealing with autonomy, benefit to the patient, information sharing and confidentiality as well as guarantees concerning the storage and use of DNA. We describe the practical applications of themes illustrated theoretically in the literature. Most of the points raised are not specific to HIV, but some of them may be more accurate due to the characteristics of the HIV population. The questions raised are not exhaustive and we conclude with specific points that remain to be defined. Our results are summarized in the memorandum and consent form presented in the appendices. This work should allow other researchers and members of evaluation committees to enrich their considerations and should stimulate discussion on this subject. PMID:12464796

  4. Observation of HIV-1 Nucleocapsid Protein induced TAR DNA melting at the single molecule level

    NASA Astrophysics Data System (ADS)

    Cosa, Gonzalo; Harbron, Elizabeth; O'Connor, Donald; Musier-Forsyth, Karin; Barbara, Paul

    2003-03-01

    Reverse transcription of the HIV-1 RNA genome involves several nucleic acid rearrangement steps, and the HIV-1 nucleocapsid protein (NC) plays a key role in this process. NC is a nucleic acid chaperone protein, which facilitates the formation of the most stable nucleic acid structures. Single molecule fluorescence resonance energy transfer (SM-FRET) measurements enable us to observe the NC-induced conformational fluctuations of a transactivation response region (TAR) DNA hairpin, which is part of the initial product of reverse transcription known as minus-strand strong-stop DNA. SM-FRET studies show that the majority of conformational fluctuations of the fluorescently-labeled TAR DNA hairpin in the presence of NC occur in <100 ms. A single molecule explores a wide range of confomations unpon NC binding, with fluctuations encompassing as many as 40 bases in both arms of the hairpin. No conformational fluctuations are observed with the dye-labeled TAR DNA hairpin in the absence of NC or when a labeled TAR DNA hairpin variant lacking bulges and internal loops is analyzed in the presence of NC. This study represents the first real-time observation of NC-mediated nucleic acid conformational fluctuations, revealing new insights into NC's nucleic acid chaperone activity.

  5. Increased Coronary Vessel Wall Thickness in HIV-Infected Young Adults

    PubMed Central

    Abd-Elmoniem, Khaled Z.; Unsal, Aylin B.; Eshera, Sarah; Matta, Jatin R.; Muldoon, Nancy; McAreavey, Dorothea; Purdy, Julia B.; Hazra, Rohan; Hadigan, Colleen; Gharib, Ahmed M.

    2014-01-01

    Background. Individuals with long-term human immunodeficiency virus (HIV) infection are at risk for premature vasculopathy and cardiovascular disease (CVD). We evaluated coronary vessel wall thickening, coronary plaque, and epicardial fat in patients infected with HIV early in life compared with healthy controls. Methods. This is a prospective cross-sectional study of 35 young adults who acquired HIV in early life and 11 healthy controls, free of CVD. Time resolved phase-sensitive dual inversion recovery black-blood vessel wall magnetic resonance imaging (TRAPD) was used to measure proximal right coronary artery (RCA) wall thickness, and multidetector computed tomography (CT) angiography was used to quantify coronary plaque and epicardial fat. Results. RCA vessel wall thickness was significantly increased in HIV-infected patients compared with sex- and race-matched controls (1.32 ± 0.21 mm vs 1.09 ± 0.14 mm, P = .002). No subject had discrete plaque on CT sufficient to cause luminal narrowing, and plaque was not related to RCA wall thickness. In multivariate regression analyses, smoking pack-years (P = .004) and HIV infection (P = .007) were independently associated with thicker RCA vessel walls. Epicardial fat did not differ between groups. Among the HIV-infected group, duration of antiretroviral therapy (ART) (P = .02), duration of stavudine exposure (P < .01), low-density lipoprotein cholesterol (P = .04), and smoking pack-years (P < .01) were positively correlated with RCA wall thickness. Conclusions. This investigation provides evidence of subclinical coronary vascular disease among individuals infected with HIV in early life. Increased duration of ART, hyperlipidemia, and smoking contributed to proximal RCA thickening, independent of atherosclerotic plaque quantified by CT. These modifiable risk factors appear to influence early atherogenesis as measured by coronary wall thickness and may be important targets for CVD risk reduction. PMID:25159580

  6. CD4+ and CD8+ T Cell Activation Are Associated with HIV DNA in Resting CD4+ T Cells

    PubMed Central

    Cockerham, Leslie R.; Siliciano, Janet D.; Sinclair, Elizabeth; O'Doherty, Una; Palmer, Sarah; Yukl, Steven A.; Strain, Matt C.; Chomont, Nicolas; Hecht, Frederick M.; Siliciano, Robert F.; Richman, Douglas D.; Deeks, Steven G.

    2014-01-01

    The association between the host immune environment and the size of the HIV reservoir during effective antiretroviral therapy is not clear. Progress has also been limited by the lack of a well-accepted assay for quantifying HIV during therapy. We examined the association between multiple measurements of HIV and T cell activation (as defined by markers including CD38, HLA-DR, CCR5 and PD-1) in 30 antiretroviral-treated HIV-infected adults. We found a consistent association between the frequency of CD4+ and CD8+ T cells expressing HLA-DR and the frequency of resting CD4+ T cells containing HIV DNA. This study highlights the need to further examine this relationship and to better characterize the biology of markers commonly used in HIV studies. These results may also have implications for reactivation strategies. PMID:25340755

  7. 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

  8. Expression of HIV gp120 protein increases sensitivity to the rewarding properties of methamphetamine in mice

    PubMed Central

    Kesby, James P.; Hubbard, David T.; Markou, Athina; Semenova, Svetlana

    2012-01-01

    Methamphetamine abuse and human immunodeficiency virus (HIV) infection induce neuropathological changes in corticolimbic brain areas involved in reward and cognitive function. Little is known about the combined effects of methamphetamine and HIV infection on cognitive and reward processes. The HIV/gp120 protein induces neurodegeneration in mice, similar to HIV-induced pathology in humans. We investigated the effects of gp120 expression on associative learning, preference for methamphetamine and non-drug reinforcers, and sensitivity to the conditioned rewarding properties of methamphetamine in transgenic (tg) mice expressing HIV/gp120 protein (gp120-tg). gp120-tg mice learned the operant response for food at the same rate as non-tg mice. In the two-bottle choice procedure with restricted access to drugs, gp120-tg mice exhibited greater preference for methamphetamine and saccharin than non-tg mice, whereas preference for quinine was similar between genotypes. Under conditions of unrestricted access to methamphetamine, the mice exhibited a decreased preference for increasing methamphetamine concentrations. However, male gp120-tg mice showed a decreased preference for methamphetamine at lower concentrations than non-tg male mice. gp120-tg mice developed methamphetamine-induced conditioned place preference at lower methamphetamine doses compared with non-tg mice. No differences in methamphetamine pharmacokinetics were found between genotypes. These results indicate that gp120-tg mice exhibit no deficits in associative learning or reward/motivational function for a natural reinforcer. Interestingly, gp120 expression resulted in increased preference for methamphetamine and a highly palatable non-drug reinforcer (saccharin) and increased sensitivity to methamphetamine-induced conditioned reward. These data suggest that HIV-positive individuals may have increased sensitivity to methamphetamine, leading to high methamphetamine abuse potential in this population. PMID

  9. Expression of HIV gp120 protein increases sensitivity to the rewarding properties of methamphetamine in mice.

    PubMed

    Kesby, James P; Hubbard, David T; Markou, Athina; Semenova, Svetlana

    2014-07-01

    Methamphetamine abuse and human immunodeficiency virus (HIV) infection induce neuropathological changes in corticolimbic brain areas involved in reward and cognitive function. Little is known about the combined effects of methamphetamine and HIV infection on cognitive and reward processes. The HIV/gp120 protein induces neurodegeneration in mice, similar to HIV-induced pathology in humans. We investigated the effects of gp120 expression on associative learning, preference for methamphetamine and non-drug reinforcers, and sensitivity to the conditioned rewarding properties of methamphetamine in transgenic (tg) mice expressing HIV/gp120 protein (gp120-tg). gp120-tg mice learned the operant response for food at the same rate as non-tg mice. In the two-bottle choice procedure with restricted access to drugs, gp120-tg mice exhibited greater preference for methamphetamine and saccharin than non-tg mice, whereas preference for quinine was similar between genotypes. Under conditions of unrestricted access to methamphetamine, the mice exhibited a decreased preference for increasing methamphetamine concentrations. However, male gp120-tg mice showed a decreased preference for methamphetamine at lower concentrations than non-tg male mice. gp120-tg mice developed methamphetamine-induced conditioned place preference at lower methamphetamine doses compared with non-tg mice. No differences in methamphetamine pharmacokinetics were found between genotypes. These results indicate that gp120-tg mice exhibit no deficits in associative learning or reward/motivational function for a natural reinforcer. Interestingly, gp120 expression resulted in increased preference for methamphetamine and a highly palatable non-drug reinforcer (saccharin) and increased sensitivity to methamphetamine-induced conditioned reward. These data suggest that HIV-positive individuals may have increased sensitivity to methamphetamine, leading to high methamphetamine abuse potential in this population. PMID

  10. Increases in sexually transmitted infections and sexual risk behaviour without a concurrent increase in HIV incidence among men who have sex with men in San Francisco: a suggestion of HIV serosorting?

    PubMed Central

    Truong, H M; Kellogg, T; Klausner, J D; Katz, M H; Dilley, J; Knapper, K; Chen, S; Prabhu, R; Grant, R M; Louie, B; McFarland, W

    2006-01-01

    Background Sexually transmitted infections (STI) and unprotected anal intercourse (UAI) have been increasing among men who have sex with men (MSM) in San Francisco. However, HIV incidence has stabilised. Objectives To describe recent trends in sexual risk behaviour, STI, and HIV incidence among MSM in San Francisco and to assess whether increases in HIV serosorting (that is, selective unprotected sex with partners of the same HIV status) may contribute to preventing further expansion of the epidemic. Methods The study applies an ecological approach and follows the principles of second generation HIV surveillance. Temporal trends in biological and behavioural measures among MSM were assessed using multiple pre‐existing data sources: STI case reporting, prevention outreach programmatic data, and voluntary HIV counselling and testing data. Results Reported STI cases among MSM rose from 1998 through 2004, although the rate of increase slowed between 2002 and 2004. Rectal gonorrhoea cases increased from 157 to 389 while early syphilis increased from nine to 492. UAI increased overall from 1998 to 2004 (p<0.001) in community based surveys; however, UAI with partners of unknown HIV serostatus decreased overall (p<0.001) among HIV negative MSM, and among HIV positive MSM it declined from 30.7% in 2001 to a low of 21.0% in 2004 (p<0.001). Any UAI, receptive UAI, and insertive UAI with a known HIV positive partner decreased overall from 1998 to 2004 (p<0.001) among MSM seeking anonymous HIV testing and at the STI clinic testing programme. HIV incidence using the serological testing algorithm for recent HIV seroconversion (STARHS) peaked in 1999 at 4.1% at the anonymous testing sites and 4.8% at the STI clinic voluntary testing programme, with rates levelling off through 2004. Conclusions HIV incidence among MSM appears to have stabilised at a plateau following several years of resurgence. Increases in the selection of sexual partners of concordant HIV serostatus may be

  11. Enhanced non-inflammasome mediated immune responses by mannosylated zwitterionic-based cationic liposomes for HIV DNA vaccines.

    PubMed

    Qiao, Chenmeng; Liu, Jiandong; Yang, Jun; Li, Yan; Weng, Jie; Shao, Yiming; Zhang, Xin

    2016-04-01

    Human immunodeficiency virus (HIV) DNA vaccine can induce cellular and humoral immunity. A safe and effective HIV DNA vaccine is urgent need to prevent the spread of acquired immune deficiency syndrome (AIDS). The major drawback of DNA vaccines is the low immunogenicity, which is caused by the poor delivery to antigen presenting cells and insufficient antigen expression. Sparked by the capability of endosomal/lysosomal escape of the zwitterionic lipid distearoyl phosphoethanol-amine-polycarboxybetaine (DSPE-PCB), we attempted to develop a zwitterionic-based cationic liposome with enhanced immunogenicity of DNA vaccines. The mannosylated zwitterionic-based cationic liposome (man-ZCL) was constructed as a DNA vaccine adjuvant for HIV vaccination. Man-ZCL could complex with DNA antigens to form a tight structure and protect them from nuclei enzyme degradation. Benefited from the capability of the specific mannose receptor mediated antigen processing cells targeting and enhanced endosomal/lysosomal escape, the man-ZCL lipoplexes were supposed to promote antigen presentation and the immunogenicity of DNA vaccines. In vitro and in vivo results revealed that man-ZCL lipoplexes showed enhanced anti-HIV immune responses and lower toxicity compared with CpG/DNA and Lipo2k/DNA, and triggered a Th1/Th2 mixed immunity. An antigen-depot effect was observed in the administration site, and this resulted in enhanced retention of DNA antigens in draining lymph nodes. Most importantly, the man-ZCL could assist to activate T cells through a non-inflammasome pathway. These findings suggested that the man-ZCL could be potentially applied as a safe and efficient DNA adjuvant for HIV vaccines. PMID:26851653

  12. Inhibition of PP2A by LIS1 increases HIV-1 gene expression

    PubMed Central

    Epie, Nicolas; Ammosova, Tatyana; Turner, Willie; Nekhai, Sergei

    2006-01-01

    Background Lissencephaly is a severe brain malformation in part caused by mutations in the LIS1 gene. LIS1 interacts with microtubule-associated proteins, and enhances transport of microtubule fragments. Previously we showed that LIS1 interacts with HIV-1 Tat protein and that this interaction was mediated by WD40 domains of LIS1. In the present study, we analyze the effect of LIS1 on Tat-mediated transcription of HIV-1 LTR. Results Tat-mediated HIV-1 transcription was upregulated in 293 cells transfected with LIS1 expression vector. The WD5 but not the N-terminal domain of LIS1 increases Tat-dependent HIV-1 transcription. The effect of LIS1 was similar to the effect of okadaic acid, an inhibitor of protein phosphatase 2A (PP2A). We then analyzed the effect of LIS1 on the activity of PP2A in vitro. We show that LIS1 and its isolated WD5 domain but not the N-terminal domain of LIS1 blocks PP2A activity. Conclusion Our results show that inhibition of PP2A by LIS1 induces HIV-1 transcription. Our results also point to a possibility that LIS1 might function in the cells as a yet unrecognized regulatory subunit of PP2A. PMID:17018134

  13. Reduced sTWEAK and Increased sCD163 Levels in HIV-Infected Patients: Modulation by Antiretroviral Treatment, HIV Replication and HCV Co-Infection

    PubMed Central

    Beltrán, Luis M.; García Morillo, José S.; Egido, Jesús; Noval, Manuel Leal; Ferrando-Martinez, Sara; Blanco-Colio, Luis M.; Genebat, Miguel; Villar, José R.; Moreno-Luna, Rafael; Moreno, Juan Antonio

    2014-01-01

    Background Patients infected with the human immunodeficiency virus (HIV) have an increased risk of cardiovascular disease due to increased inflammation and persistent immune activation. CD163 is a macrophage scavenger receptor that is involved in monocyte-macrophage activation in HIV-infected patients. CD163 interacts with TWEAK, a member of the TNF superfamily. Circulating levels of sTWEAK and sCD163 have been previously associated with cardiovascular disease, but no previous studies have fully analyzed their association with HIV. Objective The aim of this study was to analyze circulating levels of sTWEAK and sCD163 as well as other known markers of inflammation (hsCRP, IL-6 and sTNFRII) and endothelial dysfunction (sVCAM-1 and ADMA) in 26 patients with HIV before and after 48 weeks of antiretroviral treatment (ART) and 23 healthy subjects. Results Patients with HIV had reduced sTWEAK levels and increased sCD163, sVCAM-1, ADMA, hsCRP, IL-6 and sTNFRII plasma concentrations, as well as increased sCD163/sTWEAK ratio, compared with healthy subjects. Antiretroviral treatment significantly reduced the concentrations of sCD163, sVCAM-1, hsCRP and sTNFRII, although they remained elevated when compared with healthy subjects. Antiretroviral treatment had no effect on the concentrations of ADMA and sTWEAK, biomarkers associated with endothelial function. The use of protease inhibitors as part of antiretroviral therapy and the presence of HCV-HIV co-infection and/or active HIV replication attenuated the ART-mediated decrease in sCD163 plasma concentrations. Conclusion HIV-infected patients showed a proatherogenic profile characterized by increased inflammatory, immune-activation and endothelial-dysfunction biomarkers that partially improved after ART. HCV-HIV co-infection and/or active HIV replication enhanced immune activation despite ART. PMID:24594990

  14. Four-Stage Audit Demonstrating Increased Uptake of HIV Testing in Acute Neurology Admissions Using Staged Practical Interventions

    PubMed Central

    Sokhi, Dilraj Singh; Oxenham, Chantal; Coates, Rebecca; Forbes, Mhairi; Gupta, Nadi K.; Blackburn, Daniel J.

    2015-01-01

    Background UK National Guidelines (UKNG) advise HIV testing in clinically indicated neurological presentations. We audited the impact of our practical strategies to increase uptake of HIV testing at a regional acute neurology admissions unit. Methods We audited HIV testing in 4 periods over 2 years: before we designed a UKNG-based “HIV testing in Neurology” protocol (“pre-protocol”); after dissemination of the protocol alone (“post-protocol”); post-protocol dissemination combined with both a tailored departmental admissions clerking proforma to prompt for HIV testing & consenting, and regular focussed tutorials to doctors on HIV testing in neurological patients (“post-proforma”); and finally one year after the post-proforma period (“+1 year”). We also looked at the total number of HIV tests sent from the unit during the two-year period. We assessed significance using Fisher’s exact test. Results 47.8% of all acute neurology non-stroke admissions were eligible for HIV testing during all the audit periods. Testing rates were as follows: pre-protocol 21.9%; post-protocol 36.6%; post-proforma 83.3%; and at +1 year 65.4% (p<0.05 for both post-protocol and +1 year when compared to pre-protocol). Documentation of consent for HIV testing improved from 25% to 67.6% with the HIV-tailored clerking proforma. The total number of HIV tests requested from the unit doubled in the post-proforma period compared to pre-protocol (p<0.05). Conclusion In conclusion: the combination of an HIV testing protocol, a tailored departmental clerking proforma and regular focussed teaching to doctors on indications for HIV testing led to a sustained increase in HIV testing uptake in our regional acute neurology admissions unit. PMID:26335351

  15. Saccharin consumption increases sperm DNA fragmentation and apoptosis in mice

    PubMed Central

    Rahimipour, Marzieh; Talebi, Ali Reza; Anvari, Morteza; Abbasi Sarcheshmeh, Abolghasem; Omidi, Marjan

    2014-01-01

    Background: Saccharin is an artificial non-caloric sweetener that used to sweeten products such as drinks, candies, medicines, and toothpaste, but our bodies cannot metabolize it. Sodium saccharin is considered as an important factor in tumor promotion in male rats but not in humans. Objective: The objective of this study was to investigate the effect of saccharin consumption on sperm parameters and apoptosis in adult mice. Materials and Methods: Totally 14 adult male mice were divided into 2 groups. Group 1 served as control fed on basal diet and group 2 or experimental animals received distilled water containing saccharin (0.2% w/v) for 35 days. After that, the left cauda epididymis of each mouse was cut and placed in Ham’s F10. Swimmed-out spermatozoa were used to analyze count, motility, morphology (Pap-staining) and viability (eosin-Y staining). Sperm DNA integrity, as an indicator of apoptosis, was assessed by SCD (sperm chromatin dispersion) and terminal deoxynucleotidyl transferase (TUNEL) assay. Results: Following saccharin consumption, we had a reduction in sperm motility with respect to control animals (p=0.000). In addition, the sperm count diminished (17.70±1.11 in controls vs. 12.80±2.79 in case group, p=0.003) and the rate of sperm normal morphology decreased from 77.00±6.40 in control animals into 63.85±6.81 in saccharin-treated mice (p=0.001). Also, we saw a statistically significant increase in rates of sperm DNA damage and apoptosis in experimental group when compared to control one (p=0.001, p=0.002 respectively). Conclusion: Saccharin consumption may have negative effects on sperm parameters, and increases the rate of sperm DNA fragmentation and apoptosis in mice. PMID:25031574

  16. Plasma HHV8 DNA predicts relapse in individuals with HIV-associated multicentric Castleman disease.

    PubMed

    Stebbing, Justin; Adams, Caroline; Sanitt, Adam; Mletzko, Salvinia; Nelson, Mark; Gazzard, Brian; Newsom-Davis, Tom; Bower, Mark

    2011-07-14

    HIV-associated multicentric Castleman disease (HIV-MCD) is a rare lymphoproliferative disorder caused by infection with human herpesvirus-8. The disease follows a relapsing and remitting clinical course, with marked systemic symptoms during an active attack, which can prove fatal. Its incidence is rising, and new data indicate the utility of the anti-CD20 monoclonal antibody rituximab at inducing remissions in both first- and second-line settings, although biomarkers associated with relapse have not been previously identified. In 52 individuals with a histologic diagnosis of HIV-MCD, we performed univariate and multivariate analyses to predict factors associated with an HIV-MCD attack. Although a younger age (< 50 years) was associated with an attack, the strongest association was observed with plasma levels of human herpesvirus-8 DNA. Rising levels predicted an attack (hazard ratio = 2.9; 95% confidence interval, 1.3-6.7), and maintenance therapy with rituximab should be considered in these individuals. PMID:21511959

  17. Ribonuclease H/DNA Polymerase HIV-1 Reverse Transcriptase Dual Inhibitor: Mechanistic Studies on the Allosteric Mode of Action of Isatin-Based Compound RMNC6

    PubMed Central

    Corona, Angela; Meleddu, Rita; Esposito, Francesca; Distinto, Simona; Bianco, Giulia; Masaoka, Takashi; Maccioni, Elias; Menéndez-Arias, Luis; Alcaro, Stefano; Le Grice, Stuart F. J.; Tramontano, Enzo

    2016-01-01

    The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC50 values of 1.4 and 9.8 μM on HIV-1 RT-associated RNase H and polymerase activities, respectively. Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain (partially overlapping the non-nucleoside RT inhibitor [NNRTI] binding pocket), and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC50 in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 RT. PMID:26800261

  18. Integrase-independent HIV-1 infection is augmented under conditions of DNA damage and produces a viral reservoir

    SciTech Connect

    Ebina, Hirotaka Kanemura, Yuka; Suzuki, Yasutsugu; Urata, Kozue; Misawa, Naoko; Koyanagi, Yoshio

    2012-05-25

    HIV-1 possesses a viral protein, integrase (IN), which is necessary for its efficient integration in target cells. However, it has been reported that an IN-defective HIV strain is still capable of integration. Here, we assessed the ability of wild type (WT) HIV-1 to establish infection in the presence of IN inhibitors. We observed a low, yet clear infection of inhibitor-incubated cells infected with WT HIV which was identical to cells infected with IN-deficient HIV, D64A. Furthermore, the IN-independent integration could be enhanced by the pretreatment of cells with DNA-damaging agents suggesting that integration is mediated by a DNA repair system. Moreover, significantly faster viral replication kinetics with augmented viral DNA integration was observed after infection in irradiated cells treated with IN inhibitor compared to nonirradiated cells. Altogether, our results suggest that HIV DNA has integration potential in the presence of an IN inhibitor and may serve as a virus reservoir.

  19. Solution structure of the DNA binding domain of HIV-1 integrase.

    PubMed

    Lodi, P J; Ernst, J A; Kuszewski, J; Hickman, A B; Engelman, A; Craigie, R; Clore, G M; Gronenborn, A M

    1995-08-01

    The solution structure of the DNA binding domain of HIV-1 integrase (residues 220-270) has been determined by multidimensional NMR spectroscopy. The protein is a dimer in solution, and each subunit is composed of a five-stranded beta-barrel with a topology very similar to that of the SH3 domain. The dimer is formed by a stacked beta-interface comprising strands 2, 3, and 4, with the two triple-stranded antiparallel beta-sheets, one from each subunit, oriented antiparallel to each other. One surface of the dimer, bounded by the loop between strands beta 1 and beta 2, forms a saddle-shaped groove with dimensions of approximately 24 x 23 x 12 A in cross section. Lys264, which has been shown from mutational data to be involved in DNA binding, protrudes from this surface, implicating the saddle-shaped groove as the potential DNA binding site. PMID:7632683

  20. 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

  1. Increasing the Reach of HIV Testing to Young Latino MSM: Results of a Pilot Study Integrating Outreach and Services

    PubMed Central

    Erausquin, Jennifer Toller; Duan, Naihua; Grusky, Oscar; Swanson, Aimee-Noelle; Kerrone, Dustin; Rudy, Ellen T.

    2015-01-01

    Background In the U.S., HIV infections are increasing among men who have sex with men (MSM), particularly young, racial/ethnic minority MSM. Objective To examine the feasibility of increasing HIV testing among young Latino MSM by integrating tailored outreach strategies with testing, counseling, and HIV medical services. Design Descriptive study comparing demographic characteristics, behaviors, and HIV test results of clients from the intervention period with clients who tested during other time periods. Results Clients in the intervention period were younger and more likely to be Latino than those in other time periods. In addition, clients who received outreach were more likely than those who did not receive outreach to report methamphetamine use, sex with an HIV-positive person, and sex with a sex worker. Conclusion Venue-based and selective media outreach, in combination with linking rapid testing to HIV care, may help overcome some of the barriers to testing among high-risk young Latino MSM. PMID:19648703

  2. Epstein-Barr and cytomegalovirus DNA salivary shedding correlate with long-term plasma HIV RNA detection in HIV-infected men who have sex with men.

    PubMed

    Scaggiante, Renzo; Andreis, Samantha; Basso, Monica; Franchin, Elisa; Franzetti, Marzia; Del Vecchio, Claudia; Torti, Carlo; Mengoli, Carlo; Cruciani, Mario; Sarmati, Loredana; Palù, Giorgio; Parisi, Saverio Giuseppe

    2016-07-01

    The aim of the study was to evaluate cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNA salivary shedding in HIV-positive men who have sex with men (MSM) and to determine whether viro-immunological parameters and long-term (24 months) plasma HIV RNA (pHIV) detection may predict herpesviruses replication. A total of 193 HIV-positive MSM were consecutively recruited (mean CD4+ cell count 607 cells/mm(3) and mean nadir value 333 cells/mm(3) ); pHIV was analyzed for 24 months prior to saliva sampling: patients were categorized as successfully suppressed (SS) and not suppressed (NS). The EBV viral load was categorized as high viral load (HVL), intermediate (IVL), or low (LVL), CMV DNA as positive or negative. NS patients experienced both herpesviruses detectability more frequently respect to SS patients (P = 0.034); conversely, no salivary shedding was more frequent in SS patients (P = 0.014). HVL EBV was more frequent in NS patients than in SS subjects (P = 0.038 for isolated EBV detection and P = 0.001 when CMV shedding was associated). NS subjects with HVL EBV had a median pHIV of 43,820 copies/ml, significantly higher respect to IVL and LVL patients (P = 0.027 and P = 0.0005, respectively). CMV shedding was mostly associated to EBV shedding. NS patients showed a significantly higher frequency of saliva HVL EBV detection compared to SS patients; moreover, NS patients with HVL EBV had a higher pHIV respect to those with IVL and LVL shedding. Our results suggest that a successful pHIV suppression could reduce the burden of salivary EBV replication and likely the risk of herpesviruses-related cancers. PMID:26636290

  3. Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

    PubMed Central

    Taylor, N R; Kaplan, B E; Swiderski, P; Li, H; Rossi, J J

    1992-01-01

    Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents. Images PMID:1408757

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

    PubMed Central

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

    2016-01-01

    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. PMID:27358023

  5. 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

    2016-01-01

    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. PMID:27358023

  6. 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. PMID:27295571

  7. An emergency department registration kiosk can increase HIV screening in high risk patients.

    PubMed

    Hsieh, Yu-Hsiang; Gauvey-Kern, Megan; Peterson, Stephen; Woodfield, Alonzo; Deruggiero, Katherine; Gaydos, Charlotte A; Rothman, Richard E

    2014-12-01

    We evaluated the feasibility and the patient acceptability of integrating a kiosk into routine emergency department (ED) practice for offering HIV testing. The work was conducted in four phases: phase 1 was a baseline, in which external testing staff offered testing at the bedside; phase 2 was a pilot assessment of a prototype kiosk; phase 3 was a pilot implementation and phase 4 was the full implementation with automated login. Feasibility was assessed by the proportion of offering HIV tests, acceptance, completion and result reporting. During the study period, the number of ED patients and eligible patients for screening were similar in the three main phases. However, the number and proportion of patients offered testing of those eligible for screening increased significantly from phase 1 (32%) to phase 3 (37%) and phase 4 (40%). There were slightly higher prevalences of newly diagnosed HIV with kiosk versus bedside testing (phase 1, 0%; phase 3, 0.2%; phase 4, 0.5%). Compared to patients tested at the bedside, patients tested via the kiosk were significantly younger, more likely to be female, to be black, and to report high risk behaviours. ED-based HIV screening via a registration-based kiosk was feasible, yielded similar proportions of testing, and increased the proportion of engagement of higher-risk patients in testing. PMID:25316041

  8. Increased sensitivity of HIV-1 p24 ELISA using a photochemical signal amplification system

    PubMed Central

    Bystryak, Simon; Santockyte, Rasa

    2016-01-01

    In this study we describe a photochemical signal amplification method (PSAM) for increasing of the sensitivity of enzyme-linked immunosorbent assay (ELISA) for determination of HIV-1 p24 antigen. This method can be used for both commercially available and in-house ELISA tests, and has the advantage of being considerably simpler and less costly than alternative signal amplification methods. The photochemical signal amplification method is based on an autocatalytic photochemical reaction of a horseradish peroxidase (HRP) substrate, orthophenylenediamine (OPD). To compare the performance of PSAM-boosted ELISA with a conventional colorimetric ELISA for determination of HIV-1 p24 antigen we employed a PerkinElmer HIV-1 p24 ELISA kit, using conventional ELISA alongside ELISA + PSAM. In the present study, we show that PSAM technology allows one to increase the analytical sensitivity and dynamic range of a commercial HIV-1 p24 ELISA kit, with and without immune-complex disruption (ICD and Non-ICD ELISA), by a factor of approximately 40-fold. ELISA + PSAM is compatible with commercially available microtiter plate readers, requires only an inexpensive illumination device, and the PSAM amplification step takes no longer than 15 min. PMID:26090753

  9. 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.

  10. Pandemic Influenza A (H1N1) Virus Infection Increases Apoptosis and HIV-1 Replication in HIV-1 Infected Jurkat Cells

    PubMed Central

    Wang, Xue; Tan, Jiying; Biswas, Santanu; Zhao, Jiangqin; Devadas, Krishnakumar; Ye, Zhiping; Hewlett, Indira

    2016-01-01

    Influenza virus infection has a significant impact on public health, since it is a major cause of morbidity and mortality. It is not well-known whether influenza virus infection affects cell death and human immunodeficiency virus (HIV)-1 replication in HIV-1-infected patients. Using a lymphoma cell line, Jurkat, we examined the in vitro effects of pandemic influenza A (H1N1) virus (pH1N1) infection on cell death and HIV-1 RNA production in infected cells. We found that pH1N1 infection increased apoptotic cell death through Fas and Bax-mediated pathways in HIV-1-infected Jurkat cells. Infection with pH1N1 virus could promote HIV-1 RNA production by activating host transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), nuclear factor of activated T-cells (NFAT) and activator protein 1 (AP-1) through mitogen-activated protein kinases (MAPK) pathways and T-cell antigen receptor (TCR)-related pathways. The replication of HIV-1 latent infection could be reactivated by pH1N1 infection through TCR and apoptotic pathways. These data indicate that HIV-1 replication can be activated by pH1N1 virus in HIV-1-infected cells resulting in induction of cell death through apoptotic pathways. PMID:26848681

  11. The Amagugu Intervention: A Conceptual Framework for Increasing HIV Disclosure and Parent-Led Communication about Health among HIV-Infected Parents with HIV-Uninfected Primary School-Aged Children

    PubMed Central

    Rochat, Tamsen J.; Mitchell, Joanie; Stein, Alan; Mkwanazi, Ntombizodumo Brilliant; Bland, Ruth M.

    2016-01-01

    Advances in access to HIV prevention and treatment have reduced vertical transmission of HIV, with most children born to HIV-infected parents being HIV-uninfected themselves. A major challenge that HIV-infected parents face is disclosure of their HIV status to their predominantly HIV-uninfected children. Their children enter middle childhood and early adolescence facing many challenges associated with parental illness and hospitalization, often exacerbated by stigma and a lack of access to health education and support. Increasingly, evidence suggests that primary school-aged children have the developmental capacity to grasp concepts of health and illness, including HIV, and that in the absence of parent-led communication and education about these issues, HIV-exposed children may be at increased risk of psychological and social problems. The Amagugu intervention is a six-session home-based intervention, delivered by lay counselors, which aims to increase parenting capacity to disclose their HIV status and offer health education to their primary school-aged children. The intervention includes information and activities on disclosure, health care engagement, and custody planning. An uncontrolled pre–post-evaluation study with 281 families showed that the intervention was feasible, acceptable, and effective in increasing maternal disclosure. The aim of this paper is to describe the conceptual model of the Amagugu intervention, as developed post-evaluation, showing the proposed pathways of risk that Amagugu aims to disrupt through its intervention targets, mechanisms, and activities; and to present a summary of results from the large-scale evaluation study of Amagugu to demonstrate the acceptability and feasibility of the intervention model. This relatively low-intensity home-based intervention led to: increased HIV disclosure to children, improvements in mental health for mother and child, and improved health care engagement and custody planning for the child. The

  12. Effects of recombinant human growth hormone on HIV-1-specific T-cell responses, thymic output and proviral DNA in patients on HAART: 48-week follow-up

    PubMed Central

    Herasimtschuk, Anna A; Westrop, Samantha J; Moyle, Graeme J; Downey, Jocelyn S; Imami, Nesrina

    2008-01-01

    Background Efficacious immune-based therapy in treated chronic HIV-1 infection requires the induction of virus-specific CD4+ T cells and subsequent maturation and maintenance of specific memory CD8+ T cells. Concomitant daily administration of recombinant human growth hormone (rhGH) with highly active antiretroviral therapy (HAART) was used in chronically infected patients with lipodystrophy in an attempt to reconstitute these virus-specific T-cell responses. Methods Individuals with chronic HIV-1 infection on HAART were enrolled on a randomized, double-blinded, placebo-controlled study to receive rhGH therapy. We assessed HIV-1-specific proliferative CD4+ and interferon-gamma (IFN-γ)-producing CD8+ T-cell responses, quantified thymic output and proviral HIV-1 DNA at the following time points: baseline; after 12 weeks of rhGH therapy; at 24 weeks, after randomization into three groups [placebo weeks 12–24 (Group A), alternate-day dosing weeks 12–24 (Group B), and twice-per-week dosing weeks 12–24 (Group C)]; and at 48 weeks after all patients had received HAART alone for the final 24 weeks. Results We found significant increases in both proliferative CD4+ and IFN-γ-producing CD8+ HIV-1-specific T-cell responses after daily administration of rhGH. This increase was focused on HIV-1 Gag-specific T-cell responses. Following subsequent randomisation into different dosing regimens, HIV-1-specific proliferative CD4+ T-cell responses declined in patients receiving less frequent dosing of rhGH, while virus-specific IFN-γ-producing CD8+ T-cell responses were maintained for longer periods of time. There was no significant change in thymic output and the cell-associated HIV-1 DNA remained stable in most patients. An increased anti-HIV-1 Nef-specific CD4+ T-cell proliferative response was correlated to a decrease in proviral load, and an increased HIV-1 Gag-specific IFN-γ-producing CD8+ T-cell response correlated with an increase in proviral load. Conclusion The

  13. Vitamin Supplementation Increases Risk of Subclinical Mastitis in HIV-Infected Women123

    PubMed Central

    Arsenault, Joanne E.; Aboud, Said; Manji, Karim P.; Fawzi, Wafaie W.; Villamor, Eduardo

    2010-01-01

    Subclinical mastitis is common in HIV-infected women and is a risk factor for mother-to-child transmission of HIV. The purpose of this study was to examine the effect of vitamin supplementation [vitamin A + β-carotene, multivitamins (B complex, C, and E), or multivitamins, including vitamin A + β-carotene] on the risk of subclinical mastitis during the first 2 y postpartum among HIV-infected women. The study was a randomized, placebo-controlled, clinical trial including 674 HIV-infected, antiretroviral naïve Tanzanian women who were recruited during pregnancy and followed-up after delivery. Breast milk samples were obtained approximately every 3 mo. Any subclinical mastitis was defined as a ratio of the sodium to potassium (Na:K) breast milk concentrations > 0.6 and further classified as either moderate (Na:K ≥ 0.6 and ≤ 1) or severe (Na:K > 1.0). Fifty-eight percent of women had at least 1 episode of any subclinical mastitis. Women assigned to multivitamins (B complex, C, and E) had a 33% greater risk of any subclinical mastitis (P = 0.005) and a 75% greater risk of severe subclinical mastitis (P = 0.0006) than women who received the placebo. Vitamin A + β-carotene also increased the risk of severe subclinical mastitis by 45% (P = 0.03). Among women with CD4+ T-cell counts ≥ 350 cells/μL, multivitamin intake resulted in a 49% increased risk of any subclinical mastitis (P = 0.006); by contrast, there were no treatment effects among women with CD4+ T-cell counts < 350 cells/μL (P- interaction for treatment × CD4+ T-cell count = 0.10). Supplementation of HIV-infected women with vitamins increased the risk of subclinical mastitis. PMID:20739447

  14. Structural determinants of HIV-1 Vif susceptibility and DNA binding in APOBEC3F

    PubMed Central

    Siu, Karen K.; Sultana, Azmiri; Azimi, Farshad C.; Lee, Jeffrey E.

    2016-01-01

    The human APOBEC3 family of DNA cytosine deaminases serves as a front-line intrinsic immune response to inhibit the replication of diverse retroviruses. APOBEC3F and APOBEC3G are the most potent factors against HIV-1. As a countermeasure, HIV-1 viral infectivity factor (Vif) targets APOBEC3s for proteasomal degradation. Here, we report the crystal structure of the Vif-binding domain in APOBEC3F and a novel assay to assess Vif-APOBEC3 binding. Our results point to an amphipathic surface that is conserved in APOBEC3s as critical for Vif susceptibility in APOBEC3F. Electrostatic interactions likely mediate Vif binding. Moreover, structure-guided mutagenesis reveals a straight ssDNA-binding groove distinct from the Vif-binding site, and a novel ‘aromatic switch’ is proposed to explain DNA substrate specificities across the APOBEC3 family. This study opens new lines of inquiry that will further our understanding of APOBEC3-mediated retroviral restriction and provides an accurate template for structure-guided development of inhibitors targeting the APOBEC3-Vif axis. PMID:24185281

  15. T Cells Detect Intracellular DNA but Fail to Induce Type I IFN Responses: Implications for Restriction of HIV Replication

    PubMed Central

    Kofod-Olsen, Emil; Holm, Christian K.; Melchjorsen, Jesper; Jensen, David G.; Hansen, Anne Louise; Jørgensen, Louise B.; Ostergaard, Lars; Tolstrup, Martin; Larsen, Carsten S.; Paludan, Søren R.; Jakobsen, Martin R.; Mogensen, Trine H.

    2014-01-01

    HIV infects key cell types of the immune system, most notably macrophages and CD4+ T cells. Whereas macrophages represent an important viral reservoir, activated CD4+ T cells are the most permissive cell types supporting high levels of viral replication. In recent years, it has been appreciated that the innate immune system plays an important role in controlling HIV replication, e.g. via interferon (IFN)-inducible restriction factors. Moreover, innate immune responses are involved in driving chronic immune activation and the pathogenesis of progressive immunodeficiency. Several pattern recognition receptors detecting HIV have been reported, including Toll-like receptor 7 and Retinoic-inducible gene-I, which detects viral RNA. Here we report that human primary T cells fail to induce strong IFN responses, despite the fact that this cell type does express key molecules involved in DNA signaling pathways. We demonstrate that the DNA sensor IFI16 migrates to sites of foreign DNA localization in the cytoplasm and recruits the signaling molecules stimulator of IFN genes and Tank-binding kinase, but this does not result in expression of IFN and IFN-stimulated genes. Importantly, we show that cytosolic DNA fails to affect HIV replication. However, exogenous treatment of activated T cells with type I IFN has the capacity to induce expression of IFN-stimulated genes and suppress HIV replication. Our data suggest the existence of an impaired DNA signaling machinery in T cells, which may prevent this cell type from activating cell-autonomous anti-HIV responses. This phenomenon could contribute to the high permissiveness of CD4+ T cells for HIV-1. PMID:24404168

  16. Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes.

    PubMed

    Richardson, C D; Ray, G J; Bray, N L; Corn, J E

    2016-01-01

    The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9-sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous gene disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced gene disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption. PMID:27530320

  17. Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes

    PubMed Central

    Richardson, C. D.; Ray, G. J.; Bray, N. L.; Corn, J. E.

    2016-01-01

    The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9–sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous gene disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced gene disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption. PMID:27530320

  18. Increased utilisation of PEPFAR-supported laboratory services by non-HIV patents in Tanzania

    PubMed Central

    McNairy, Margaret L.; Gwynn, Charon; Rabkin, Miriam; Antelman, Gretchen; Wu, Yingfeng; Alemayehu, Bereket; Lim, Travis; Imtiaz, Rubina; Mosha, Fausta; Mwasekaga, Michael; Othman, Asha A.; Justman, Jessica

    2016-01-01

    Background It is unknown to what extent the non-HIV population utilises laboratories supported by the President's Emergency Plan for AIDS Relief (PEPFAR). Objectives We aimed to describe the number and proportion of laboratory tests performed in 2009 and 2011 for patients referred from HIV and non-HIV services (NHSs) in a convenience sample collected from 127 laboratories supported by PEPFAR in Tanzania. We then compared changes in the proportions of tests performed for patients referred from NHSs in 2009 vs 2011. Methods Haematology, chemistry, tuberculosis and syphilis test data were collected from available laboratory registers. Referral sources, including HIV services, NHSs, or lack of a documented referral source, were recorded. A generalised linear mixed model reported the odds that a test was from a NHS. Results A total of 94 132 tests from 94 laboratories in 2009 and 157 343 tests from 101 laboratories in 2011 were recorded. Half of all tests lacked a documented referral source. Tests from NHSs constituted 42% (66 084) of all tests in 2011, compared with 31% (29 181) in 2009. A test in 2011 was twice as likely to have been referred from a NHS as in 2009 (adjusted odds ratio: 2.0 [95% confidence interval: 2.0–2.1]). Conclusion Between 2009 and 2011, the number and proportion of tests from NHSs increased across all types of test. This finding may reflect increased documentation of NHS referrals or that the laboratory scale-up originally intended to service the HIV-positive population in Tanzania may be associated with a ‘spillover effect’ amongst the general population. PMID:26962475

  19. Curriculum Development to Increase Minority Research Literacy for HIV Prevention Research: A CBPR Approach

    PubMed Central

    Isler, Malika Roman; Brown, Andre L.; Eley, Natalie; Mathews, Allison; Batten, Kendra; Rogers, Randy; Powell, Noah; White, Caressa; Underwood, Rosalee; MacQueen, Kathleen M.

    2015-01-01

    Background Minority engagement in HIV prevention research can improve the process and products of research. Using community-based participatory research (CBPR) to develop capacity-building tools can promote community awareness of HIV prevention, clinical research, and community roles in research. Objectives We sought to describe a CBPR approach to curriculum development to increase HIV prevention research literacy among Blacks ages 18 to 30. Methods Community members and researchers documented the iterative and participatory nature of curriculum development and lessons learned. Results/Lessons Learned We used specific strategies to support and verify multi-stakeholder engagement, team building, capacity building, and shared decision making. Objective or formal assessments of baseline capacity, ongoing stakeholder engagement, and reinforcing the value of multiple perspectives can promote further equity in curriculum development between researchers and community members. Conclusions The iterative process of shared discussion, development, and consensus building strengthened collaboration between stakeholder groups and produced a stronger, more culturally appropriate curriculum to promote HIV prevention research engagement among young Blacks. PMID:25727984

  20. Increasing the acceptability of HIV counseling and testing with three C's: convenience, confidentiality and credibility.

    PubMed

    Angotti, Nicole; Bula, Agatha; Gaydosh, Lauren; Kimchi, Eitan Zeev; Thornton, Rebecca L; Yeatman, Sara E

    2009-06-01

    Agencies engaged in humanitarian efforts to prevent the further spread of HIV have emphasized the importance of voluntary counseling and testing (VCT), and most high-prevalence countries now have facilities that offer testing free of charge. The utilization of these services is disappointingly low, however, despite high numbers reporting that they would like to be tested. Explanations of this discrepancy typically rely on responses to hypothetical questions posed in terms of psychological or social barriers; often, the explanation is that people fear learning that they are infected with a disease that they understand to be fatal and stigmatizing. Yet when we offered door-to-door rapid blood testing for HIV as part of a longitudinal study in rural Malawi, the overwhelming majority agreed to be tested and to receive their results immediately. Thus, in this paper, we ask: why are more people not getting tested? Using an explanatory research design, we find that rural Malawians are responsive to door-to-door HIV testing for the following reasons: it is convenient, confidential, and the rapid blood test is credible. Our study suggests that attention to these factors in VCT strategies may mitigate the fear of HIV testing, and ultimately increase uptake in rural African settings. PMID:19375208

  1. Syringe Sharing and HIV Incidence Among Injection Drug Users and Increased Access to Sterile Syringes

    PubMed Central

    Small, Will; Buchner, Chris; Zhang, Ruth; Li, Kathy; Montaner, Julio; Wood, Evan

    2010-01-01

    Objectives. We assessed the effects of syringe exchange program (SEP) policy on rates of HIV risk behavior and HIV incidence among injection drug users. Methods. Using a multivariate generalized estimating equation and Cox regression methods, we examined syringe borrowing, syringe lending, and HIV incidence among a prospective cohort of 1228 injection drug users in Vancouver, British Columbia. Results. We observed substantial declines in rates of syringe borrowing (from 20.1% in 1998 to 9.2% in 2003) and syringe lending (from 19.1% in 1998 to 6.8% in 2003) following SEP policy change. These declines coincided with a statistically significant increase in the proportion of participants accessing sterile syringes from nontraditional SEP sources (P < .001). In multivariate analyses, the period following the change in SEP policy was independently associated with a greater than 40% reduction in syringe borrowing (adjusted odds ratio [AOR] = 0.57; 95% confidence interval [CI] = 0.49, 0.65) and lending (AOR = 0.52; 95% CI = 0.45, 0.60), as well as declining HIV incidence (adjusted hazard ratio = 0.13; 95% CI = 0.06, 0.31). Conclusions. Widespread syringe distribution appears to be a more effective SEP policy than do more restrictive SEP policies that limit syringe access. Efforts should be made to ensure that SEP policies and program design serve to maximize rather than hinder syringe access. PMID:20558797

  2. Increased mortality associated with treated active tuberculosis in HIV-infected adults in Tanzania.

    PubMed

    Kabali, Conrad; Mtei, Lillian; Brooks, Daniel R; Waddell, Richard; Bakari, Muhammad; Matee, Mecky; Arbeit, Robert D; Pallangyo, Kisali; von Reyn, C Fordham; Horsburgh, C Robert

    2013-07-01

    Active tuberculosis (TB) among HIV-infected patients, even when successfully treated, may be associated with excess mortality. We conducted a prospective cohort study nested in a randomized TB vaccine trial to compare mortality between HIV-infected patients diagnosed and treated for TB (TB, n = 77) and HIV-infected patients within the same CD4 range, who were not diagnosed with or treated for active TB (non-TB, n = 308) in the period 2001-2008. Only twenty four subjects (6%) were on antiretroviral therapy at the beginning of this study. After accounting for covariate effects including use of antiretroviral therapy, isoniazid preventive therapy, and receipt of vaccine, we found a four-fold increase in mortality in TB patients compared with non-TB patients (adjusted Hazard Ratio 4.61; 95% Confidence Interval (CI): 1.63, 13.05). These findings suggest that treatment for TB alone is not sufficient to avert the excess mortality associated with HIV-related TB and that prevention of TB may provide a mortality benefit. PMID:23523641

  3. Increased mortality associated with treated active tuberculosis in HIV-infected adults in Tanzania

    PubMed Central

    Kabali, Conrad; Mtei, Lillian; Brooks, Daniel R.; Waddell, Richard; Bakari, Muhammad; Matee, Mecky; Arbeit, Robert D.; Pallangyo, Kisali; von Reyn, C. Fordham; Horsburgh, C. Robert

    2013-01-01

    SUMMARY Active tuberculosis (TB) among HIV-infected patients, even when successfully treated, may be associated with excess mortality. We conducted a prospective cohort study nested in a randomized TB vaccine trial to compare mortality between HIV-infected patients diagnosed and treated for TB (TB, n=77) and HIV-infected patients within the same CD4 range, who were not diagnosed with or treated for active TB (non-TB, n=308) in the period 2001–2008. Only twenty four subjects (6%) were on antiretroviral therapy at the beginning of this study. After accounting for covariate effects including use of antiretroviral therapy, isoniazid preventive therapy, and receipt of vaccine, we found a four-fold increase in mortality in TB patients compared with non-TB patients (adjusted Hazard Ratio 4.61; 95% Confidence Interval (CI): 1.63, 13.05). These findings suggest that treatment for TB alone is not sufficient to avert the excess mortality associated with HIV-related TB and that prevention of TB may provide a mortality benefit. PMID:23523641

  4. 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.

  5. 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.

  6. Gene polymorphisms and increased DNA damage in morbidly obese women.

    PubMed

    Luperini, B C O; Almeida, D C; Porto, M P; Marcondes, J P C; Prado, R P; Rasera, I; Oliveira, M R M; Salvadori, D M F

    2015-06-01

    Obesity is characterized by increased adipose tissue mass resulting from a chronic imbalance between energy intake and expenditure. Furthermore, there is a clearly defined relationship among fat mass expansion, chronic low-grade systemic inflammation and reactive oxygen species (ROS) generation; leading to ROS-related pathological events. In the past years, genome-wide association studies have generated convincing evidence associating genetic variation at multiple regions of the genome with traits that reflect obesity. Therefore, the present study aimed to evaluate the relationships among the gene polymorphisms ghrelin (GHRL-rs26802), ghrelin receptor (GHSR-rs572169), leptin (LEP-rs7799039), leptin receptor (LEPR-rs1137101) and fat mass and obesity-associated (FTO-rs9939609) and obesity. The relationships among these gene variants and the amount of DNA damage were also investigated. Three hundred Caucasian morbidly obese and 300 eutrophic (controls) women were recruited. In summary, the results demonstrated that the frequencies of the GHRL, GHSR, LEP and LEPR polymorphisms were not different between Brazilian white morbidly obese and eutrophic women. Exceptions were the AA-FTO genotype and allele A, which were significantly more frequent in obese women than in the controls (0.23% vs. 0.10%; 0.46 vs. 0.36, respectively), and the TT-FTO genotype and the T allele, which were less frequent in morbidly obese women (p<0.01). Furthermore, significant differences in the amount of genetic lesions associated with FTO variants were observed only in obese women. In conclusion, this study demonstrated that the analyzed SNPs were not closely associated with morbid obesity, suggesting they are not the major contributors to obesity. Therefore, our data indicated that these gene variants are not good biomarkers for predicting risk susceptibility for obesity, whereas ROS generated by the inflammatory status might be one of the causes of DNA damage in obese women, favoring

  7. Fetal exposure to HIV-1 alters chemokine receptor expression by CD4+T cells and increases susceptibility to HIV-1.

    PubMed

    Bunders, Madeleine J; van Hamme, John L; Jansen, Machiel H; Boer, Kees; Kootstra, Neeltje A; Kuijpers, Taco W

    2014-01-01

    Absolute numbers of lymphocytes are decreased in uninfected infants born to HIV-1-infected women (HIV-1-exposed). Although the exact mechanism is unknown, fetal exposure to maternal HIV-1-infection could prime the immune system and affect T cell trafficking. We compared the expression of chemokine receptors on cord blood CD4(+) T cells from HIV-1-exposed children and healthy controls. At baseline CD4(+) T cells had a largely naïve phenotype. However, stimulation with cytokines resulted in an upregulation of inflammatory response-related chemokine receptors on CD4(+) T cells, with HIV-1-exposed infants having a significantly higher frequency of CD4(+) T cells expressing, in particularly Th2 associated chemokine receptors (CCR3 p < 0.01, CCR8 p = 0.03). Numbers of naive CCR7(+) CD4(+) T cells were reduced (p = 0.01) in HIV-1-exposed infants. We further assessed whether the inflammatory phenotype was associated with susceptibility to HIV-1 and detected higher levels of p24 upon in in vitro infection of stimulated CD4(+) T cells of HIV-1-exposed infants. In summary, fetal exposure to HIV-1 primes the immune system in the infant leading to an enhanced immune activation and altered T cell homing, with potential ramifications regarding T cell responses and the acquisition of HIV-1 as an infant. PMID:25341640

  8. Structural Studies of the HIV-1 Integrase Protein: Compound Screening and Characterization of a DNA-Binding Inhibitor

    PubMed Central

    Hassounah, Said; Mesplède, Thibault; Wainberg, Mark A.

    2015-01-01

    Understanding the HIV integrase protein and mechanisms of resistance to HIV integrase inhibitors is complicated by the lack of a full length HIV integrase crystal structure. Moreover, a lentiviral integrase structure with co-crystallised DNA has not been described. For these reasons, we have developed a structural method that utilizes free software to create quaternary HIV integrase homology models, based partially on available full-length prototype foamy virus integrase structures as well as several structures of truncated HIV integrase. We have tested the utility of these models in screening of small anti-integrase compounds using randomly selected molecules from the ZINC database as well as a well characterized IN:DNA binding inhibitor, FZ41, and a putative IN:DNA binding inhibitor, HDS1. Docking studies showed that the ZINC compounds that had the best binding energies bound at the IN:IN dimer interface and that the FZ41 and HDS1 compounds docked at approximately the same location in integrase, i.e. behind the DNA binding domain, although there is some overlap with the IN:IN dimer interface to which the ZINC compounds bind. Thus, we have revealed two possible locations in integrase that could potentially be targeted by allosteric integrase inhibitors, that are distinct from the binding sites of other allosteric molecules such as LEDGF inhibitors. Virological and biochemical studies confirmed that HDS1 and FZ41 share a similar activity profile and that both can inhibit each of integrase and reverse transcriptase activities. The inhibitory mechanism of HDS1 for HIV integrase seems to be at the DNA binding step and not at either of the strand transfer or 3' processing steps of the integrase reaction. Furthermore, HDS1 does not directly interact with DNA. The modeling and docking methodology described here will be useful for future screening of integrase inhibitors as well as for the generation of models for the study of integrase drug resistance. PMID:26046987

  9. Fetal Cord Blood Mononuclear Cells Collected At Term from HIV-1 Infected Women Harbor Transcriptionally Active Integrated Proviral DNA

    PubMed Central

    ELLIS, Jane E.; HAIR, Greg A.; LINDSAY, Michael K.; ANSARI, Aftab A.; SUNDSTROM, J. Bruce

    2007-01-01

    Objective To determine levels of intrauterine infection and transcriptional activity in cord blood mononuclear cells collected at term from fetuses born to HIV-infected pregnant women on highly active anti-retroviral therapy (HAART). Methods RNA and DNA were isolated from maternal placental tissues and fetal cord blood specimens obtained at term from HIV-infected pregnant women on HAART. Levels of integrated HIV provirus and mRNA transcripts were determined by real-time PCR. Results Detectable levels of transcriptionally active integrated provirus were present in approximately 27% of cord blood samples (n=22) collected from fetuses, born to HIV-positive mothers on HAART. Levels of HIV-p24 antigen in cultures detected in randomly selected cord blood samples confirmed the presence of inducible infectious virus. Conclusions These findings suggest that some fetuses from HIV-infected mothers on HAART who may present as HIV-negative infants postpartum, can harbor circulating leukocytes that are productively infected by intrauterine transmission (IUT) of HIV. PMID:17904964

  10. Lymphocyte protein synthesis is increased with the progression of HIV-associated disease to AIDS.

    PubMed

    Caso, G; Garlick, P J; Gelato, M C; McNurlan, M A

    2001-12-01

    HIV infection has been shown to affect lymphocyte function and to reduce lymphocyte responsiveness in vitro to mitogenic stimulation, but little is known about lymphocyte metabolism in vivo and how it is affected during the course of the disease. This study investigated the metabolic activity of lymphocytes in vivo through the progression of HIV-associated disease. Lymphocyte protein synthesis was measured with L-[(2)H(5)]phenylalanine (45 mg/kg body weight) in healthy volunteers (n=7), in patients who were HIV-positive (n=7) but asymptomatic, and in patients with AIDS (n=8). The rates of lymphocyte protein synthesis [expressed as a percentage of lymphocyte protein, i.e. fractional synthesis rate (FSR)] were not altered in HIV-positive patients compared with healthy controls (7.9+/-1.28% and 9.1+/-0.53%/day respectively), but were significantly elevated in AIDS patients (14.0+/-1.16%/day; P<0.05). The serum concentration of the cytokine tumour necrosis factor-alpha (TNF-alpha) increased with the progression of the disease, and TNF-alpha levels were significantly higher in AIDS patients (6.81+/-0.88 ng/l) than in healthy controls (3.09+/-0.27 ng/l; P<0.05). Lymphocyte protein FSR was positively correlated with serum TNF-alpha concentration (r=0.55, P=0.009) and negatively correlated with CD4(+) lymphocyte count (r=-0.70, P=0.004). The elevation of lymphocyte protein synthesis in AIDS patients suggests a higher rate of turnover of lymphocytes. This may be associated with a generalized activation of the immune system, which is also reflected by the elevated serum TNF-alpha concentration in the late stages of HIV-associated disease. PMID:11724643

  11. Increased lung epithelial permeability in HIV-infected patients with isolated cytotoxic T-lymphocytic alveolitis

    SciTech Connect

    Meignan, M.; Guillon, J.M.; Denis, M.; Joly, P.; Rosso, J.; Carette, M.F.; Baud, L.; Parquin, F.; Plata, F.; Debre, P. )

    1990-05-01

    HIV-related lymphocytic alveolitis is common in HIV-seropositive patients without lung infection or tumor. In some of them a fraction of alveolar lymphocytes are HIV-specific cytotoxic T-lymphocytes (CTL) bearing the CD8 and D44 cell surface markers and capable of killing HIV-infected alveolar macrophages. In order to evaluate the in vivo effect of these CTL on lung function, we measured the pulmonary clearance of aerosolized 99mTc-diethylene triamine penta-acetate (DTPA-CI) on 24 occasions in 22 patients with lymphocytic alveolitis. DTPA-CI has been selected as a highly sensitive test to detect injury of the lung epithelium. In 13 of the patients, we found a high DTPA-CI of 4.56 +/- 2.54%.min-1 (mean +/- SD), suggesting an increase of the epithelial permeability. The lymphocytic alveolitis was then characterized by a high cellularity, a high proportion of lymphocytes (59 +/- 18%), mainly composed of CD8+D44+ T-lymphocytes (149 +/- 109 cells/mm3), which spontaneously exhibited a cytolytic activity against the autologous alveolar macrophages in a standard 51Cr release assay. In the remaining 11 patients, DTPA-CI was normal (less than 1.78%.min-1), lymphocytic alveolitis being characterized by a low number or an absence of CD8+D44+ alveolar lymphocytes (9 +/- 13 cells/mm3) with no significant cytolytic activity. In the whole group, a significant correlation (r = 0.74, p = 0.0004) was found between the DTPA-CI and the number of CD8+D44+ lymphocytes and their cytotoxic activity against alveolar macrophages. Altogether, these results suggest that an injury of the lung epithelium could result from a HIV-specific CTL-induced immunologic conflict.

  12. Increased metallothionein gene expression, zinc, and zinc-dependent resistance to apoptosis in circulating monocytes during HIV viremia

    PubMed Central

    Raymond, Andrea D.; Gekonge, Bethsebah; Giri, Malavika S.; Hancock, Aidan; Papasavvas, Emmanouil; Chehimi, Jihed; Kossevkov, Andrew V.; Nicols, Calen; Yousef, Malik; Mounzer, Karam; Shull, Jane; Kostman, Jay; Showe, Louise; Montaner, Luis J.

    2010-01-01

    Circulating monocytes exhibit an apoptotic resistance phenotype during HIV viremia in association with increased MT expression. MTs are known to play an important role in zinc metabolism and immune function. We now show, in a cross-sectional study using peripheral monocytes, that expression of MT1 isoforms E, G, H, and X is increased significantly in circulating monocyte cells from HIV+ subjects during chronic viremic episodes as compared with uninfected subjects. This increase in expression is also observed during acute viremia following interruption of suppressive ART. Circulating monocytes from HIV+ donors were also found to have elevated zinc importer gene Zip8 expression in conjunction with elevated intracellular zinc levels in contrast to CD4+T-lymphocytes. In vitro HIV-1 infection studies with elutriated MDM confirm a direct relation between HIV-1 infection and increased MDM MT1 (isoform G) gene expression and increased intracellular zinc levels. A direct link between elevated zinc levels and apoptosis resistance was established using a cell-permeable zinc chelator TPEN, which reversed apoptosis resistance effectively in monocytes from HIV-infected to levels comparable with uninfected controls. Taken together, increases in MT gene expression and intracellular zinc levels may contribute directly to maintenance of an immune-activated monocyte by mediating an increased resistance to apoptosis during active HIV-1 viremia. PMID:20551211

  13. Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab.

    PubMed

    Muthumani, Kar; Flingai, Seleeke; Wise, Megan; Tingey, Colleen; Ugen, Kenneth E; Weiner, David B

    2013-10-01

    Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an "enhanced and optimized" DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This "enhanced" DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of "adaptive" in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection, against diverse

  14. Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab

    PubMed Central

    Muthumani, Kar; Flingai, Seleeke; Wise, Megan; Tingey, Colleen; Ugen, Kenneth E; Weiner, David B

    2013-01-01

    Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an “enhanced and optimized” DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This “enhanced” DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of “adaptive” in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection

  15. The Level of DING Proteins Is Increased in HIV-Infected Patients: In Vitro and In Vivo Studies

    PubMed Central

    Djeghader, Ahmed; Aragonès, Gerard; Darbinian, Nune; Elias, Mikael; Gonzalez, Daniel; García-Heredia, Anabel; Beltrán-Debón, Raúl; Kaminski, Rafal; Gotthard, Guillaume; Hiblot, Julien; Rull, Anna; Rohr, Olivier; Schwartz, Christian; Alonso-Villaverde, Carlos; Joven, Jorge; Camps, Jordi; Chabriere, Eric

    2012-01-01

    DING proteins constitute an interesting family, owing to their intriguing and important activities. However, after a decade of research, little is known about these proteins. In humans, at least five different DING proteins have been identified, which were implicated in important biological processes and diseases, including HIV. Indeed, recent data from different research groups have highlighted the anti-HIV activity of some DING representatives. These proteins share the ability to inhibit the transcriptional step of HIV-1, a key step of the viral cycle that is not yet targeted by the current therapies. Since such proteins have been isolated from humans, we undertook a comprehensive study that focuses on the relationship between these proteins and HIV-infection in an infectious context. Hence, we developed a home-made ELISA for the quantification of the concentration of DING proteins in human serum. Using this method, we were able to determine the concentration of DING proteins in healthy and HIV-infected patients. Interestingly, we observed a significant increase of the concentration of DING proteins in non treated and treated HIV-infected patients compared to controls. In addition, cell cultures infected with HIV also show an increased expression of DING proteins, ruling out the possible role of antiretroviral treatment in the increase of the expression of DING proteins. In conclusion, results from this study show that the organism reacts to HIV-infection by an overexpression of DING proteins. PMID:22427948

  16. Vascular endothelial growth factor (VEGF) is increased in serum, but not in cerebrospinal fluid in HIV associated CNS diseases.

    PubMed

    Sporer, B; Koedel, U; Paul, R; Eberle, J; Arendt, G; Pfister, H-W

    2004-02-01

    Vascular endothelial growth factor (VEGF) is a potent angiogenic and mitogenic peptide, which also induces several mediators that may play a role in HIV induced CNS damage. VEGF levels were determined in cerebrospinal fluid (CSF) and serum samples from patients with (n = 8) and without (n = 19) directly HIV associated CNS disorders and HIV negative control patients (n = 18). VEGF serum but not CSF levels were significantly increased in HIV infected patients with (381.1 (78.9) pg/ml) HIV associated CNS diseases compared with those without (120.8 (13.1) pg/ml) and HIV negative control patients (133.1(14.8) pg/ml). Serum samples from patients with untreated HIV associated encephalopathy (HIVE, n = 3) contained the highest VEGF levels (583.9 (71.5) pg/ml). In two patients VEGF serum levels were reduced during antiretroviral therapy. However, regardless of effective viral suppression, patients with HIVE still had higher levels compared with HIV infected patients without HIVE. A relevant increase of serum VEGF was not observed in patients without HIVE though high HI viral load. We conclude that HIVE is associated with increased serum VEGF levels. Further studies are warranted to elucidate the role of VEGF in HIVE. PMID:14742610

  17. Immune Activation Is Associated With Increased Gut Microbial Translocation in Treatment-Naive, HIV-Infected Children in a Resource-Limited Setting

    PubMed Central

    Pilakka-Kanthikeel, Sudheesh; Kris, Arheart; Selvaraj, Anbalagan; Swaminathan, Soumya; Pahwa, Savita

    2015-01-01

    Background Gut damage resulting in microbial translocation (MT) is considered a major cause of immune activation (IA) in HIV infection, but data in children are limited, particularly in the absence of antiretroviral therapy. Methods Sixty perinatally HIV-infected, antiretroviral therapy–naive children, aged 2–12 years, were evaluated for plasma levels of lipopolysaccharide, DNA sequences encoding bacterial ribosomal 16 second (16S) RNA (16S rDNA) and soluble CD14 concurrently with markers of CD4 and CD8 T-cell IA and immune exhaustion (IE), CD4 counts, and plasma viral load. At study entry, participants were classified into immune categories (ICs): IC1 (CD4% > 25), IC2 (CD4% 15–25), and IC3 (CD4% < 15). Age-matched HIV-uninfected children served as controls. Data were evaluated at study entry and at 12 months. Results Levels of MT, IA, and IE were increased in patients as compared with controls, were highest in patients in IC3 group, and did not change over 12 months. MT products lipopolysaccharide and 16S rDNA correlated with each other and each correlated with plasma viral load, soluble CD14, and T-cell IA and IE. There was a correlation of IA with IE. CD4 counts and percentage were inversely correlated with MT products and underlying CD4 activation. Conclusions In a natural history cohort of HIV-infected children not on therapy, MT was more pronounced in the most severely immunocompromised patients and was associated with IA. Strategies to reduce MT may help to reduce IA and prevent CD4 depletion. PMID:24378729

  18. HIV-1 Integrase Strand Transfer Inhibitors Stabilize an Integrase-Single Blunt-Ended DNA Complex

    PubMed Central

    Bera, Sibes; Pandey, Krishan K.; Vora, Ajaykumar C.; Grandgenett, Duane P.

    2011-01-01

    Summary Integration of HIV (human immunodeficiency virus) cDNA ends by integrase (IN) into host chromosomes involves a concerted integration mechanism. IN juxtaposes two DNA blunt-ends to form the synaptic complex (SC) which is the intermediate in the concerted integration pathway. SC is inactivated by strand transfer inhibitors (STI) with IC50 values of ~20 nM for inhibition of concerted integration. We detected a new nucleoprotein complex on native agarose that was produced in the presence of STI >200 nM, termed IN-single DNA (ISD) complex. Two IN dimers appear to bind in a parallel fashion at the DNA terminus producing a ~32 bp DNaseI protective footprint. In the presence of Raltegravir, MK-2048 and L-841,411, IN incorporated ~20 to 25% of the input blunt-ended DNA substrate into the stabilized ISD complex. Seven other STI also produced the ISD complex (≤ 5% of input DNA). The formation of the ISD complex was not dependent upon 3’ OH processing and the DNA was predominately blunt-ended in the complex. Raltegravir-resistant IN mutant N155H weakly form the ISD complex in the presence of Raltegravir at ~25% level of wild type IN. In contrast, MK-2048 and L-841,411 produced ~3 to 5-fold more ISD than Raltegravir with N155H IN, which is susceptible to these two inhibitors. The results suggest STI are slow binding inhibitors and the potency to form and stabilize the ISD complex is not always related to inhibition of concerted integration. Rather, the apparent binding and dissociation properties of each STI influenced the production of the ISD complex. PMID:21295584

  19. Contributions of an intensive HIV prevention programme in increasing HIV testing among men who have sex with men in Andhra Pradesh, India.

    PubMed

    Ramesh, Sowmya; Mehrotra, Purnima; Saggurti, Niranjan

    2015-01-01

    The objective of this study was to identify the factors associated with uptake of HIV testing and to assess their relative contributions in increasing HIV testing. Data are drawn from two rounds of cross-sectional Integrated Behavioural and Biological Assessment (IBBA) surveys of self-identified men who have sex with men (MSM) from Andhra Pradesh, India, recruited through probability-based sampling in 2005-2006 and 2009-2010 (IBBA1, n = 1621; IBBA2, n = 1608, respectively). Logistic regression model was used to assess the relationship between socio-demographic characteristics, sexual behaviours, programme exposure and HIV testing. Significant factors were further parsed using decomposition analysis to examine the contribution of different components of that factor towards the change in HIV testing. There was a significant increase in the proportion of MSM reporting HIV testing from IBBA1 to IBBA2. Higher literacy levels, being 25-34 years old, being a kothi (predominantly receptive), engaging in both commercial and non-commercial sexual relationships and intervention programme exposure contributed the most to the increase in HIV testing. PMID:25635532

  20. Induction of human immunodeficiency virus type-1-specific immunity with a novel gene transport unit (GTU)-MultiHIV DNA vaccine.

    PubMed

    Blazevic, Vesna; Männik, Andres; Malm, Maria; Sikut, Rein; Valtavaara, Minna; Toots, Urve; Ustav, Mart; Krohn, Kai

    2006-07-01

    A multiHIV fusion gene expressing an antigenic fusion protein composed of regulatory HIV-1 proteins Rev, Nef, and Tat, as well as Gag p17/p24 and a stretch of 11 cytotoxic T lymphocyte (CTL) epitope clusters from Pol and Env, was cloned into a novel DNA vector named the Gene Transport Unit (GTU). A mouse H-2(d)-restricted HIV-1 gp120 epitope (RGPGRAFVTI) was cloned into the fusion gene as well. In addition to the HIV- 1 genes the GTU codes for a nuclear anchoring protein (bovine papilloma virus E2), ensuring the long maintenance of the vector and a high expression level of the selected immunogens. BALB/c mice were immunized with the GTU-MultiHIV DNA construct by different routes and regimens of immunization to assess the immunogenicity of the DNA vaccine in vivo. Mice developed strong CD8(+) CTL responses to HIV-1 Env and Gag measured by an ELISPOT-IFN-gamma assay and chromium release assay. In addition, T cell responses to regulatory proteins Rev, Nef, and Tat were induced. Antibody responses were detected to each of the HIV antigens encoded by the DNA construct. Minimal doses of the GTU-MultiHIV DNA delivered by gene gun were potent in inducing significant HIV-specific CTL responses. The equivalent doses of the conventional plasmid expressing MultiHIV DNA delivered by gene gun failed to do so. An ideal DNA vaccine should yield high expression of the viral antigens for a prolonged period of time, and expression of the multiple viral antigens is probably required for the induction of a broad and protective immune response. The GTU-MultiHIV DNA vaccine described is a good vaccine candidate that meets the above criteria. PMID:16831091

  1. Structural Factors That Increase HIV/STI Vulnerability Among Indigenous People in the Peruvian Amazon

    PubMed Central

    Orellana, E. Roberto; Alva, Isaac E.; Cárcamo, Cesar P.; García, Patricia J.

    2015-01-01

    We examined structural factors—social, political, economic, and environmental—that increase vulnerability to HIV among indigenous people in the Peruvian Amazon. Indigenous adults belonging to 12 different ethnic groups were purposively recruited in four Amazonian river ports and 16 indigenous villages. Qualitative data revealed a complex set of structural factors that give rise to environments of risk where health is constantly challenged. Ferryboats that cross Amazonian rivers are settings where unprotected sex—including transactional sex between passengers and boat crew and commercial sex work—often take place. Population mobility and mixing also occurs in settings like the river docks, mining sites, and other resource extraction camps, where heavy drinking and unprotected sex work are common. Multilevel, combination prevention strategies that integrate empirically based interventions with indigenous knowledge are urgently needed, not only to reduce vulnerability to HIV transmission, but also to eliminate the structural determinants of indigenous people’s health. PMID:23925407

  2. Ethnicity and Smoking-Associated DNA Methylation Changes at HIV Co-Receptor GPR15

    PubMed Central

    Dogan, Meeshanthini V.; Xiang, Jinhua; Beach, Steven R. H.; Cutrona, Carolyn; Gibbons, Frederick X.; Simons, Ronald L.; Brody, Gene H.; Stapleton, Jack T.; Philibert, Robert A.

    2015-01-01

    Smoking is associated with poorer health outcomes for both African and European Americans. In order to better understand whether ethnic-specific genetic variation may underlie some of these differences, we compared the smoking-associated genome-wide methylation signatures of African Americans with those of European Americans, and followed up this analysis with a focused examination of the most ethnically divergent locus, cg19859270, at the GPR15 gene. We examined the association of methylation at this locus to the rs2230344 SNP and GPR15 gene and protein expression. Consistent with prior analyses, AHRR residue cg05575921 was the most differentially methylated residue in both African Americans and European Americans. However, the second most differentially methylated locus in African Americans, cg19859270, was only modestly differentially methylated in European Americans. Interrogation of the methylation status of this CpG residue found in GPR15, a chemokine receptor involved in HIV pathogenesis, showed a significant interaction of ethnicity with smoking as well as a marginal effect of genotype at rs2230344, a neighboring non-synonymous SNP, but only among African Americans. Gene and protein expression analyses showed that demethylation at cg19859270 was associated with an increase in both mRNA and protein levels. Since GPR15 is involved in the early stages of viral replication for some HIV-1 and HIV-2 isolates, and the prevalence of HIV is increased in African Americans and smokers, these data support a possible role for GPR15 in the ethnically dependent differential prevalence of HIV. PMID:26441693

  3. Why Are the Benefits of Increased Resources Not Impacting the Risk of HIV Infection for High SES Women in Cameroon?

    PubMed Central

    Mumah, Joyce N.; Jackson-Smith, Douglas

    2014-01-01

    Background Despite evidence of a positive SES-HIV gradient in some SSA countries, researchers and policy-makers frequently assume that a range of protective interventions – increasing awareness of mechanisms of HIV transmission, techniques for prevention, greater access to health care facilities, and greater availability of condoms – will reduce the likelihood of contracting HIV, even among higher SES populations. We therefore explore the relationships between SES and these intervening behaviors to illuminate the complex factors that link SES and HIV among women in Cameroon. Methods We use bivariate and multivariate statistical analysis to examine patterns among the 5, 155 women aged 15–49 who participated in the 2004 CDHS. Results The results show a strong pattern where higher SES women have greater access to and use of health care facilities, higher levels of condom use, more HIV knowledge, and command higher power within their relationships, yet also have higher rates of HIV. These traditionally protective factors appear to be offset by riskier sexual behaviors on the part of women with increased resources, most notably longer years of premarital sexual experience, multiple partners in last 12 months, and sexual encounters outside of relationship. Multivariate analyses suggests net of the effect of other factors, women who command higher decision-making power, have greater access to health care, more negative attitudes toward wife beating, longer years of premarital sexual exposure, and partners with professional/white collar jobs (characteristics associated with rising SES) had higher odds of testing positive for HIV. Conclusion Results show that higher riskier sexual practices on part of high SES women offset benefits that may have accrued from their increased access to resources. The results suggest that traditional approaches to HIV prevention which rely on poverty reduction, improving access to health care, improving HIV knowledge, and boosting women

  4. Increased Sensitivity to Binge Alcohol-Induced Gut Leakiness and Inflammatory Liver Disease in HIV Transgenic Rats

    PubMed Central

    Banerjee, Atrayee; Abdelmegeed, Mohamed A.; Jang, Sehwan; Song, Byoung-Joon

    2015-01-01

    The mechanisms of alcohol-mediated advanced liver injury in HIV-infected individuals are poorly understood. Thus, this study was aimed to investigate the effect of binge alcohol on the inflammatory liver disease in HIV transgenic rats as a model for simulating human conditions. Female wild-type (WT) or HIV transgenic rats were treated with three consecutive doses of binge ethanol (EtOH) (3.5 g/kg/dose oral gavages at 12-h intervals) or dextrose (Control). Blood and liver tissues were collected at 1 or 6-h following the last dose of ethanol or dextrose for the measurements of serum endotoxin and liver pathology, respectively. Compared to the WT, the HIV rats showed increased sensitivity to alcohol-mediated gut leakiness, hepatic steatosis and inflammation, as evidenced with the significantly elevated levels of serum endotoxin, hepatic triglycerides, histological fat accumulation and F4/80 staining. Real-time PCR analysis revealed that hepatic levels of toll-like receptor-4 (TLR4), leptin and the downstream target monocyte chemoattractant protein-1 (MCP-1) were significantly up-regulated in the HIV-EtOH rats, compared to all other groups. Subsequent experiments with primary cultured cells showed that both hepatocytes and hepatic Kupffer cells were the sources of the elevated MCP-1 in HIV-EtOH rats. Further, TLR4 and MCP-1 were found to be upregulated by leptin. Collectively, these results show that HIV rats, similar to HIV-infected people being treated with the highly active anti-retroviral therapy (HAART), are more susceptible to binge alcohol-induced gut leakiness and inflammatory liver disease than the corresponding WT, possibly due to additive or synergistic interaction between binge alcohol exposure and HIV infection. Based on these results, HIV transgenic rats can be used as a surrogate model to study the molecular mechanisms of many disease states caused by heavy alcohol intake in HIV-infected people on HAART. PMID:26484872

  5. The MSHA strain of Pseudomonas aeruginosa activated TLR pathway and enhanced HIV-1 DNA vaccine immunoreactivity.

    PubMed

    Hou, Jue; Liu, Yong; Liu, Ying; Shao, Yiming

    2012-01-01

    The mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa (PA-MSHA) has been shown to trigger naïve immune responses through the activation of monocytes, macrophages, natural killer cells (NK cells) and antigen presenting cells (APCs). Based on the hypothesis that PA-MSHA activates natural immunity through the Toll-like receptor (TLR) pathway, we scanned several critical TLR pathway molecules in mouse splenocytes using high-throughput real-time QRT-PCR and co-stimulatory molecule in bone marrow-derived dendritic cells (BMDCs) following in vitro stimulation by PA-MSHA. PA-MSHA enabled activation of the TLR pathway mediated by NF-κB and JNK signaling in splenocytes, and the co-stimulatory molecule CD86 was up-regulated in BMDCs. We then assessed the adjuvant effect of PA-MSHA for HIV-1 DNA vaccines. In comparison to DNA inoculation alone, co-inoculation with low dosage of PA-MSHA enhanced specific immunoreactivity against HIV-1 Env in both cellular and humoral responses, and promoted antibody avidity maturation. However, high doses of adjuvant resulted in an immunosuppressive effect; a two- or three-inoculation regimen yielded low antibody responses and the two-inoculation regimen exhibited only a slight cellular immunity response. To our knowledge, this is the first report demonstrating the utility of PA-MSHA as an adjuvant to a DNA vaccine. Further research is needed to investigate the exact mechanisms through which PA-MSHA achieves its adjuvant effects on innate immune responses, especially on dendritic cells. PMID:23077664

  6. Increasing access to oral health care for people living with HIV/AIDS in rural Oregon.

    PubMed

    Jones, Jill; Mofidi, Mahyar; Bednarsh, Helene; Gambrell, Alan; Tobias, Carol R

    2012-05-01

    Access to oral health care for people living with HIV/AIDS is a severe problem. This article describes the design and impact of an Innovations in Oral Health Care Initiative program, funded through the Health Resources and Services Administration HIV/AIDS Bureau's Special Projects of National Significance (SPNS) program, that expanded oral health-care services for these individuals in rural Oregon. From April 2007 to August 2010, 473 patients received dental care (exceeding the target goal of 410 patients) and 153 dental hygiene students were trained to deliver oral health care to HIV-positive patients. The proportion of patients receiving oral health care increased from 10% to 65%, while the no-show rate declined from 40% to 10%. Key implementation components were leveraging SPNS funding and services to create an integrated delivery system, collaborations that resulted in improved service delivery systems, using dental hygiene students to deliver oral health care, enhanced care coordination through the services of a dental case manager, and program capacity to adjust to unanticipated needs. PMID:22547878

  7. Incident Major Depressive Episodes increase the severity and risk of apathy in HIV infection

    PubMed Central

    Kamat, Rujvi; Cattie, Jordan E.; Marcotte, Thomas D.; Woods, Steven Paul; Franklin, Donald R.; Corkran, Stephanie H.; Ellis, Ronald J.; Grant, Igor; Heaton, Robert K.

    2015-01-01

    Apathy and depression are inter-related yet separable and prevalent neuropsychiatric disturbances in persons infected with HIV. In the present study of 225 HIV+ persons, we investigated the role of an incident depressive episode in changes in apathy. Participants completed the apathy subscale of the Frontal Systems Behavior Scale during a detailed neuropsychiatric and neuromedical evaluation at visit 1 and again at approximately a 14 month follow-up. The Composite International Diagnostic Interview was used to obtain diagnoses of a new major depressive disorder. At their follow-up visit, participants were classified into four groups depending on their visit 1 elevation in apathy and new major depressive episode (MDE) status. Apathetic participants at baseline with a new MDE (n=23) were at risk for continued, clinically elevated apathy at follow-up, although severity of symptoms did not increase. Of the 144 participants without clinically elevated apathy at visit 1, those who developed a new MDE (n=16) had greater apathy symptomatology at follow-up than those without MDE. These findings suggest that HIV+ individuals, who do not as yet present with elevated apathy, may be at greater risk of elevated psychiatric distress should they experience a new/recurrent depressive episode. Thus, in the context of previous findings, it appears that although apathy and depression are separable constructs, they interact such that a new depressive episode is a risk factor for incident apathy. PMID:25679203

  8. Prevalence and risk factors for oral DNA tumor viruses in HIV-infected youth.

    PubMed

    Kahn, Jessica A; Rudy, Bret J; Xu, Jiahong; Kapogiannis, Bill; Secord, Elizabeth; Gillison, Maura

    2016-11-01

    Human papillomavirus (HPV), Epstein-Barr virus (EBV), and Kaposi sarcoma-associated herpes virus (KSHV) may promote oral cancers, especially among immunosuppressed individuals. The aims of this study were to examine whether demographic characteristics, medical history, sexual behaviors, substance use, CD4+ T-cell count, HIV viral load, and HPV vaccination were associated with HPV, EBV, and KSHV infection and viral load. Multivariable modeling using logistic or linear regression examined associations between independent variables and infection or viral load, respectively. Among 272 HIV-infected 12-24-year-old youth, 19.5% were positive for oral HPV, 88.2% for EBV, and 11.8% for KSHV. In multivariable models, recent marijuana use (OR 1.97, 95%CI 1.02-3.82) and lower CD4+ T-cell count (<350 vs. ≥350 cells/mm(3) : OR 1.92, 95%CI 1.003-3.69) were associated with HPV infection; lifetime tobacco use (estimated coefficient [EC] 1.55, standard error [SE] 0.53, P = 0.0052) with HPV viral load; recent tobacco use (OR 2.90, 95%CI 1.06-7.97), and higher HIV viral load (>400 vs. <400 copies/ml: OR 3.98, 95%CI 1.84-8.74) with EBV infection; Black versus White race (EC 1.18, SE 0.37, P = 0.0023), and lower CD4+ T-cell count (EC 0.70, SE 0.28, P = 0.017) with EBV viral load, male versus female gender (OR 10, 95%CI 1.32-100) with KSHV infection, and younger age at HIV diagnosis (1-14 vs. 18-20 years: EC 0.33, SE 0.16, P = 0.049; 15-17 vs. 18-20 years: EC 0.35, SE 0.13, P = 0.0099) with KSHV viral load. In conclusion, substance use and immunosuppression are associated with oral DNA tumor viruses in HIV-infected youth. J. Med. Virol. 88:1944-1952, 2016. © 2016 Wiley Periodicals, Inc. PMID:27096166

  9. 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

  10. 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

    Gray, Glenda E; 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

    2016-06-01

    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 × 10(9) 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

  11. Increasing polymer diffusivity by increasing the contour length: The surprising effect of YOYO-1 on DNA dynamics

    NASA Astrophysics Data System (ADS)

    Shin, Seunghwan; Dorfman, Kevin; Cheng, Xiang

    2015-03-01

    Double-stranded DNA (dsDNA) labeled with cyanine dyes such as YOYO-1 has been extensively used as a model to study equilibrium and dynamic properties of semiflexible polyelectrolytes. The ability to directly visualize the polymer dynamics is an attractive feature of these experiments, but positively charged cyanine dyes affect the physical properties of dsDNA, distorting the double helix and counterbalancing the intrinsic negative charge of the backbone. A variety of studies have been conducted to reveal the effect of the dye on the contour length and the persistence length of dsDNA. However, fewer efforts have been made to directly quantify the effect of dye on the diffusion behavior of dsDNA. In order to resolve this issue, we measured the in-plane diffusion coefficient of unconfined dsDNA using confocal microscopy. Although there is widespread consensus that intercalation increases the contour length of dsDNA, we find that increasing the dye:base pair ratio for YOYO-1 actually enhances the diffusion of dsDNA. This enhancement is more significant at lower ionic strengths, which implies that the increase in the diffusion coefficient by dye-DNA intercalation is mainly due to a reduction of excluded volume effect resulting from charge neutralization on the backbone.

  12. DNA-dependent protein kinase interacts functionally with the RNA polymerase II complex recruited at the human immunodeficiency virus (HIV) long terminal repeat and plays an important role in HIV gene expression.

    PubMed

    Tyagi, Shilpi; Ochem, Alex; Tyagi, Mudit

    2011-07-01

    DNA-dependent protein kinase (DNA-PK), a nuclear protein kinase that specifically requires association with DNA for its kinase activity, plays important roles in the regulation of different DNA transactions, including transcription, replication and DNA repair, as well as in the maintenance of telomeres. Due to its large size, DNA-PK is also known to facilitate the activities of other factors by providing the docking platform at their site of action. In this study, by running several chromatin immunoprecipitation assays, we demonstrate the parallel distribution of DNA-PK with RNA polymerase II (RNAP II) along the human immunodeficiency virus (HIV) provirus before and after activation with tumour necrosis factor alpha. The association between DNA-PK and RNAP II is also long-lasting, at least for up to 4 h (the duration analysed in this study). Knockdown of endogenous DNA-PK using specific small hairpin RNAs expressed from lentiviral vectors resulted in significant reduction in HIV gene expression and replication, demonstrating the importance of DNA-PK for HIV gene expression. Sequence analysis of the HIV-1 Tat protein revealed three potential target sites for phosphorylation by DNA-PK and, by using kinase assays, we confirmed that Tat is an effective substrate of DNA-PK. Through peptide mapping, we found that two of these three potential phosphorylation sites are recognized and phosphorylated by DNA-PK. Mutational studies on the DNA-PK target sites of Tat further demonstrated the functional significance of the Tat-DNA-PK interaction. Thus, overall our results clearly demonstrate the functional interaction between DNA-PK and RNAP II during HIV transcription. PMID:21450944

  13. Using Social Media to Increase HIV Testing Among Gay and Bisexual Men, Other Men Who Have Sex With Men, and Transgender Persons: Outcomes From a Randomized Community Trial.

    PubMed

    Rhodes, Scott D; McCoy, Thomas P; Tanner, Amanda E; Stowers, Jason; Bachmann, Laura H; Nguyen, Annie L; Ross, Michael W

    2016-06-01

    We tested an intervention designed to increase human immunodeficiency virus (HIV) testing among men who have sex with men and transgender persons within existing and commonly used social media. At follow-up, intervention communities had significantly higher past 12-month HIV testing than the comparison communities. Findings suggest that promoting HIV testing via social media can increase testing. PMID:26980878

  14. Innate Immune Activity Correlates with CD4 T Cell-Associated HIV-1 DNA Decline during Latency-Reversing Treatment with Panobinostat

    PubMed Central

    Olesen, Rikke; Vigano, Selena; Rasmussen, Thomas A.; Søgaard, Ole S.; Ouyang, Zhengyu; Buzon, Maria; Bashirova, Arman; Carrington, Mary; Palmer, Sarah; Brinkmann, Christel R.; Yu, Xu G.; Østergaard, Lars; Tolstrup, Martin

    2015-01-01

    ABSTRACT The pharmaceutical reactivation of dormant HIV-1 proviruses by histone deacetylase inhibitors (HDACi) represents a possible strategy to reduce the reservoir of HIV-1-infected cells in individuals treated with suppressive combination antiretroviral therapy (cART). However, the effects of such latency-reversing agents on the viral reservoir size are likely to be influenced by host immune responses. Here, we analyzed the immune factors associated with changes in proviral HIV-1 DNA levels during treatment with the potent HDACi panobinostat in a human clinical trial involving 15 cART-treated HIV-1-infected patients. We observed that the magnitude, breadth, and cytokine secretion profile of HIV-1-specific CD8 T cell responses were unrelated to changes in HIV-1 DNA levels in CD4 T cells during panobinostat treatment. In contrast, the proportions of CD3− CD56+ total NK cells and CD16+ CD56dim NK cells were inversely correlated with HIV-1 DNA levels throughout the study, and changes in HIV-1 DNA levels during panobinostat treatment were negatively associated with the corresponding changes in CD69+ NK cells. Decreasing levels of HIV-1 DNA during latency-reversing treatment were also related to the proportions of plasmacytoid dendritic cells, to distinct expression patterns of interferon-stimulated genes, and to the expression of the IL28B CC genotype. Together, these data suggest that innate immune activity can critically modulate the effects of latency-reversing agents on the viral reservoir and may represent a target for future immunotherapeutic interventions in HIV-1 eradication studies. IMPORTANCE Currently available antiretroviral drugs are highly effective in suppressing HIV-1 replication, but the virus persists, despite treatment, in a latent form that does not actively express HIV-1 gene products. One approach to eliminate these cells, colloquially termed the “shock-and-kill” strategy, focuses on the use of latency-reversing agents that induce active

  15. Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein

    PubMed Central

    Cruceanu, Margareta; Stephen, Andrew G.; Beuning, Penny J.; Gorelick, Robert J.; Fisher, Robert J.; Williams, Mark C.

    2006-01-01

    We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single λ-DNA molecules through the helix-to-coil transition in the presence of NCp7 and various chemical compounds. The change in the helix-coil transition width induced by wild-type NCp7 and its zinc finger variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound-NC interaction measured here reduces NCp7’s capability to alter the transition width. Purified compounds from the NCI Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10 nM, 25 nM, and 100 nM concentration, respectively. Similarly, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC’s nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins, and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity. PMID:17034752

  16. Types of HIV/AIDS Antiretroviral Drugs

    MedlinePlus

    ... reverse transcriptase (RT) from converting single-stranded HIV RNA into double-stranded HIV DNA―a process called ... RT, interfering with its ability to convert HIV RNA into HIV DNA Integrase Inhibitors block the HIV ...

  17. Estimating the Impact of Earlier ART Initiation and Increased Testing Coverage on HIV Transmission among Men Who Have Sex with Men in Mexico using a Mathematical Model

    PubMed Central

    Caro-Vega, Yanink; del Rio, Carlos; Dias Lima, Viviane; Lopez-Cervantes, Malaquias; Crabtree-Ramirez, Brenda; Bautista-Arredondo, Sergio; Colchero, M. Arantxa; Sierra-Madero, Juan

    2015-01-01

    Objective To estimate the impact of late ART initiation on HIV transmission among men who have sex with men (MSM) in Mexico. Methods An HIV transmission model was built to estimate the number of infections transmitted by HIV-infected men who have sex with men (MSM-HIV+) MSM-HIV+ in the short and long term. Sexual risk behavior data were estimated from a nationwide study of MSM. CD4+ counts at ART initiation from a representative national cohort were used to estimate time since infection. Number of MSM-HIV+ on treatment and suppressed were estimated from surveillance and government reports. Status quo scenario (SQ), and scenarios of early ART initiation and increased HIV testing were modeled. Results We estimated 14239 new HIV infections per year from MSM-HIV+ in Mexico. In SQ, MSM take an average 7.4 years since infection to initiate treatment with a median CD4+ count of 148 cells/mm3(25th-75th percentiles 52–266). In SQ, 68% of MSM-HIV+ are not aware of their HIV status and transmit 78% of new infections. Increasing the CD4+ count at ART initiation to 350 cells/mm3 shortened the time since infection to 2.8 years. Increasing HIV testing to cover 80% of undiagnosed MSM resulted in a reduction of 70% in new infections in 20 years. Initiating ART at 500 cells/mm3 and increasing HIV testing the reduction would be of 75% in 20 years. Conclusion A substantial number of new HIV infections in Mexico are transmitted by undiagnosed and untreated MSM-HIV+. An aggressive increase in HIV testing coverage and initiating ART at a CD4 count of 500 cells/mm3 in this population would significantly benefit individuals and decrease the number of new HIV infections in Mexico. PMID:26302044

  18. Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs.

    PubMed

    Alian, Akram; Griner, Sarah L; Chiang, Vicki; Tsiang, Manuel; Jones, Gregg; Birkus, Gabriel; Geleziunas, Romas; Leavitt, Andrew D; Stroud, Robert M

    2009-05-19

    HIV-1 integration into the host cell genome is a multistep process catalyzed by the virally-encoded integrase (IN) protein. In view of the difficulty of obtaining a stable DNA-bound IN at high concentration as required for structure determination, we selected IN-DNA complexes that form disulfide linkages between 5'-thiolated DNA and several single mutations to cysteine around the catalytic site of IN. Mild reducing conditions allowed for selection of the most thermodynamically-stable disulfide-linked species. The most stable complexes induce tetramer formation of IN, as happens during the physiological integration reaction, and are able to catalyze the strand transfer step of retroviral integration. One of these complexes also binds strand-transfer inhibitors of HIV antiviral drugs, making it uniquely valuable among the mutants of this set for understanding portions of the integration reaction. This novel complex may help define substrate interactions and delineate the mechanism of action of known integration inhibitors. PMID:19416821

  19. Quantitation of cytomegalovirus (CMV) DNA by real-time PCR for occurrence of CMV disease in HIV-infected patients receiving highly active antiretroviral therapy.

    PubMed

    Gourlain, Karine; Salmon, Dominique; Gault, Elyanne; Leport, Catherine; Katlama, Christine; Matheron, Sophie; Costagliola, Dominique; Mazeron, Marie-Christine; Fillet, Anne-Marie

    2003-03-01

    In HIV-infected patients treated with highly active antiretroviral therapy (HAART) included in the Predivir cohort, we have evaluated the usefulness of CMV DNA quantitation by a TaqMan PCR assay from peripheral blood leukocytes (PBLs) to predict CMV disease occurrence. In parallel with the immune restoration after treatment by HAART, the percentage of positive samples decreased progressively from 7.3% at Day 0 to 3.5% at Month 12. Among the CMV markers, the smallest concordance with PBL CMV TaqMan PCR, as evaluated by kappa, was observed with pp65 antigenemia, whereas concordance with all other CMV markers was high. Among the 16 patients with CMV DNA copies at least once >100/150,000 cells, CMV disease occurred in six during follow-up, whereas among the 159 patients with CMV DNA copies always <10/150,000 cells, CMV disease occurred in three and among the seven patients with CMV DNA copies >10 and <100 occurred in only one. In univariate Cox models, all the CMV markers including PBL CMV TaqMan PCR >10/150,000 cells (RR: 27.6, IC95: 7.1-107.2), the CD4 cell count <75 cells/mm(3) and the HIV viral load >100,000 copies/ml were predictive for CMV disease. In a stepwise multivariate analysis, which should be interpreted with caution due to the small number of events (n = 10), three covariates were associated independently with CMV disease: pp65 antigenemia >100 nuclei/200,000, PBL CMV TaqMan PCR >10 copies/150,000 cells and HIV viral load remaining or increasing >100,000 copies/ml. PMID:12526052

  20. Mitochondrial DNA mutations in blood samples from HIV-1-infected children undergoing long-term antiretroviral therapy.

    PubMed

    Ouyang, Yabo; Qiao, Luxin; Liu, Kai; Zang, Yunjin; Sun, Yu; Dong, Yaowu; Liu, Daojie; Guo, Xianghua; Wei, Feili; Lin, Minghua; Zhang, Fujie; Chen, Dexi

    2016-07-01

    We have analyzed mutations in whole mitochondrial (mt) genomes of blood samples from HIV-1-infected children treated with long-term antiretroviral therapy (ART), who had an excellent virological response. HIV-1-infected children who have undergone ART for 4 y with an excellent virological response (group A; 15 children) and ten healthy children (controls) without HIV-1 infection were enrolled retrospectively. Peripheral blood mononuclear cells (PBMCs) were obtained and mt DNA mutations were studied. The total number of mtDNA mutations in group A was 3 H more than in the controls (59 vs. 19, P<0.001) and the same trend was seen in all mtDNA regions. Among these mtDNA mutations, 140 and 28 mutations were detected in group A and the controls, respectively. The D-loop, CYTB and 12s rRNA were the three most common mutation regions in both groups, with significant differences between the groups observed at nucleotide positions C309CC, T489C CA514deletion, T16249C and G16474GG (D-loop); T14783C, G15043A, G15301A, and A15662G (CYTB); and G709A (12s rRNA). G15043A and A15662G had been associated with mitochondrial diseases. Our findings suggest that mtDNA mutations occur frequently in long-term ART-treated, HIV-1-infected children who have an excellent virological response, although they did not have obvious current symptoms. The CYTB region may play an important role in mtDNA mutation during ART, which might contribute to the development of subsequent mitochondrial diseases. PMID:27402477

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

    SciTech Connect

    Ordonhez Rigato, Paula; Maciel, Milton; Goldoni, Adriana Leticia; Piubelli, Orlando; Alves de Brito, Cyro; Fusaro, Ana Elisa; Eurico de Alencar, Liciana Xavier; August, Thomas; Torres Azevedo Marques, Ernesto; Silva Duarte, Alberto Jose da; Sato, Maria Notomi

    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, 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.

  2. 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 Central

    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

    Background 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. Methods 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. Results 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. Conclusions 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. Trial Registration World Health

  3. Increased HIV disclosure three months after an online video intervention for men who have sex with men (MSM).

    PubMed

    Chiasson, Mary Ann; Shaw, Francine Shuchat; Humberstone, Mike; Hirshfield, Sabina; Hartel, Diana

    2009-09-01

    A behavioral intervention for men who have sex with men (MSM) was created for online delivery. The nine-minute video drama "The Morning After" (www.hivbigdeal.org) was designed to promote critical thinking about HIV risk. MSM were recruited for the evaluation through banner ads on a subscription-based gay sexual meeting web site. Participants viewed the intervention online and completed online behavioral questionnaires at enrollment and three month follow-up. Each participant served as his own control. No participant incentives were provided. Follow-up was completed by 522 (54%) of 971 eligible men. Men completing follow-up were similar to those who did not by age and prevalence of unprotected anal intercourse (UAI) but reported more lifetime sex partners. They also differed somewhat by race, education, and HIV testing. In the three months after the intervention, men were significantly more likely to disclose HIV status to partners (odds ratio [OR] = 3.37, p<0.001) and less likely to report a casual partner or UAI in their most recent sexual encounter than at baseline. Of 120 men HIV tested during follow-up, 17 (14%) reported being HIV seropositive. These findings show that MSM at high risk for HIV will participate in a brief online video intervention designed to engage critical thinking and that significant, self-reported increases in HIV disclosure and decreases in risk behavior occurred three months after MSM viewed the video. PMID:20024766

  4. Continued Follow-Up of Phambili Phase 2b Randomized HIV-1 Vaccine Trial Participants Supports Increased HIV-1 Acquisition among Vaccinated Men

    PubMed Central

    Moodie, Zoe; Metch, Barbara; Bekker, Linda-Gail; Churchyard, Gavin; Nchabeleng, Maphoshane; Mlisana, Koleka; Laher, Fatima; Roux, Surita; Mngadi, Kathryn; Innes, Craig; Mathebula, Matsontso; Allen, Mary; Bentley, Carter; Gilbert, Peter B.; Robertson, Michael; Kublin, James; Corey, Lawrence; Gray, Glenda E.

    2015-01-01

    Background The Phase 2b double-blinded, randomized Phambili/HVTN 503 trial evaluated safety and efficacy of the MRK Ad5 gag/pol/nef subtype B HIV-1 preventive vaccine vs placebo in sexually active HIV-1 seronegative participants in South Africa. Enrollment and vaccinations stopped and participants were unblinded but continued follow-up when the Step study evaluating the same vaccine in the Americas, Caribbean, and Australia was unblinded for non-efficacy. Final Phambili analyses found more HIV-1 infections amongst vaccine than placebo recipients, impelling the HVTN 503-S recall study. Methods HVTN 503-S sought to enroll all 695 HIV-1 uninfected Phambili participants, provide HIV testing, risk reduction counseling, physical examination, risk behavior assessment and treatment assignment recall. After adding HVTN 503-S data, HIV-1 infection hazard ratios (HR vaccine vs. placebo) were estimated by Cox models. Results Of the 695 eligible, 465 (67%) enrolled with 230 from the vaccine group and 235 from the placebo group. 38% of the 184 Phambili dropouts were enrolled. Enrollment did not differ by treatment group, gender, or baseline HSV-2. With the additional 1286 person years of 503-S follow-up, the estimated HR over Phambili and HVTN 503-S follow-up was 1.52 (95% CI 1.08–2.15, p = 0.02, 82 vaccine/54 placebo infections). The HR was significant for men (HR = 2.75, 95% CI 1.49, 5.06, p = 0.001) but not for women (HR = 1.12, 95% CI 0.73, 1.72, p = 0.62). Conclusion The additional follow-up from HVTN 503-S supported the Phambili finding of increased HIV-1 acquisition among vaccinated men and strengthened the evidence of lack of vaccine effect among women. Trial Registration clinicaltrials.gov NCT00413725 SA National Health Research Database DOH-27-0207-1539 PMID:26368824

  5. HIV prevalence and risk behaviour among prostitutes and clients in Amsterdam: migrants at increased risk for HIV infection.

    PubMed Central

    van Haastrecht, H J; Fennema, J S; Coutinho, R A; van der Helm, T C; Kint, J A; van den Hoek, J A

    1993-01-01

    OBJECTIVES--To study groups of prostitutes and clients of prostitutes in order (i) to determine HIV prevalence and sexual risk behaviour, (ii) to determine differences between samples recruited within and outside a clinic for sexually transmitted diseases (STD) and (iii) to determine correlates of inconsistent condom use (ICU) among both groups. DESIGN--Participants were interviewed and anonymously tested for HIV-antibody; approximately half were recruited at a clinic for sexually transmitted diseases (STD) and half at prostitute working places. SETTING--An STD clinic and prostitute working places in Amsterdam in 1991. SUBJECTS--201 female prostitutes without a history of injecting drugs and 213 male clients of female prostitutes. MAIN OUTCOME MEASURES--antibodies to HIV, consistency of condom use in commercial vaginal contacts in the preceding 6 months. RESULTS--HIV prevalence was low: three prostitutes (1.5%; 95% CI 0.5-4.6%) and one client (0.5%; 95% CI 0.1-3.3%) were infected. All three HIV positive prostitutes originated from AIDS-endemic countries, came to the Netherlands only recently and were recruited outside the STD clinic. Large differences between subgroups resulted from the two recruitment methods: while clients of prostitutes with relatively high risk behaviour were strongly represented among the STD clinic sample, high risk prostitutes were underrepresented in this sample. Consistent condom use (with 100% of contacts) was reported by 66% of prostitutes and 56% of clients of prostitutes. Inconsistent condom use was found to be high among prostitutes who had migrated from Latin America and among migrant clients of prostitutes. CONCLUSIONS--When monitoring HIV infection one must take into account imported cases. HIV prevention efforts should be particularly focused at prostitutes from Latin America and at clients of prostitutes who migrated to the Netherlands. PMID:7721282

  6. Increased Sensitivity of DNA Damage Response-Deficient Cells to Stimulated Microgravity-Induced DNA Lesions

    PubMed Central

    Li, Nan; An, Lili; Hang, Haiying

    2015-01-01

    Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG). In this study we used mouse embryonic stem (MES) and mouse embryonic fibroblast (MEF) cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs) in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS). Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR) defects. PMID:25915950

  7. Increasing Parent Involvement in Youth HIV Prevention: A Randomized Caribbean Study

    ERIC Educational Resources Information Center

    Baptiste, Donna R.; Kapungu, Chisina; Miller, Steve; Crown, Laurel; Henry, David; Da Costa Martinez, Dona; Jo-Bennett, Karen

    2009-01-01

    This article presents preliminary findings of a randomized HIV prevention study in Trinidad and Tobago in the Caribbean. The study centers on a family HIV workshop aimed at strengthening parenting skills that are empirically linked to reducing adolescent HIV exposure and other sexual risks. These skills include parental monitoring; educating youth…

  8. Storytelling for Empowerment for Latino Teens: Increasing HIV Prevention Knowledge and Attitudes

    ERIC Educational Resources Information Center

    Nelson, Annabelle; Cordova, David; Walters, Andrew S.; Szecsy, Elsie

    2016-01-01

    Latino adolescents are disproportionately impacted by HIV, but researchers have documented few programs to prevent and reduce HIV risk. The Storytelling for Empowerment (SFE) "HIV StoryBook" was designed with an innovative ecodevelopment approach combining empowerment, family communication, and positive cultural identity. A mixed method…

  9. Increases in Human Papillomavirus Detection During Early HIV Infection Among Women in Zimbabwe

    PubMed Central

    Nowak, Rebecca G.; Morrison, Charles S.; Gange, Stephen J.; Kwok, Cynthia; Oliver, Amy E.; Howard, Roslyn; Van Der Pol, Barbara; Salata, Robert A.; Padian, Nancy S.; Chipato, Tsungai; Munjoma, Marshall; Celentano, David D.

    2011-01-01

    Background. Individuals who acquire human immunodeficiency virus (HIV) may experience an immediate disruption of genital tract immunity, altering the ability to mount a local and effective immune response. This study examined the impact of early HIV infection on new detection of human papillomavirus (HPV). Methods. One hundred fifty-five Zimbabwean women with observation periods before and after HIV acquisition and 486 HIV-uninfected women were selected from a cohort study evaluating hormonal contraceptive use and risk of HIV acquisition. Study visits occurred at 3-month intervals. Cervical swab samples available from up to 6 months before, at, and up to 6 months after the visit when HIV was first detected were typed for 37 HPV genotypes or subtypes. Results. We observed ∼5-fold higher odds of multiple (≥2) new HPV detections only after HIV acquisition, relative to HIV-negative women after adjusting for sexual behavior and concurrent genital tract infections. We also observed ∼2.5-fold higher odds of single new HPV detections at visits before and after HIV acquisition, relative to HIV-uninfected women in multivariable models. Conclusions. These findings suggest that HIV infection has an immediate impact on genital tract immunity, as evidenced by the high risk of multiple new HPV detections immediately after HIV acquisition. PMID:21451006

  10. A new activity of anti-HIV and anti-tumor protein GAP31: DNA adenosine glycosidase - Structural and modeling insight into its functions

    SciTech Connect

    Li, Hui-Guang; Huang, Philip L.; Zhang, Dawei; Sun, Yongtao; Chen, Hao-Chia; Zhang, John; Huang, Paul L.; Kong, Xiang-Peng; Lee-Huang, Sylvia

    2010-01-01

    We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

  11. A New Activity of Anti-HIV and Anti-tumor Protein GAP31: DNA Adenosine Glycosidase – Structural and Modeling Insight into its Functions

    SciTech Connect

    Li, H.; Huang, P; Zhang, D; Sun, Y; Chen, H; Zhang, J; Huang, P; Kong, X; Lee-Huang, S

    2010-01-01

    We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

  12. Using social and behavior change communication to increase HIV testing and condom use: the Malawi BRIDGE Project.

    PubMed

    Kaufman, Michelle R; Rimal, Rajiv N; Carrasco, Maria; Fajobi, OlaOluwa; Soko, Anthony; Limaye, Rupali; Mkandawire, Glory

    2014-01-01

    While overall HIV prevalence in Malawi has decreased, it is still high in the southern region of the country. Behavioral prevention activities are crucial to continue the reduction in HIV prevalence. Behavior change is influenced by many factors. Previous work indicates knowledge about HIV transmission, self-efficacy to protect oneself from exposure, and accurate risk perception of one's susceptibility all impact sexual behavior. The current study looks at the effects of a behavior change communication program in Malawi called the BRIDGE II Project on psychosocial and behavioral variables. The program sought to address barriers to individual action and confront societal norms related to sexual risk behavior through a mix of community-based activities and mass media messages delivered through local radio stations. Using cohort data (n = 594), we examined the effect of BRIDGE exposure on three variables that affect HIV behaviors: knowledge, self-efficacy, and risk perception, as well as two behavioral outcomes: HIV testing and condom use at last sex. Data were collected at baseline and for a midterm evaluation. Regression analyses showed exposure to BRIDGE was significantly associated with knowledge level (β = 0.20, p < .001) and self-efficacy (β = 0.35, p < .001) at midterm when controlling for baseline scores, but not risk perception. Psychosocial variables did not show a significant relationship to either behavioral outcome. However, program exposure was a significant predictor of both HIV testing in the past year (odds ratio [OR] = 1.40, p < .001) and condom use at last sex (OR = 1.26, p < .05). This study suggests such a communication intervention may play an important role in not only affecting HIV-related behaviors themselves, but also critical factors that affect HIV behaviors, including knowledge and self-efficacy. It is recommended that communication efforts around HIV risk reduction be increased. PMID:24735337

  13. The Community Liaison Program: a health education pilot program to increase minority awareness of HIV and acceptance of HIV vaccine trials.

    PubMed

    Kelley, R T; Hannans, A; Kreps, G L; Johnson, K

    2012-08-01

    This paper describes a 16-month health education pilot program based on diffusion of innovation and social network theories. The program was implemented by volunteer community liaisons for the purposes of increasing awareness of and support for HIV vaccine research in minority populations. This theoretically driven pilot program allowed the liaisons to integrate delivery of the HIV vaccine research messages created for the program into their existing activities and routines. Through training in participatory engagement, volunteers were able to tailor and adapt an HIV prevention message for their communities. Process evaluation data showed that the acceptance of participatory engagement and HIV vaccine message dissemination far exceeded expectations. The anticipated number of community members to receive the message was estimated at 500 with 10 volunteer liaisons or 50 per person. However, the actual number of people reached was 644, with only 7 volunteer liaisons, or an average of 92 persons per liaison, almost double the original number. Further research is recommended to analyze the specific behavioral changes that can come from the use of social networks in HIV vaccine research awareness within minority populations. PMID:22327809

  14. Retinoic acid imprints a mucosal-like phenotype on dendritic cells with an increased ability to fuel HIV-1 infection.

    PubMed

    Guerra-Pérez, Natalia; Frank, Ines; Veglia, Filippo; Aravantinou, Meropi; Goode, Diana; Blanchard, James L; Gettie, Agegnehu; Robbiani, Melissa; Martinelli, Elena

    2015-03-01

    The tissue microenvironment shapes the characteristics and functions of dendritic cells (DCs), which are important players in HIV infection and dissemination. Notably, DCs in the gut have the daunting task of orchestrating the balance between immune response and tolerance. They produce retinoic acid (RA), which imprints a gut-homing phenotype and influences surrounding DCs. To investigate how the gut microenvironment impacts the ability of DCs to drive HIV infection, we conditioned human immature monocyte-derived DCs (moDCs) with RA (RA-DCs), before pulsing them with HIV and mixing them with autologous T cells. RA-DCs showed a semimature, mucosal-like phenotype and released higher amounts of TGF-β1 and CCL2. Using flow cytometry, Western blot, and microscopy, we determined that moDCs express the cell adhesion molecule mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and that RA increases its expression. MAdCAM-1 was also detected on a small population of DCs in rhesus macaque (Macaca mulata) mesenteric lymph node. RA-DCs formed more DC-T cell conjugates and promoted significantly higher HIV replication in DC-T cell mixtures compared with moDCs. This correlated with the increase in MAdCAM-1 expression. Blocking MAdCAM-1 partially inhibited the enhanced HIV replication. In summary, RA influences DC phenotype, increasing their ability to exacerbate HIV infection. We describe a previously unknown mechanism that may contribute to rapid HIV spread in the gut, a major site of HIV replication after mucosal exposure. PMID:25624458

  15. Increases in Recent HIV Testing Among Men Who Have Sex With Men Coincide With the Centers for Disease Control and Prevention's Expanded Testing Initiative

    PubMed Central

    Cooley, Laura A.; Wejnert, Cyprian; Rose, Charles E.; Paz-Bailey, Gabriela; Taussig, Jennifer; Gern, Robert; Hoyte, Tamika; Salazar, Laura; White, Jianglan; Todd, Jeff; Bautista, Greg; Flynn, Colin; Sifakis, Frangiscos; German, Danielle; Isenberg, Debbie; Driscoll, Maura; Hurwitz, Elizabeth; Doherty, Rose; Wittke, Chris; Prachand, Nikhil; Benbow, Nanette; Melville, Sharon; Pannala, Praveen; Yeager, Richard; Sayegh, Aaron; Dyer, Jim; Sheu, Shane; Novoa, Alicia; Thrun, Mark; Al-Tayyib, Alia; Wilmoth, Ralph; Higgins, Emily; Griffin, Vivian; Mokotoff, Eve; MacMaster, Karen; Wolverton, Marcia; Risser, Jan; Rehman, Hafeez; Padgett, Paige; Bingham, Trista; Sey, Ekow Kwa; LaLota, Marlene; Metsch, Lisa; Forrest, David; Beck, Dano; Cardenas, Gabriel; Nemeth, Chris; Anderson, Bridget J.; Watson, Carol-Ann; Smith, Lou; Robinson, William T.; Gruber, DeAnn; Barak, Narquis; Murrill, Chris; Neaigus, Alan; Jenness, Samuel; Hagan, Holly; Reilly, Kathleen H.; Wendel, Travis; Cross, Helene; Bolden, Barbara; D'Errico, Sally; Wogayehu, Afework; Godette, Henry; Brady, Kathleen A.; Kirkland, Althea; Sifferman, Andrea; Miguelino-Keasling, Vanessa; Velasco, Al; Tovar, Veronica; Raymond, H. Fisher; De León, Sandra Miranda; Rolón-Colón, Yadira; Marzan, Melissa; Courogen, Maria; Jaenicke, Tom; Thiede, Hanne; Burt, Richard; Jia, Yujiang; Opoku, Jenevieve; Sansone, Marie; West, Tiffany; Magnus, Manya; Kuo, Irene

    2015-01-01

    According to National HIV Behavioral Surveillance system data, human immunodeficiency virus (HIV) testing increased among gay, bisexual, and other men who have sex with men from 2008 to 2011 in cities funded by the Centers for Disease Control and Prevention's Expanded Testing Initiative, suggesting that focused HIV testing initiatives might have positive effects. PMID:25352589

  16. Brief Training of HIV Medical Providers Increases Their Frequency of Delivering Prevention Counselling to Patients at Risk of Transmitting HIV to Others

    ERIC Educational Resources Information Center

    Patel, Shilpa N.; Marks, Gary; Gardner, Lytt; Golin, Carol E.; Shinde, Sanjyot; O'Daniels, Christine; Wilson, Tracey E.; Quinlivan, E. Byrd; Banderas, Julie W.

    2013-01-01

    Objective: The aim of this study was to examine whether brief training of human immunodeficiency virus (HIV) medical providers increased the frequency with which they routinely delivered prevention counselling to patients, and whether patient characteristics were associated with receipt of that counselling. Design: Longitudinal. Setting: Seven HIV…

  17. The Community Liaison Program: A Health Education Pilot Program to Increase Minority Awareness of HIV and Acceptance of HIV Vaccine Trials

    ERIC Educational Resources Information Center

    Kelley, R. T.; Hannans, A.; Kreps, G. L.; Johnson, K.

    2012-01-01

    This paper describes a 16-month health education pilot program based on diffusion of innovation and social network theories. The program was implemented by volunteer community liaisons for the purposes of increasing awareness of and support for HIV vaccine research in minority populations. This theoretically driven pilot program allowed the…

  18. Are HIV drug advertisements contributing to increases in risk behavior among men in San Francisco, 2001?

    PubMed

    Klausner, Jeffrey D; Kim, Andrea; Kent, Charlotte

    2002-11-22

    In winter 2001, we quantified exposure to HIV medication direct-to-consumer advertising and its relationship to attitudes and behaviors among sexually transmitted disease clinic attendees. We found that homosexual men frequently saw HIV medication advertisements. HIV-positive homosexual men with greater advertisement exposure believed HIV was a less serious disease. Men believed these advertisements influenced an individual's decision to have unprotected sex. HIV-positive homosexual men with greater advertisement exposure may be more likely to have unprotected anal intercourse. PMID:12441813

  19. Ultrasensitive Visual Detection of HIV DNA Biomarkers via a Multi-amplification Nanoplatform

    PubMed Central

    Long, Yuyin; Zhou, Cuisong; Wang, Congmin; Cai, Honglian; Yin, Cuiyun; Yang, Qiufang; Xiao, Dan

    2016-01-01

    Methodologies to detect disease biomarkers at ultralow concentrations can potentially improve the standard of living. A facile and label-free multi-amplification strategy is proposed for the ultrasensitive visual detection of HIV DNA biomarkers in real physiological media. This multi-amplification strategy not only exhibits a signficantly low detection limit down to 4.8 pM but also provides a label-free, cost-effective and facile technique for visualizing a few molecules of nucleic acid analyte with the naked eye. Importantly, the biosensor is capable of discriminating single-based mismatch lower than 5.0 nM in human serum samples. Moreover, the visual sensing platform exhibits excellent specificity, acceptable reusability and a long-term stability. All these advantages could be attributed to the nanofibrous sensing platform that 1) has a high surface-area-to-volume provided by electrospun nanofibrous membrane, and 2) combines glucose oxidase (GOx) biocatalysis, DNAzyme-catalyzed colorimetric reaction and catalytic hairpin assembly (CHA) recycling amplification together. This multi-amplification nanoplatform promises label-free and visual single-based mismatch DNA monitoring with high sensitivity and specificity, suggesting wide applications that range from virus detection to genetic disease diagnosis. PMID:27032385

  20. Ultrasensitive Visual Detection of HIV DNA Biomarkers via a Multi-amplification Nanoplatform.

    PubMed

    Long, Yuyin; Zhou, Cuisong; Wang, Congmin; Cai, Honglian; Yin, Cuiyun; Yang, Qiufang; Xiao, Dan

    2016-01-01

    Methodologies to detect disease biomarkers at ultralow concentrations can potentially improve the standard of living. A facile and label-free multi-amplification strategy is proposed for the ultrasensitive visual detection of HIV DNA biomarkers in real physiological media. This multi-amplification strategy not only exhibits a signficantly low detection limit down to 4.8 pM but also provides a label-free, cost-effective and facile technique for visualizing a few molecules of nucleic acid analyte with the naked eye. Importantly, the biosensor is capable of discriminating single-based mismatch lower than 5.0 nM in human serum samples. Moreover, the visual sensing platform exhibits excellent specificity, acceptable reusability and a long-term stability. All these advantages could be attributed to the nanofibrous sensing platform that 1) has a high surface-area-to-volume provided by electrospun nanofibrous membrane, and 2) combines glucose oxidase (GOx) biocatalysis, DNAzyme-catalyzed colorimetric reaction and catalytic hairpin assembly (CHA) recycling amplification together. This multi-amplification nanoplatform promises label-free and visual single-based mismatch DNA monitoring with high sensitivity and specificity, suggesting wide applications that range from virus detection to genetic disease diagnosis. PMID:27032385

  1. Lymphotropic herpes virus (EBV, HHV-6, HHV-8) DNA sequences in HIV negative Castleman's disease

    PubMed Central

    Barozzi, P; Luppi, M; Masini, L; Marasca, R; Savarino, M; Morselli, M; Ferrari, M G; Bevini, M; Bonacorsi, G; Torelli, G

    1996-01-01

    Aim—To evaluate the possible involvement of lymphotropic herpes viruses in Castleman's disease. Methods—Archival formalin fixed, paraffin wax embedded biopsy specimens from 16 HIV negative patients (11 with localised and five of multicentric disease) were studied. Epstein-Barr virus (EBV), human herpes virus-6 (HHV-6) and human herpes virus-8 (HHV-8) DNA was detected using PCR. PCR was also used to characterise the EBV genomes and the clonal status of the lesions. Results—EBV sequences were identified in nine (56%) cases. The main EBV genotype detected was type 1. Two (12%) cases were positive for both HHV-6 and EBV sequences. HHV-8 sequences were detected in one case of localised Castleman's disease, the sequence of which differed from that of the HHV-8 prototype. No clonal immunoglobulin gene rearrangements were found. Conclusions—EBV DNA was detected in a substantial proportion of cases, suggesting that it may have a role in the pathogenesis of Castleman's disease, unlike HHV-6 which was detected rarely. This is the first report of HHV-8 specific sequences in the localised from of the disease. Images PMID:16696081

  2. HIV/AIDS status disclosure increases support, behavioural change and, HIV prevention in the long term: a case for an Urban Clinic, Kampala, Uganda

    PubMed Central

    2014-01-01

    Background Disclosure of HIV status supports risk reduction and facilitates access to prevention and care services, but can be inhibited by the fear of negative repercussions. We explored the short and long-term outcomes of disclosure among clients attending an urban HIV clinic in Uganda. Methods Qualitative semi-structured interviews were administered to a purposeful sample of 40 adult HIV clients that was stratified by gender. The information elicited included their lived experiences and outcomes of disclosure in the short and long term. A text data management software (ATLAS.ti) was used for data analysis. Codes were exported to MS Excel and pivot tables, and code counts made to generate statistical data. Results Of the 134 short-term responses elicited during the interview regarding disclosure events, most responses were supportive including encouragement, advice and support regarding HIV care and treatment. The results show on-disclosing to spouse, there was more trust, and use of condoms for HIV prevention. Only one third were negative responses, like emotional shock and feeling of distress. The negative reactions to the spouses included rejection, shock and distress in the short term. Even then, none of these events led to drastic change such as divorce. Other responses reflected HIV prevention and call for behavioural change and advice to change sexual behaviour, recipient seeking HIV testing or care. Women reported more responses of encouragement compared to men. Men reported more preventive behaviour compared to women. Of the 137 long-term outcomes elicited during disclosure, three quarters were positive followed by behavioral change and prevention, and then negative responses. Men reported increased care and support when they disclosed to fellow men compared to when women disclosed to women. There was better or not change in relationship when women disclosed to women than when women disclosed to men. Conclusions There is overwhelming support to

  3. Increasing HIV-1 Drug Resistance Between 2010 and 2012 in Adults Participating in Population-Based HIV Surveillance in Rural KwaZulu-Natal, South Africa.

    PubMed

    Manasa, Justen; Danaviah, Siva; Lessells, Richard; Elshareef, Muna; Tanser, Frank; Wilkinson, Eduan; Pillay, Sureshnee; Mthiyane, Hloniphile; Mwambi, Henry; Pillay, Deenan; de Oliveira, Tulio

    2016-08-01

    As more human immunodeficiency virus (HIV)-infected patients access combination antiretroviral therapy (cART), higher proportions of newly infected patients may be infected with drug-resistant viruses. Regular surveillance of transmitted drug resistance (TDR) is required in southern Africa where high rates of transmission persist despite rapid expansion of ART. Dried blood spot samples from cART-naive participants from two rounds of an annual population-based HIV surveillance program in rural KwaZulu-Natal were tested for HIV RNA, and samples with HIV RNA >10,000 copies/ml were genotyped for drug resistance. The 2009 surveillance of drug resistance mutation (SDRM) list was used for drug resistance interpretation. The data were added to previously published data from the same program, and the χ(2) test for trend was used to test for trend in estimated prevalence of any TDR. Seven hundred and one participants' data were analyzed: 67 (2010), 381 (2011), and 253 (2012). No TDR was detected in 2010. Years 2011 and 2012 had 18 participants with SDRMs 4.7% and 7.1%, respectively (p = .02, χ(2) test for trend). The nonnucleoside reverse transcriptase inhibitor mutation, K103N, was the most common mutation, occurring in 27 (3.8%) of the participants, while nucleoside reverse transcriptase inhibitor (NRTI) SDRMs were detected in 10 (1.4%) of the participants, of whom eight had only a single NRTI SDRM. The increase in levels of drug resistance observed in this population could be a signal of increasing transmission of drug-resistant HIV. Thus, continued surveillance is critical to inform public health policies around HIV treatment and prevention. PMID:27002368

  4. Increasing HIV-1 Drug Resistance Between 2010 and 2012 in Adults Participating in Population-Based HIV Surveillance in Rural KwaZulu-Natal, South Africa

    PubMed Central

    Danaviah, Siva; Lessells, Richard; Elshareef, Muna; Tanser, Frank; Wilkinson, Eduan; Pillay, Sureshnee; Mthiyane, Hloniphile; Mwambi, Henry; Pillay, Deenan; de Oliveira, Tulio

    2016-01-01

    Abstract As more human immunodeficiency virus (HIV)–infected patients access combination antiretroviral therapy (cART), higher proportions of newly infected patients may be infected with drug-resistant viruses. Regular surveillance of transmitted drug resistance (TDR) is required in southern Africa where high rates of transmission persist despite rapid expansion of ART. Dried blood spot samples from cART-naive participants from two rounds of an annual population-based HIV surveillance program in rural KwaZulu-Natal were tested for HIV RNA, and samples with HIV RNA >10,000 copies/ml were genotyped for drug resistance. The 2009 surveillance of drug resistance mutation (SDRM) list was used for drug resistance interpretation. The data were added to previously published data from the same program, and the χ2 test for trend was used to test for trend in estimated prevalence of any TDR. Seven hundred and one participants' data were analyzed: 67 (2010), 381 (2011), and 253 (2012). No TDR was detected in 2010. Years 2011 and 2012 had 18 participants with SDRMs 4.7% and 7.1%, respectively (p = .02, χ2 test for trend). The nonnucleoside reverse transcriptase inhibitor mutation, K103N, was the most common mutation, occurring in 27 (3.8%) of the participants, while nucleoside reverse transcriptase inhibitor (NRTI) SDRMs were detected in 10 (1.4%) of the participants, of whom eight had only a single NRTI SDRM. The increase in levels of drug resistance observed in this population could be a signal of increasing transmission of drug-resistant HIV. Thus, continued surveillance is critical to inform public health policies around HIV treatment and prevention. PMID:27002368

  5. Increased adrenal androgen secretion with inhibition of 11beta-hydroxylase in HIV-infected women.

    PubMed

    Koutkia, Polyxeni; Berry, Jacqueline; Eaton, Kristina; Breu, Jeff; Grinspoon, Steven

    2006-05-01

    Adrenal androgen production is reduced in association with disease severity in HIV-infected women. This response may be maladaptive in terms of maintenance of lean body mass, functional status, and immune function. The aim of this study was to assess whether the use of an adrenal enzyme inhibitor of 11beta-hydroxylase might increase androgen production in this population. We conducted a randomized, double-blind, placebo-controlled study of metyrapone (500 mg p.o. qid) or placebo for 2 wk in 10 HIV-infected women with AIDS wasting [weight <90% ideal body weight (IBW) or weight loss >10%] and reduced androgen levels. Basal and ACTH-stimulated androgen, mineralocorticoid, and glucocorticoid levels were measured at baseline and after 14 days of treatment. Subjects were similar in age (40.9 +/- 0.9 yr), weight (91.7 +/- 3.5% IBW) and hormone concentrations at study entry. Total testosterone (84 +/- 54 vs. -0.4 +/- 2 ng/dl, P = 0.024), free testosterone (6.5 +/- 2.8 vs. 0.1 +/- 0.1 pg/ml, P = 0.024), DHEA (5.0 +/- 3.2 vs. -0.6 +/- 0.5 microg/l, P = 0.024), and 11-deoxycortisol (2,145 +/- 820 vs. -14 +/- 22 ng/dl, P = 0.024) levels increased in response to metyrapone compared with placebo treatment. In response to ACTH, significant increases in the DHEA/cortisol ratio (174 +/- 48 vs. 3 +/- 3, P = 0.008) were seen in the metyrapone group compared with placebo. Blood pressure and electrolytes did not change, and signs of adrenal insufficiency were not apparent. These data demonstrate that inhibition of 11beta-hydroxylase with metyrapone increases adrenal androgen secretion in HIV-infected women. Further studies are needed to assess the physiological effects of this strategy to increase anabolic hormone levels in severe stress, including detailed testing to rule out the potential risk of concomitant adrenal insufficiency. PMID:16303845

  6. Impact of the terminal bulges of HIV-1 cTAR DNA on its stability and the destabilizing activity of the nucleocapsid protein NCp7.

    PubMed

    Beltz, Hervé; Azoulay, Joel; Bernacchi, Serena; Clamme, Jean-Pierre; Ficheux, Damien; Roques, Bernard; Darlix, Jean-Luc; Mély, Yves

    2003-04-18

    Reverse transcription of HIV-1 genomic RNA to double-stranded DNA by reverse transcriptase (RT) is a critical step in HIV-1 replication. This process relies on two viral proteins, the RT enzyme and nucleocapsid protein NCp7 that has well documented nucleic acid chaperone properties. At the beginning of the linear DNA synthesis, the newly made minus-strand strong-stop DNA ((-)ssDNA) is transferred to the 3'end of the genomic RNA by means of an hybridization reaction between transactivation response element (TAR) RNA and cTAR DNA sequences. Since both TAR sequences exhibit stable hairpin structures, NCp7 needs to destabilize the TAR structures in order to chaperone their hybridization. To further characterize the relationships between TAR stability and NC-mediated destabilization, the role of the A(49) and G(52) bulged residues in cTAR DNA stability was investigated. The stability of cTAR and mutants where one or the two terminal bulges were replaced by base-pairs as well as the NCp7-mediated destabilization of these cTAR sequences were examined. Thermodynamic data indicate that the two bulges cooperatively destabilize cTAR by reducing the stacking interactions between the bases. This causes a free energy change of about 6.4 kcal/mol and seems to be critical for NC activity. Time-resolved fluorescence data of doubly labelled cTAR derivatives suggest that NC-mediated melting of cTAR ends propagates up to the 10C.A(44) mismatch or T(40) bulge. Fluorescence correlation spectroscopy using two-photon excitation was also used to monitor cTAR ends fraying by NC. Results show that NC causes a very significant increase of cTAR ends fraying, probably limited to the terminal base-pair in the case of cTAR mutants. Since the TAR RNA and cTAR DNA bulges or mismatches appear well conserved among all HIV-1 strains, the present data support the notion of a co-evolutionary relationship between TAR and NC activity. PMID:12684000

  7. Developing strategies to increase plasmid DNA production in Escherichia coli DH5α using batch culture.

    PubMed

    Islas-Lugo, Fabiola; Vega-Estrada, Jesús; Alvis, Christian Ariel; Ortega-López, Jaime; Del Carmen Montes-Horcasitas, María

    2016-09-10

    Plasmid DNA (pDNA) production has recently increased as a result of advances in DNA vaccines. The practical development of pDNA vaccines requires high yield and productivity of supercoiled plasmid DNA (sc-pDNA). The yield and productivity are influenced by the host strain, the plasmid, the production process, and especially by growth conditions, such as the culture type and medium. We evaluated different strategies to increase pDNA production by Escherichia coli DH5α in batch culture. The strategies were driven by the development of a four single-factor experimental design and were based on the change of culture media composition in terms of carbon and nitrogen and the modification of the pH control by using NaOH or NH4OH. The results revealed the carbon (50g/L of glycerol) and nitrogen (8.34g/L of YESP) concentration in the culture medium and starting pH control with NH4OH when most of the organic nitrogen was consumed. Under these conditions, we obtained a volumetric yield of 213mg pDNA/L, a specific yield of 10mg pDNA/g DCW (dry cell weight), 92% of sc-pDNA and a productivity of 17.6mg pDNA/(Lh). The pDNA productivities reached were 42% higher than the productivities reported by other authors applying similar conditions. PMID:27374404

  8. The Histone Deacetylase Inhibitor Vorinostat (SAHA) Increases the Susceptibility of Uninfected CD4+ T Cells to HIV by Increasing the Kinetics and Efficiency of Postentry Viral Events

    PubMed Central

    Lucera, Mark B.; Tilton, Carisa A.; Mao, Hongxia; Dobrowolski, Curtis; Tabler, Caroline O.; Haqqani, Aiman A.; Karn, Jonathan

    2014-01-01

    ABSTRACT Latently infected cells remain a primary barrier to eradication of HIV-1. Over the past decade, a better understanding of the molecular mechanisms by which latency is established and maintained has led to the discovery of a number of compounds that selectively reactivate latent proviruses without inducing polyclonal T cell activation. Recently, the histone deacetylase (HDAC) inhibitor vorinostat has been demonstrated to induce HIV transcription from latently infected cells when administered to patients. While vorinostat will be given in the context of antiretroviral therapy (ART), infection of new cells by induced virus remains a clinical concern. Here, we demonstrate that vorinostat significantly increases the susceptibility of CD4+ T cells to infection by HIV in a dose- and time-dependent manner that is independent of receptor and coreceptor usage. Vorinostat does not enhance viral fusion with cells but rather enhances the kinetics and efficiency of postentry viral events, including reverse transcription, nuclear import, and integration, and enhances viral production in a spreading-infection assay. Selective inhibition of the cytoplasmic class IIb HDAC6 with tubacin recapitulated the effect of vorinostat. These findings reveal a previously unknown cytoplasmic effect of HDAC inhibitors promoting productive infection of CD4+ T cells that is distinct from their well-characterized effects on nuclear histone acetylation and long-terminal-repeat (LTR) transcription. Our results indicate that careful monitoring of patients and ART intensification are warranted during vorinostat treatment and indicate that HDAC inhibitors that selectively target nuclear class I HDACs could reactivate latent HIV without increasing the susceptibility of uninfected cells to HIV. IMPORTANCE HDAC inhibitors, particularly vorinostat, are currently being investigated clinically as part of a “shock-and-kill” strategy to purge latent reservoirs of HIV. We demonstrate here that

  9. DNA prime-protein boost using subtype consensus Env was effective in eliciting neutralizing antibody responses against subtype BC HIV-1 viruses circulating in China

    PubMed Central

    Zhang, Mingshun; Zhang, Lu; Zhang, Chunhua; Hong, Kunxue; Shao, Yiming; Huang, Zuhu; Wang, Shixia; Lu, Shan

    2012-01-01

    Previously, we have shown that DNA prime-protein boost is effective in eliciting neutralizing antibodies (NAb) against randomly selected HIV-1 isolates. Given the genetic diversity of HIV-1 viruses and the unique predominant subtypes in different geographic regions, it is critical to test the DNA prime-protein boost approach against circulating viral isolates in key HIV endemic areas. In the current study, the same DNA prime-protein boost vaccine was used as in previous studies to investigate the induction of NAb responses against HIV-1 clade BC, a major subtype circulating in China. A codon optimized gp120-BC DNA vaccine, based on the consensus envelope (Env) antigen sequence of clade BC, was constructed and a stable CHO cell line expressing the same consensus BC gp120 protein was produced. The immunogenicity of this consensus gp120-BC was examined in New Zealand White rabbits by either DNA prime-protein boost or protein alone vaccination approaches. High levels of Env-specific antibody responses were elicited by both approaches. However, DNA prime-protein boost but not the protein alone immune sera contained significant levels of NAb against pseudotyped viruses expressing HIV-1 BC Env antigens. Furthermore, high frequencies of CD4 binding site-targeted antibodies were found in the DNA prime- protein boost rabbit sera indicating that the positive NAb may be the result of antibodies against conformationally sensitive epitopes on HIV-1 Env. The findings support that DNA prime-protein boost was effective in eliciting NAb against a key HIV-1 virus subtype in China. This result may lead to the development of regional HIV vaccines through this approach. PMID:23111170

  10. Plasma Mitochondrial DNA Levels as a Biomarker of Lipodystrophy Among HIV-infected Patients Treated with Highly Active Antiretroviral Therapy (HAART).

    PubMed

    Dai, Z; Cai, W; Hu, F; Lan, Y; Li, L; Chung, C; Caughey, B; Zhang, K; Tang, X

    2015-01-01

    Lipodystrophy is a common complication in HIV-infected patients taking highly active antiretroviral therapy. Its early diagnosis is crucial for timely modification of antiretroviral therapy. We hypothesize that mitochondrial DNA in plasma may be a potential marker of LD in HIV-infected individuals. In this study, we compared plasma mitochondrial DNA levels in HIV-infected individuals and non-HIV-infected individuals to investigate its potential diagnostic value. Total plasma DNA was extracted from 67 HIV-infected patients at baseline and 12, 24 and 30 months after initiating antiretroviral therapy. Real-time quantitative PCR was used to determine the mitochondrial DNA levels in plasma. Lipodystrophy was defined by the physician-assessed presence of lipoatrophy or lipohypertrophy in one or more body regions. The mitochondrial DNA levels in plasma were significantly higher at baseline in HIV-infected individuals than in non-HIV-infected individuals (p<0.05). At month 30, 33 out of 67 patients (49.2%) showed at least one sign of lipodystrophy. The mean plasma mitochondrial DNA levels in lipodystrophy patients were significantly higher compared to those without lipodystrophy at month 24 (p<0.001). The receiver operating curve analysis demonstrated that using plasma mitochondrial DNA level (with cut-off value <5.09 log10 copies/ml) as a molecular marker allowed identification of patients with lipodystrophy with a sensitivity of 64.2% and a specificity of 73.0%. Our data suggest that mitochondrial DNA levels may help to guide therapy selection with regards to HIV lipodystrophy risk. PMID:26592244