Science.gov

Sample records for ad5 neutralizing antibodies

  1. Differential Specificity and Immunogenicity of Adenovirus Type 5 Neutralizing Antibodies Elicited by Natural Infection or Immunization▿

    PubMed Central

    Cheng, Cheng; Gall, Jason G. D.; Nason, Martha; King, C. Richter; Koup, Richard A.; Roederer, Mario; McElrath, M. Juliana; Morgan, Cecilia A.; Churchyard, Gavin; Baden, Lindsey R.; Duerr, Ann C.; Keefer, Michael C.; Graham, Barney S.; Nabel, Gary J.

    2010-01-01

    A recent clinical trial of a T-cell-based AIDS vaccine delivered with recombinant adenovirus type 5 (rAd5) vectors showed no efficacy in lowering viral load and was associated with increased risk of human immunodeficiency virus type 1 (HIV-1) infection. Preexisting immunity to Ad5 in humans could therefore affect both immunogenicity and vaccine efficacy. We hypothesized that vaccine-induced immunity is differentially affected, depending on whether subjects were exposed to Ad5 by natural infection or by vaccination. Serum samples from vaccine trial subjects receiving a DNA/rAd5 AIDS vaccine with or without prior immunity to Ad5 were examined for the specificity of their Ad5 neutralizing antibodies and their effect on HIV-1 immune responses. Here, we report that rAd5 neutralizing antibodies were directed to different components of the virion, depending on whether they were elicited by natural infection or vaccination in HIV vaccine trial subjects. Neutralizing antibodies elicited by natural infection were directed largely to the Ad5 fiber, while exposure to rAd5 through vaccination elicited antibodies primarily to capsid proteins other than fiber. Notably, preexisting immunity to Ad5 fiber from natural infection significantly reduced the CD4 and CD8 cell responses to HIV Gag after DNA/rAd5 vaccination. The specificity of Ad5 neutralizing antibodies therefore differs depending on the route of exposure, and natural Ad5 infection compromises Ad5 vaccine-induced immunity to weak immunogens, such as HIV-1 Gag. These results have implications for future AIDS vaccine trials and the design of next-generation gene-based vaccine vectors. PMID:19846512

  2. Broadly Neutralizing Antibodies for HIV Eradication.

    PubMed

    Stephenson, Kathryn E; Barouch, Dan H

    2016-02-01

    Passive transfer of antibodies has long been considered a potential treatment modality for infectious diseases, including HIV. Early efforts to use antibodies to suppress HIV replication, however, were largely unsuccessful, as the antibodies that were studied neutralized only a relatively narrow spectrum of viral strains and were not very potent. Recent advances have led to the discovery of a large portfolio of human monoclonal antibodies that are broadly neutralizing across many HIV-1 subtypes and are also substantially more potent. These antibodies target multiple different epitopes on the HIV envelope, thus allowing for the development of antibody combinations. In this review, we discuss the application of broadly neutralizing antibodies (bNAbs) for HIV treatment and HIV eradication strategies. We highlight bNAbs that target key epitopes, such as the CD4 binding site and the V2/V3-glycan-dependent sites, and we discuss several bNAbs that are currently in the clinical development pipeline.

  3. Sensitive neutralization test for rubella antibody.

    PubMed Central

    Sato, H; Albrecht, P; Krugman, S; Ennis, F A

    1979-01-01

    A modified rubella virus plaque neutralization test for measuring rubella antibody was developed based on the potentiation of the virus-antibody complex by heterologous anti-immunoglobulin. The test is highly sensitive, yielding titers on the average 50 to 100 times higher than the haemagglutination inhibition test or the conventional plaque neutralization test. The sensitivity of this enhanced neutralization test is somewhat limited by the existence of a prozone phenomenon which precludes testing of low-titered sera below a dilution of 1:16. No prozone effect was observed with cerebrospinal fluids. The specificity of the enhanced neutralization test was determined by seroconversion of individuals receiving rubella vaccine. Although the rubella hemagglutination inhibition test remains the test of choice in routine diagnostic and surveillance work, the enhanced rubella neutralization test is particularly useful in monitoring low-level antibody in the cerebrospinal fluid in patients with neurological disorders and in certain instances of vaccine failure. PMID:107192

  4. Cross-reactive broadly neutralizing antibodies: timing is everything.

    PubMed

    Euler, Zelda; Schuitemaker, Hanneke

    2012-01-01

    The recent surge of research into new broadly neutralizing antibodies in HIV-1 infection has recharged the field of HIV-1 vaccinology. In this review we discuss the currently known broadly neutralizing antibodies and focus on factors that may shape these antibodies in natural infection. We further discuss the role of these antibodies in the clinical course of the infection and consider immunological obstacles in inducing broadly neutralizing antibodies with a vaccine.

  5. Antibody neutralization of retargeted measles viruses.

    PubMed

    Lech, Patrycja J; Pappoe, Roland; Nakamura, Takafumi; Tobin, Gregory J; Nara, Peter L; Russell, Stephen J

    2014-04-01

    The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization.

  6. Neutralizing antibodies to HIV-1 induced by immunization

    PubMed Central

    McCoy, Laura E.

    2013-01-01

    Most neutralizing antibodies act at the earliest steps of viral infection and block interaction of the virus with cellular receptors to prevent entry into host cells. The inability to induce neutralizing antibodies to HIV has been a major obstacle to HIV vaccine research since the early days of the epidemic. However, in the past three years, the definition of a neutralizing antibody against HIV has been revolutionized by the isolation of extremely broad and potent neutralizing antibodies from HIV-infected individuals. Considerable hurdles remain for inducing neutralizing antibodies to a protective level after immunization. Meanwhile, novel technologies to bypass the induction of antibodies are being explored to provide prophylactic antibody-based interventions. This review addresses the challenge of inducing HIV neutralizing antibodies upon immunization and considers notable recent advances in the field. A greater understanding of the successes and failures for inducing a neutralizing response upon immunization is required to accelerate the development of an effective HIV vaccine. PMID:23401570

  7. Homologous Boosting with Adenoviral Serotype 5 HIV Vaccine (rAd5) Vector Can Boost Antibody Responses despite Preexisting Vector-Specific Immunity in a Randomized Phase I Clinical Trial

    PubMed Central

    Sarwar, Uzma N.; Novik, Laura; Enama, Mary E.; Plummer, Sarah A.; Koup, Richard A.; Nason, Martha C.; Bailer, Robert T.; McDermott, Adrian B.; Roederer, Mario; Mascola, John R.; Ledgerwood, Julie E.; Graham, Barney S.

    2014-01-01

    Background Needle-free delivery improves the immunogenicity of DNA vaccines but is also associated with more local reactogenicity. Here we report the first comparison of Biojector and needle administration of a candidate rAd5 HIV vaccine. Methods Thirty-one adults, 18–55 years, 20 naive and 11 prior rAd5 vaccine recipients were randomized to receive single rAd5 vaccine via needle or Biojector IM injection at 1010 PU in a Phase I open label clinical trial. Solicited reactogenicity was collected for 5 days; clinical safety and immunogenicity follow-up was continued for 24 weeks. Results Overall, injections by either method were well tolerated. There were no serious adverse events. Frequency of any local reactogenicity was 16/16 (100%) for Biojector compared to 11/15 (73%) for needle injections. There was no difference in HIV Env-specific antibody response between Biojector and needle delivery. Env-specific antibody responses were more than 10-fold higher in subjects receiving a booster dose of rAd5 vaccine than after a single dose delivered by either method regardless of interval between prime and boost. Conclusions Biojector delivery did not improve antibody responses to the rAd5 vaccine compared to needle administration. Homologous boosting with rAd5 gene-based vectors can boost insert-specific antibody responses despite pre-existing vector-specific immunity. Trial Registration Clinicaltrials.gov NCT00709605 NCT00709605 PMID:25264782

  8. Mechanism of human antibody-mediated neutralization of Marburg virus.

    PubMed

    Flyak, Andrew I; Ilinykh, Philipp A; Murin, Charles D; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L; Hashiguchi, Takao; Bornholdt, Zachary A; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Ward, Andrew B; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E

    2015-02-26

    The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition.

  9. Mechanism of Human Antibody-Mediated Neutralization of Marburg Virus

    PubMed Central

    Flyak, Andrew I.; Ilinykh, Philipp A.; Murin, Charles D.; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L.; Hashiguchi, Takao; Bornholdt, Zachary A.; Slaughter, James C.; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G.; Ward, Andrew B.; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E.

    2015-01-01

    Summary The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of Antibody-GP complexes reveals that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP but not to MARV GP. The data suggest that MARV neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition. PMID:25723164

  10. Incomplete Neutralization and Deviation from Sigmoidal Neutralization Curves for HIV Broadly Neutralizing Monoclonal Antibodies.

    PubMed

    McCoy, Laura E; Falkowska, Emilia; Doores, Katie J; Le, Khoa; Sok, Devin; van Gils, Marit J; Euler, Zelda; Burger, Judith A; Seaman, Michael S; Sanders, Rogier W; Schuitemaker, Hanneke; Poignard, Pascal; Wrin, Terri; Burton, Dennis R

    2015-08-01

    The broadly neutralizing HIV monoclonal antibodies (bnMAbs) PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4) assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs) Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC)-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design.

  11. Elicitation of broadly neutralizing influenza antibodies in animals with previous influenza exposure.

    PubMed

    Wei, Chih-Jen; Yassine, Hadi M; McTamney, Patrick M; Gall, Jason G D; Whittle, James R R; Boyington, Jeffrey C; Nabel, Gary J

    2012-08-15

    The immune system responds to influenza infection by producing neutralizing antibodies to the viral surface protein, hemagglutinin (HA), which regularly changes its antigenic structure. Antibodies that target the highly conserved stem region of HA neutralize diverse influenza viruses and can be elicited through vaccination in animals and humans. Efforts to develop universal influenza vaccines have focused on strategies to elicit such antibodies; however, the concern has been raised that previous influenza immunity may abrogate the induction of such broadly protective antibodies. We show here that prime-boost immunization can induce broadly neutralizing antibody responses in influenza-immune mice and ferrets that were previously infected or vaccinated. HA stem-directed antibodies were elicited in mice primed with a DNA vaccine and boosted with inactivated vaccine from H1N1 A/New Caledonia/20/1999 (1999 NC) HA regardless of preexposure. Similarly, gene-based vaccination with replication-defective adenovirus 28 (rAd28) and 5 (rAd5) vectors encoding 1999 NC HA elicited stem-directed neutralizing antibodies and conferred protection against unmatched 1934 and 2007 H1N1 virus challenge in influenza-immune ferrets. Indeed, previous exposure to certain strains could enhance immunogenicity: The strongest HA stem-directed immune response was observed in ferrets previously infected with a divergent 1934 H1N1 virus. These findings suggest that broadly neutralizing antibodies against the conserved stem region of HA can be elicited through vaccination despite previous influenza exposure, which supports the feasibility of developing stem-directed universal influenza vaccines for humans.

  12. Recent Progress toward Engineering HIV-1-Specific Neutralizing Monoclonal Antibodies

    PubMed Central

    Sun, Ming; Li, Yue; Zheng, Huiwen; Shao, Yiming

    2016-01-01

    The recent discoveries of broadly potent neutralizing human monoclonal antibodies represent a new generation of antiretrovirals for the treatment and prophylaxis. Antibodies are generally considered more effective and safer and have been proved to provide passive protection against mucosal challenge in humanized mice and macaques. Several neutralizing Abs could protect animals against HIV-1 but are not effective when used in an established infected model for therapy. In order to overcome the limitation of antiviral activities, multiple antibody-engineering technologies have been explored to generate “the better” neutralizing antibodies against HIV-1 since bNAbs attack viral entry by various mechanisms. Thus, a promising direction of research is to discover and exploit rational antibody combination or engineered antibodies (eAbs) as potential candidate therapeutics against HIV-1. It has been reported that inclusion of fusion-neutralizing antibodies in a set of bNAbs could improve their overall activities and neutralizing spectrum. Here, we review several routes for engineering bNAbs, such as design and generation of bispecific antibodies, specific glycosylation of antibodies to enhance antiviral activity, and variable region-specific modification guided by structure and computer, as well as reviewing antibody-delivery technologies by non-viral vector, viral vector, and human hematopoietic stem/progenitor cells transduced with a lentiviral construct. We also discuss the optimized antiviral activities and benefits of these strategy and potential mechanisms. PMID:27746780

  13. Human-like antibodies neutralizing Western equine encephalitis virus

    PubMed Central

    Hülseweh, Birgit; Rülker, Torsten; Pelat, Thibaut; Langermann, Claudia; Frenzel, Andrè; Schirrmann, Thomas; Dübel, Stefan; Thullier, Philippe; Hust, Michael

    2014-01-01

    This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69–3A2) neutralized 50% of 5x104 TCID50/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69–3A2 antibody is a promising candidate for future passive vaccine development. PMID:24518197

  14. Structure of HCMV glycoprotein B in the postfusion conformation bound to a neutralizing human antibody

    PubMed Central

    Chandramouli, Sumana; Ciferri, Claudio; Nikitin, Pavel A.; Caló, Stefano; Gerrein, Rachel; Balabanis, Kara; Monroe, James; Hebner, Christy; Lilja, Anders E.; Settembre, Ethan C.; Carfi, Andrea

    2015-01-01

    Human cytomegalovirus (HCMV) poses a significant threat to immunocompromised individuals and neonates infected in utero. Glycoprotein B (gB), the herpesvirus fusion protein, is a target for neutralizing antibodies and a vaccine candidate due to its indispensable role in infection. Here we show the crystal structure of the HCMV gB ectodomain bound to the Fab fragment of 1G2, a neutralizing human monoclonal antibody isolated from a seropositive subject. The gB/1G2 interaction is dominated by aromatic residues in the 1G2 heavy chain CDR3 protruding into a hydrophobic cleft in the gB antigenic domain 5 (AD-5). Structural analysis and comparison with HSV gB suggest the location of additional neutralizing antibody binding sites on HCMV gB. Finally, immunoprecipitation experiments reveal that 1G2 can bind to HCMV virion gB suggesting that its epitope is exposed and accessible on the virus surface. Our data will support the development of vaccines and therapeutic antibodies against HCMV infection. PMID:26365435

  15. Structure of HCMV glycoprotein B in the postfusion conformation bound to a neutralizing human antibody.

    PubMed

    Chandramouli, Sumana; Ciferri, Claudio; Nikitin, Pavel A; Caló, Stefano; Gerrein, Rachel; Balabanis, Kara; Monroe, James; Hebner, Christy; Lilja, Anders E; Settembre, Ethan C; Carfi, Andrea

    2015-09-14

    Human cytomegalovirus (HCMV) poses a significant threat to immunocompromised individuals and neonates infected in utero. Glycoprotein B (gB), the herpesvirus fusion protein, is a target for neutralizing antibodies and a vaccine candidate due to its indispensable role in infection. Here we show the crystal structure of the HCMV gB ectodomain bound to the Fab fragment of 1G2, a neutralizing human monoclonal antibody isolated from a seropositive subject. The gB/1G2 interaction is dominated by aromatic residues in the 1G2 heavy chain CDR3 protruding into a hydrophobic cleft in the gB antigenic domain 5 (AD-5). Structural analysis and comparison with HSV gB suggest the location of additional neutralizing antibody binding sites on HCMV gB. Finally, immunoprecipitation experiments reveal that 1G2 can bind to HCMV virion gB suggesting that its epitope is exposed and accessible on the virus surface. Our data will support the development of vaccines and therapeutic antibodies against HCMV infection.

  16. Neutralizing antibodies decrease the envelope fluidity of HIV-1

    SciTech Connect

    Harada, Shinji Monde, Kazuaki; Tanaka, Yuetsu; Kimura, Tetsuya; Maeda, Yosuke; Yusa, Keisuke

    2008-01-05

    For successful penetration of HIV-1, the formation of a fusion pore may be required in order to accumulate critical numbers of fusion-activated gp41 with the help of fluidization of the plasma membrane and viral envelope. An increase in temperature to 40 {sup o}C after viral adsorption at 25 {sup o}C enhanced the infectivity by 1.4-fold. The enhanced infectivity was inhibited by an anti-CXCR4 peptide, T140, and anti-V3 monoclonal antibodies (0.5{beta} and 694/98-D) by post-attachment neutralization, but not by non-neutralizing antibodies (670-30D and 246-D) specific for the C5 of gp120 and cluster I of gp41, respectively. Anti-HLA-II and an anti-HTLV-I gp46 antibody, LAT27, neutralized the molecule-carrying HIV-1{sub C-2(MT-2)}. The anti-V3 antibodies suppressed the fluidity of the HIV-1{sub C-2} envelope, whereas the non-neutralizing antibodies did not. The anti-HLA-II antibody decreased the envelope fluidity of HIV-1{sub C-2(MT-2)}, but not that of HIV-1{sub C-2}. Therefore, fluidity suppression by these antibodies represents an important neutralization mechanism, in addition to inhibition of viral attachment.

  17. Antiviral Therapy by HIV-1 Broadly Neutralizing and Inhibitory Antibodies.

    PubMed

    Zhang, Zhiqing; Li, Shaowei; Gu, Ying; Xia, Ningshao

    2016-11-18

    Human immunodeficiency virus type 1 (HIV-1) infection causes acquired immune deficiency syndrome (AIDS), a global epidemic for more than three decades. HIV-1 replication is primarily controlled through antiretroviral therapy (ART) but this treatment does not cure HIV-1 infection. Furthermore, there is increasing viral resistance to ART, and side effects associated with long-term therapy. Consequently, there is a need of alternative candidates for HIV-1 prevention and therapy. Recent advances have discovered multiple broadly neutralizing antibodies against HIV-1. In this review, we describe the key epitopes on the HIV-1 Env protein and the reciprocal broadly neutralizing antibodies, and discuss the ongoing clinical trials of broadly neutralizing and inhibitory antibody therapy as well as antibody combinations, bispecific antibodies, and methods that improve therapeutic efficacy by combining broadly neutralizing antibodies (bNAbs) with latency reversing agents. Compared with ART, HIV-1 therapeutics that incorporate these broadly neutralizing and inhibitory antibodies offer the advantage of decreasing virus load and clearing infected cells, which is a promising prospect in HIV-1 prevention and treatment.

  18. Antiviral Therapy by HIV-1 Broadly Neutralizing and Inhibitory Antibodies

    PubMed Central

    Zhang, Zhiqing; Li, Shaowei; Gu, Ying; Xia, Ningshao

    2016-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection causes acquired immune deficiency syndrome (AIDS), a global epidemic for more than three decades. HIV-1 replication is primarily controlled through antiretroviral therapy (ART) but this treatment does not cure HIV-1 infection. Furthermore, there is increasing viral resistance to ART, and side effects associated with long-term therapy. Consequently, there is a need of alternative candidates for HIV-1 prevention and therapy. Recent advances have discovered multiple broadly neutralizing antibodies against HIV-1. In this review, we describe the key epitopes on the HIV-1 Env protein and the reciprocal broadly neutralizing antibodies, and discuss the ongoing clinical trials of broadly neutralizing and inhibitory antibody therapy as well as antibody combinations, bispecific antibodies, and methods that improve therapeutic efficacy by combining broadly neutralizing antibodies (bNAbs) with latency reversing agents. Compared with ART, HIV-1 therapeutics that incorporate these broadly neutralizing and inhibitory antibodies offer the advantage of decreasing virus load and clearing infected cells, which is a promising prospect in HIV-1 prevention and treatment. PMID:27869733

  19. Complete mapping of viral escape from neutralizing antibodies

    PubMed Central

    Hensley, Scott E.

    2017-01-01

    Identifying viral mutations that confer escape from antibodies is crucial for understanding the interplay between immunity and viral evolution. We describe a high-throughput approach to quantify the selection that monoclonal antibodies exert on all single amino-acid mutations to a viral protein. This approach, mutational antigenic profiling, involves creating all replication-competent protein variants of a virus, selecting with antibody, and using deep sequencing to identify enriched mutations. We use mutational antigenic profiling to comprehensively identify mutations that enable influenza virus to escape four monoclonal antibodies targeting hemagglutinin, and validate key findings with neutralization assays. We find remarkable mutation-level idiosyncrasy in antibody escape: for instance, at a single residue targeted by two antibodies, some mutations escape both antibodies while other mutations escape only one or the other. Because mutational antigenic profiling rapidly maps all mutations selected by an antibody, it is useful for elucidating immune specificities and interpreting the antigenic consequences of viral genetic variation. PMID:28288189

  20. Higher Throughput Quantification of Neutralizing Antibody to Herpes Simplex Viruses.

    PubMed

    Blevins, Tamara P; Mitchell, Michelle C; Korom, Maria; Wang, Hong; Yu, Yinyi; Morrison, Lynda A; Belshe, Robert B

    2015-01-01

    We report a rapid, higher throughput method for measuring neutralizing antibody to herpes simplex virus (HSV) in human sera. Clinical isolates and sera from the Herpevac Trial for Women were used in a colorimetric assay in which infection of tissue culture (lack of neutralization) was indicated by substrate metabolism by beta-galactosidase induced in the ELVIS cell line. The neutralization assay was optimized by addition of guinea pig complement, which particularly enhanced neutralizing antibody titers to HSV-2. Higher neutralizing antibody titers were also achieved using virus particles isolated from the supernatant of infected cells rather than lysate of infected cells as the source of virus. The effect of assay incubation time and incubation time with substrate were also optimized. We found that incubating with substrate until a standard optical density of 1.0 was reached permitted a better comparison among virus isolates, and achieved reliable measurement of neutralizing antibody activity. Interestingly, in contrast to results in the absence of complement, addition of complement allowed sera from HSV-2 gD-vaccinated subjects to neutralize HSV-1 and HSV-2 clinical and laboratory isolates with equal potency.

  1. Thermodynamic mechanism for the evasion of antibody neutralization in flaviviruses.

    PubMed

    Maillard, Rodrigo A; Liu, Tong; Beasley, David W C; Barrett, Alan D T; Hilser, Vincent J; Lee, J Ching

    2014-07-23

    Mutations in the epitopes of antigenic proteins can confer viral resistance to antibody-mediated neutralization. However, the fundamental properties that characterize epitope residues and how mutations affect antibody binding to alter virus susceptibility to neutralization remain largely unknown. To address these questions, we used an ensemble-based algorithm to characterize the effects of mutations on the thermodynamics of protein conformational fluctuations. We applied this method to the envelope protein domain III (ED3) of two medically important flaviviruses: West Nile and dengue 2. We determined an intimate relationship between the susceptibility of a residue to thermodynamic perturbations and epitope location. This relationship allows the successful identification of the primary epitopes in each ED3, despite their high sequence and structural similarity. Mutations that allow the ED3 to evade detection by the antibody either increase or decrease conformational fluctuations of the epitopes through local effects or long-range interactions. Spatially distant interactions originate in the redistribution of conformations of the ED3 ensembles, not through a mechanically connected array of contiguous amino acids. These results reconcile previous observations of evasion of neutralization by mutations at a distance from the epitopes. Finally, we established a quantitative correlation between subtle changes in the conformational fluctuations of the epitope and large defects in antibody binding affinity. This correlation suggests that mutations that allow viral growth, while reducing neutralization, do not generate significant structural changes and underscores the importance of protein fluctuations and long-range interactions in the mechanism of antibody-mediated neutralization resistance.

  2. Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection

    PubMed Central

    Malinge, Pauline; Magistrelli, Giovanni; Fischer, Nicolas; Sahin, Mehmet; Bergthaler, Andreas; Igonet, Sebastien; ter Meulen, Jan; Rigo, Dorothée; Meda, Paolo; Rabah, Nadia; Coutard, Bruno; Bowden, Thomas A.; Lambert, Paul-Henri; Siegrist, Claire-Anne; Pinschewer, Daniel D.

    2015-01-01

    Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein’s globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy. PMID:26587982

  3. Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection.

    PubMed

    Sommerstein, Rami; Flatz, Lukas; Remy, Melissa M; Malinge, Pauline; Magistrelli, Giovanni; Fischer, Nicolas; Sahin, Mehmet; Bergthaler, Andreas; Igonet, Sebastien; Ter Meulen, Jan; Rigo, Dorothée; Meda, Paolo; Rabah, Nadia; Coutard, Bruno; Bowden, Thomas A; Lambert, Paul-Henri; Siegrist, Claire-Anne; Pinschewer, Daniel D

    2015-11-01

    Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein's globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy.

  4. Influenza virus resistance to human neutralizing antibodies.

    PubMed

    Crowe, James E

    2012-01-01

    The human antibody repertoire has an exceptionally large capacity to recognize new or changing antigens through combinatorial and junctional diversity established at the time of V(D)J recombination and through somatic hypermutation. Influenza viruses exhibit a relentless capacity to escape the human antibody response by altering the amino acids of their surface proteins in hypervariable domains that exhibit a high level of structural plasticity. Both parties in this high-stakes game of shape shifting drive structural evolution of their functional proteins (the B cell receptor/antibody on one side and the viral hemagglutinin and neuraminidase proteins on the other) using error-prone polymerase systems. It is likely that most of the genetic mutations that occur in these systems are deleterious, resulting in the failure of the B cell or virus with mutations to propagate in the immune repertoire or viral quasispecies. A subset of mutations is tolerated in functional surface proteins that enter the B cell or virus progeny pool. In both cases, selection occurs in the population of mutated and unmutated species. In cases where the functional avidity of the B cell receptor is increased significantly, that clone may be selected for preferential expansion. In contrast, an influenza virus that "escapes" the inhibitory effect of secreted antibodies may represent a high proportion of the progeny virus in that host. The recent paper by O'Donnell et al. [C. D. O'Donnell et al., mBio 3(3):e00120-12, 2012] identifies a mechanism for antibody resistance that does not require escape from binding but rather achieves a greater efficiency in replication.

  5. Computational Prediction of Broadly Neutralizing HIV-1 Antibody Epitopes from Neutralization Activity Data

    PubMed Central

    Ferguson, Andrew L.; Falkowska, Emilia; Walker, Laura M.; Seaman, Michael S.; Burton, Dennis R.; Chakraborty, Arup K.

    2013-01-01

    Broadly neutralizing monoclonal antibodies effective against the majority of circulating isolates of HIV-1 have been isolated from a small number of infected individuals. Definition of the conformational epitopes on the HIV spike to which these antibodies bind is of great value in defining targets for vaccine and drug design. Drawing on techniques from compressed sensing and information theory, we developed a computational methodology to predict key residues constituting the conformational epitopes on the viral spike from cross-clade neutralization activity data. Our approach does not require the availability of structural information for either the antibody or antigen. Predictions of the conformational epitopes of ten broadly neutralizing HIV-1 antibodies are shown to be in good agreement with new and existing experimental data. Our findings suggest that our approach offers a means to accelerate epitope identification for diverse pathogenic antigens. PMID:24312481

  6. TRIM21-dependent intracellular antibody neutralization of virus infection.

    PubMed

    McEwan, William A; James, Leo C

    2015-01-01

    The ability of antibodies to prevent viral infection has long been recognized. In vitro neutralization assays, which take place in the absence of professional immune effector mechanisms, have demonstrated that the process of neutralization can occur by a variety of molecular mechanisms. Most known mechanisms involve the blocking of an event essential for infection, for instance, the steric inhibition of attachment to entry receptors. As such, neutralization is often thought of as a passive process that can occur without the need for host effector machinery. In contrast to this view, it has recently been demonstrated that neutralization can depend on the widely expressed cytosolic Fc binding protein TRIM21. This unique and novel Ig receptor directs the ubiquitin and proteasome-dependent degradation of intracellular antibody-bound viral particles and prevents infection. It has been further demonstrated that detection of cytosolic antibody by TRIM21 activates inflammatory signaling pathways and promotes the production of cytokines and chemokines. Studies in a TRIM21-null mouse demonstrate the importance of these activities: homozygous knockouts suffer fatal viral infection where wild-type mice survive. Though there is much to be learned about the role of TRIM21 in immunity, it is clear that there is a hitherto unappreciated role for antibodies in the intracellular environment.

  7. HIV-1 resistance to neutralizing antibodies: Determination of antibody concentrations leading to escape mutant evolution.

    PubMed

    Magnus, Carsten; Reh, Lucia; Trkola, Alexandra

    2016-06-15

    Broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) are considered vital components of novel therapeutics and blueprints for vaccine research. Yet escape to even the most potent of these antibodies is imminent in natural infection. Measures to define antibody efficacy and prevent mutant selection are thus urgently needed. Here, we derive a mathematical framework to predict the concentration ranges for which antibody escape variants can outcompete their viral ancestors, referred to as mutant selection window (MSW). When determining the MSW, we focus on the differential efficacy of neutralizing antibodies against HIV-1 in two canonical infection routes, free-virus infection and cell-cell transmission. The latter has proven highly effective in vitro suggesting its importance for both in vivo spread as well as for escaping targeted intervention strategies. We observed a range of MSW patterns that highlight the potential of mutants to arise in both transmission pathways and over wide concentration ranges. Most importantly, we found that only when the arising mutant has both, residual sensitivity to the neutralizing antibody and reduced infectivity compared to the parental virus, antibody dosing outside of the MSW to restrict mutant selection is possible. Emergence of mutants that provide complete escape and have no considerable fitness loss cannot be prevented by adjusting antibody doses. The latter may in part explain the ubiquitous resistance to neutralizing antibodies observed in natural infection and antibody treatment. Based on our findings, combinations of antibodies targeting different epitopes should be favored for antibody-based interventions as this may render complete resistance less likely to occur and also increase chances that multiple escapes result in severe fitness loss of the virus making longer-term antibody treatment more feasible.

  8. [Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ].

    PubMed

    Larina, M V; Aliev, T K; Solopova, O N; Pozdnyakova, L P; Korobova, S V; Yakimov, S A; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P

    2015-01-01

    Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

  9. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    PubMed Central

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  10. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

    PubMed

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-09-11

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  11. Recognition determinants of broadly neutralizing human antibodies against dengue viruses.

    PubMed

    Rouvinski, Alexander; Guardado-Calvo, Pablo; Barba-Spaeth, Giovanna; Duquerroy, Stéphane; Vaney, Marie-Christine; Kikuti, Carlos M; Navarro Sanchez, M Erika; Dejnirattisai, Wanwisa; Wongwiwat, Wiyada; Haouz, Ahmed; Girard-Blanc, Christine; Petres, Stéphane; Shepard, William E; Desprès, Philippe; Arenzana-Seisdedos, Fernando; Dussart, Philippe; Mongkolsapaya, Juthathip; Screaton, Gavin R; Rey, Félix A

    2015-04-02

    Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.

  12. Structural basis of hepatitis C virus neutralization by broadly neutralizing antibody HCV1

    SciTech Connect

    Kong, Leopold; Giang, Erick; Robbins, Justin B.; Stanfield, Robyn L.; Burton, Dennis R.; Wilson, Ian A.; Law, Mansun

    2012-10-29

    Hepatitis C virus (HCV) infects more than 2% of the global population and is a leading cause of liver cirrhosis, hepatocellular carcinoma, and end-stage liver diseases. Circulating HCV is genetically diverse, and therefore a broadly effective vaccine must target conserved T- and B-cell epitopes of the virus. Human mAb HCV1 has broad neutralizing activity against HCV isolates from at least four major genotypes and protects in the chimpanzee model from primary HCV challenge. The antibody targets a conserved antigenic site (residues 412-423) on the virus E2 envelope glycoprotein. Two crystal structures of HCV1 Fab in complex with an epitope peptide at 1.8-{angstrom} resolution reveal that the epitope is a {beta}-hairpin displaying a hydrophilic face and a hydrophobic face on opposing sides of the hairpin. The antibody predominantly interacts with E2 residues Leu{sup 413} and Trp{sup 420} on the hydrophobic face of the epitope, thus providing an explanation for how HCV isolates bearing mutations at Asn{sup 415} on the same binding face escape neutralization by this antibody. The results provide structural information for a neutralizing epitope on the HCV E2 glycoprotein and should help guide rational design of HCV immunogens to elicit similar broadly neutralizing antibodies through vaccination.

  13. JC polyomavirus mutants escape antibody-mediated neutralization.

    PubMed

    Ray, Upasana; Cinque, Paola; Gerevini, Simonetta; Longo, Valeria; Lazzarin, Adriano; Schippling, Sven; Martin, Roland; Buck, Christopher B; Pastrana, Diana V

    2015-09-23

    JC polyomavirus (JCV) persistently infects the urinary tract of most adults. Under conditions of immune impairment, JCV causes an opportunistic brain disease, progressive multifocal leukoencephalopathy (PML). JCV strains found in the cerebrospinal fluid of PML patients contain distinctive mutations in surface loops of the major capsid protein, VP1. We hypothesized that VP1 mutations might allow the virus to evade antibody-mediated neutralization. Consistent with this hypothesis, neutralization serology revealed that plasma samples from PML patients neutralized wild-type JCV strains but failed to neutralize patient-cognate PML-mutant JCV strains. This contrasted with serological results for healthy individuals, most of whom robustly cross-neutralized all tested JCV variants. Mice administered a JCV virus-like particle (VLP) vaccine initially showed neutralizing "blind spots" (akin to those observed in PML patients) that closed after booster immunization. A PML patient administered an experimental JCV VLP vaccine likewise showed markedly increased neutralizing titer against her cognate PML-mutant JCV. The results indicate that deficient humoral immunity is a common aspect of PML pathogenesis and that vaccination may overcome this humoral deficiency. Thus, vaccination with JCV VLPs might prevent the development of PML.

  14. Neutralizing Monoclonal Antibodies against Biological Toxins. Phase 1

    DTIC Science & Technology

    1993-08-14

    the original copy. AD-B’i76 29`8 CONTRACT NO: DAMD17-93-C-3083 TITLE: NEUTRALIZING MONOCLONAL ANTIBODIES AGAINST BIOLOGIC4JL TOXINS PRINCIPAL...Biological ’ Toxins DAMDl7-93-C-308.3 6. AUTHOR($) 65502A 30665502MB02. S4 .274 Mark C. Glassy, Ph.D. WUDA336206 7. PERFORMING ORGANIZATION NAMI(S...NUMBER OF PAGES RA I, SBIR, Antibody, Toxins , BL2, BD 16. PRICE COUE 17. SEC.URIly (LASSIFICATION 18. SECURITY CLASSIMICATION 19. SECURITY

  15. Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody

    PubMed Central

    Nowakowski, A.; Wang, C.; Powers, D. B.; Amersdorfer, P.; Smith, T. J.; Montgomery, V. A.; Sheridan, R.; Blake, R.; Smith, L. A.; Marks, J. D.

    2002-01-01

    The botulinum neurotoxins (BoNTs) cause the paralytic human disease botulism and are one of the highest-risk threat agents for bioterrorism. To generate a pharmaceutical to prevent or treat botulism, monoclonal antibodies (mAbs) were generated by phage display and evaluated for neutralization of BoNT serotype A (BoNT/A) in vivo. Although no single mAb significantly neutralized toxin, a combination of three mAbs (oligoclonal Ab) neutralized 450,000 50% lethal doses of BoNT/A, a potency 90 times greater than human hyperimmune globulin. The potency of oligoclonal Ab was primarily due to a large increase in functional Ab binding affinity. The results indicate that the potency of the polyclonal humoral immune response can be deconvoluted to a few mAbs binding nonoverlapping epitopes, providing a route to drugs for preventing and treating botulism and diseases caused by other pathogens and biologic threat agents. PMID:12177434

  16. Neutralization mechanism of a highly potent antibody against Zika virus

    PubMed Central

    Zhang, Shuijun; Kostyuchenko, Victor A.; Ng, Thiam-Seng; Lim, Xin-Ni; Ooi, Justin S. G.; Lambert, Sebastian; Tan, Ter Yong; Widman, Douglas G.; Shi, Jian; Baric, Ralph S.; Lok, Shee-Mei

    2016-01-01

    The rapid spread of Zika virus (ZIKV), which causes microcephaly and Guillain-Barré syndrome, signals an urgency to identify therapeutics. Recent efforts to rescreen dengue virus human antibodies for ZIKV cross-neutralization activity showed antibody C10 as one of the most potent. To investigate the ability of the antibody to block fusion, we determined the cryoEM structures of the C10-ZIKV complex at pH levels mimicking the extracellular (pH8.0), early (pH6.5) and late endosomal (pH5.0) environments. The 4.0 Å resolution pH8.0 complex structure shows that the antibody binds to E proteins residues at the intra-dimer interface, and the virus quaternary structure-dependent inter-dimer and inter-raft interfaces. At pH6.5, antibody C10 locks all virus surface E proteins, and at pH5.0, it locks the E protein raft structure, suggesting that it prevents the structural rearrangement of the E proteins during the fusion event—a vital step for infection. This suggests antibody C10 could be a good therapeutic candidate. PMID:27882950

  17. Intracellular Neutralization of Virus by Immunoglobulin A Antibodies

    NASA Astrophysics Data System (ADS)

    Mazanec, Mary B.; Kaetzel, Charlotte S.; Lamm, Michael E.; Fletcher, David; Nedrud, John G.

    1992-08-01

    IgA is thought to neutralize viruses at the epithelial surface of mucous membranes by preventing their attachment. Since IgA, a polymeric immunoglobulin, is transported through the lining of epithelial cells by the polymeric-immunoglobulin receptor and since viruses are obligate intracellular parasites, we hypothesized that IgA antibodies may also interfere with viral replication by binding to newly synthesized viral proteins within infected cells. Polarized monolayers of Madin-Darby canine kidney epithelial cells expressing the polymeric-immunoglobulin receptor were infected on the apical surface with Sendai virus. Anti-Sendai virus IgA monoclonal antibody delivered from the basolateral surface colocalized with viral protein within the cell, as documented by immunofluorescence. More importantly, anti-viral IgA reduced virus titers >1000-fold (P < 0.0001) in apical supernatants and >10-fold (P < 0.0001) in cell lysates from monolayers treated with anti-viral IgA compared with those treated with either anti-viral IgG or an irrelevant IgA monoclonal antibody. We believe that the differences in viral titers between cell layers treated with specific IgA, which enters the epithelial cell by binding to the polymeric-immunoglobulin receptor, and those treated with specific IgG, which does not enter the cells, or irrelevant IgA indicate that specific intracellular IgA antibodies can inhibit viral replication. Thus, in addition to the classical role of humoral antibodies in extracellular defense, IgA antibody may be able to neutralize microbial pathogens intracellularly, giving IgA a role in host defense that has traditionally been reserved for cell-mediated immunity.

  18. Structural basis for the antibody neutralization of Herpes simplex virus

    SciTech Connect

    Lee, Cheng-Chung; Lin, Li-Ling; Chan, Woan-Eng; Ko, Tzu-Ping; Lai, Jiann-Shiun; Wang, Andrew H.-J.

    2013-10-01

    The gD–E317-Fab complex crystal revealed the conformational epitope of human mAb E317 on HSV gD, providing a molecular basis for understanding the viral neutralization mechanism. Glycoprotein D (gD) of Herpes simplex virus (HSV) binds to a host cell surface receptor, which is required to trigger membrane fusion for virion entry into the host cell. gD has become a validated anti-HSV target for therapeutic antibody development. The highly inhibitory human monoclonal antibody E317 (mAb E317) was previously raised against HSV gD for viral neutralization. To understand the structural basis of antibody neutralization, crystals of the gD ectodomain bound to the E317 Fab domain were obtained. The structure of the complex reveals that E317 interacts with gD mainly through the heavy chain, which covers a large area for epitope recognition on gD, with a flexible N-terminal and C-terminal conformation. The epitope core structure maps to the external surface of gD, corresponding to the binding sites of two receptors, herpesvirus entry mediator (HVEM) and nectin-1, which mediate HSV infection. E317 directly recognizes the gD–nectin-1 interface and occludes the HVEM contact site of gD to block its binding to either receptor. The binding of E317 to gD also prohibits the formation of the N-terminal hairpin of gD for HVEM recognition. The major E317-binding site on gD overlaps with either the nectin-1-binding residues or the neutralizing antigenic sites identified thus far (Tyr38, Asp215, Arg222 and Phe223). The epitopes of gD for E317 binding are highly conserved between two types of human herpesvirus (HSV-1 and HSV-2). This study enables the virus-neutralizing epitopes to be correlated with the receptor-binding regions. The results further strengthen the previously demonstrated therapeutic and diagnostic potential of the E317 antibody.

  19. Aptamer-facilitated Protection of Oncolytic Virus from Neutralizing Antibodies

    PubMed Central

    Muharemagic, Darija; Zamay, Anna; Ghobadloo, Shahrokh M; Evgin, Laura; Savitskaya, Anna; Bell, John C; Berezovski, Maxim V

    2014-01-01

    Oncolytic viruses promise to significantly improve current cancer treatments through their tumor-selective replication and multimodal attack against cancer cells. However, one of the biggest setbacks for oncolytic virus therapy is the intravenous delivery of the virus, as it can be cleared from the bloodstream by neutralizing antibodies before it reaches the tumor cells. We have selected DNA aptamers against an oncolytic virus, vesicular stomatitis virus, using a competitive binding approach, as well as against the antigen binding fragment (Fab) of antivesicular stomatitis virus polyclonal antibodies, in order to shield the virus from nAbs and enhance its in vivo survival. We used flow cytometry to identify these aptamers and evaluated their efficiency to shield vesicular stomatitis virus in a cell-based plaque forming assay. These oligonucleotides were then modified to obtain multivalent binders, which led to a decrease of viral aggregation, an increase in its infectivity and an increase in its stability in serum. The aptamers were also incubated in nondiluted serum, showing their effectiveness under conditions mimicking those in vivo. With this approach, we were able to increase viral infectivity by more than 70% in the presence of neutralizing antibodies. Thus, this method has the potential to enhance the delivery of vesicular stomatitis virus through the bloodstream without compromising the patient's immune system. PMID:24892725

  20. Anti-idiotypic antibodies induce neutralizing antibodies to bovine herpesvirus 1.

    PubMed Central

    Srikumaran, S; Onisk, D V; Borca, M V; Nataraj, C; Zamb, T J

    1990-01-01

    A neutralizing murine monoclonal antibody (mAb) of the IgG2a isotype (MM-113), specific for bovine herpesvirus 1 (BHV-1) glycoprotein gIV, was used to develop anti-idiotypic antibodies (anti-Id) in a calf. The bovine anti-Id were isolated from the serum of the immunized calf by affinity chromatography on an MM-113-Sepharose column, followed by repeated adsorption on a murine IgG2a column. The anti-Id thus obtained specifically reacted with MM-113, but not with isotype-matched controls. They also inhibited the binding of MM-113 to BHV-1 in a concentration-dependent manner. Mice immunized with the anti-Id produced neutralizing antibodies to BHV-1. The anti-Id bound to cells permissive to BHV-1 in a cell-binding radioimmunoassay (RIA). PMID:2165998

  1. Towards HIV-1 remission: potential roles for broadly neutralizing antibodies

    PubMed Central

    Halper-Stromberg, Ariel; Nussenzweig, Michel C.

    2016-01-01

    Current antiretroviral drug therapies do not cure HIV-1 because they do not eliminate a pool of long-lived cells harboring immunologically silent but replication-competent proviruses — termed the latent reservoir. Eliminating this reservoir and stimulating the immune response to control infection in the absence of therapy remain important but unsolved goals of HIV-1 cure research. Recently discovered broadly neutralizing antibodies (bNAbs) exhibit remarkable breadth and potency in their ability to neutralize HIV-1 in vitro, and recent studies have demonstrated new therapeutic applications for passively administered bNAbs in vivo. This Review discusses the roles bNAbs might play in HIV-1 treatment regimens, including prevention, therapy, and cure. PMID:26752643

  2. Neutralizing antibodies against the peptide antibiotic AS-48: immunocytological studies.

    PubMed Central

    Maqueda, M; Gálvez, A; Martínez-Bueno, M; Guerra, I; Valdivia, E

    1993-01-01

    Antisera against the broad-spectrum peptide antibiotic AS-48 produced by Enterococcus faecalis were obtained from immunized rabbits. Appreciable antibody titers were obtained only after repeated immunization, suggesting a feeble antigenicity for AS-48. Upon incubation with AS-48, the antisera neutralized its bacteriolytic action on E. faecalis S-47, although the simultaneous addition of AS-48 and serum did not prevent lysis. Crude serum cross-reacted with outer envelope components of enterococci, although specific anti-AS-48 antibodies, purified by affinity chromatography, reacted only with AS-48-treated cells. Labelling with immunofluorescence and colloidal gold particles was carried out on sensitive and resistant bacterial species to determine the interaction of AS-48 with cell structures. Images PMID:8431014

  3. Elimination of HIV-1-infected cells by broadly neutralizing antibodies

    PubMed Central

    Bruel, Timothée; Guivel-Benhassine, Florence; Amraoui, Sonia; Malbec, Marine; Richard, Léa; Bourdic, Katia; Donahue, Daniel Aaron; Lorin, Valérie; Casartelli, Nicoletta; Noël, Nicolas; Lambotte, Olivier; Mouquet, Hugo; Schwartz, Olivier

    2016-01-01

    The Fc region of HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) is required for suppressing viraemia, through mechanisms which remain poorly understood. Here, we identify bNAbs that exert antibody-dependent cellular cytotoxicity (ADCC) in cell culture and kill HIV-1-infected lymphocytes through natural killer (NK) engagement. These antibodies target the CD4-binding site, the glycans/V3 and V1/V2 loops on gp120, or the gp41 moiety. The landscape of Env epitope exposure at the surface and the sensitivity of infected cells to ADCC vary considerably between viral strains. Efficient ADCC requires sustained cell surface binding of bNAbs to Env, and combining bNAbs allows a potent killing activity. Furthermore, reactivated infected cells from HIV-positive individuals expose heterogeneous Env epitope patterns, with levels that are often but not always sufficient to trigger killing by bNAbs. Our study delineates the parameters controlling ADCC activity of bNAbs, and supports the use of the most potent antibodies to clear the viral reservoir. PMID:26936020

  4. Interaction between hexon and L4-100K determines virus rescue and growth of hexon-chimeric recombinant Ad5 vectors

    PubMed Central

    Yan, Jingyi; Dong, Jianing; Wu, Jiaxin; Zhu, Rui; Wang, Zhen; Wang, Baoming; Wang, Lizheng; Wang, Zixuan; Zhang, Haihong; Wu, Hui; Yu, Bin; Kong, Wei; Yu, Xianghui

    2016-01-01

    The immunogenicity of recombinant adenovirus serotype 5 (rAd5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against hexon hypervariable regions (HVRs). Preexisting immunity can be circumvented by replacing HVRs of rAd5 hexon with those derived from alternate adenovirus serotypes. However, chimeric modification of rAd5 hexon HVRs tends to cause low packaging efficiency or low proliferation of rAd5 vectors, but the related mechanism remains unclear. In this study, several Ad5-based vectors with precise replacement of HVRs with those derived from Ad37 and Ad43 were generated. We first observed that a HVR-exchanged rAd5 vector displayed a higher efficacy of the recombinant virus rescue and growth improvement compared with the rAd5 vector, although most hexon-chimeric rAd5 vectors constructed by us and other groups have proven to be nonviable or growth defective. We therefore evaluated the structural stability of the chimeric hexons and their interactions with the L4-100K chaperone. We showed that the viability of hexon-chimeric Ad5 vectors was not attributed to the structural stability of the chimeric hexon, but rather to the hexon maturation which was assisted by L4-100K. Our results suggested that the intricate interaction between hexon and L4-100K would determine the virus rescue and proliferation efficiency of hexon-chimeric rAd5 vectors. PMID:26934960

  5. Interaction between hexon and L4-100K determines virus rescue and growth of hexon-chimeric recombinant Ad5 vectors.

    PubMed

    Yan, Jingyi; Dong, Jianing; Wu, Jiaxin; Zhu, Rui; Wang, Zhen; Wang, Baoming; Wang, Lizheng; Wang, Zixuan; Zhang, Haihong; Wu, Hui; Yu, Bin; Kong, Wei; Yu, Xianghui

    2016-03-03

    The immunogenicity of recombinant adenovirus serotype 5 (rAd5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against hexon hypervariable regions (HVRs). Preexisting immunity can be circumvented by replacing HVRs of rAd5 hexon with those derived from alternate adenovirus serotypes. However, chimeric modification of rAd5 hexon HVRs tends to cause low packaging efficiency or low proliferation of rAd5 vectors, but the related mechanism remains unclear. In this study, several Ad5-based vectors with precise replacement of HVRs with those derived from Ad37 and Ad43 were generated. We first observed that a HVR-exchanged rAd5 vector displayed a higher efficacy of the recombinant virus rescue and growth improvement compared with the rAd5 vector, although most hexon-chimeric rAd5 vectors constructed by us and other groups have proven to be nonviable or growth defective. We therefore evaluated the structural stability of the chimeric hexons and their interactions with the L4-100K chaperone. We showed that the viability of hexon-chimeric Ad5 vectors was not attributed to the structural stability of the chimeric hexon, but rather to the hexon maturation which was assisted by L4-100K. Our results suggested that the intricate interaction between hexon and L4-100K would determine the virus rescue and proliferation efficiency of hexon-chimeric rAd5 vectors.

  6. Structural basis of anthrax edema factor neutralization by a neutralizing antibody

    PubMed Central

    Makiya, Michelle; Dolan, Michael; Agulto, Liane; Purcell, Robert; Chen, Zhaochun

    2012-01-01

    Fine epitope mapping of EF13D, ahighly potent neutralizing monoclonal antibody specific for the anthrax edema factor (EF), was accomplished through random mutagenesis and yeast surface display. A yeast-displayed library of single point mutants of an EF domain III (DIII), comprising amino acids624-800, was constructed by random mutagenesis and screened for reduced binding to EF13D. With this method, residues Leu 667, Ser 668, Arg 671, and Arg 672 were identified as key residues important for EF13D binding. They form a contiguous patch on a solvent-exposed surface at one end of the four-helixbundle of DIII. Computational protein-protein docking experiments between an EF13D model and a crystal structure of EF indicate that the EF13D heavy chain complementarity-determining region 3 (HCDR3) is deeply buried within a hydrophobic cleft between two helices of DIII and interacts directly with residues Leu 667, Ser 668, Arg 671 and Arg 672, providing an explanation for the high binding affinity. In addition, they show that the HCDR3 binding site overlaps with the binding site of the N-terminal lobe of calmodulin (CaM), an EF enzymatic activator, consistent with a previous finding showing direct competition with CaM that results in neutralization of EF. Identifying the neutralization epitope of EF13D on EF improves our understanding of the neutralization mechanism and has implications for vaccine development. PMID:22155239

  7. Broadly neutralizing antibodies: An approach to control HIV-1 infection.

    PubMed

    Yaseen, Mahmoud Mohammad; Yaseen, Mohammad Mahmoud; Alqudah, Mohammad Ali

    2017-01-02

    Although available antiretroviral therapy (ART) has changed human immunodeficiency virus (HIV)-1 infection to a non-fatal chronic disease, the economic burden of lifelong therapy, severe adverse ART effects, daily ART adherence, and emergence of ART-resistant HIV-1 mutants require prospecting for alternative therapeutic modalities. Indeed, a growing body of evidence suggests that broadly neutralizing anti-HIV-1 antibodies (BNAbs) may offer one such feasible alternative. To evaluate their therapeutic potential in established HIV-1 infection, we sought to address recent advances in pre-clinical and clinical investigations in this area of HIV-1 research. In addition, we addressed the obstacles that may impede the success of such immunotherapeutic approach, suggested strategic solutions, and briefly compared this approach with the currently used ART to open new insights for potential future passive immunotherapy for HIV-1 infection.

  8. Broadly neutralizing antibodies developed by an HIV-positive elite neutralizer exact a replication fitness cost on the contemporaneous virus.

    PubMed

    Sather, D Noah; Carbonetti, Sara; Kehayia, Jenny; Kraft, Zane; Mikell, Iliyana; Scheid, Johannes F; Klein, Florian; Stamatatos, Leonidas

    2012-12-01

    Approximately 1% of those infected with HIV-1 develop broad and potent serum cross-neutralizing antibody activities. It is unknown whether or not the development of such immune responses affects the replication of the contemporaneous autologous virus. Here, we defined a pathway of autologous viral escape from contemporaneous potent and broad serum neutralizing antibodies developed by an elite HIV-1-positive (HIV-1(+)) neutralizer. These antibodies potently neutralize diverse isolates from different clades and target primarily the CD4-binding site (CD4-BS) of the viral envelope glycoprotein. Viral escape required mutations in the viral envelope glycoprotein which limited the accessibility of the CD4-binding site to the autologous broadly neutralizing anti-CD4-BS antibodies but which allowed the virus to infect cells by utilizing CD4 receptors on their surface. The acquisition of neutralization resistance, however, resulted in reduced cell entry potential and slower viral replication kinetics. Our results indicate that in vivo escape from autologous broadly neutralizing antibodies exacts fitness costs to HIV-1.

  9. The neutralization of interferons by antibody. I. Quantitative and theoretical analyses of the neutralization reaction in different bioassay systems.

    PubMed

    Grossberg, S E; Kawade, Y; Kohase, M; Yokoyama, H; Finter, N

    2001-09-01

    The highly specific ability of antibodies to inhibit the biologic activity of cytokines or other therapeutic proteins is widely used in research and a subject of increasing clinical importance. The need exists for a standardized approach to the reporting of neutralizing antibody potency soundly based on theoretical and practical considerations and tested by experimental data. Pursuant to the original studies of Kawade on the theoretical and functional aspects of neutralization of interferons (IFN), experimental data were obtained by different laboratories employing varied methodology to address two hypotheses concerning the nature of IFN neutralization reactions, based on a derived formula that allows expression of neutralizing power as the reduction of 10 laboratory units (LU)/ml to 1 LU/ml, the end point of most bioassays. Two hypotheses are posed: (1) antibody acts to neutralize a fixed amount of biologically active IFN molecules, or (2) antibody reduces IFN activity in a set ratio of added/residual biologically active IFN. The first, or fixed amount, hypothesis relates to the reactivity of high-affinity antibodies neutralizing equimolar amounts of antigen, whereas the second, or constant proportion, hypothesis postulates a reduction in the ratio of total added IFN to residual active IFN molecules, such as a low-affinity antibody might exhibit. Analyses of data of the neutralization of IFN-alpha and IFN-beta are presented, employing human polyclonal antibodies and murine monoclonal antibodies (mAb). The theoretical constructs of Kawade are extended in the Appendix and correlated with new experimental data in the text. The data clearly indicate that the low-antibody affinity, constant proportion hypothesis, rather than the high-antibody affinity, fixed amount hypothesis, is applicable, if the bioassay is sensitive to IFN. The findings presented here and in the following paper (pp. 743-755, this issue) taken together provide the basis for a standardized method of

  10. Neutralizing antibodies against rotavirus produced in transgenically labelled purple tomatoes.

    PubMed

    Juárez, Paloma; Presa, Silvia; Espí, Joaquín; Pineda, Benito; Antón, María T; Moreno, Vicente; Buesa, Javier; Granell, Antonio; Orzaez, Diego

    2012-04-01

    Edible fruits are inexpensive biofactories for human health-promoting molecules that can be ingested as crude extracts or partially purified formulations. We show here the production of a model human antibody for passive protection against the enteric pathogen rotavirus in transgenically labelled tomato fruits. Transgenic tomato plants expressing a recombinant human immunoglobulin A (hIgA_2A1) selected against the VP8* peptide of rotavirus SA11 strain were obtained. The amount of hIgA_2A1 protein reached 3.6 ± 0.8% of the total soluble protein in the fruit of the transformed plants. Minimally processed fruit-derived products suitable for oral intake showed anti-VP8* binding activity and strongly inhibited virus infection in an in vitro virus neutralization assay. In order to make tomatoes expressing hIgA_2A1 easily distinguishable from wild-type tomatoes, lines expressing hIgA_2A1 transgenes were sexually crossed with a transgenic tomato line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors, which confer purple colour to the fruit. Consequently, transgenically labelled purple tomato fruits expressing hIgA_2A1 have been developed. The resulting purple-coloured extracts from these fruits contain high levels of recombinant anti-rotavirus neutralizing human IgA in combination with increased amounts of health-promoting anthocyanins.

  11. Potent neutralizing monoclonal antibodies against Ebola virus infection

    PubMed Central

    Zhang, Qi; Gui, Miao; Niu, Xuefeng; He, Shihua; Wang, Ruoke; Feng, Yupeng; Kroeker, Andrea; Zuo, Yanan; Wang, Hua; Wang, Ying; Li, Jiade; Li, Chufang; Shi, Yi; Shi, Xuanling; Gao, George F.; Xiang, Ye; Qiu, Xiangguo; Chen, Ling; Zhang, Linqi

    2016-01-01

    Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection. PMID:27181584

  12. Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody.

    PubMed

    Derman, Yağmur; Selby, Katja; Miethe, Sebastian; Frenzel, André; Liu, Yvonne; Rasetti-Escargueil, Christine; Avril, Arnaud; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Thullier, Philippe; Sesardic, Dorothea; Lindström, Miia; Hust, Michael; Korkeala, Hannu

    2016-09-12

    Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans.

  13. Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody

    PubMed Central

    Derman, Yağmur; Selby, Katja; Miethe, Sebastian; Frenzel, André; Liu, Yvonne; Rasetti-Escargueil, Christine; Avril, Arnaud; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Thullier, Philippe; Sesardic, Dorothea; Lindström, Miia; Hust, Michael; Korkeala, Hannu

    2016-01-01

    Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans. PMID:27626446

  14. HIV-1 envelope glycoprotein immunogens to induce broadly neutralizing antibodies.

    PubMed

    Sliepen, Kwinten; Sanders, Rogier W

    2016-01-01

    The long pursuit for a vaccine against human immunodeficiency virus 1 (HIV-1) has recently been boosted by a number of exciting developments. An HIV-1 subunit vaccine ideally should elicit potent broadly neutralizing antibodies (bNAbs), but raising bNAbs by vaccination has proved extremely difficult because of the characteristics of the HIV-1 envelope glycoprotein complex (Env). However, the isolation of bNAbs from HIV-1-infected patients demonstrates that the human humoral immune system is capable of making such antibodies. Therefore, a focus of HIV-1 vaccinology is the elicitation of bNAbs by engineered immunogens and by using vaccination strategies aimed at mimicking the bNAb maturation pathways in HIV-infected patients. Important clues can also be taken from the successful subunit vaccines against hepatitis B virus and human papillomavirus. Here, we review the different types of HIV-1 immunogens and vaccination strategies that are being explored in the search for an HIV-1 vaccine that induces bNAbs.

  15. Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1.

    PubMed Central

    Collins, J K; Butcher, A C; Riegel, C A; McGrane, V; Blair, C D; Teramoto, Y A; Winston, S

    1984-01-01

    Monoclonal antibodies were used to study neutralizing determinants on polypeptides of bovine herpesvirus 1. Two of three monoclonal antibodies which recognized nonoverlapping epitopes on a glycoprotein of 82,000 daltons were found to neutralize. A second group of monoclonal antibodies that individually precipitated five viral glycopolypeptides ranging in size from 102,000 to 55,000 daltons also neutralized. Two monoclonal antibodies which were the most efficient in neutralization recognized a non-glycosylated protein of 115,000 daltons which was the major polypeptide on the virus. A fourth group of monoclonal antibodies precipitated a non-glycosylated polypeptide of 91,000 daltons and several smaller polypeptides, but these antibodies demonstrated only limited neutralizing activity. Images PMID:6208375

  16. Trimerization of the HIV Transmembrane Domain in Lipid Bilayers Modulates Broadly Neutralizing Antibody Binding.

    PubMed

    Reichart, Timothy M; Baksh, Michael M; Rhee, Jin-Kyu; Fiedler, Jason D; Sligar, Stephen G; Finn, M G; Zwick, Michael B; Dawson, Philip E

    2016-02-18

    The membrane-proximal external region (MPER) of HIV gp41 is an established target of antibodies that neutralize a broad range of HIV isolates. To evaluate the role of the transmembrane (TM) domain, synthetic MPER-derived peptides were incorporated into lipid nanoparticles using natural and designed TM domains, and antibody affinity was measured using immobilized and solution-based techniques. Peptides incorporating the native HIV TM domain exhibit significantly stronger interactions with neutralizing antibodies than peptides with a monomeric TM domain. Furthermore, a peptide with a trimeric, three-helix bundle TM domain recapitulates the binding profile of the native sequence. These studies suggest that neutralizing antibodies can bind the MPER when the TM domain is a three-helix bundle and this presentation could influence the binding of neutralizing antibodies to the virus. Lipid-bilayer presentation of viral antigens in Nanodiscs is a new platform for evaluating neutralizing antibodies.

  17. In vitro neutralization of prions with PrP(Sc)-specific antibodies.

    PubMed

    Taschuk, Ryan; Van der Merwe, Jacques; Marciniuk, Kristen; Potter, Andrew; Cashman, Neil; Griebel, Philip; Napper, Scott

    2015-01-01

    Prion diseases reflect the misfolding of a self-protein (PrP(C)) into an infectious, pathological isomer (PrP(Sc)). By targeting epitopes uniquely exposed by misfolding, our group developed PrP(Sc)-specific vaccines to 3 disease specific epitopes (DSEs). Here, antibodies induced by individual DSE vaccines are evaluated for their capacity to neutralize prions in vitro. For both purified antibodies and immunoreactive sera, the PrP(Sc)-specific antibodies were equally effective in neutralizing prions. Further, there was no significant increase in neutralizing activity when multiple DSEs were targeted within an assay. At a low antibody concentration, the PrP(Sc)-specific antibodies matched the neutralization achieved by an antibody that may act via both PrP(C) and PrP(Sc). At higher doses, however, this pan-specific antibody was more effective, potentially due to a combined deactivation of PrP(Sc) and depletion of PrP(C).

  18. In vitro neutralization of prions with PrPSc-specific antibodies

    PubMed Central

    Taschuk, Ryan; Van der Merwe, Jacques; Marciniuk, Kristen; Potter, Andrew; Cashman, Neil; Griebel, Philip; Napper, Scott

    2015-01-01

    Abstract Prion diseases reflect the misfolding of a self-protein (PrPC) into an infectious, pathological isomer (PrPSc). By targeting epitopes uniquely exposed by misfolding, our group developed PrPSc-specific vaccines to 3 disease specific epitopes (DSEs). Here, antibodies induced by individual DSE vaccines are evaluated for their capacity to neutralize prions in vitro. For both purified antibodies and immunoreactive sera, the PrPSc-specific antibodies were equally effective in neutralizing prions. Further, there was no significant increase in neutralizing activity when multiple DSEs were targeted within an assay. At a low antibody concentration, the PrPSc-specific antibodies matched the neutralization achieved by an antibody that may act via both PrPC and PrPSc. At higher doses, however, this pan-specific antibody was more effective, potentially due to a combined deactivation of PrPSc and depletion of PrPC. PMID:26284508

  19. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

    PubMed

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I

    2015-11-01

    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function.

  20. Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-Specific Monoclonal Antibodies

    PubMed Central

    von Bredow, Benjamin; Arias, Juan F.; Heyer, Lisa N.; Moldt, Brian; Le, Khoa; Robinson, James E.; Burton, Dennis R.

    2016-01-01

    ABSTRACT Although antibodies to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data are currently available on nonneutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and nonneutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus (SHIV) adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO. ADCC activity generally correlated with antibody binding to Env on the surfaces of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection. IMPORTANCE This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection. PMID:27122574

  1. Pharmacokinetics and pharmacodynamics of VEGF-neutralizing antibodies

    PubMed Central

    2011-01-01

    Background Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis, and its role in cancer biology has been widely studied. Many cancer therapies target angiogenesis, with a focus being on VEGF-mediated signaling such as antibodies to VEGF. However, it is difficult to predict the effects of VEGF-neutralizing agents. We have developed a whole-body model of VEGF kinetics and transport under pathological conditions (in the presence of breast tumor). The model includes two major VEGF isoforms VEGF121 and VEGF165, receptors VEGFR1, VEGFR2 and co-receptors Neuropilin-1 and Neuropilin-2. We have added receptors on parenchymal cells (muscle fibers and tumor cells), and incorporated experimental data for the cell surface density of receptors on the endothelial cells, myocytes, and tumor cells. The model is applied to investigate the action of VEGF-neutralizing agents (called "anti-VEGF") in the treatment of cancer. Results Through a sensitivity study, we examine how model parameters influence the level of free VEGF in the tumor, a measure of the response to VEGF-neutralizing drugs. We investigate the effects of systemic properties such as microvascular permeability and lymphatic flow, and of drug characteristics such as the clearance rate and binding affinity. We predict that increasing microvascular permeability in the tumor above 10-5 cm/s elicits the undesired effect of increasing tumor interstitial VEGF concentration beyond even the baseline level. We also examine the impact of the tumor microenvironment, including receptor expression and internalization, as well as VEGF secretion. We find that following anti-VEGF treatment, the concentration of free VEGF in the tumor can vary between 7 and 233 pM, with a dependence on both the density of VEGF receptors and co-receptors and the rate of neuropilin internalization on tumor cells. Finally, we predict that free VEGF in the tumor is reduced following anti-VEGF treatment when VEGF121 comprises at least

  2. Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

    SciTech Connect

    Moody, M.  Anthony; Gao, Feng; Gurley, Thaddeus  C.; Amos, Joshua  D.; Kumar, Amit; Hora, Bhavna; Marshall, Dawn  J.; Whitesides, John  F.; Xia, Shi-Mao; Parks, Robert; Lloyd, Krissey  E.; Hwang, Kwan-Ki; Lu, Xiaozhi; Bonsignori, Mattia; Finzi, Andrés; Vandergrift, Nathan  A.; Alam, S.  Munir; Ferrari, Guido; Shen, Xiaoying; Tomaras, Georgia  D.; Kamanga, Gift; Cohen, Myron  S.; Sam, Noel  E.; Kapiga, Saidi; Gray, Elin S.; Tumba, Nancy  L.; Morris, Lynn; Zolla-Pazner, Susan; Gorny, Miroslaw  K.; Mascola, John  R.; Hahn, Beatrice H.; Shaw, George  M.; Sodroski, Joseph  G.; Liao, Hua-Xin; Montefiori, David C.; Hraber, Peter T.; Korber, Bette T.; Haynes, Barton F.

    2015-09-09

    The third variable (V3) loop and the CD4 binding site (CD4bs) of the viral envelope are frequently targeted by neutralizing antibodies (nAbs) in HIV-1-infected individuals. In chronic infection, virus escape mutants repopulate the plasma and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.

  3. Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

    DOE PAGES

    Moody, M.  Anthony; Gao, Feng; Gurley, Thaddeus  C.; ...

    2015-09-09

    The third variable (V3) loop and the CD4 binding site (CD4bs) of the viral envelope are frequently targeted by neutralizing antibodies (nAbs) in HIV-1-infected individuals. In chronic infection, virus escape mutants repopulate the plasma and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tiermore » 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.« less

  4. Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses.

    PubMed

    Moody, M Anthony; Gao, Feng; Gurley, Thaddeus C; Amos, Joshua D; Kumar, Amit; Hora, Bhavna; Marshall, Dawn J; Whitesides, John F; Xia, Shi-Mao; Parks, Robert; Lloyd, Krissey E; Hwang, Kwan-Ki; Lu, Xiaozhi; Bonsignori, Mattia; Finzi, Andrés; Vandergrift, Nathan A; Alam, S Munir; Ferrari, Guido; Shen, Xiaoying; Tomaras, Georgia D; Kamanga, Gift; Cohen, Myron S; Sam, Noel E; Kapiga, Saidi; Gray, Elin S; Tumba, Nancy L; Morris, Lynn; Zolla-Pazner, Susan; Gorny, Miroslaw K; Mascola, John R; Hahn, Beatrice H; Shaw, George M; Sodroski, Joseph G; Liao, Hua-Xin; Montefiori, David C; Hraber, Peter T; Korber, Bette T; Haynes, Barton F

    2015-09-09

    The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3 and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.

  5. Detection of Bovine viral diarrhea virus-specific neutralizing antibodies in fresh colostrum: a modification of the virus neutralization test.

    PubMed

    Bedekovic, Tomislav; Mihaljevic, Zeljko; Jungic, Andreja; Lemo, Nina; Lojkic, Ivana; Cvetnic, Zeljko; Cac, Zeljko

    2013-03-01

    To eliminate cytotoxic effects of colostrum on cells, a modified virus neutralization test (VNT) for the detection of Bovine viral diarrhea virus-specific neutralizing antibodies in colostrum was developed. The new test was compared to the World Organization for Animal Health-recommended VNT and the results evaluated. The agreement of the new test compared to the standard VNT was determined to be 98%, whereas sensitivity and specificity of the modified VNT compared to the standard VNT were 100%. Bovine viral diarrhea virus-specific antibodies were detected in 42 sera samples and 38 colostrum samples. The antibody titers in serum and colostrum showed a high correlation (n = 56, r = 0.9719, P < 0.001). The modified virus neutralization technique described herein succeeds in eliminating cytotoxic effects and can be readily applied for the detection of specific antibodies against other infectious agents in colostrum.

  6. Vaccine Elicitation of High Mannose-Dependent Neutralizing Antibodies against the V3-Glycan Broadly Neutralizing Epitope in Nonhuman Primates.

    PubMed

    Saunders, Kevin O; Nicely, Nathan I; Wiehe, Kevin; Bonsignori, Mattia; Meyerhoff, R Ryan; Parks, Robert; Walkowicz, William E; Aussedat, Baptiste; Wu, Nelson R; Cai, Fangping; Vohra, Yusuf; Park, Peter K; Eaton, Amanda; Go, Eden P; Sutherland, Laura L; Scearce, Richard M; Barouch, Dan H; Zhang, Ruijun; Von Holle, Tarra; Overman, R Glenn; Anasti, Kara; Sanders, Rogier W; Moody, M Anthony; Kepler, Thomas B; Korber, Bette; Desaire, Heather; Santra, Sampa; Letvin, Norman L; Nabel, Gary J; Montefiori, David C; Tomaras, Georgia D; Liao, Hua-Xin; Alam, S Munir; Danishefsky, Samuel J; Haynes, Barton F

    2017-02-28

    Induction of broadly neutralizing antibodies (bnAbs) that target HIV-1 envelope (Env) is a goal of HIV-1 vaccine development. A bnAb target is the Env third variable loop (V3)-glycan site. To determine whether immunization could induce antibodies to the V3-glycan bnAb binding site, we repetitively immunized macaques over a 4-year period with an Env expressing V3-high mannose glycans. Env immunizations elicited plasma antibodies that neutralized HIV-1 expressing only high-mannose glycans-a characteristic shared by early bnAb B cell lineage members. A rhesus recombinant monoclonal antibody from a vaccinated macaque bound to the V3-glycan site at the same amino acids as broadly neutralizing antibodies. A structure of the antibody bound to glycan revealed that the three variable heavy-chain complementarity-determining regions formed a cavity into which glycan could insert and neutralized multiple HIV-1 isolates with high-mannose glycans. Thus, HIV-1 Env vaccination induced mannose-dependent antibodies with characteristics of V3-glycan bnAb precursors.

  7. Stability of isolated antibody-antigen complexes as a predictive tool for selecting toxin neutralizing antibodies

    PubMed Central

    Legler, Patricia M.; Compton, Jaimee R.; Hale, Martha L.; Anderson, George P.; Olson, Mark A.; Millard, Charles B.; Goldman, Ellen R.

    2017-01-01

    ABSTRACT Ricin is an A-B ribosome inactivating protein (RIP) toxin composed of an A-chain subunit (RTA) that contains a catalytic N-glycosidase and a B-chain (RTB) lectin domain that binds cell surface glycans. Ricin exploits retrograde transport to enter into the Golgi and the endoplasmic reticulum, and then dislocates into the cytoplasm where it can reach its substrate, the rRNA. A subset of isolated antibodies (Abs) raised against the RTA subunit protect against ricin intoxication, and RTA-based vaccine immunogens have been shown to provide long-lasting protective immunity against the holotoxin. Anti-RTA Abs are unlikely to cross a membrane and reach the cytoplasm to inhibit the enzymatic activity of the A-chain. Moreover, there is not a strict correlation between the apparent binding affinity (Ka) of anti-RTA Abs and their ability to successfully neutralize ricin toxicity. Some anti-RTA antibodies are toxin-neutralizing, whereas others are not. We hypothesize that neutralizing anti-RTA Abs may interfere selectively with conformational change(s) or partial unfolding required for toxin internalization. To test this hypothesis, we measured the melting temperatures (Tm) of neutralizing single-domain Ab (sdAb)-antigen (Ag) complexes relative to the Tm of the free antigen (Tm-shift = Tmcomplex – TmAg), and observed increases in the Tmcomplex of 9–20 degrees. In contrast, non-neutralizing sdAb-Ag complexes shifted the TmComplex by only 6–7 degrees. A strong linear correlation (r2 = 0.992) was observed between the magnitude of the Tm-shift and the viability of living cells treated with the sdAb and ricin holotoxin. The Tm-shift of the sdAb-Ag complex provided a quantitative biophysical parameter that could be used to predict and rank-order the toxin-neutralizing activities of Abs. We determined the first structure of an sdAb-RTA1-33/44-198 complex, and examined other sdAb-RTA complexes. We found that neutralizing sdAb bound to regions involved in the early stages

  8. Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones

    PubMed Central

    He, Wenqian; Mullarkey, Caitlin E.; Duty, J. Andrew; Moran, Thomas M.; Palese, Peter

    2015-01-01

    ABSTRACT Current influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of “universal” influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior in vitro but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during “universal” influenza virus vaccine design. IMPORTANCE Influenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the

  9. Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus.

    PubMed

    Liao, Hua-Xin; Lynch, Rebecca; Zhou, Tongqing; Gao, Feng; Alam, S Munir; Boyd, Scott D; Fire, Andrew Z; Roskin, Krishna M; Schramm, Chaim A; Zhang, Zhenhai; Zhu, Jiang; Shapiro, Lawrence; Mullikin, James C; Gnanakaran, S; Hraber, Peter; Wiehe, Kevin; Kelsoe, Garnett; Yang, Guang; Xia, Shi-Mao; Montefiori, David C; Parks, Robert; Lloyd, Krissey E; Scearce, Richard M; Soderberg, Kelly A; Cohen, Myron; Kamanga, Gift; Louder, Mark K; Tran, Lillian M; Chen, Yue; Cai, Fangping; Chen, Sheri; Moquin, Stephanie; Du, Xiulian; Joyce, M Gordon; Srivatsan, Sanjay; Zhang, Baoshan; Zheng, Anqi; Shaw, George M; Hahn, Beatrice H; Kepler, Thomas B; Korber, Bette T M; Kwong, Peter D; Mascola, John R; Haynes, Barton F

    2013-04-25

    Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.

  10. Cross-neutralizing human anti-poliovirus antibodies bind the recognition site for cellular receptor

    PubMed Central

    Chen, Zhaochun; Fischer, Elizabeth R.; Kouiavskaia, Diana; Hansen, Bryan T.; Ludtke, Steven J.; Bidzhieva, Bella; Makiya, Michelle; Agulto, Liane; Purcell, Robert H.; Chumakov, Konstantin

    2013-01-01

    Most structural information about poliovirus interaction with neutralizing antibodies was obtained in the 1980s in studies of mouse monoclonal antibodies. Recently we have isolated a number of human/chimpanzee anti-poliovirus antibodies and demonstrated that one of them, MAb A12, could neutralize polioviruses of both serotypes 1 and 2. This communication presents data on isolation of an additional cross-neutralizing antibody (F12) and identification of a previously unknown epitope on the surface of poliovirus virions. Epitope mapping was performed by sequencing of antibody-resistant mutants and by cryo-EM of complexes of virions with Fab fragments. The results have demonstrated that both cross-neutralizing antibodies bind the site located at the bottom of the canyon surrounding the fivefold axis of symmetry that was previously shown to interact with cellular poliovirus receptor CD155. However, the same antibody binds to serotypes 1 and 2 through different specific interactions. It was also shown to interact with type 3 poliovirus, albeit with about 10-fold lower affinity, insufficient for effective neutralization. Antibody interaction with the binding site of the cellular receptor may explain its broad reactivity and suggest that further screening or antibody engineering could lead to a universal antibody capable of neutralizing all three serotypes of poliovirus. PMID:24277851

  11. Neutralizing Antibody Blocks Adenovirus Infection by Arresting Microtubule-Dependent Cytoplasmic Transport▿

    PubMed Central

    Smith, Jason G.; Cassany, Aurelia; Gerace, Larry; Ralston, Robert; Nemerow, Glen R.

    2008-01-01

    Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry. PMID:18448546

  12. Neutralizing antibody blocks adenovirus infection by arresting microtubule-dependent cytoplasmic transport.

    PubMed

    Smith, Jason G; Cassany, Aurelia; Gerace, Larry; Ralston, Robert; Nemerow, Glen R

    2008-07-01

    Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry.

  13. Bispecific Antibodies Targeting Different Epitopes on the HIV-1 Envelope Exhibit Broad and Potent Neutralization

    PubMed Central

    Asokan, M.; Rudicell, R. S.; Louder, M.; McKee, K.; O'Dell, S.; Stewart-Jones, G.; Wang, K.; Xu, L.; Chen, X.; Choe, M.; Chuang, G.; Georgiev, I. S.; Joyce, M. G.; Kirys, T.; Ko, S.; Pegu, A.; Shi, W.; Todd, J. P.; Yang, Z.; Bailer, R. T.; Rao, S.; Kwong, P. D.; Nabel, G. J.

    2015-01-01

    ABSTRACT The potency and breadth of the recently isolated neutralizing human monoclonal antibodies to HIV-1 have stimulated interest in their use to prevent or to treat HIV-1 infection. Due to the antigenically diverse nature of the HIV-1 envelope (Env), no single antibody is highly active against all viral strains. While the physical combination of two broadly neutralizing antibodies (bNAbs) can improve coverage against the majority of viruses, the clinical-grade manufacturing and testing of two independent antibody products are time and resource intensive. In this study, we constructed bispecific immunoglobulins (IgGs) composed of independent antigen-binding fragments with a common Fc region. We developed four different bispecific IgG variants that included antibodies targeting four major sites of HIV-1 neutralization. We show that these bispecific IgGs display features of both antibody specificities and, in some cases, display improved coverage over the individual parental antibodies. All four bispecific IgGs neutralized 94% to 97% of antigenically diverse viruses in a panel of 206 HIV-1 strains. Among the bispecific IgGs tested, VRC07 × PG9-16 displayed the most favorable neutralization profile. It was superior in breadth to either of the individual antibodies, neutralizing 97% of viruses with a median 50% inhibitory concentration (IC50) of 0.055 μg/ml. This bispecific IgG also demonstrated in vivo pharmacokinetic parameters comparable to those of the parental bNAbs when administered to rhesus macaques. These results suggest that IgG-based bispecific antibodies are promising candidates for the prevention and treatment of HIV-1 infection in humans. IMPORTANCE To prevent or treat HIV-1 infection, antibodies must potently neutralize nearly all strains of HIV-1. Thus, the physical combination of two or more antibodies may be needed to broaden neutralization coverage and diminish the possibility of viral resistance. A bispecific antibody that has two different

  14. Envelope Variants Circulating as Initial Neutralization Breadth Developed in Two HIV-Infected Subjects Stimulate Multiclade Neutralizing Antibodies in Rabbits

    PubMed Central

    Malherbe, Delphine C.; Pissani, Franco; Sather, D. Noah; Guo, Biwei; Pandey, Shilpi; Sutton, William F.; Stuart, Andrew B.; Robins, Harlan; Park, Byung; Krebs, Shelly J.; Schuman, Jason T.; Kalams, Spyros; Hessell, Ann J.

    2014-01-01

    ABSTRACT Identifying characteristics of the human immunodeficiency virus type 1 (HIV-1) envelope that are effective in generating broad, protective antibodies remains a hurdle to HIV vaccine design. Emerging evidence of the development of broad and potent neutralizing antibodies in HIV-infected subjects suggests that founder and subsequent progeny viruses may express unique antigenic motifs that contribute to this developmental pathway. We hypothesize that over the course of natural infection, B cells are programmed to develop broad antibodies by exposure to select populations of emerging envelope quasispecies variants. To test this hypothesis, we identified two unrelated subjects whose antibodies demonstrated increasing neutralization breadth against a panel of HIV-1 isolates over time. Full-length functional env genes were cloned longitudinally from these subjects from months after infection through 2.6 to 5.8 years of infection. Motifs associated with the development of breadth in published, cross-sectional studies were found in both subjects. We compared the immunogenicity of envelope vaccines derived from time points obtained during and after broadening of neutralization activity within these subjects. Rabbits were coimmunized four times with selected multiple gp160 DNAs and gp140-trimeric envelope proteins. The affinity of the polyclonal response increased as a function of boosting. The most rapid and persistent neutralization of multiclade tier 1 viruses was elicited by envelopes that were circulating in plasma at time points prior to the development of 50% neutralization breadth in both human subjects. The breadth elicited in rabbits was not improved by exposure to later envelope variants. These data have implications for vaccine development in describing a target time point to identify optimal envelope immunogens. IMPORTANCE Vaccine protection against viral infections correlates with the presence of neutralizing antibodies; thus, vaccine components capable

  15. Irreproducibility of neutralization of herpes simplex virus under conditions where antibody is not in excess.

    PubMed

    Shariff, D; Hallworth, J A; Buchan, A; Skinner, G R

    1987-01-01

    Neutralizing activity against herpes simplex virus was significantly reduced if initial virus titers were greater than 10(6) PFU/ml; there was no significant neutralization when initial virus titers approached 10(8) PFU/ml. This was a result of utilization of all available antibody by virus particles and 'free' virus antigen and emphasizes the importance of conducting virus neutralization tests under conditions of antibody excess.

  16. Paradoxical suppression of poly-specific broadly neutralizing antibodies in the presence of strain-specific neutralizing antibodies following HIV infection.

    PubMed

    Ciupe, Stanca M; De Leenheer, Patrick; Kepler, Thomas B

    2011-05-21

    One of the first immunologic responses against HIV infection is the presence of neutralizing antibodies that seem able to inactivate several HIV strains. Moreover, in vitro studies have shown the existence of monoclonal antibodies that exhibit broad crossclade neutralizing potential. Yet their number is low and slow to develop in vivo. In this paper, we investigate the potential benefits of inducing poly-specific neutralizing antibodies in vivo throughout immunization. We develop a mathematical model that considers the activation of families of B lymphocytes producing poly-specific and strain-specific antibodies and use it to demonstrate that, even if such families are successful in producing neutralizing antibodies, the competition between them may limit the poly-specific response allowing the virus to escape. We modify this model to account for viral evolution under the pressure of antibody responses in natural HIV infection. The model can reproduce viral escape under certain conditions of B lymphocyte competition. Using these models we provide explanations for the observed antibody failure in controlling natural infection and predict quantitative measures that need to be satisfied for long-term control of HIV infection.

  17. Evaluation of the impact of neutralizing antibodies on IFNβ response.

    PubMed

    Bertolotto, Antonio

    2015-09-20

    IFNβ therapeutic action depends on a sequence of biological steps: i) the interaction between interferon beta (IFNβ) and its receptor (IFNAR) located at the cell surface of peripheral blood mononuclear cells; ii) activation of second messengers; iii) transcription of several genes containing specific ISRE regions (Interferon Stimulated Response Elements); and iv) synthesis of specific proteins. Although IFNβ therapy has improved treatment options of patients with multiple sclerosis (MS), the long-term efficacy of IFNβs can be compromised due to the development of neutralizing antibodies (NAbs). High titer NAbs develop in about 15% of patients; they abolish IFNβ biological activity and consequently the therapeutic action of IFNβ. Different IFNβ preparations carry different risks of developing NAbs, ranging from 3 to 28%. The risk of inducing NAbs must be considered in the selection of treatment. Guidelines for NAbs testing and the therapeutic decision in case of NAbs positivity have been established. NAbs positivity predicts MRI and clinical activity. Precocious identification of Nabs-positive patients and switch to alternative treatments can improve the percentage of responders and allow a better allocation of relevant economical resources.

  18. Focused Evolution of HIV-1 Neutralizing Antibodies Revealed by Structures and Deep Sequencing

    SciTech Connect

    Wu, Xueling; Zhou, Tongqing; Zhu, Jiang; Zhang, Baoshan; Georgiev, Ivelin; Wang, Charlene; Chen, Xuejun; Longo, Nancy S.; Louder, Mark; McKee, Krisha; O’Dell, Sijy; Perfetto, Stephen; Schmidt, Stephen D.; Shi, Wei; Wu, Lan; Yang, Yongping; Yang, Zhi-Yong; Yang, Zhongjia; Zhang, Zhenhai; Bonsignori, Mattia; Crump, John A.; Kapiga, Saidi H.; Sam, Noel E.; Haynes, Barton F.; Simek, Melissa; Burton, Dennis R.; Koff, Wayne C.; Doria-Rose, Nicole A.; Connors, Mark; Mullikin, James C.; Nabel, Gary J.; Roederer, Mario; Shapiro, Lawrence; Kwong, Peter D.; Mascola, John R.

    2013-03-04

    Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.

  19. Neutralization of Plasmodium falciparum merozoites by antibodies against PfRH5

    PubMed Central

    Douglas, Alexander D.; Williams, Andrew R.; Knuepfer, Ellen; Illingworth, Joseph J.; Furze, Julie M.; Crosnier, Cécile; Choudhary, Prateek; Bustamante, Leyla Y.; Zakutansky, Sara E.; Awuah, Dennis K.; Alanine, Daniel G. W.; Theron, Michel; Worth, Andrew; Shimkets, Richard; Rayner, Julian C.; Holder, Anthony A.; Wright, Gavin J.; Draper, Simon J.

    2013-01-01

    There is intense interest in induction and characterization of strain-transcending neutralizing antibody against antigenically variable human pathogens. We have recently identified the human malaria parasite Plasmodium falciparum reticulocyte-binding protein homologue 5 (PfRH5) as a target of broadly-neutralizing antibodies, but there is little information regarding the functional mechanism(s) of antibody-mediated neutralization. Here, we report that vaccine-induced polyclonal anti-PfRH5 antibodies inhibit the tight attachment of merozoites to erythrocytes, and are capable of blocking the interaction of PfRH5 with its receptor basigin. Furthermore, by developing anti-PfRH5 monoclonal antibodies (mAbs), we provide evidence that i) the ability to block the PfRH5-basigin interaction in vitro is predictive of functional activity, but absence of blockade does not predict absence of functional activity; ii) neutralizing mAbs bind spatially-related epitopes on the folded protein, involving at least two defined regions of the PfRH5 primary sequence; iii) a brief exposure window of PfRH5 is likely to necessitate rapid binding of antibody to neutralize parasites; and iv) intact bivalent IgG contributes to but is not necessary for parasite neutralization. These data provide important insight into the mechanisms of broadly-neutralizing anti-malaria antibodies and further encourage anti-PfRH5 based malaria prevention efforts. PMID:24293631

  20. Signature Biochemical Properties of Broadly Cross-Reactive HIV-1 Neutralizing Antibodies in Human Plasma

    PubMed Central

    Sajadi, Mohammad M.; Lewis, George K.; Seaman, Michael S.; Guan, Yongjun; Redfield, Robert R.

    2012-01-01

    The common properties of broadly cross-reactive HIV-1 neutralization antibodies found in certain HIV-1-infected individuals holds significant value for understanding natural and vaccine-mediated anti-HIV immunity. Recent efforts have addressed this question by deriving neutralizing monoclonal anti-envelope antibodies from memory B cell pools of selected subjects. However, it has been more difficult to identify whether broadly neutralizing antibodies circulating in plasma possess shared characteristics among individuals. To address this question, we used affinity chromatography and isoelectric focusing to fractionate plasma immunoglobulin from 10 HIV-1-infected subjects (5 subjects with broad HIV-1 neutralizing activity and 5 controls). We find that plasma neutralizing activity typically partitions into at least two subsets of antibodies. Antibodies with restricted neutralization breadth have relatively neutral isoelectric points and preferentially bind to envelope monomers and trimers versus core antigens from which variable loops and other domains have been deleted. In comparison, broadly neutralizing antibodies account for a minor fraction of the total anti-envelope response. They are consistently distinguished by more basic isoelectric points and specificity for epitopes shared by monomeric gp120, gp120 core, or CD4-induced structures. Such biochemical properties might be exploited to reliably predict or produce broad anti-HIV immunity. PMID:22379105

  1. Signature biochemical properties of broadly cross-reactive HIV-1 neutralizing antibodies in human plasma.

    PubMed

    Sajadi, Mohammad M; Lewis, George K; Seaman, Michael S; Guan, Yongjun; Redfield, Robert R; DeVico, Anthony L

    2012-05-01

    The common properties of broadly cross-reactive HIV-1 neutralization antibodies found in certain HIV-1-infected individuals holds significant value for understanding natural and vaccine-mediated anti-HIV immunity. Recent efforts have addressed this question by deriving neutralizing monoclonal anti-envelope antibodies from memory B cell pools of selected subjects. However, it has been more difficult to identify whether broadly neutralizing antibodies circulating in plasma possess shared characteristics among individuals. To address this question, we used affinity chromatography and isoelectric focusing to fractionate plasma immunoglobulin from 10 HIV-1-infected subjects (5 subjects with broad HIV-1 neutralizing activity and 5 controls). We find that plasma neutralizing activity typically partitions into at least two subsets of antibodies. Antibodies with restricted neutralization breadth have relatively neutral isoelectric points and preferentially bind to envelope monomers and trimers versus core antigens from which variable loops and other domains have been deleted. In comparison, broadly neutralizing antibodies account for a minor fraction of the total anti-envelope response. They are consistently distinguished by more basic isoelectric points and specificity for epitopes shared by monomeric gp120, gp120 core, or CD4-induced structures. Such biochemical properties might be exploited to reliably predict or produce broad anti-HIV immunity.

  2. A humanized neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein.

    PubMed

    Li, Yan; Wan, Yuhua; Liu, Peipei; Zhao, Jincun; Lu, Guangwen; Qi, Jianxun; Wang, Qihui; Lu, Xuancheng; Wu, Ying; Liu, Wenjun; Zhang, Buchang; Yuen, Kwok-Yung; Perlman, Stanley; Gao, George F; Yan, Jinghua

    2015-11-01

    The newly-emerging Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans. Despite global efforts, the potential for an associated pandemic in the future cannot be excluded. The development of effective counter-measures is urgent. MERS-CoV-specific anti-viral drugs or vaccines are not yet available. Using the spike receptor-binding domain of MERS-CoV (MERS-RBD) to immunize mice, we identified two neutralizing monoclonal antibodies (mAbs) 4C2 and 2E6. Both mAbs potently bind to MERS-RBD and block virus entry in vitro with high efficacy. We further investigated their mechanisms of neutralization by crystallizing the complex between the Fab fragments and the RBD, and solved the structure of the 4C2 Fab/MERS-RBD complex. The structure showed that 4C2 recognizes an epitope that partially overlaps the receptor-binding footprint in MERS-RBD, thereby interfering with the virus/receptor interactions by both steric hindrance and interface-residue competition. 2E6 also blocks receptor binding, and competes with 4C2 for binding to MERS-RBD. Based on the structure, we further humanized 4C2 by preserving only the paratope residues and substituting the remaining amino acids with the counterparts from human immunoglobulins. The humanized 4C2 (4C2h) antibody sustained similar neutralizing activity and biochemical characteristics to the parental mouse antibody. Finally, we showed that 4C2h can significantly abate the virus titers in lungs of Ad5-hCD26-transduced mice infected with MERS-CoV, therefore representing a promising agent for prophylaxis and therapy in clinical settings.

  3. Production of a neutralizing mouse-human chimeric antibody against botulinum neurotoxin serotype E.

    PubMed

    Mukamoto, Masafumi; Maeda, Hiroaki; Kohda, Tomoko; Nozaki, Chikateru; Takahashi, Motohide; Kozaki, Shunji

    2013-01-01

    A mouse-human chimeric antibody that can neutralize botulinum neurotoxin serotype E (BoNT/E) was developed. Variable regions of heavy and light chains obtained using a mouse hybridoma clone (E9-4) cDNA, which was selected on the basis of neutralizing activity against BoNT/E, were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO-DG44 cells were transfected with these plasmids and a mouse-human chimeric antibody (EC94) was purified to examine binding and neutralizing activity against BoNT/E. EC94 exhibited the same levels of binding activities against BoNT/E as those of a parent mouse monoclonal antibody and neutralized more than 4,000 LD(50)/mg antibody. This chimeric antibody seems to be a useful candidate for infant botulism in which the use of passive immunotherapy is not planned so as to avoid serious events such as anaphylactic shock. We designed shuffling chimeric antibodies with replacement of V(H) or V(L) of EC94 with that of a chimeric antibody (AC24) that possessed neutralizing activity against BoNT/A. These shuffling antibodies did not exhibit neutralizing activity against either BoNT/E or BoNT/A.

  4. The mechanism of differential neutralization of dengue serotype 3 strains by monoclonal antibody 8A1.

    PubMed

    Zhou, Yang; Austin, S Kyle; Fremont, Daved H; Yount, Boyd L; Huynh, Jeremy P; de Silva, Aravinda M; Baric, Ralph S; Messer, William B

    2013-04-25

    While previous studies have demonstrated that envelope (E) glycoprotein variation between dengue viruses (DENV) genotypes can influence antibody neutralization potency, the mechanisms of variable neutralization remain incompletely understood. Here we characterize epitope antibody interactions of a DENV-3 EDIII binding mouse mAb 8A1 which displays highly variable neutralizing activity against DENV-3 genotypes. Using a DENV-3 reverse genetics platform, we characterize ability of 8A1 to bind and neutralize naturally occurring DENV-3 E genotypic variant viruses. Introduction of single and multiple amino acid mutations into the parental clone background demonstrates that mutations at positions 301 and 383 on EDIII are responsible for 8A1 differential neutralization phenotypes. ELISA and surface plasmon resonance (SPR) studies indicate differences in binding are responsible for the variable neutralization. Variability at position 301 primarily determined binding difference through influencing antibody-EDIII dissociation rate. Our findings are relevant to many groups focusing on DENV EDIII as a vaccine target.

  5. Escape From Monoclonal Antibody Neutralization Affects Henipavirus Fitness In Vitro and In Vivo.

    PubMed

    Borisevich, Viktoriya; Lee, Benhur; Hickey, Andrew; DeBuysscher, Blair; Broder, Christopher C; Feldmann, Heinz; Rockx, Barry

    2016-02-01

    Henipaviruses are zoonotic viruses that can cause severe and acute respiratory diseases and encephalitis in humans. To date, no vaccine or treatments are approved for human use. The presence of neutralizing antibodies is a strong correlate of protection against lethal disease in animals. However, since RNA viruses are prone to high mutation rates, the possibility that these viruses will escape neutralization remains a potential concern. In the present study, we generated neutralization-escape mutants, using 6 different monoclonal antibodies, and studied the effect of these neutralization-escape mutations on in vitro and in vivo fitness. These data provide a mechanism for overcoming neutralization escape by use of cocktails of cross-neutralizing monoclonal antibodies that recognize residues within the glycoprotein that are important for virus replication and virulence.

  6. Escape From Monoclonal Antibody Neutralization Affects Henipavirus Fitness In Vitro and In Vivo

    PubMed Central

    Borisevich, Viktoriya; Lee, Benhur; Hickey, Andrew; DeBuysscher, Blair; Broder, Christopher C.; Feldmann, Heinz; Rockx, Barry

    2016-01-01

    Henipaviruses are zoonotic viruses that can cause severe and acute respiratory diseases and encephalitis in humans. To date, no vaccine or treatments are approved for human use. The presence of neutralizing antibodies is a strong correlate of protection against lethal disease in animals. However, since RNA viruses are prone to high mutation rates, the possibility that these viruses will escape neutralization remains a potential concern. In the present study, we generated neutralization-escape mutants, using 6 different monoclonal antibodies, and studied the effect of these neutralization-escape mutations on in vitro and in vivo fitness. These data provide a mechanism for overcoming neutralization escape by use of cocktails of cross-neutralizing monoclonal antibodies that recognize residues within the glycoprotein that are important for virus replication and virulence. PMID:26357909

  7. Preclinical development of a humanized neutralizing antibody targeting HGF

    PubMed Central

    Kim, Hyori; Hong, Sung Hee; Kim, Jung Yong; Kim, In-Chull; Park, Young-Whan; Lee, Song-Jae; Song, Seong-Won; Kim, Jung Ju; Park, Gunwoo; Kim, Tae Min; Kim, Yun-Hee; Park, Jong Bae; Chung, Junho; Kim, In-Hoo

    2017-01-01

    Hepatocyte growth factor (HGF) and its receptor, cMET, play critical roles in cell proliferation, angiogenesis and invasion in a wide variety of cancers. We therefore examined the anti-tumor activity of the humanized monoclonal anti-HGF antibody, YYB-101, in nude mice bearing human glioblastoma xenografts as a single agent or in combination with temozolomide. HGF neutralization, The extracellular signal-related kinases 1 and 2 (ERK1/2) phosphorylation, and HGF-induced scattering were assessed in HGF-expressing cell lines treated with YYB-101. To support clinical development, we also evaluated the preclinical pharmacokinetics and toxicokinetics in cynomolgus monkeys, and human and cynomolgus monkey tissue was stained with YYB-101 to test tissue cross-reactivity. We found that YYB-101 inhibited cMET activation in vitro and suppressed tumor growth in the orthotopic mouse model of human glioblastoma. Combination treatment with YYB-101 and temozolomide decreased tumor growth and increased overall survival compared with the effects of either agent alone. Five cancer-related genes (TMEM119, FST, RSPO3, ROS1 and NBL1) were overexpressed in YYB-101-treated mice that showed tumor regrowth. In the tissue cross-reactivity assay, critical cross-reactivity was not observed. The terminal elimination half-life was 21.7 days. Taken together, the in vitro and in vivo data demonstrated the anti-tumor efficacy of YYB-101, which appeared to be mediated by blocking the HGF/cMET interaction. The preclinical pharmacokinetics, toxicokinetics and tissue cross-reactivity data support the clinical development of YYB-101 for advanced cancer. PMID:28336956

  8. Feline immunodeficiency virus (FIV) vaccine efficacy and FIV neutralizing antibodies

    PubMed Central

    Coleman, James K.; Pu, Ruiyu; Martin, Marcus M.; Noon-Song, Ezra N.; Zwijnenberg, Raphael; Yamamoto, Janet K.

    2013-01-01

    A HIV-1 tier system has been developed to categorize the various subtype viruses based on their sensitivity to vaccine-induced neutralizing antibodies (NAbs): tier 1 with greatest sensitivity, tier 2 being moderately sensitive, and tier 3 being the least sensitive to NAbs (Mascola et al., J Virol 2005; 79:10103-7). Here, we define an FIV tier system using two related FIV dual-subtype (A+D) vaccines: the commercially available inactivated infected-cell vaccine (Fel-O-Vax® FIV) and its prototype vaccine solely composed of inactivated whole viruses. Both vaccines afforded combined protection rates of 100% against subtype-A tier-1 FIVPet, 89% against subtype-B tier-3 FIVFC1, 61% against recombinant subtype-A/B tier-2 FIVBang, 62% against recombinant subtype-F′/C tier-3 FIVNZ1, and 40% against subtype-A tier-2 FIVUK8 in short-duration (37–41 weeks) studies. In long-duration (76–80 weeks) studies, the commercial vaccine afforded a combined protection rate of at least 46% against the tier-2 and tier-3 viruses. Notably, protection rates observed here are far better than recently reported HIV-1 vaccine trials (Sanou et al., The Open AIDS 2012; 6:246-60). Prototype vaccine protection against two tier-3 and one tier-2 viruses was more effective than commercial vaccine. Such protection did not correlate with the presence of vaccine-induced NAbs to challenge viruses. This is the first large-scale (228 laboratory cats) study characterizing short- and long-duration efficacies of dual-subtype FIV vaccines against heterologous subtype and recombinant viruses, as well as FIV tiers based on in vitro NAb analysis and in vivo passive-transfer studies. These studies demonstrate that not all vaccine protection is mediated by vaccine-induced NAbs. PMID:23800540

  9. Broadly neutralizing antibodies against the rapidly evolving porcine reproductive and respiratory syndrome virus.

    PubMed

    Robinson, Sally R; Li, Juan; Nelson, Eric A; Murtaugh, Michael P

    2015-05-04

    Neutralizing antibodies are a critical part of the immune armory for defense against viruses, and the mechanism by which many effective vaccines work to protect against viral infections. However, infections by rapidly evolving and genetically diverse viruses are often characterized by ineffective neutralizing antibody responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly genetically diverse RNA virus that causes PRRS, the most significant disease of pigs worldwide. The prevailing view of immunity to PRRSV is characterized by delayed and ineffectual production of neutralizing antibodies lacking cross-reactivity that is necessary for vaccine efficacy. Using an ELISA-based neutralizing assay developed to analyze PRRSV growth in porcine alveolar macrophages, the naturally permissive cell of PRRSV, we showed that sera from previously infected commercial sows had high levels of neutralizing activity against diverse PRRSV strains, including across distinct genotypes of PRRSV. Fifty percent cross-neutralization titers in excess of 1/1024 were observed. Neutralizing activity was dose-dependent and was maintained in the immunoglobulin fraction. Presence of high-titer, anti-PRRSV antibody activity that cross-neutralizes diverse strains of virus has prompted reevaluation of the role of neutralizing antibodies for cross-protection against PRRSV under field conditions. Understanding conditions that favor development of cross-neutralizing activity will be crucial for improved strategies to enhance cross-protection against PRRSV. More detailed studies are expected to elucidate mechanisms of neutralizing antibody production and maturation and to investigate conserved epitope targets of cross-neutralization in this rapidly evolving virus.

  10. The transduction of Coxsackie and Adenovirus Receptor-negative cells and protection against neutralizing antibodies by HPMA-co-oligolysine copolymer-coated adenovirus

    PubMed Central

    Wang, Chung-Huei K.; Chan, Leslie W.; Johnson, Russell N.; Chu, David S.H.; Shi, Julie; Schellinger, Joan G.; Lieber, Andre; Pun, Suzie H.

    2011-01-01

    Adenoviral (AdV) gene vectors offer efficient nucleic acid transfer into both dividing and non-dividing cells. However issues such as vector immunogenicity, toxicity and restricted transduction to receptor-expressing cells have prevented broad clinical translation of these constructs. To address this issue, engineered AdV have been prepared by both genetic and chemical manipulation. In this work, a polymer-coated Ad5 formulation is optimized by evaluating a series of N-(2-hydroxypropyl) methacrylamide (HPMA)-co-oligolysine copolymers synthesized by living polymerization techniques. This synthesis approach was used to generate highly controlled and well-defined polymers with varying peptide length (K5, K10 and K15), polymer molecular weight, and degradability to coat the viral capsid. The optimal formulation was not affected by the presence of serum during transduction and significantly increased Ad5 transduction of several cell types that lack the Coxsackie and Adenovirus Receptor (CAR) by up to 6-fold compared to unmodified AdV. Polymer-coated Ad5 also retained high transduction capability in the presence of Ad5 neutralizing antibodies. The critical role of heparan sulfate proteoglycans (HSPGs) in mediating cell binding and internalization of polymer-coated AdV was also demonstrated by evaluating transduction in HSPG-defective recombinant CHO cells. The formulations developed here are attractive vectors for ex vivo gene transfer in applications such as cell therapy. In addition, this platform for adenoviral modification allows for facile introduction of alternative targeting ligands. PMID:21959008

  11. Respiratory Syncytial Virus (RSV): Neutralizing Antibody, a Correlate of Immune Protection.

    PubMed

    Piedra, Pedro A; Hause, Anne M; Aideyan, Letisha

    2016-01-01

    Assays that measure RSV-specific neutralizing antibody activity are very useful for evaluating vaccine candidates, performing seroprevalence studies, and detecting infection. Neutralizing antibody activity is normally measured by a plaque reduction neutralization assay or by a microneutralization assay with or without complement. These assays measure the functional capacity of serum (or other fluids) to neutralize virus infectivity in cells as compared to ELISA assays that only measure the binding capacity against an antigen. This chapter discusses important elements in standardization of the RSV-specific microneutralization assay for use in the laboratory.

  12. Structural repertoire of HIV-1-neutralizing antibodies targeting the CD4 supersite in 14 donors

    PubMed Central

    Zhou, Tongqing; Lynch, Rebecca M.; Chen, Lei; Acharya, Priyamvada; Wu, Xueling; Doria-Rose, Nicole A.; Joyce, M. Gordon; Lingwood, Daniel; Soto, Cinque; Bailer, Robert T.; Ernandes, Michael J.; Kong, Rui; Longo, Nancy S.; Louder, Mark K.; McKee, Krisha; O’Dell, Sijy; Schmidt, Stephen D.; Tran, Lillian; Yang, Zhongjia; Druz, Aliaksandr; Luongo, Timothy S.; Moquin, Stephanie; Srivatsan, Sanjay; Yang, Yongping; Zhang, Baoshan; Zheng, Anqi; Pancera, Marie; Kirys, Tatsiana; Georgiev, Ivelin S.; Gindin, Tatyana; Peng, Hung-Pin; Yang, An-Suei; Mullikin, James C.; Gray, Matthew D.; Stamatatos, Leonidas; Burton, Dennis R.; Koff, Wayne C.; Cohen, Myron S.; Haynes, Barton F.; Casazza, Joseph P.; Connors, Mark; Corti, Davide; Lanzavecchia, Antonio; Sattentau, Quentin J.; Weiss, Robin A.; West, Anthony P.; Bjorkman, Pamela J.; Scheid, Johannes F.; Nussenzweig, Michel C.; Shapiro, Lawrence; Mascola, John R.; Kwong, Peter D.

    2015-01-01

    The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures –8 determined here– of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies. PMID:26004070

  13. Neutralizing antibody responses in acute human immunodeficiency virus type 1 subtype C infection.

    PubMed

    Gray, E S; Moore, P L; Choge, I A; Decker, J M; Bibollet-Ruche, F; Li, H; Leseka, N; Treurnicht, F; Mlisana, K; Shaw, G M; Karim, S S Abdool; Williamson, C; Morris, L

    2007-06-01

    The study of the evolution and specificities of neutralizing antibodies during the course of human immunodeficiency virus type 1 (HIV-1) infection may be important in the discovery of possible targets for vaccine design. In this study, we assessed the autologous and heterologous neutralization responses of 14 HIV-1 subtype C-infected individuals, using envelope clones obtained within the first 2 months postinfection. Our data show that potent but relatively strain-specific neutralizing antibodies develop within 3 to 12 months of HIV-1 infection. The magnitude of this response was associated with shorter V1-to-V5 envelope lengths and fewer glycosylation sites, particularly in the V1-V2 region. Anti-MPER antibodies were detected in 4 of 14 individuals within a year of infection, while antibodies to CD4-induced (CD4i) epitopes developed to high titers in 12 participants, in most cases before the development of autologous neutralizing antibodies. However, neither anti-MPER nor anti-CD4i antibody specificity conferred neutralization breadth. These data provide insights into the kinetics, potency, breadth, and epitope specificity of neutralizing antibody responses in acute HIV-1 subtype C infection.

  14. A pilot study on an attenuated Chinese EIAV vaccine inducing broadly neutralizing antibodies.

    PubMed

    Meng, Qinglai; Lin, Yuezhi; Ma, Jian; Ma, Yan; Zhao, Liping; Li, Shenwei; Liang, Hua; Zhou, Jianhua; Shen, Rongxian; Zhang, Xiaoyan; Shao, Yiming

    2011-08-01

    The attenuated Chinese equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. In this pilot study, to determine whether this attenuated vaccine can induce broadly neutralizing antibodies, we immunized four horses with the attenuated Chinese vaccine strain EIAVFDDV and then observed the evolution of neutralizing antibodies against different EIAV strains. During the vaccination phase, all vaccinees rapidly developed high levels of neutralizing antibodies against the homologous vaccine strain (pLGFD3V), and 3 out of 4 horses showed a gradual increase in serum neutralizing activity against two relatively heterologous virulent variants of the challenge strain (pLGFD3Mu12V and DLV34). After challenge, the three horses that had developed high levels of neutralizing antibodies against pLGFD3Mu12V and DLV34 did not show signs of infection, which was demonstrated by immune suppression, while the one horse producing serum that could only neutralize pLGFD3V developed a febrile episode during the 8-month observation period. To assess whether the broadly neutralizing activity is associated with immune protection, sera drawn on the day of challenge from these four vaccinees and an additional four EIAVFDDV-vaccinated horses were analyzed for neutralizing antibodies against pLGFD3V, pLGFD3Mu12V and DLV34. Although there was no significant correlation between protection from infection and serum neutralizing activity against any of these three viral strains, protection from infection was observed to correlate better with serum neutralizing activity against the two heterologous virulent strains than against the homologous vaccine strain. These data indicate that EIAVFDDV induced broadly neutralizing antibodies, which might confer enhanced protection of vaccinees from infection by the challenge virus.

  15. Two Escape Mechanisms of Influenza A Virus to a Broadly Neutralizing Stalk-Binding Antibody.

    PubMed

    Chai, Ning; Swem, Lee R; Reichelt, Mike; Chen-Harris, Haiyin; Luis, Elizabeth; Park, Summer; Fouts, Ashley; Lupardus, Patrick; Wu, Thomas D; Li, Olga; McBride, Jacqueline; Lawrence, Michael; Xu, Min; Tan, Man-Wah

    2016-06-01

    Broadly neutralizing antibodies targeting the stalk region of influenza A virus (IAV) hemagglutinin (HA) are effective in blocking virus infection both in vitro and in vivo. The highly conserved epitopes recognized by these antibodies are critical for the membrane fusion function of HA and therefore less likely to be permissive for virus mutational escape. Here we report three resistant viruses of the A/Perth/16/2009 strain that were selected in the presence of a broadly neutralizing stalk-binding antibody. The three resistant viruses harbor three different mutations in the HA stalk: (1) Gln387Lys; (2) Asp391Tyr; (3) Asp391Gly. The Gln387Lys mutation completely abolishes binding of the antibody to the HA stalk epitope. The other two mutations, Asp391Tyr and Asp391Gly, do not affect antibody binding at neutral pH and only slightly reduce binding at low pH. Interestingly, they enhance the fusion ability of the HA, representing a novel mechanism that allows productive membrane fusion even in the presence of antibody and hence virus escape from antibody neutralization. Therefore, these mutations illustrate two different resistance mechanisms used by IAV to escape broadly neutralizing stalk-binding antibodies. Compared to the wild type virus, the resistant viruses release fewer progeny viral particles during replication and are more sensitive to Tamiflu, suggesting reduced viral fitness.

  16. Two Escape Mechanisms of Influenza A Virus to a Broadly Neutralizing Stalk-Binding Antibody

    PubMed Central

    Chai, Ning; Swem, Lee R.; Reichelt, Mike; Chen-Harris, Haiyin; Luis, Elizabeth; Park, Summer; Fouts, Ashley; Lupardus, Patrick; Wu, Thomas D.; Li, Olga; McBride, Jacqueline; Lawrence, Michael; Xu, Min; Tan, Man-Wah

    2016-01-01

    Broadly neutralizing antibodies targeting the stalk region of influenza A virus (IAV) hemagglutinin (HA) are effective in blocking virus infection both in vitro and in vivo. The highly conserved epitopes recognized by these antibodies are critical for the membrane fusion function of HA and therefore less likely to be permissive for virus mutational escape. Here we report three resistant viruses of the A/Perth/16/2009 strain that were selected in the presence of a broadly neutralizing stalk-binding antibody. The three resistant viruses harbor three different mutations in the HA stalk: (1) Gln387Lys; (2) Asp391Tyr; (3) Asp391Gly. The Gln387Lys mutation completely abolishes binding of the antibody to the HA stalk epitope. The other two mutations, Asp391Tyr and Asp391Gly, do not affect antibody binding at neutral pH and only slightly reduce binding at low pH. Interestingly, they enhance the fusion ability of the HA, representing a novel mechanism that allows productive membrane fusion even in the presence of antibody and hence virus escape from antibody neutralization. Therefore, these mutations illustrate two different resistance mechanisms used by IAV to escape broadly neutralizing stalk-binding antibodies. Compared to the wild type virus, the resistant viruses release fewer progeny viral particles during replication and are more sensitive to Tamiflu, suggesting reduced viral fitness. PMID:27351973

  17. Development and optimization of a novel assay to measure neutralizing antibodies against Clostridium difficile toxins.

    PubMed

    Xie, Jinfu; Zorman, Julie; Indrawati, Lani; Horton, Melanie; Soring, Keri; Antonello, Joseph M; Zhang, Yuhua; Secore, Susan; Miezeiewski, Matthew; Wang, Su; Kanavage, Anthony D; Skinner, Julie M; Rogers, Irene; Bodmer, Jean-Luc; Heinrichs, Jon H

    2013-04-01

    Clostridium difficile produces two major virulence toxins, toxin A (TcdA) and toxin B (TcdB). Antitoxin antibodies, especially neutralizing antibodies, have been shown to be associated with a lower incidence of C. difficile infection (CDI) recurrence, and antibody levels are predictive of asymptomatic colonization. The development of an assay to detect the presence of neutralizing antibodies in animal and human sera for the evaluation of vaccine efficacy is highly desired. We have developed such an assay, which allows for the quantification of the effect of toxins on eukaryotic cells in an automated manner. We describe here the optimization of this assay to measure toxin potency as well as neutralizing antibody (NAb) activity against C. difficile toxins using a design-of-experiment (DOE) methodology. Toxin concentration and source, cell seeding density, and serum-toxin preincubation time were optimized in the assay using Vero cells. The assay was shown to be robust and to produce linear results across a range of antibody concentrations. It can be used to quantify neutralizing antibodies in sera of monkeys and hamsters immunized with C. difficile toxoid vaccines. This assay was shown to correlate strongly with traditional assays which rely on labor-intensive methods of determining neutralizing antibody titers by visual microscopic inspection of intoxicated-cell monolayers. This assay has utility for the selection and optimization of C. difficile vaccine candidates.

  18. Egg yolk antibodies for detection and neutralization of Clostridium botulinum type A neurotoxin.

    PubMed

    Trott, D L; Yang, M; Gonzalez, J; Larson, A E; Tepp, W H; Johnson, E A; Cook, M E

    2009-05-01

    The objective of this research project was to determine the usefulness of an egg antibody platform for producing materials for the detection and neutralization of botulinum type A neurotoxin. Yield estimates for detection and neutralizing antibodies produced using methods described were calculated. Antibody specific to botulinum toxoid A (aToxoid) and toxin A (aBoNT/A) was produced by immunizing hens with botulinum toxoid A (toxoid) followed by increasing amounts of botulinum neurotoxin A (BoNT/A) in Freund incomplete adjuvant. Egg yolks were extracted with polyethylene glycol (PEG) for antibody detection and neutralization experiments. A model aToxoid/toxoid immunoassay using only egg yolk antibody was developed and had a detection limit of 1 pg/ml of toxoid. In an indirect enzyme-linked immunosorbent assay of BoNT/A-specific antibody, the aBoNT/A contained more BoNT/A-specific antibody than did the aToxoid, and aBoNT/A was as effective as commercial rabbit antibody. The aToxoid provided no protection against BoNT/A in a standard mouse neutralization assay; however, 1 mg of PEG-extracted aBoNT/A neutralized 4,000 lethal doses of BoNT/A injected intraperitoneally. Based on these results, we calculated that in 1 month one hen could produce more than 100 liters of antibody detection reagents or enough antibody to neutralize approximately 11.6 million mouse lethal doses of botulinum toxin. Utilization of an egg antibody platform is potentially rapid (28 to 70 days) and scalable to kilogram quantities using current egg production facilities with as few as 1,000 hens.

  19. Serum virus neutralization assay for detection and quantitation of serum neutralizing antibodies to influenza A virus in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The serum virus neutralization (SVN) assay is a serological test to detect the presence and magnitude of functional systemic antibodies that prevent infectivity of a virus. The SVN assay is a highly sensitive and specific test that may be applied to influenza A viruses (IAV) in swine to measure the ...

  20. Characterization of a Novel Neutralizing Monoclonal Antibody Against Ebola Virus GP.

    PubMed

    Reynard, Olivier; Volchkov, Viktor E

    2015-10-01

    Ebola virus is the etiological agent of a severe hemorrhagic fever with a high mortality rate. As the only protein exposed on the surface of viral particles, the spike glycoprotein GP is the unique target for neutralizing monoclonal antibodies. In this study, we demonstrate the strong neutralization capacity of the monoclonal antibody #3327 and characterize its activity. GP residues that are required for recognition and neutralization were found to be located both in the internal fusion loop and in the receptor-binding domain. Analysis of Ebola virus entry in the presence of #3327 allows us to hypothesize that this antibody binds to the virus particle before internalization and endosomal processing of GP and likely prevents the final viral fusion step. Importantly, #3327 is able to block entry of virions bearing GP that contain the Q508 escape mutation common to a number of virus-neutralizing antibodies, and therefore provides future perspectives for treatment strategies against Ebola virus infection.

  1. Immunogenic peptides comprising a T-helper epitope and a B-cell neutralizing antibody epitope

    DOEpatents

    Haynes, Barton F.; Korber, Bette T.; De Lorimier, Robert M.

    2006-12-26

    The present invention relates, generally, to a polyvalent immunogen and, more particularly, to a method of inducing neutralizing antibodies against HIV and to a polyvalent immunogen suitable for use in such a method.

  2. Neutralizing human antibodies prevent Zika virus replication and fetal disease in mice.

    PubMed

    Sapparapu, Gopal; Fernandez, Estefania; Kose, Nurgun; Bin Cao; Fox, Julie M; Bombardi, Robin G; Zhao, Haiyan; Nelson, Christopher A; Bryan, Aubrey L; Barnes, Trevor; Davidson, Edgar; Mysorekar, Indira U; Fremont, Daved H; Doranz, Benjamin J; Diamond, Michael S; Crowe, James E

    2016-12-15

    Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defects during pregnancy. To develop candidate therapeutic agents against ZIKV, we isolated a panel of human monoclonal antibodies from subjects that were previously infected with ZIKV. We show that a subset of antibodies recognize diverse epitopes on the envelope (E) protein and exhibit potent neutralizing activity. One of the most inhibitory antibodies, ZIKV-117, broadly neutralized infection of ZIKV strains corresponding to African and Asian-American lineages. Epitope mapping studies revealed that ZIKV-117 recognized a unique quaternary epitope on the E protein dimer-dimer interface. We evaluated the therapeutic efficacy of ZIKV-117 in pregnant and non-pregnant mice. Monoclonal antibody treatment markedly reduced tissue pathology, placental and fetal infection, and mortality in mice. Thus, neutralizing human antibodies can protect against maternal-fetal transmission, infection and disease, and reveal important determinants for structure-based rational vaccine design efforts.

  3. Latency of Herpes Simplex Virus in Absence of Neutralizing Antibody: Model for Reactivation

    NASA Astrophysics Data System (ADS)

    Sekizawa, Tsuyoshi; Openshaw, Harry; Wohlenberg, Charles; Notkins, Abner Louis

    1980-11-01

    Mice inoculated with herpes simplex virus (type 1) by the lip or corneal route and then passively immunized with rabbit antibody to herpes simplex virus developed a latent infection in the trigeminal ganglia within 96 hours. Neutralizing antibody to herpes simplex virus was cleared from the circulation and could not be detected in most of these mice after 2 months. Examination of ganglia from the antibody-negative mice revealed latent virus in over 90 percent of the animals, indicating that serum neutralizing antibody is not necessary to maintain the latent state. When the lips or corneas of these mice were traumatized, viral reactivation occurred in up to 90 percent of the mice, as demonstrated by the appearance of neutralizing antibody. This study provides a model for identifying factors that trigger viral reactivation.

  4. Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies.

    PubMed

    Doria-Rose, Nicole A; Schramm, Chaim A; Gorman, Jason; Moore, Penny L; Bhiman, Jinal N; DeKosky, Brandon J; Ernandes, Michael J; Georgiev, Ivelin S; Kim, Helen J; Pancera, Marie; Staupe, Ryan P; Altae-Tran, Han R; Bailer, Robert T; Crooks, Ema T; Cupo, Albert; Druz, Aliaksandr; Garrett, Nigel J; Hoi, Kam H; Kong, Rui; Louder, Mark K; Longo, Nancy S; McKee, Krisha; Nonyane, Molati; O'Dell, Sijy; Roark, Ryan S; Rudicell, Rebecca S; Schmidt, Stephen D; Sheward, Daniel J; Soto, Cinque; Wibmer, Constantinos Kurt; Yang, Yongping; Zhang, Zhenhai; Mullikin, James C; Binley, James M; Sanders, Rogier W; Wilson, Ian A; Moore, John P; Ward, Andrew B; Georgiou, George; Williamson, Carolyn; Abdool Karim, Salim S; Morris, Lynn; Kwong, Peter D; Shapiro, Lawrence; Mascola, John R

    2014-05-01

    Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development.

  5. Cross-clade neutralizing antibodies against HIV-1 induced in rabbits by focusing the immune response on a neutralizing epitope

    SciTech Connect

    Zolla-Pazner, Susan; Cohen, Sandra; Pinter, Abraham; Krachmarov, Chavdar; Wrin, Terri; Wang Shixia; Lu Shan

    2009-09-15

    Studies were performed to induce cross-clade neutralizing antibodies (Abs) by testing various combinations of prime and boost constructs that focus the immune response on structurally-conserved epitopes in the V3 loop of HIV-1 gp120. Rabbits were immunized with gp120 DNA containing a V3 loop characterized by the GPGR motif at its tip, and/or with gp120 DNA with a V3 loop carrying the GPGQ motif. Priming was followed by boosts with V3-fusion proteins (V3-FPs) carrying the V3 sequence from a subtype B virus (GPGR motif), and/or with V3 sequences from subtypes A and C (GPGQ motif). The broadest and most consistent neutralizing responses were generated when using a clade C gp120 DNA prime and with the V3{sub B}-FP boost. Immune sera displayed neutralizing activity in three assays against pseudoviruses and primary isolates from subtypes A, AG, B, C, and D. Polyclonal Abs in the immune rabbit sera neutralized viruses that were not neutralized by pools of human anti-V3 monoclonal Abs. Greater than 80% of the neutralizing Abs were specific for V3, showing that the immune response could be focused on a neutralizing epitope and that vaccine-induced anti-V3 Abs have cross-clade neutralizing activity.

  6. In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes.

    PubMed

    Bartosch, Birke; Bukh, Jens; Meunier, Jean-Christophe; Granier, Christelle; Engle, Ronald E; Blackwelder, William C; Emerson, Suzanne U; Cosset, François-Loïc; Purcell, Robert H

    2003-11-25

    Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C.

  7. In vitro assay for neutralizing antibody to hepatitis C virus: Evidence for broadly conserved neutralization epitopes

    PubMed Central

    Bartosch, Birke; Bukh, Jens; Meunier, Jean-Christophe; Granier, Christelle; Engle, Ronald E.; Blackwelder, William C.; Emerson, Suzanne U.; Cosset, François-Loïc; Purcell, Robert H.

    2003-01-01

    Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C. PMID:14617769

  8. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus

    PubMed Central

    Marcobal, Angela; Liu, Xiaowen; Zhang, Wenlei; Dimitrov, Antony S.; Jia, Letong; Lee, Peter P.; Fouts, Timothy R.; Parks, Thomas P.

    2016-01-01

    Abstract Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 μg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission. PMID:26950606

  9. Antibody to gp41 MPER alters functional properties of HIV-1 Env without complete neutralization.

    PubMed

    Kim, Arthur S; Leaman, Daniel P; Zwick, Michael B

    2014-07-01

    Human antibody 10E8 targets the conserved membrane proximal external region (MPER) of envelope glycoprotein (Env) subunit gp41 and neutralizes HIV-1 with exceptional potency. Remarkably, HIV-1 containing mutations that reportedly knockout 10E8 binding to linear MPER peptides are partially neutralized by 10E8, producing a local plateau in the dose response curve. Here, we found that virus partially neutralized by 10E8 becomes significantly less neutralization sensitive to various MPER antibodies and to soluble CD4 while becoming significantly more sensitive to antibodies and fusion inhibitors against the heptad repeats of gp41. Thus, 10E8 modulates sensitivity of Env to ligands both pre- and post-receptor engagement without complete neutralization. Partial neutralization by 10E8 was influenced at least in part by perturbing Env glycosylation. With unliganded Env, 10E8 bound with lower apparent affinity and lower subunit occupancy to MPER mutant compared to wild type trimers. However, 10E8 decreased functional stability of wild type Env while it had an opposite, stabilizing effect on MPER mutant Envs. Clade C isolates with natural MPER polymorphisms also showed partial neutralization by 10E8 with altered sensitivity to various gp41-targeted ligands. Our findings suggest a novel mechanism of virus neutralization by demonstrating how antibody binding to the base of a trimeric spike cross talks with adjacent subunits to modulate Env structure and function. The ability of an antibody to stabilize, destabilize, partially neutralize as well as alter neutralization sensitivity of a virion spike pre- and post-receptor engagement may have implications for immunotherapy and vaccine design.

  10. Molecular evolution of broadly neutralizing Llama antibodies to the CD4-binding site of HIV-1.

    PubMed

    McCoy, Laura E; Rutten, Lucy; Frampton, Dan; Anderson, Ian; Granger, Luke; Bashford-Rogers, Rachael; Dekkers, Gillian; Strokappe, Nika M; Seaman, Michael S; Koh, Willie; Grippo, Vanina; Kliche, Alexander; Verrips, Theo; Kellam, Paul; Fassati, Ariberto; Weiss, Robin A

    2014-12-01

    To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.

  11. The neutralizing human recombinant antibodies to pathogenic Orthopoxviruses derived from a phage display immune library.

    PubMed

    Tikunova, Nina; Dubrovskaya, Viktoriya; Morozova, Vera; Yun, Tatiana; Khlusevich, Yana; Bormotov, Nikolai; Laman, Aleksandr; Brovko, Fedor; Shvalov, Aleksandr; Belanov, Eugeni

    2012-01-01

    A panel of recombinant human antibodies to orthopoxviruses was isolated from a combinatorial phage display library of human scFv antibodies constructed from the Vh and Vl genes cloned from the peripheral blood lymphocytes of Vaccinia virus (VACV) immune donors. Plaque-reduction neutralization tests showed that seven selected phage-displaying scFv antibodies (pdAbs) neutralized both CPXV and VACV, and five of them neutralized Monkeypox virus (MPXV). Western blot analysis of VACV and CPXV proteins demonstrated that seven neutralizing antibodies recognized a 35 kDa protein. To identify this target protein, we produced a recombinant J3L protein of CPXV and showed that all the selected neutralizing antibodies recognized this protein. Neutralizing pdAb b9 was converted into fully human mAb b9 (fh b9), and scFv b9 displayed high binding affinities (K(d) of 0.7 and 3.2 nM). The fh b9 reduced VACV plaque formation in a dose-dependent manner.

  12. Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

    PubMed Central

    McCoy, Laura E.; Rutten, Lucy; Frampton, Dan; Anderson, Ian; Granger, Luke; Bashford-Rogers, Rachael; Dekkers, Gillian; Strokappe, Nika M.; Seaman, Michael S.; Koh, Willie; Grippo, Vanina; Kliche, Alexander; Verrips, Theo; Kellam, Paul; Fassati, Ariberto; Weiss, Robin A.

    2014-01-01

    To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols. PMID:25522326

  13. Preventive and therapeutic applications of neutralizing antibodies to Human Immunodeficiency Virus Type 1 (HIV-1)

    PubMed Central

    Ringe, Rajesh

    2013-01-01

    The development of a preventive vaccine to neutralize the highly variable and antigenically diverse human immunodeficiency virus type 1 (HIV-1) has been an indomitable goal. The recent discovery of a number of cross-neutralizing and potent monoclonal antibodies from elite neutralizers has provided important insights in this field. Neutralizing antibodies (NAbs) are useful in identifying neutralizing epitopes of vaccine utility and for understanding the mechanism of potent and broad cross-neutralization thus providing a modality of preventive and therapeutic value. In this article we review the current understanding on the potential use of broadly neutralizing antibodies (bNAbs) in their full-length IgG structure, engineered domain antibody or bispecific versions towards preventive and therapeutic applications. The potential implications of NAbs are discussed in the light of the recent developments as key components in vaccination against HIV-1. The development of a vaccine immunogen which elicits bNAbs and confers protective immunity remains a real challenge. PMID:24757516

  14. Structural Basis for Broad and Potent Neutralization of HIV-1 by Antibody VRC01

    SciTech Connect

    Zhou, Tongqing; Georgiev, Ivelin; Wu, Xueling; Yang, Zhi-Yong; Dai, Kaifan; Finzi, Andrés; Kwon, Young Do; Scheid, Johannes F.; Shi, Wei; Xu, Ling; Yang, Yongping; Zhu, Jiang; Nussenzweig, Michel C.; Sodroski, Joseph; Shapiro, Lawrence; Nabel, Gary J.; Mascola, John R.; Kwong, Peter D.

    2010-08-26

    During HIV-1 infection, antibodies are generated against the region of the viral gp120 envelope glycoprotein that binds CD4, the primary receptor for HIV-1. Among these antibodies, VRC01 achieves broad neutralization of diverse viral strains. We determined the crystal structure of VRC01 in complex with a human immunodeficiency virus HIV-1 gp120 core. VRC01 partially mimics CD4 interaction with gp120. A shift from the CD4-defined orientation, however, focuses VRC01 onto the vulnerable site of initial CD4 attachment, allowing it to overcome the glycan and conformational masking that diminishes the neutralization potency of most CD4-binding-site antibodies. To achieve this recognition, VRC01 contacts gp120 mainly through immunoglobulin V-gene regions substantially altered from their genomic precursors. Partial receptor mimicry and extensive affinity maturation thus facilitate neutralization of HIV-1 by natural human antibodies.

  15. NEUTRALIZING AND COMPLEMENT-FIXING ANTIBODY PRODUCTION AND RESISTANCE FOLLOWING VACCINATION IN EXPERIMENTAL ENCEPHALITIS INFECTIONS

    PubMed Central

    Casals, J.

    1943-01-01

    In mice vaccinated subcutaneously with different doses of virulent W.E.E. virus or with formolized vaccine, neutralizing and complement-fixing antibodies paralleled resistance to some extent yet appeared in groups in which resistance remained undetectable, persisted at a similar maximum level in spite of different titers of resistance, and after resistance had become negligible. In mice vaccinated subcutaneously with different doses of virulent St. Louis encephalitis virus or with formolized vaccine, neutralizing and complement-fixing antibodies bore little relation to resistance. Neutralizing antibodies appeared only in the group showing resistance but not until resistance was diminishing. Complement-fixing antibodies developed equally well in groups with or without resistance. PMID:19871341

  16. CATNAP: a tool to compile, analyze and tally neutralizing antibody panels.

    PubMed

    Yoon, Hyejin; Macke, Jennifer; West, Anthony P; Foley, Brian; Bjorkman, Pamela J; Korber, Bette; Yusim, Karina

    2015-07-01

    CATNAP (Compile, Analyze and Tally NAb Panels) is a new web server at Los Alamos HIV Database, created to respond to the newest advances in HIV neutralizing antibody research. It is a comprehensive platform focusing on neutralizing antibody potencies in conjunction with viral sequences. CATNAP integrates neutralization and sequence data from published studies, and allows users to analyze that data for each HIV Envelope protein sequence position and each antibody. The tool has multiple data retrieval and analysis options. As input, the user can pick specific antibodies and viruses, choose a panel from a published study, or supply their own data. The output superimposes neutralization panel data, virus epidemiological data, and viral protein sequence alignments on one page, and provides further information and analyses. The user can highlight alignment positions, or select antibody contact residues and view position-specific information from the HIV databases. The tool calculates tallies of amino acids and N-linked glycosylation motifs, counts of antibody-sensitive and -resistant viruses in conjunction with each amino acid or N-glycosylation motif, and performs Fisher's exact test to detect potential positive or negative amino acid associations for the selected antibody. Website name: CATNAP (Compile, Analyze and Tally NAb Panels). Website address: http://hiv.lanl.gov/catnap.

  17. Neutralization of Virus Infectivity by Antibodies: Old Problems in New Perspectives

    PubMed Central

    Klasse, P. J.

    2016-01-01

    Neutralizing antibodies (NAbs) can be both sufficient and necessary for protection against viral infections, although they sometimes act in concert with cellular immunity. Successful vaccines against viruses induce NAbs but vaccine candidates against some major viral pathogens, including HIV-1, have failed to induce potent and effective such responses. Theories of how antibodies neutralize virus infectivity have been formulated and experimentally tested since the 1930s; and controversies about the mechanistic and quantitative bases for neutralization have continually arisen. Soluble versions of native oligomeric viral proteins that mimic the functional targets of neutralizing antibodies now allow the measurement of the relevant affinities of NAbs. Thereby the neutralizing occupancies on virions can be estimated and related to the potency of the NAbs. Furthermore, the kinetics and stoichiometry of NAb binding can be compared with neutralizing efficacy. Recently, the fundamental discovery that the intracellular factor TRIM21 determines the degree of neutralization of adenovirus has provided new mechanistic and quantitative insights. Since TRIM21 resides in the cytoplasm, it would not affect the neutralization of enveloped viruses, but its range of activity against naked viruses will be important to uncover. These developments bring together the old problems of virus neutralization—mechanism, stoichiometry, kinetics, and efficacy—from surprising new angles. PMID:27099867

  18. Discovering neutralizing antibodies targeting the stem epitope of H1N1 influenza hemagglutinin with synthetic phage-displayed antibody libraries.

    PubMed

    Tung, Chao-Ping; Chen, Ing-Chien; Yu, Chung-Ming; Peng, Hung-Pin; Jian, Jhih-Wei; Ma, Shiou-Hwa; Lee, Yu-Ching; Jan, Jia-Tsrong; Yang, An-Suei

    2015-10-12

    Broadly neutralizing antibodies developed from the IGHV1-69 germline gene are known to bind to the stem region of hemagglutinin in diverse influenza viruses but the sequence determinants for the antigen recognition, including neutralization potency and binding affinity, are not clearly understood. Such understanding could inform designs of synthetic antibody libraries targeting the stem epitope on hemagglutinin, leading to artificially designed antibodies that are functionally advantageous over antibodies from natural antibody repertoires. In this work, the sequence space of the complementarity determining regions of a broadly neutralizing antibody (F10) targeting the stem epitope on the hemagglutinin of a strain of H1N1 influenza virus was systematically explored; the elucidated antibody-hemagglutinin recognition principles were used to design a phage-displayed antibody library, which was then used to discover neutralizing antibodies against another strain of H1N1 virus. More than 1000 functional antibody candidates were selected from the antibody library and were shown to neutralize the corresponding strain of influenza virus with up to 7 folds higher potency comparing with the parent F10 antibody. The antibody library could be used to discover functionally effective antibodies against other H1N1 influenza viruses, supporting the notion that target-specific antibody libraries can be designed and constructed with systematic sequence-function information.

  19. Enhanced clearance of HIV-1-infected cells by broadly neutralizing antibodies against HIV-1 in vivo.

    PubMed

    Lu, Ching-Lan; Murakowski, Dariusz K; Bournazos, Stylianos; Schoofs, Till; Sarkar, Debolina; Halper-Stromberg, Ariel; Horwitz, Joshua A; Nogueira, Lilian; Golijanin, Jovana; Gazumyan, Anna; Ravetch, Jeffrey V; Caskey, Marina; Chakraborty, Arup K; Nussenzweig, Michel C

    2016-05-20

    Antiretroviral drugs and antibodies limit HIV-1 infection by interfering with the viral life cycle. In addition, antibodies also have the potential to guide host immune effector cells to kill HIV-1-infected cells. Examination of the kinetics of HIV-1 suppression in infected individuals by passively administered 3BNC117, a broadly neutralizing antibody, suggested that the effects of the antibody are not limited to free viral clearance and blocking new infection but also include acceleration of infected cell clearance. Consistent with these observations, we find that broadly neutralizing antibodies can target CD4(+) T cells infected with patient viruses and can decrease their in vivo half-lives by a mechanism that requires Fcγ receptor engagement in a humanized mouse model. The results indicate that passive immunotherapy can accelerate elimination of HIV-1-infected cells.

  20. Roles of glycans in interactions between gp120 and HIV broadly neutralizing antibodies.

    PubMed

    Qi, Yifei; Jo, Sunhwan; Im, Wonpil

    2016-03-01

    Many novel broadly neutralizing antibodies against human immunodeficiency virus (HIV) have been identified during the past decade, providing promising templates for the development of an effective HIV-1 vaccine. Structural studies reveal that the epitopes of some of these antibodies involve one or more crucial glycans, without which the binding is completely abolished. In this study, we have investigated the critical roles of glycans in interactions between HIV-1 gp120 and two broadly neutralizing antibodies PG9 (targeting V1/V2) and PGT128 (targeting V3) that are able to neutralize more than 70% of HIV-1 isolates. We have performed molecular dynamics simulations of a number of systems including antibody-gp120 complex with and without glycans, antibody, gp120 with and without glycans, and glycan-only systems. The simulation results show that the complex structures are stabilized by the glycans, and the multivalent interactions between the antibody and gp120 promote cooperativities to further enhance the binding. In the free gp120, the glycans increase the flexibility of the V1/V2 and V3 loops, which likely increases the entropy cost of the antibody recognition. However, the antibodies are able to bind the flexible interface by recognizing the preexisting glycan conformation, and penetrating the glycan shield with flexible complementarity determining region loops that sample the bound conformations occasionally.

  1. Characterization of Neutralizing Antibodies and Identification of Neutralizing Epitope Mimics on the Clostridium botulinum Neurotoxin Type A

    PubMed Central

    Wu, Han-Chung; Yeh, Chia-Tsui; Huang, Yue-Ling; Tarn, Lih-Jeng; Lung, Chien-Cheng

    2001-01-01

    Clostridium botulinum neurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 103 and 104 times the 50% lethal dose, respectively. Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic acid (D5) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K5). Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A. These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies. In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine. PMID:11425742

  2. Global Structure of HIV-1 Neutralizing Antibody IgG1 b12 is Asymmetric

    SciTech Connect

    Ashish, F.; Solanki, A; Boone, C; Krueger, J

    2010-01-01

    Human antibody IgG1 b12 is one of the four antibodies known to neutralize a broad range of human immunodeficiency virus-1. The crystal structure of this antibody displayed an asymmetric disposition of the Fab arms relative to its Fc portion. Comparison of structures solved for other IgG1 antibodies led to a notion that crystal packing forces entrapped a 'snap-shot' of different conformations accessible to this antibody. To elucidate global structure of this unique antibody, we acquired small-angle X-ray scattering data from its dilute solution. Data analysis indicated that b12 adopts a bilobal globular structure in solution with a radius of gyration and a maximum linear dimension of {approx}54 and {approx}180 {angstrom}, respectively. Extreme similarity between its solution and crystal structure concludes that non-flexible, asymmetric shape is an inherent property of this rare antibody.

  3. Escape from neutralization by the respiratory syncytial virus-specific neutralizing monoclonal antibody palivizumab is driven by changes in on-rate of binding to the fusion protein.

    PubMed

    Bates, John T; Keefer, Christopher J; Slaughter, James C; Kulp, Daniel W; Schief, William R; Crowe, James E

    2014-04-01

    The role of binding kinetics in determining neutralizing potency for antiviral antibodies is poorly understood. While it is believed that increased steady-state affinity correlates positively with increased virus-neutralizing activity, the relationship between association or dissociation rate and neutralization potency is unclear. We investigated the effect of naturally-occurring antibody resistance mutations in the RSV F protein on the kinetics of binding to palivizumab. Escape from palivizumab-mediated neutralization of RSV occurred with reduced association rate (Kon) for binding to RSV F protein, while alteration of dissociation rate (Koff) did not significantly affect neutralizing activity. Interestingly, linkage of reduced Kon with reduced potency mirrored the effect of increased Kon found in a high-affinity enhanced potency palivizumab variant (motavizumab). These data suggest that association rate is the dominant factor driving neutralization potency for antibodies to RSV F protein antigenic site A and determines the potency of antibody somatic variants or efficiency of escape of viral glycoprotein variants.

  4. Prevalence of neutralizing antibodies to 9 rotavirus strains representing 7 G-serotypes in sheep sera.

    PubMed

    Muñoz, M; Lanza, I; Alvarez, M; Cármenes, P

    1995-08-01

    Neutralizing antibodies to 9 rotavirus strains representing serotypes G1, G3, G5, G6, G8, G9, and G10 were investigated in 212 ovine serum samples from 3 age groups, 1-week-old lambs, 2- to 3-months-old lambs and adult sheep. All sera from 1-week-old lambs had neutralizing antibodies to all 9 rotavirus strains. Both neutralizing antibody titers and prevalences to all 9 strains markedly decreased in the 2- to 3-months-old lamb group and increased again in the adult sheep group. Also, adult sheep sera neutralized a larger number of rotavirus strains than 2- to 3-months-old lamb sera. The highest neutralizing antibody titers and prevalences were found to strains B223 and K923, representing serotype G10, to strain RRV, representing serotype G3, and to strain NCDV, representing serotype G6, indicating that these could be the predominant 3 rotavirus serotypes in Spanish sheep. The rotavirus serotypes infecting sheep observed by us differ from those described for cattle, where G6 is the most prevalent serotype followed by G10, and G3 has been seldom found. Very low prevalences were observed for strains WA and OSU representing serotypes G1 and G5 respectively, suggesting that they probably do not infect sheep and neutralizing antibodies found are derived from heterotypic responses to other serotypes. Intermediate prevalences and titers were found to strains UK (serotype G6), 69M (serotype G8) and WI61 (serotype G9). Neutralizing antibodies distinguished between different strains sharing their VP7 specificity: B223 and K923, a bovine and an ovine serotype G10 strains, and NCDV and UK, two serotype G6 bovine rotavirus strains with different VP4 antigen.

  5. Kinetics of the neutralizing antibody response to respiratory syncytial virus infections in a birth cohort.

    PubMed

    Sande, C J; Mutunga, M N; Okiro, E A; Medley, G F; Cane, P A; Nokes, D J

    2013-11-01

    The kinetics of respiratory syncytial virus (RSV) neutralizing antibodies following birth, primary and secondary infections are poorly defined. The aims of the study were to measure and compare neutralizing antibody responses at different time points in a birth cohort followed-up over three RSV epidemics. Rural Kenyan children, recruited at birth between 2002 and 2003, were monitored for RSV infection over three epidemic seasons. Cord and 3-monthly sera, and acute and convalescent sera following RSV infection, were assayed in 28 children by plaque reduction neutralization test (PRNT). Relative to the neutralizing antibody titers of pre-exposure control sera (1.8 log10 PRNT), antibody titers following primary infection were (i) no different in sera collected between 0 and 0.4 months post-infection (1.9 log10 PRNT, P=0.146), (ii) higher in sera collected between 0.5 and 0.9 (2.8 log10 PRNT, P<0.0001), 1.0-1.9 (2.5 log10 PRNT, P<0.0001), and 2.0-2.9 (2.3 log10 PRNT, P<0.001) months post-infection, and (iii) no different in sera collected at between 3.0 and 3.9 months post-infection (2.0 log10 PRNT, P=0.052). The early serum neutralizing response to secondary infection (3.02 log10 PRNT) was significantly greater than the early primary response (1.9 log10 PRNT, P<0.0001). Variation in population-level virus transmission corresponded with changes in the mean cohort-level neutralizing titers. It is concluded that following primary RSV infection the neutralizing antibody response declines to pre-infection levels rapidly (~3 months) which may facilitate repeat infection. The kinetics of the aggregate levels of acquired antibody reflect seasonal RSV occurrence, age, and infection history.

  6. Characterization of Two Human Monoclonal Antibodies Neutralizing Influenza A H7N9 Viruses

    PubMed Central

    Wang, Jianmin; Chen, Zhe; Bao, Linlin; Zhang, Weijia; Xue, Ying; Pang, XingHuo; Zhang, Xi

    2015-01-01

    H7N9 was a cause of significant global health concern due to its severe infection and approximately 35% mortality in humans. By screening a Fab antibody phage library derived from patients who recovered from H7N9 infections, we characterized two human monoclonal antibodies (HuMAbs), HNIgGD5 and HNIgGH8. The epitope of these two antibodies was dependent on two residues in the receptor binding site at positions V186 and L226 of the hemagglutinin glycoprotein. Both antibodies possessed high neutralizing activity. PMID:26063436

  7. A fusion intermediate gp41 immunogen elicits neutralizing antibodies to HIV-1.

    PubMed

    Lai, Rachel P J; Hock, Miriam; Radzimanowski, Jens; Tonks, Paul; Hulsik, David Lutje; Effantin, Gregory; Seilly, David J; Dreja, Hanna; Kliche, Alexander; Wagner, Ralf; Barnett, Susan W; Tumba, Nancy; Morris, Lynn; LaBranche, Celia C; Montefiori, David C; Seaman, Michael S; Heeney, Jonathan L; Weissenhorn, Winfried

    2014-10-24

    The membrane-proximal external region (MPER) of the human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein subunit gp41 is targeted by potent broadly neutralizing antibodies 2F5, 4E10, and 10E8. These antibodies recognize linear epitopes and have been suggested to target the fusion intermediate conformation of gp41 that bridges viral and cellular membranes. Anti-MPER antibodies exert different degrees of membrane interaction, which is considered to be the limiting factor for the generation of such antibodies by immunization. Here we characterize a fusion intermediate conformation of gp41 (gp41(int)-Cys) and show that it folds into an elongated ∼ 12-nm-long extended structure based on small angle x-ray scattering data. Gp41(int)-Cys was covalently linked to liposomes via its C-terminal cysteine and used as immunogen. The gp41(int)-Cys proteoliposomes were administered alone or in prime-boost regimen with trimeric envelope gp140(CA018) in guinea pigs and elicited high anti-gp41 IgG titers. The sera interacted with a peptide spanning the MPER region, demonstrated competition with broadly neutralizing antibodies 2F5 and 4E10, and exerted modest lipid binding, indicating the presence of MPER-specific antibodies. Although the neutralization potency generated solely by gp140(CA018) was higher than that induced by gp41(int)-Cys, the majority of animals immunized with gp41(int)-Cys proteoliposomes induced modest breadth and potency in neutralizing tier 1 pseudoviruses and replication-competent simian/human immunodeficiency viruses in the TZM-bl assay as well as responses against tier 2 HIV-1 in the A3R5 neutralization assay. Our data thus demonstrate that liposomal gp41 MPER formulation can induce neutralization activity, and the strategy serves to improve breadth and potency of such antibodies by improved vaccination protocols.

  8. Complexity of Neutralizing Antibodies against Multiple Dengue Virus Serotypes after Heterotypic Immunization and Secondary Infection Revealed by In-Depth Analysis of Cross-Reactive Antibodies

    PubMed Central

    Tsai, Wen-Yang; Durbin, Anna; Tsai, Jih-Jin; Hsieh, Szu-Chia; Whitehead, Stephen

    2015-01-01

    ABSTRACT The four serotypes of dengue virus (DENV) cause the most important and rapidly emerging arboviral diseases in humans. The recent phase 2b and 3 studies of a tetravalent dengue vaccine reported a moderate efficacy despite the presence of neutralizing antibodies, highlighting the need for a better understanding of neutralizing antibodies in polyclonal human sera. Certain type-specific (TS) antibodies were recently discovered to account for the monotypic neutralizing activity and protection after primary DENV infection. The nature of neutralizing antibodies after secondary DENV infection remains largely unknown. In this study, we examined sera from 10 vaccinees with well-documented exposure to first and second DENV serotypes through heterotypic immunization with live-attenuated vaccines. Higher serum IgG avidities to both exposed and nonexposed serotypes were found after secondary immunization than after primary immunization. Using a two-step depletion protocol to remove different anti-envelope antibodies, including group-reactive (GR) and complex-reactive (CR) antibodies separately, we found GR and CR antibodies together contributed to more than 50% of neutralizing activities against multiple serotypes after secondary immunization. Similar findings were demonstrated in patients after secondary infection. Anti-envelope antibodies recognizing previously exposed serotypes consisted of a large proportion of GR antibodies, CR antibodies, and a small proportion of TS antibodies, whereas those recognizing nonexposed serotypes consisted of GR and CR antibodies. These findings have implications for sequential heterotypic immunization or primary immunization of DENV-primed individuals as alternative strategies for DENV vaccination. The complexity of neutralizing antibodies after secondary infection provides new insights into the difficulty of their application as surrogates of protection. IMPORTANCE The four serotypes of dengue virus (DENV) are the leading cause of

  9. Broadening the neutralizing capacity of a family of antibody fragments against different toxins from Mexican scorpions.

    PubMed

    Rodríguez-Rodríguez, Everardo Remi; Olamendi-Portugal, Timoteo; Serrano-Posada, Hugo; Arredondo-López, Jonathan Noé; Gómez-Ramírez, Ilse; Fernández-Taboada, Guillermo; Possani, Lourival D; Anguiano-Vega, Gerardo Alfonso; Riaño-Umbarila, Lidia; Becerril, Baltazar

    2016-09-01

    New approaches aimed at neutralizing the primary toxic components present in scorpion venoms, represent a promising alternative to the use of antivenoms of equine origin in humans. New potential therapeutics developed by these approaches correspond to neutralizing antibody fragments obtained by selection and maturation processes from libraries of human origin. The high sequence identity shared among scorpion toxins is associated with an important level of cross reactivity exhibited by these antibody fragments. We have exploited the cross reactivity showed by single chain variable antibody fragments (scFvs) of human origin to re-direct the neutralizing capacity toward various other scorpion toxins. As expected, during these evolving processes several variants derived from a parental scFv exhibited the capacity to simultaneously recognize and neutralize different toxins from Centruroides scorpion venoms. A sequence analyses of the cross reacting scFvs revealed that specific mutations are responsible for broadening their neutralizing capacity. In this work, we generated a set of new scFvs that resulted from the combinatorial insertion of these point mutations. These scFvs are potential candidates to be part of a novel recombinant antivenom of human origin that could confer protection against scorpion stings. A remarkable property of one of these new scFvs (ER-5) is its capacity to neutralize at least three different toxins and its complementary capacity to neutralize the whole venom from Centruroides suffusus in combination with a second scFv (LR), which binds to a different epitope shared by Centruroides scorpion toxins.

  10. Genetic Signatures in the Envelope Glycoproteins of HIV-1 that Associate with Broadly Neutralizing Antibodies

    PubMed Central

    Gnanakaran, S.; Daniels, Marcus G.; Bhattacharya, Tanmoy; Lapedes, Alan S.; Sethi, Anurag; Li, Ming; Tang, Haili; Greene, Kelli; Gao, Hongmei; Haynes, Barton F.; Cohen, Myron S.; Shaw, George M.; Seaman, Michael S.; Kumar, Amit; Gao, Feng; Montefiori, David C.; Korber, Bette

    2010-01-01

    A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env) is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an important role for

  11. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies

    PubMed Central

    Chervyakova, Olga V.; Zaitsev, Valentin L.; Iskakov, Bulat K.; Tailakova, Elmira T.; Strochkov, Vitaliy M.; Sultankulova, Kulyaisan T.; Sandybayev, Nurlan T.; Stanbekova, Gulshan E.; Beisenov, Daniyar K.; Abduraimov, Yergali O.; Mambetaliyev, Muratbay; Sansyzbay, Abylay R.; Kovalskaya, Natalia Y.; Nemchinov, Lev. G.; Hammond, Rosemarie W.

    2016-01-01

    The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. PMID:27338444

  12. Broadly Neutralizing Antibodies Display Potential for Prevention of HIV-1 Infection of Mucosal Tissue Superior to That of Nonneutralizing Antibodies.

    PubMed

    Cheeseman, Hannah M; Olejniczak, Natalia J; Rogers, Paul M; Evans, Abbey B; King, Deborah F L; Ziprin, Paul; Liao, Hua-Xin; Haynes, Barton F; Shattock, Robin J

    2017-01-01

    Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralization is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Nonneutralizing antibodies (nnAbs) may also impact mucosal transmission events through Fc-gamma receptor (FcγR)-mediated inhibition. In this study, a panel of broadly neutralizing antibodies (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for the ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralization potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4-binding site (CD4bs)-specific bnAbs, in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. Membrane-proximal external region (MPER) (2F5) and outer domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) was more variable. In contrast, nnAbs alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest that tissue resident effector cell numbers and low FcγR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over that of nonneutralizing antibodies for preventing HIV-1 infection at the mucosal portals of infection.

  13. Crystal structure of human antibody 2909 reveals conserved features of quaternary structure-specific antibodies that potently neutralize HIV-1.

    PubMed

    Changela, Anita; Wu, Xueling; Yang, Yongping; Zhang, Baoshan; Zhu, Jiang; Nardone, Glenn A; O'Dell, Sijy; Pancera, Marie; Gorny, Miroslaw K; Phogat, Sanjay; Robinson, James E; Stamatatos, Leonidas; Zolla-Pazner, Susan; Mascola, John R; Kwong, Peter D

    2011-03-01

    Monoclonal antibody 2909 belongs to a class of potently neutralizing antibodies that recognize quaternary epitopes on HIV-1. Some members of this class, such as 2909, are strain specific, while others, such as antibody PG16, are broadly neutralizing; all, however, recognize a region on the gp120 envelope glycoprotein that includes two loops (V2 and V3) and forms appropriately only in the oligomeric HIV-1 spike (gp120(3)/gp41(3)). Here we present the crystal structure of 2909 and report structure-function analysis with antibody chimeras composed of 2909 and other members of this antibody class. The 2909 structure was dominated by a heavy-chain third-complementarity-determining region (CDR H3) of 21 residues, which comprised 36% of the combining surface and formed a β-hairpin club extending ∼20 Å beyond the rest of the antibody. Sequence analysis and mass spectrometry identified sites of tyrosine sulfation at the middle and top of CDR H3; substitutions with phenylalanine either ablated (middle substitution) or substantially diminished (top substitution) neutralization. Chimeric antibodies composed of heavy and light chains, exchanged between 2909 and other members of the class, indicated a substantial lack of complementation. Comparison of 2909 to PG16 (which is tyrosine sulfated and the only other member of the class for which a structure has previously been reported) showed that both utilize protruding, anionic CDR H3s for recognition. Thus, despite some diversity, members of this class share structural and functional similarities, with conserved features of the CDR H3 subdomain likely reflecting prevalent solutions by the human immune system for recognition of a quaternary site of HIV-1 vulnerability.

  14. Autophagy-associated dengue vesicles promote viral transmission avoiding antibody neutralization

    PubMed Central

    Wu, Yan-Wei; Mettling, Clément; Wu, Shang-Rung; Yu, Chia-Yi; Perng, Guey-Chuen; Lin, Yee-Shin; Lin, Yea-Lih

    2016-01-01

    One of the major defense mechanisms against virus spread in vivo is the blocking of viral infectibility by neutralizing antibodies. We describe here the identification of infectious autophagy-associated dengue vesicles released from infected cells. These vesicles contain viral proteins E, NS1, prM/M, and viral RNA, as well as host lipid droplets and LC3-II, an autophagy marker. The viral RNA can be protected within the autophagic organelles since anti-dengue neutralizing antibodies do not have an effect on the vesicle-mediated transmission that is able to initiate a new round of infection in target cells. Importantly, such infectious vesicles were also detected in a patient serum. Our study suggests that autophagy machinery plays a new role in dengue virus transmission. This discovery explains the inefficiency of neutralizing antibody upon dengue infection as a potential immune evasion mechanism in vivo. PMID:27558165

  15. Neutralizing Antibodies and Sin Nombre Virus RNA after Recovery from Hantavirus Cardiopulmonary Syndrome

    PubMed Central

    Ye, Chunyan; Prescott, Joseph; Nofchissey, Robert; Goade, Diane

    2004-01-01

    Patients who later have a mild course of hantavirus cardiopulmonary syndrome (HCPS) are more likely to exhibit a high titer of neutralizing antibodies against Sin Nombre virus (SNV), the etiologic agent of HCPS, at the time of hospital admission. Because administering plasma from patients who have recovered from HCPS to those in the early stages of disease may be an advantageous form of passive immunotherapy, we examined the neutralizing antibody titers of 21 patients who had recovered from SNV infection. Even 1,000 days after admission to the hospital, 6 of 10 patients had titers of 800 or higher, with one sample retaining a titer of 3,200 after more than 1,400 days. None of the convalescent-phase serum samples contained detectable viral RNA. These results confirm that patients retain high titers of neutralizing antibodies long after recovery from SNV infection. PMID:15109416

  16. Selection of Peptide Mimics of HIV-1 Epitope Recognized by Neutralizing Antibody VRC01

    PubMed Central

    Chikaev, Anton N.; Bakulina, Anastasiya Yu.; Burdick, Ryan C.; Karpenko, Larisa I.; Pathak, Vinay K.; Ilyichev, Alexander A.

    2015-01-01

    The ability to induce anti-HIV-1 antibodies that can neutralize a broad spectrum of viral isolates from different subtypes seems to be a key requirement for development of an effective HIV-1 vaccine. The epitopes recognized by the most potent broadly neutralizing antibodies that have been characterized are largely discontinuous. Mimetics of such conformational epitopes could be potentially used as components of a synthetic immunogen that can elicit neutralizing antibodies. Here we used phage display technology to identify peptide motifs that mimic the epitope recognized by monoclonal antibody VRC01, which is able to neutralize up to 91% of circulating primary isolates. Three rounds of biopanning were performed against 2 different phage peptide libraries for this purpose. The binding specificity of selected phage clones to monoclonal antibody VRC01 was estimated using dot blot analysis. The putative peptide mimics exposed on the surface of selected phages were analyzed for conformational and linear homology to the surface of HIV-1 gp120 fragment using computational analysis. Corresponding peptides were synthesized and checked for their ability to interfere with neutralization activity of VRC01 in a competitive inhibition assay. One of the most common peptides selected from 12-mer phage library was found to partially mimic a CD4-binding loop fragment, whereas none of the circular C7C-mer peptides was able to mimic any HIV-1 domains. However, peptides identified from both the 12-mer and C7C-mer peptide libraries showed rescue of HIV-1 infectivity in the competitive inhibition assay. The identification of epitope mimics may lead to novel immunogens capable of inducing broadly reactive neutralizing antibodies. PMID:25785734

  17. Neutralizing activities of caprine antibodies towards conserved regions of the HCV envelope glycoprotein E2

    PubMed Central

    2011-01-01

    Anti HCV vaccine is not currently available and the present antiviral therapies fail to cure approximately half of the treated HCV patients. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 and test their neutralizing activities in a step towards developing therapeutic and/or prophylactic immunogens against HCV infection. Antibodies were generated by vaccination of goats with synthetic peptides derived from HCV E2. Viral neutralizing capacity of the generated anti E2 antibodies was tested using in vitro assays. Goats immunized with E2 synthetic peptides termed p412 [a.a 412-419], p430 [a.a 430-447] and p517 [a.a 517-531] generated high titers of antibody responses 2 to 4.5 fold higher than comparable titers of antibodies to the same epitopes in chronic HCV patients. In post infection experiments of native HCV into cultured Huh7.5 cells anti p412 and anti p 517 were proven to be neutralizing to HCV genotype 4a from patients' sera (87.5% and 75% respectively). On the contrary anti p430 exhibited weak viral neutralization capacity on the same samples (31.25%). Furthermore Ab mixes containing anti p430 exhibited reduced viral neutralization properties. From these experiments one could predict that neutralization by Abs towards different E2-epitopes varies considerably and success in the enrichment of neutralization epitope-specific antibodies may be accompanied by favorable results in combating HCV infection. Also, E2 conserved peptides p517 and p412 represent potential components of a candidate peptide vaccine against HCV infection. PMID:21819575

  18. Improved Protection of Rhesus Macaques against Intrarectal Simian Immunodeficiency Virus SIVmac251 Challenge by a Replication-Competent Ad5hr-SIVenv/rev and Ad5hr-SIVgag Recombinant Priming/gp120 Boosting Regimen

    PubMed Central

    Zhao, Jun; Pinczewski, Joel; Gómez-Román, Victor R.; Venzon, David; Kalyanaraman, V. S.; Markham, Phillip D.; Aldrich, Kristine; Moake, Matthew; Montefiori, David C.; Lou, Yuanmei; Pavlakis, George N.; Robert-Guroff, Marjorie

    2003-01-01

    In this study we investigated the ability of a replication-competent Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinant priming/gp120 boosting regimen to induce protective immunity in rhesus macaques against pathogenic simian immunodeficiency virusmac251. Immunization of macaques by two sequential administrations of the same recombinants by the same route resulted in boosting and persistence of SIV-specific cellular immune responses for 42 weeks past the initial immunization. Anti-SIV gp120 immunoglobulin G (IgG) and IgA antibodies were induced in secretory fluids, and all macaques exhibited serum neutralizing antibody activity. After intrarectal SIVmac251 challenge, all of the macaques became infected. However, relative protection, as assessed by statistically significant lower SIV viral loads in plasma at both acute infection and set point, was observed in 8 out of 12 immunized non-Mamu-A∗01 animals. Elevated mean cellular immune responses to Gag and Env, neutralizing antibody activity, and IgG and IgA binding antibody levels were observed in the eight protected macaques. Statistically significant correlations with protective outcome were observed for cellular immune responses to SIV Env and Gag and for SIV gp120-specific IgG antibodies in nasal and vaginal fluids. Two macaques that exhibited the greatest and most persistent viremia control also exhibited strong CD8+ T-cell antiviral activity. The results suggest that a spectrum of immune responses may be necessary for adequate control of viral replication and disease progression and highlight a potential role for nonneutralizing antibodies at mucosal sites. PMID:12857905

  19. Structure of a Human Astrovirus Capsid-Antibody Complex and Mechanistic Insights into Virus Neutralization

    SciTech Connect

    Bogdanoff, Walter A.; Campos, Jocelyn; Perez, Edmundo I.; Yin, Lu; Alexander, David L.; DuBois, Rebecca M.; López, Susana

    2016-11-02

    ABSTRACT

    Human astroviruses (HAstVs) are a leading cause of viral diarrhea in young children, the immunocompromised, and the elderly. There are no vaccines or antiviral therapies against HAstV disease. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against reinfection by HAstV. However, development of anti-HAstV therapies is hampered by the gap in knowledge of protective antibody epitopes on the HAstV capsid surface. Here, we report the structure of the HAstV capsid spike domain bound to the neutralizing monoclonal antibody PL-2. The antibody uses all six complementarity-determining regions to bind to a quaternary epitope on each side of the dimeric capsid spike. We provide evidence that the HAstV capsid spike is a receptor-binding domain and that the antibody neutralizes HAstV by blocking virus attachment to cells. We identify patches of conserved amino acids that overlap the antibody epitope and may comprise a receptor-binding site. Our studies provide a foundation for the development of therapies to prevent and treat HAstV diarrheal disease.

    IMPORTANCEHuman astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies in healthy adults protect them against reinfection. Here, we determined the crystal structure of a complex of the HAstV capsid protein and a virus-neutralizing antibody. We show that the antibody binds to the outermost spike domain of the capsid, and we provide evidence that the antibody blocks virus attachment to human cells. Importantly, our findings suggest that a subunit-based vaccine focusing the immune system on the HAstV capsid spike domain could be effective in protecting children against HAstV disease.

  20. Structural Insights into Reovirus σ1 Interactions with Two Neutralizing Antibodies.

    PubMed

    Dietrich, Melanie H; Ogden, Kristen M; Katen, Sarah P; Reiss, Kerstin; Sutherland, Danica M; Carnahan, Robert H; Goff, Matthew; Cooper, Tracy; Dermody, Terence S; Stehle, Thilo

    2017-02-15

    Reovirus attachment protein σ1 engages glycan receptors and junctional adhesion molecule-A (JAM-A) and is thought to undergo a conformational change during the proteolytic disassembly of virions to infectious subvirion particles (ISVPs) that accompanies cell entry. The σ1 protein is also the primary target of neutralizing antibodies. Here, we present a structural and functional characterization of two neutralizing antibodies that target σ1 of serotype 1 (T1) and serotype 3 (T3) reoviruses. The crystal structures revealed that each antibody engages its cognate σ1 protein within the head domain via epitopes distinct from the JAM-A-binding site. Surface plasmon resonance and cell-binding assays indicated that both antibodies likely interfere with JAM-A engagement by steric hindrance. To define the interplay between the carbohydrate receptor and antibody binding, we conducted hemagglutination inhibition assays using virions and ISVPs. The glycan-binding site of T1 σ1 is located in the head domain and is partly occluded by the bound Fab in the crystal structure. The T1-specific antibody inhibited hemagglutination by virions and ISVPs, probably via direct interference with glycan engagement. In contrast to T1 σ1, the carbohydrate-binding site of T3 σ1 is located in the tail domain, distal to the antibody epitope. The T3-specific antibody inhibited hemagglutination by T3 virions but not ISVPs, indicating that the antibody- and glycan-binding sites in σ1 are in closer spatial proximity on virions than on ISVPs. Our results provide direct evidence for a structural rearrangement of σ1 during virion-to-ISVP conversion and contribute new information about the mechanisms of antibody-mediated neutralization of reovirus.

  1. Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus.

    PubMed

    Wang, Qihui; Yang, Huabing; Liu, Xiaoqing; Dai, Lianpan; Ma, Tong; Qi, Jianxun; Wong, Gary; Peng, Ruchao; Liu, Sheng; Li, Junfu; Li, Shihua; Song, Jian; Liu, Jianying; He, Jianhua; Yuan, Hui; Xiong, Ying; Liao, Yong; Li, Jianhua; Yang, Jianping; Tong, Zhou; Griffin, Bryan D; Bi, Yuhai; Liang, Mifang; Xu, Xiaoning; Qin, Chuan; Cheng, Gong; Zhang, Xinzheng; Wang, Peiyi; Qiu, Xiangguo; Kobinger, Gary; Shi, Yi; Yan, Jinghua; Gao, George F

    2016-12-14

    The 2015-2016 outbreak of Zika virus (ZIKV) disease has affected many countries and is a major public health concern. ZIKV is associated with fetal microcephaly and neurological complications, and countermeasures are needed to treat and prevent ZIKV infection. We report the isolation of 13 specific human monoclonal antibodies from a single patient infected with ZIKV. Two of the isolated antibodies (Z23 and Z3L1) demonstrated potent ZIKV-specific neutralization in vitro without binding or neutralizing activity against strains 1 to 4 of dengue virus, the closest relative to ZIKV. These two antibodies provided postexposure protection to mice in vivo. Structural studies revealed that Z23 and Z3L1 bound to tertiary epitopes in envelope protein domain I, II, or III, indicating potential targets for ZIKV-specific therapy. Our results suggest the potential of antibody-based therapeutics and provide a structure-based rationale for the design of future ZIKV-specific vaccines.

  2. Virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions.

    PubMed Central

    Holland, J J; de la Torre, J C; Steinhauer, D A; Clarke, D; Duarte, E; Domingo, E

    1989-01-01

    Monoclonal antibody-resistant mutants have been widely used to estimate virus mutation frequencies. We demonstrate that standard virion neutralization inevitably underestimates monoclonal antibody-resistant mutant genome frequencies of vesicular stomatitis virus, due to phenotypic masking-mixing when wild-type (wt) virions are present in thousandsfold greater numbers. We show that incorporation of antibody into the plaque overlay medium (after virus penetration at 37 degrees C) can provide accurate estimates of genome frequencies of neutral monoclonal antibody-resistant mutant viruses in wt clones. By using this method, we have observed two adjacent G----A base transition frequencies in the I3 epitope to be of the order of 10(-4) in a wt glycine codon. This appears to be slightly lower than the frequencies observed at other sites for total (viable and nonviable) virus genomes when using a direct sequence approach. Images PMID:2479770

  3. Isolation of potent neutralizing antibodies from a survivor of the 2014 Ebola virus outbreak

    PubMed Central

    Bornholdt, Zachary A.; Turner, Hannah L.; Murin, Charles D.; Li, Wen; Sok, Devin; Souders, Colby A.; Piper, Ashley E.; Goff, Arthur; Shamblin, Joshua D.; Wollen, Suzanne E.; Sprague, Thomas R.; Fusco, Marnie L.; Pommert, Kathleen B.J.; Cavacini, Lisa A.; Smith, Heidi L.; Klempner, Mark; Reimann, Keith A.; Krauland, Eric; Gerngross, Tillman U.; Wittrup, Dane K.; Saphire, Erica Ollmann; Burton, Dennis R.; Glass, Pamela J.; Ward, Andrew B.; Walker, Laura M.

    2016-01-01

    Antibodies targeting the Ebola virus surface glycoprotein (EBOV GP) are implicated in protection against lethal disease, but the characteristics of the human antibody response to EBOV GP remain poorly understood. Here we isolated and characterized 349 GP-specific monoclonal antibodies (mAbs) from the peripheral B cells of a convalescent donor who survived the 2014 EBOV Zaire outbreak. Remarkably, 77% of the mAbs neutralize live EBOV and several mAbs exhibit unprecedented potency. Structures of selected mAbs in complex with GP reveal a site of vulnerability located in the GP stalk region proximal to the viral membrane. Neutralizing antibodies (NAbs) targeting this site show potent therapeutic efficacy against lethal EBOV challenge in mice. The results provide a framework for the design of new EBOV vaccine candidates and immunotherapies. PMID:26912366

  4. Molecular Basis for Antibody-Mediated Neutralization of New World Hemorrhagic Fever Mammarenaviruses.

    PubMed

    Mahmutovic, Selma; Clark, Lars; Levis, Silvana C; Briggiler, Ana M; Enria, Delia A; Harrison, Stephen C; Abraham, Jonathan

    2015-12-09

    In the Western hemisphere, at least five mammarenaviruses cause human viral hemorrhagic fevers with high case fatality rates. Junín virus (JUNV) is the only hemorrhagic fever virus for which transfusion of survivor immune plasma that contains neutralizing antibodies ("passive immunity") is an established treatment. Here, we report the structure of the JUNV surface glycoprotein receptor-binding subunit (GP1) bound to a neutralizing monoclonal antibody. The antibody engages the GP1 site that binds transferrin receptor 1 (TfR1)-the host cell surface receptor for all New World hemorrhagic fever mammarenaviruses-and mimics an important receptor contact. We show that survivor immune plasma contains antibodies that bind the same epitope. We propose that viral receptor-binding site accessibility explains the success of passive immunity against JUNV and that this functionally conserved epitope is a potential target for therapeutics and vaccines to limit infection by all New World hemorrhagic fever mammarenaviruses.

  5. Molecular basis for antibody-mediated neutralization of New World hemorrhagic fever mammarenaviruses

    PubMed Central

    Mahmutovic, Selma; Clark, Lars; Levis, Silvana C.; Briggiler, Ana M.; Enria, Delia A.; Harrison, Stephen C.; Abraham, Jonathan

    2015-01-01

    SUMMARY In the Western hemisphere, at least five mammarenaviruses cause human viral hemorrhagic fevers with high case fatality rates. Junín virus (JUNV) is the only hemorrhagic fever virus for which transfusion of survivor immune plasma that contains neutralizing antibodies (‘passive immunity’) is an established treatment. Here, we report the structure of the JUNV surface glycoprotein receptor-binding subunit (GP1) bound to a neutralizing monoclonal antibody. The antibody engages the GP1 site that binds transferrin receptor 1 (TfR1) – the host cell surface receptor for all New World hemorrhagic fever mammarenaviruses - and mimics an important receptor contact. We show that survivor immune plasma contains antibodies that bind the same epitope. We propose that viral receptor-binding site accessibility explains the success of passive immunity against JUNV and that this functionally conserved epitope is a potential target for therapeutics and vaccines to limit infection by all New World hemorrhagic fever mammarenaviruses. PMID:26651946

  6. Cross-Reactivity of Neutralizing Antibodies among Malignant Catarrhal Fever Viruses

    PubMed Central

    Taus, Naomi S.; Cunha, Cristina W.; Marquard, Jana; O’Toole, Donal; Li, Hong

    2015-01-01

    Some members of the gamma herpesvirus genus Macavirus are maintained in nature as subclinical infections in well-adapted ungulate hosts. Transmission of these viruses to poorly adapted hosts, such as American bison and cattle, can result in the frequently fatal disease malignant catarrhal fever (MCF). Based on phylogenetic analysis, the MCF viruses (MCFV) cluster into two subgroups corresponding to the reservoir hosts’ subfamilies: Alcelaphinae/Hippotraginae and Caprinae. Antibody cross-reactivity among MCFVs has been demonstrated using techniques such as enzyme linked immunosorbent and immunofluorescence assays. However, minimal information is available as to whether virus neutralizing antibodies generated against one MCFV cross react with other members of the genus. This study tested the neutralizing activity of serum and plasma from select MCFV-infected reservoir hosts against alcelaphine herpesvirus 1 (AlHV-1) and ovine herpesvirus 2 (OvHV-2). Neutralizing antibody activity against AlHV-1 was detected in samples from infected hosts in the Alcelaphinae and Hippotraginae subfamilies, but not from hosts in the Caprinae subfamily. OvHV-2 neutralizing activity was demonstrated in samples from goats (Caprinae) but not from wildebeest (Alcelaphinae). These results show that neutralizing antibody cross reactivity is present to MCFVs within a virus subgroup but not between subgroups. This information is important for diagnosing infection with MCFVs and in the development of vaccines against MCF. PMID:26658281

  7. Interactions between HIV-1 Neutralizing Antibodies and Model Lipid Membranes imaged with AFM

    NASA Astrophysics Data System (ADS)

    Zauscher, Stefan; Hardy, Gregory; Alam, Munir; Shapter, Joseph

    2012-02-01

    Lipid membrane interactions with rare, broadly neutralizing antibodies (NAbs), 2F5 and 4E10, play a critical role in HIV-1 neutralization. Our research is motivated by recent immunization studies that have shown that induction of antibodies that avidly bind the gp41-MPER antigen is not sufficient for neutralization. Rather, it is required that antigen designs induce polyreactive antibodies that recognize MPER antigens as well as the viral lipid membrane. However, the mechanistic details of how membrane properties influence NAb-lipid and NAb-antigen interactions remain unknown. Furthermore, it is well established that the native viral membrane is heterogeneous, representing a mosaic of lipid rafts and protein clustering. However, the size, physical properties, and dynamics of these regions are poorly characterized and their potential roles in HIV-1 neutralization are also unknown. To understand how membrane properties contribute to 2F5/4E10 membrane interactions, we have engineered biomimetic supported lipid bilayers (SLBs) and use atomic force microscopy to visualize membrane domains, antigen clustering, and antibody-membrane interactions at sub-nanometer z-resolution. Our results show that localized binding of HIV-1 antigens and NAbs occur preferentially with the most fluid membrane domain. This supports the theory that NAbs may interact with regions of low lateral lipid forces that allow antibody insertion into the bilayer.

  8. Broadly Neutralizing Antibodies against HIV-1 As a Novel Aspect of the Immune Response.

    PubMed

    Shcherbakov, D N; Bakulina, A Y; Karpenko, L I; Ilyichev, A A

    2015-01-01

    The human immunodeficiency virus-1 (HIV-1) has the ability to evade the adaptive immune response due to high mutation rates. Soon after the discovery of HIV-1, it was originally proposed that neutralizing of antibodies to the virus occurs rarely or cannot be elicited at all. In the 1990s, there appeared reports that sera of select HIV-1-infected individuals contained antibodies capable of neutralizing different virus subtypes. Such antibodies were named broadly neutralizing antibodies (bNAbs). Since 2009, the development of new cell technologies has intensified research efforts directed at identifying new bNAbs with a neutralization potency of over 90% of primary HIV-1 isolates. These antibodies have unique characteristics which include high levels of somatic mutations and unusually long variable loops that penetrate through the glycan shield of HIV-1 Env to contact the protein surface. In this review, we will attempt to summarize the latest data on bNAbs against HIV-1 in terms of their interactions with the sites of vulnerability on HIV-1 glycoproteins.

  9. Prevalence of hemagglutination-inhibition and neutralizing antibodies to arboviruses in horses of java.

    PubMed

    Widjaja, S; Soekotjo, W; Hartati, S; Jennings, G B; Corwin, A L

    1995-03-01

    A study was conducted to measure the prevalence of hemagglutination-inhibition (HI) and neutralizing antibodies against two arboviruses (Chikungunya and Japanese encephalitis virus) in horses of Java, Indonesia. Blood specimens were collected from a sample of 112 horses at two stables: Pulo Mas, a racing track-horse complex, located in a residential area in North Jakarta, and Pamulang, a riding school, located in a rural environment of West Jaya. Sera were tested by the HI assay and plaque reduction neutralization test. JEV antibodies were detected by HI in 58 (52%) of the horses, while only 11 (10%) had Chikungunya antibodies by HI. The proportion of Pamulang horses infected with JEV (66%) was significantly higher than found among Pulo Mas horses (40%) screened (p < 0.01). Of the 58 horses with JEV antibodies by HI, 52 (90%) were found to have specific neutralization antibodies to JEV. HI and neutralization tests on horse sera indicated that the risk to alpha virus infections was minimal in horses surveyed from Java. However, there was a high risk of JEV infection among the same population.

  10. Role of Human Immunodeficiency Virus Type 1 Envelope Structure in the Induction of Broadly Neutralizing Antibodies

    PubMed Central

    Benjelloun, F.; Lawrence, P.; Verrier, B.; Genin, C.

    2012-01-01

    Very soon after the discovery of neutralizing antibodies (NAbs) toward human immunodeficiency virus type 1 (HIV-1) infection, it became apparent that characterization of these NAbs would be an important step in finding a cure for or a vaccine to eradicate HIV-1. Since the initial description of broadly cross-clade NAbs naturally produced in HIV-1 patients, numerous studies have described new viral targets for these antibodies. More recently, studies concerning new groups of patients able to control their viremia, such as long-term nonprogressors (LTNPs) or elite controllers, have described the generation of numerous envelope-targeted NAbs. Recent studies have marked a new stage in research on NAbs with the description of antibodies obtained from a worldwide screening of HIV-positive patients. These studies have permitted the discovery of NAb families with great potential for both neutralization and neutralization breadth, such as PG, PGT, CH, and highly active agonistic anti-CD4 binding site antibodies (HAADs), of which VRC01 and its variants are members. These antibodies are able to neutralize more than 80% of circulating strains without any autoreactivity and can be rapidly integrated into clinical trials in order to test their protective potential. In this review, we will focus on new insights into HIV-1 envelope structure and their implications for the generation of potent NAbs. PMID:23015715

  11. Broadly Neutralizing Antibodies against HIV-1 As a Novel Aspect of the Immune Response

    PubMed Central

    Shcherbakov, D. N.; Bakulina, A. Y.; Karpenko, L. I.; Ilyichev, A. A.

    2015-01-01

    The human immunodeficiency virus-1 (HIV-1) has the ability to evade the adaptive immune response due to high mutation rates. Soon after the discovery of HIV-1, it was originally proposed that neutralizing of antibodies to the virus occurs rarely or cannot be elicited at all. In the 1990s, there appeared reports that sera of select HIV-1-infected individuals contained antibodies capable of neutralizing different virus subtypes. Such antibodies were named broadly neutralizing antibodies (bNAbs). Since 2009, the development of new cell technologies has intensified research efforts directed at identifying new bNAbs with a neutralization potency of over 90% of primary HIV-1 isolates. These antibodies have unique characteristics which include high levels of somatic mutations and unusually long variable loops that penetrate through the glycan shield of HIV-1 Env to contact the protein surface. In this review, we will attempt to summarize the latest data on bNAbs against HIV-1 in terms of their interactions with the sites of vulnerability on HIV-1 glycoproteins. PMID:26798488

  12. Broadly Neutralizing Antibodies Display Potential for Prevention of HIV-1 Infection of Mucosal Tissue Superior to That of Nonneutralizing Antibodies

    PubMed Central

    Cheeseman, Hannah M.; Olejniczak, Natalia J.; Rogers, Paul M.; Evans, Abbey B.; King, Deborah F. L.; Ziprin, Paul; Liao, Hua-Xin; Haynes, Barton F.

    2016-01-01

    ABSTRACT Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralization is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Nonneutralizing antibodies (nnAbs) may also impact mucosal transmission events through Fc-gamma receptor (FcγR)-mediated inhibition. In this study, a panel of broadly neutralizing antibodies (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for the ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralization potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4-binding site (CD4bs)-specific bnAbs, in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. Membrane-proximal external region (MPER) (2F5) and outer domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) was more variable. In contrast, nnAbs alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest that tissue resident effector cell numbers and low FcγR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over that of nonneutralizing antibodies for preventing HIV-1 infection at the mucosal portals of infection. IMPORTANCE Key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal

  13. Structural basis for antibody cross-neutralization of respiratory syncytial virus and human metapneumovirus.

    PubMed

    Wen, Xiaolin; Mousa, Jarrod J; Bates, John T; Lamb, Robert A; Crowe, James E; Jardetzky, Theodore S

    2017-01-30

    Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are two closely related viruses that cause bronchiolitis and pneumonia in infants and the elderly(1), with a significant health burden(2-6). There are no licensed vaccines or small-molecule antiviral treatments specific to these two viruses at present. A humanized murine monoclonal antibody (palivizumab) is approved to treat high-risk infants for RSV infection(7,8), but other treatments, as well as vaccines, for both viruses are still in development. Recent epidemiological modelling suggests that cross-immunity between RSV, HMPV and human parainfluenzaviruses may contribute to their periodic outbreaks(9), suggesting that a deeper understanding of host immunity to these viruses may lead to enhanced strategies for their control. Cross-reactive neutralizing antibodies to the RSV and HMPV fusion (F) proteins have been identified(10,11). Here, we examine the structural basis for cross-reactive antibody binding to RSV and HMPV F protein by two related, independently isolated antibodies, MPE8 and 25P13. We solved the structure of the MPE8 antibody bound to RSV F protein and identified the 25P13 antibody from an independent blood donor. Our results indicate that both antibodies use germline residues to interact with a conserved surface on F protein that could guide the emergence of cross-reactivity. The induction of similar cross-reactive neutralizing antibodies using structural vaccinology approaches could enhance intrinsic cross-immunity to these paramyxoviruses and approaches to controlling recurring outbreaks.

  14. Crystal structures of ricin toxin's enzymatic subunit (RTA) in complex with neutralizing and non-neutralizing single-chain antibodies.

    PubMed

    Rudolph, Michael J; Vance, David J; Cheung, Jonah; Franklin, Matthew C; Burshteyn, Fiana; Cassidy, Michael S; Gary, Ebony N; Herrera, Cristina; Shoemaker, Charles B; Mantis, Nicholas J

    2014-08-26

    Ricin is a select agent toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins. In this study, we determined X-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA's RNA N-glycosidase activity or induced notable allosteric changes in the toxin's subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 (CDR, complementarity-determining region) elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines.

  15. Generation and characterization of neutralizing human recombinant antibodies against antigenic site II of rabies virus glycoprotein.

    PubMed

    Sun, Lina; Chen, Zhe; Yu, Li; Wei, Jingshuang; Li, Chuan; Jin, Jing; Shen, Xinxin; Lv, Xinjun; Tang, Qing; Li, Dexin; Liang, Mifang

    2012-10-01

    The currently recommended treatment for individuals exposed to rabies virus (RV) is post-exposure prophylaxis (PEP) through the combined administration of rabies vaccine and rabies immune globulin (RIG). Human monoclonal antibodies (mAbs) that neutralize RV offer an opportunity to replace RIG for rabies PEP. Here, a combinatorial human Fab library was constructed using antibody genes derived from the blood of RV-vaccinated donors. Selections of this library against purified RV virions resulted in the identification of 11 unique Fab antibodies specific for RV glycoprotein. Of the Fab antibodies, five were converted to full human IgG1 format. The human IgG antibodies revealed high binding affinity and neutralizing activities against RV fixed strains through a rapid fluorescent focus inhibition test in vitro as well as the early stage protective function after exposure to RV infection in vivo. Furthermore, epitope mapping and binding competition analysis showed that all of obtained human neutralizing and protective antibodies were directed to the antigenic site II of RV glycoprotein. Our results provide not only important insight into the protective immune response to RV in humans, but also more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.

  16. Naturally enveloped AAV vectors for shielding neutralizing antibodies and robust gene delivery in vivo.

    PubMed

    György, Bence; Fitzpatrick, Zachary; Crommentuijn, Matheus H W; Mu, Dakai; Maguire, Casey A

    2014-08-01

    Recently adeno-associated virus (AAV) became the first clinically approved gene therapy product in the western world. To develop AAV for future clinical application in a widespread patient base, particularly in therapies which require intravenous (i.v.) administration of vector, the virus must be able to evade pre-existing antibodies to the wild type virus. Here we demonstrate that in mice, AAV vectors associated with extracellular vesicles (EVs) can evade human anti-AAV neutralizing antibodies. We observed different antibody evasion and gene transfer abilities with populations of EVs isolated by different centrifugal forces. EV-associated AAV vector (ev-AAV) was up to 136-fold more resistant over a range of neutralizing antibody concentrations relative to standard AAV vector in vitro. Importantly in mice, at a concentration of passively transferred human antibodies which decreased i.v. administered standard AAV transduction of brain by 80%, transduction of ev-AAV transduction was not reduced and was 4000-fold higher. Finally, we show that expressing a brain targeting peptide on the EV surface allowed significant enhancement of transduction compared to untargeted ev-AAV. Using ev-AAV represents an effective, clinically relevant approach to evade human neutralizing anti-AAV antibodies after systemic administration of vector.

  17. Neutralizing Antibodies Against AAV Serotypes 1, 2, 6, and 9 in Sera of Commonly Used Animal Models

    PubMed Central

    Rapti, Kleopatra; Louis-Jeune, Vedell; Kohlbrenner, Erik; Ishikawa, Kiyotake; Ladage, Dennis; Zolotukhin, Sergei; Hajjar, Roger J; Weber, Thomas

    2012-01-01

    Adeno-associated virus (AAV)-based vectors are promising gene delivery vehicles for human gene transfer. One significant obstacle to AAV-based gene therapy is the high prevalence of neutralizing antibodies in humans. Until now, it was thought that, except for nonhuman primates, pre-existing neutralizing antibodies are not a problem in small or large animal models for gene therapy. Here, we demonstrate that sera of several animal models of cardiovascular diseases harbor pre-existing antibodies against the cardiotropic AAV serotypes AAV1, AAV6, and AAV9 and against AAV2. The neutralizing antibody titers vary widely both between species and between serotypes. Of all species tested, rats displayed the lowest levels of neutralizing antibodies. Surprisingly, naive mice obtained directly from commercial vendors harbored neutralizing antibodies. Of the large animal models tested, the neutralization of AAV6 transduction by dog sera was especially pronounced. Sera of sheep and rabbits showed modest neutralization of AAV transduction whereas porcine sera strongly inhibited transduction by all AAV serotypes and displayed the largest variation between individual animals. Importantly, neutralizing antibody titers as low as 1/4 completely prevented in vivo transduction by AAV9 in rats. Our results suggest that prescreening of animals for neutralizing antibodies will be important for future gene transfer experiments in these animal models. PMID:21915102

  18. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody

    PubMed Central

    Ku, Zhiqiang; Zuo, Teng; Kong, Liangliang; Zhang, Chao; Shi, Jinping; Liu, Qingwei; Chen, Tan; Zhang, Yingyi; Jiang, Wen; Zhang, Linqi; Huang, Zhong; Cong, Yao

    2016-01-01

    Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection. PMID:26938634

  19. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

    PubMed

    Ye, Xiaohua; Fan, Chen; Ku, Zhiqiang; Zuo, Teng; Kong, Liangliang; Zhang, Chao; Shi, Jinping; Liu, Qingwei; Chen, Tan; Zhang, Yingyi; Jiang, Wen; Zhang, Linqi; Huang, Zhong; Cong, Yao

    2016-03-01

    Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.

  20. Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization.

    PubMed

    Smith, T J; Lou, J; Geren, I N; Forsyth, C M; Tsai, R; Laporte, S L; Tepp, W H; Bradshaw, M; Johnson, E A; Smith, L A; Marks, J D

    2005-09-01

    The botulinum neurotoxins (BoNTs) are category A biothreat agents which have been the focus of intensive efforts to develop vaccines and antibody-based prophylaxis and treatment. Such approaches must take into account the extensive BoNT sequence variability; the seven BoNT serotypes differ by up to 70% at the amino acid level. Here, we have analyzed 49 complete published sequences of BoNTs and show that all toxins also exhibit variability within serotypes ranging between 2.6 and 31.6%. To determine the impact of such sequence differences on immune recognition, we studied the binding and neutralization capacity of six BoNT serotype A (BoNT/A) monoclonal antibodies (MAbs) to BoNT/A1 and BoNT/A2, which differ by 10% at the amino acid level. While all six MAbs bound BoNT/A1 with high affinity, three of the six MAbs showed a marked reduction in binding affinity of 500- to more than 1,000-fold to BoNT/A2 toxin. Binding results predicted in vivo toxin neutralization; MAbs or MAb combinations that potently neutralized A1 toxin but did not bind A2 toxin had minimal neutralizing capacity for A2 toxin. This was most striking for a combination of three binding domain MAbs which together neutralized >40,000 mouse 50% lethal doses (LD(50)s) of A1 toxin but less than 500 LD(50)s of A2 toxin. Combining three MAbs which bound both A1 and A2 toxins potently neutralized both toxins. We conclude that sequence variability exists within all toxin serotypes, and this impacts monoclonal antibody binding and neutralization. Such subtype sequence variability must be accounted for when generating and evaluating diagnostic and therapeutic antibodies.

  1. Sequence Variation within Botulinum Neurotoxin Serotypes Impacts Antibody Binding and Neutralization

    PubMed Central

    Smith, T. J.; Lou, J.; Geren, I. N.; Forsyth, C. M.; Tsai, R.; LaPorte, S. L.; Tepp, W. H.; Bradshaw, M.; Johnson, E. A.; Smith, L. A.; Marks, J. D.

    2005-01-01

    The botulinum neurotoxins (BoNTs) are category A biothreat agents which have been the focus of intensive efforts to develop vaccines and antibody-based prophylaxis and treatment. Such approaches must take into account the extensive BoNT sequence variability; the seven BoNT serotypes differ by up to 70% at the amino acid level. Here, we have analyzed 49 complete published sequences of BoNTs and show that all toxins also exhibit variability within serotypes ranging between 2.6 and 31.6%. To determine the impact of such sequence differences on immune recognition, we studied the binding and neutralization capacity of six BoNT serotype A (BoNT/A) monoclonal antibodies (MAbs) to BoNT/A1 and BoNT/A2, which differ by 10% at the amino acid level. While all six MAbs bound BoNT/A1 with high affinity, three of the six MAbs showed a marked reduction in binding affinity of 500- to more than 1,000-fold to BoNT/A2 toxin. Binding results predicted in vivo toxin neutralization; MAbs or MAb combinations that potently neutralized A1 toxin but did not bind A2 toxin had minimal neutralizing capacity for A2 toxin. This was most striking for a combination of three binding domain MAbs which together neutralized >40,000 mouse 50% lethal doses (LD50s) of A1 toxin but less than 500 LD50s of A2 toxin. Combining three MAbs which bound both A1 and A2 toxins potently neutralized both toxins. We conclude that sequence variability exists within all toxin serotypes, and this impacts monoclonal antibody binding and neutralization. Such subtype sequence variability must be accounted for when generating and evaluating diagnostic and therapeutic antibodies. PMID:16113261

  2. Neutralizing monoclonal antibodies recognize antigenic variants among isolates of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Winton, J.R.; Arakawa, C.N.; Lannan, C.N.; Fryer, J.L.

    1988-01-01

    eutralizing monoclonal antibodies were developed against strains of infectious hematopoietic necrosis virus (IHNV) from steelhead trout Salmo gairdneri in the Deschutes River of Oregon, chinook salmon Oncorhynchus tshawytscha in the Sacramento River of California, and rainbow trout Salmo gairdneri reared in the Hagerman Valley of Idaho, USA. These antibodies were tested for neutralization of 12 IHNV isolates obtained from salmonids in Japan, Alaska, Washington, Oregon, California, and Idaho. The antibodies recognized antigenic variants among the isolates and could be used to separate the viruses into 4 groups. The members of each group tended to be related by geographic area rather than by source host species, virulence, or date of isolation.

  3. Neutralizing Monoclonal Antibodies to Fight HIV-1: On the Threshold of Success

    PubMed Central

    Jaworski, Juan Pablo; Vendrell, Alejandrina; Chiavenna, Sebastián Matias

    2017-01-01

    Anti-human immunodeficiency virus type-1 (anti-HIV-1) neutralizing monoclonal antibodies are broadening the spectrum of pre- and post-exposure treatment against HIV-1. A better understanding of how these antibodies develop and interact with particular regions of the viral envelope protein is guiding a more rational structure-based immunogen design. The aim of this article is to review the most recent advances in the field, from the development of these particular antibodies during natural HIV-1 infection, to their role preventing infection, boosting endogenous immune responses and clearing both free viral particles and persistently infected cells. PMID:28123384

  4. Engineering Venom’s Toxin-Neutralizing Antibody Fragments and Its Therapeutic Potential

    PubMed Central

    Alvarenga, Larissa M.; Zahid, Muhammad; di Tommaso, Anne; Juste, Matthieu O.; Aubrey, Nicolas; Billiald, Philippe; Muzard, Julien

    2014-01-01

    Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety. PMID:25153256

  5. Neutralizing antibody responses against autologous and heterologous viruses in acute versus chronic human immunodeficiency virus (HIV) infection: evidence for a constraint on the ability of HIV to completely evade neutralizing antibody responses.

    PubMed

    Deeks, Steven G; Schweighardt, Becky; Wrin, Terri; Galovich, Justin; Hoh, Rebecca; Sinclair, Elizabeth; Hunt, Peter; McCune, Joseph M; Martin, Jeffrey N; Petropoulos, Christos J; Hecht, Frederick M

    2006-06-01

    Acute human immunodeficiency virus (HIV) infection is associated with the rapid development of neutralization escape mutations. The degree to which viral evolution persists in chronic infection has not been well characterized, nor is it clear if all patients develop high-level neutralization antibody escape. We therefore measured neutralizing antibody responses against autologous and heterologous viruses in a cohort of acutely and chronically infected subjects (n = 65). Neutralizing antibody responses against both autologous virus and heterologous viruses were lower among individuals with acute infection than among those with chronic infection. Among chronically infected individuals, there was a negative correlation between the level of neutralizing antibodies against autologous virus and the level of viremia. In contrast, there was a positive correlation between the level of neutralizing antibodies against a panel of heterologous viruses and the level of viremia. Viral evolution, as defined by the presence of higher neutralizing titers directed against earlier viruses than against contemporaneous viruses, was evident for subjects with recent infection but absent for those with chronic infection. In summary, neutralizing antibody responses against contemporaneous autologous viruses are absent in early HIV infection but can be detected at low levels in chronic infection, particularly among those controlling HIV in the absence of therapy. HIV replication either directly or indirectly drives the production of increasing levels of antibodies that cross-neutralize heterologous primary isolates. Collectively, these observations indicate that although HIV continuously drives the production of neutralizing antibodies, there may be limits to the capacity of the virus to evolve continuously in response to these antibodies. These observations also suggest that the neutralizing antibody response may contribute to the long-term control of HIV in some patients while protecting

  6. Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection.

    PubMed

    Tan, Gene S; Leon, Paul E; Albrecht, Randy A; Margine, Irina; Hirsh, Ariana; Bahl, Justin; Krammer, Florian

    2016-04-01

    In the early spring of 2013, Chinese health authorities reported several cases of H7N9 influenza virus infections in humans. Since then the virus has established itself at the human-animal interface in Eastern China and continues to cause several hundred infections annually. In order to characterize the antibody response to the H7N9 virus we generated several mouse monoclonal antibodies against the hemagglutinin of the A/Shanghai/1/13 (H7N9) virus. Of particular note are two monoclonal antibodies, 1B2 and 1H5, that show broad reactivity to divergent H7 hemagglutinins. Monoclonal antibody 1B2 binds to viruses of the Eurasian and North American H7 lineages and monoclonal antibody 1H5 reacts broadly to virus isolates of the Eurasian lineage. Interestingly, 1B2 shows broad hemagglutination inhibiting and neutralizing activity, while 1H5 fails to inhibit hemagglutination and demonstrates no neutralizing activity in vitro. However, both monoclonal antibodies were highly protective in an in vivo passive transfer challenge model in mice, even at low doses. Experiments using mutant antibodies that lack the ability for Fc/Fc-receptor and Fc/complement interactions suggest that the protection provided by mAb 1H5 is, at least in part, mediated by the Fc-fragment of the mAb. These findings highlight that a protective response to a pathogen may not only be due to neutralizing antibodies, but can also be the result of highly efficacious non-neutralizing antibodies not readily detected by classical in vitro neutralization or hemagglutination inhibition assays. This is of interest because H7 influenza virus vaccines induce only low hemagglutination inhibiting antibody titers while eliciting robust antibody titers as measured by ELISA. Our data suggest that these binding but non-neutralizing antibodies contribute to protection in vivo.

  7. Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection

    PubMed Central

    Albrecht, Randy A.; Margine, Irina; Hirsh, Ariana; Bahl, Justin; Krammer, Florian

    2016-01-01

    In the early spring of 2013, Chinese health authorities reported several cases of H7N9 influenza virus infections in humans. Since then the virus has established itself at the human-animal interface in Eastern China and continues to cause several hundred infections annually. In order to characterize the antibody response to the H7N9 virus we generated several mouse monoclonal antibodies against the hemagglutinin of the A/Shanghai/1/13 (H7N9) virus. Of particular note are two monoclonal antibodies, 1B2 and 1H5, that show broad reactivity to divergent H7 hemagglutinins. Monoclonal antibody 1B2 binds to viruses of the Eurasian and North American H7 lineages and monoclonal antibody 1H5 reacts broadly to virus isolates of the Eurasian lineage. Interestingly, 1B2 shows broad hemagglutination inhibiting and neutralizing activity, while 1H5 fails to inhibit hemagglutination and demonstrates no neutralizing activity in vitro. However, both monoclonal antibodies were highly protective in an in vivo passive transfer challenge model in mice, even at low doses. Experiments using mutant antibodies that lack the ability for Fc/Fc-receptor and Fc/complement interactions suggest that the protection provided by mAb 1H5 is, at least in part, mediated by the Fc-fragment of the mAb. These findings highlight that a protective response to a pathogen may not only be due to neutralizing antibodies, but can also be the result of highly efficacious non-neutralizing antibodies not readily detected by classical in vitro neutralization or hemagglutination inhibition assays. This is of interest because H7 influenza virus vaccines induce only low hemagglutination inhibiting antibody titers while eliciting robust antibody titers as measured by ELISA. Our data suggest that these binding but non-neutralizing antibodies contribute to protection in vivo. PMID:27081859

  8. Safety, pharmacokinetics and neutralization of the broadly neutralizing HIV-1 human monoclonal antibody VRC01 in healthy adults.

    PubMed

    Ledgerwood, J E; Coates, E E; Yamshchikov, G; Saunders, J G; Holman, L; Enama, M E; DeZure, A; Lynch, R M; Gordon, I; Plummer, S; Hendel, C S; Pegu, A; Conan-Cibotti, M; Sitar, S; Bailer, R T; Narpala, S; McDermott, A; Louder, M; O'Dell, S; Mohan, S; Pandey, J P; Schwartz, R M; Hu, Z; Koup, R A; Capparelli, E; Mascola, J R; Graham, B S

    2015-12-01

    VRC-HIVMAB060-00-AB (VRC01) is a broadly neutralizing HIV-1 monoclonal antibody (mAb) isolated from the B cells of an HIV-infected patient. It is directed against the HIV-1 CD4 binding site and is capable of potently neutralizing the majority of diverse HIV-1 strains. This Phase I dose-escalation study in healthy adults was conducted at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Primary objectives were the safety, tolerability and pharmacokinetics (PK) of VRC01 intravenous (i.v.) infusion at 5, 20 or 40 mg/kg, given either once (20 mg/kg) or twice 28 days apart (all doses), and of subcutaneous (s.c.) delivery at 5 mg/kg compared to s.c. placebo given twice, 28 days apart. Cumulatively, 28 subjects received 43 VRC01 and nine received placebo administrations. There were no serious adverse events or dose-limiting toxicities. Mean 28-day serum trough concentrations after the first infusion were 35 and 57 μg/ml for groups infused with 20 mg/kg (n = 8) and 40 mg/kg (n = 5) doses, respectively. Mean 28-day trough concentrations after the second infusion were 56 and 89 μg/ml for the same two doses. Over the 5-40 mg/kg i.v. dose range (n = 18), the clearance was 0.016 l/h and terminal half-life was 15 days. After infusion VRC01 retained expected neutralizing activity in serum, and anti-VRC01 antibody responses were not detected. The human monoclonal antibody (mAb) VRC01 was well tolerated when delivered i.v. or s.c. The mAb demonstrated expected half-life and pharmacokinetics for a human immunoglobulin G. The safety and PK results support and inform VRC01 dosing schedules for planning HIV-1 prevention efficacy studies.

  9. Structural insights into the neutralization mechanism of a higher primate antibody against dengue virus

    PubMed Central

    Cockburn, Joseph JB; Navarro Sanchez, M Erika; Goncalvez, Ana P; Zaitseva, Elena; Stura, Enrico A; Kikuti, Carlos M; Duquerroy, Stéphane; Dussart, Philippe; Chernomordik, Leonid V; Lai, Ching-Juh; Rey, Felix A

    2012-01-01

    The four serotypes of dengue virus (DENV-1 to -4) cause the most important emerging viral disease. Protein E, the principal viral envelope glycoprotein, mediates fusion of the viral and endosomal membranes during virus entry and is the target of neutralizing antibodies. However, the epitopes of strongly neutralizing human antibodies have not been described despite their importance to vaccine development. The chimpanzee Mab 5H2 potently neutralizes DENV-4 by binding to domain I of E. The crystal structure of Fab 5H2 bound to E from DENV-4 shows that antibody binding prevents formation of the fusogenic hairpin conformation of E, which together with in-vitro assays, demonstrates that 5H2 neutralizes by blocking membrane fusion in the endosome. Furthermore, we show that human sera from patients recovering from DENV-4 infection contain antibodies that bind to the 5H2 epitope region on domain I. This study, thus, provides new information and tools for effective vaccine design to prevent dengue disease. PMID:22139356

  10. Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies.

    PubMed

    Herrera, Cristina; Tremblay, Jacqueline M; Shoemaker, Charles B; Mantis, Nicholas J

    2015-11-13

    Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax, and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC50 values in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors.

  11. Escape from neutralization by the respiratory syncytial virus-specific neutralizing monoclonal antibody palivizumab is driven by changes in on-rate of binding to the fusion protein

    SciTech Connect

    Bates, John T.; Keefer, Christopher J.; Slaughter, James C.; Kulp, Daniel W.; Schief, William R.

    2014-04-15

    The role of binding kinetics in determining neutralizing potency for antiviral antibodies is poorly understood. While it is believed that increased steady-state affinity correlates positively with increased virus-neutralizing activity, the relationship between association or dissociation rate and neutralization potency is unclear. We investigated the effect of naturally-occurring antibody resistance mutations in the RSV F protein on the kinetics of binding to palivizumab. Escape from palivizumab-mediated neutralization of RSV occurred with reduced association rate (K{sub on}) for binding to RSV F protein, while alteration of dissociation rate (K{sub off}) did not significantly affect neutralizing activity. Interestingly, linkage of reduced K{sub on} with reduced potency mirrored the effect of increased K{sub on} found in a high-affinity enhanced potency palivizumab variant (motavizumab). These data suggest that association rate is the dominant factor driving neutralization potency for antibodies to RSV F protein antigenic site A and determines the potency of antibody somatic variants or efficiency of escape of viral glycoprotein variants. - Highlights: • The relationship of affinity to neutralization for virus antibodies is uncertain. • Palivizumab binds to RSV escape mutant fusion proteins, but with reduced affinity. • Association rate (K{sub on}) correlated well with the potency of neutralization.

  12. Mapping the Human Memory B Cell and Serum Neutralizing Antibody Responses to Dengue Virus Serotype 4 Infection and Vaccination.

    PubMed

    Nivarthi, Usha K; Kose, Nurgun; Sapparapu, Gopal; Widman, Douglas; Gallichotte, Emily; Pfaff, Jennifer M; Doranz, Benjamin J; Weiskopf, Daniela; Sette, Alessandro; Durbin, Anna P; Whitehead, Steve S; Baric, Ralph; Crowe, James E; de Silva, Aravinda M

    2017-03-01

    The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses responsible for dengue fever and dengue hemorrhagic fever. People exposed to DENV develop antibodies (Abs) that strongly neutralize the serotype responsible for infection. Historically, infection with DENV serotype 4 (DENV4) has been less common and less studied than infections with the other three serotypes. However, DENV4 has been responsible for recent large and sustained epidemics in Asia and Latin America. The neutralizing antibody responses and the epitopes targeted against DENV4 have not been characterized in human infection. In this study, we mapped and characterized epitopes on DENV4 recognized by neutralizing antibodies in people previously exposed to DENV4 infections or to a live attenuated DENV4 vaccine. To study the fine specificity of DENV4 neutralizing human antibodies, B cells from two people exposed to DENV4 were immortalized and screened to identify DENV-specific clones. Two human monoclonal antibodies (MAbs) that neutralized DENV4 were isolated, and their epitopes were finely mapped using recombinant viruses and alanine scan mutation array techniques. Both antibodies bound to quaternary structure epitopes near the hinge region between envelope protein domain I (EDI) and EDII. In parallel, to characterize the serum neutralizing antibody responses, convalescence-phase serum samples from people previously exposed to primary DENV4 natural infections or a monovalent DENV4 vaccine were analyzed. Natural infection and vaccination also induced serum-neutralizing antibodies that targeted similar epitope domains at the EDI/II hinge region. These studies defined a target of neutralizing antigenic site on DENV4 targeted by human antibodies following natural infection or vaccination.IMPORTANCE The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses

  13. Antibody-mediated neutralization of Ebola virus can occur by two distinct mechanisms

    SciTech Connect

    Shedlock, Devon J.; Bailey, Michael A.; Popernack, Paul M.; Cunningham, James M.; Burton, Dennis R.; Sullivan, Nancy J.

    2010-06-05

    Human Ebola virus causes severe hemorrhagic fever disease with high mortality and there is no vaccine or treatment. Antibodies in survivors occur early, are sustained, and can delay infection when transferred into nonhuman primates. Monoclonal antibodies (mAbs) from survivors exhibit potent neutralizing activity in vitro and are protective in rodents. To better understand targets and mechanisms of neutralization, we investigated a panel of mAbs shown previously to react with the envelope glycoprotein (GP). While one non-neutralizing mAb recognized a GP epitope in the nonessential mucin-like domain, the rest were specific for GP1, were neutralizing, and could be further distinguished by reactivity with secreted GP. We show that survivor antibodies, human KZ52 and monkey JP3K11, were specific for conformation-dependent epitopes comprising residues in GP1 and GP2 and that neutralization occurred by two distinct mechanisms; KZ52 inhibited cathepsin cleavage of GP whereas JP3K11 recognized the cleaved, fusion-active form of GP.

  14. Cooperation of B cell lineages in induction of HIV-1-broadly neutralizing antibodies.

    PubMed

    Gao, Feng; Bonsignori, Mattia; Liao, Hua-Xin; Kumar, Amit; Xia, Shi-Mao; Lu, Xiaozhi; Cai, Fangping; Hwang, Kwan-Ki; Song, Hongshuo; Zhou, Tongqing; Lynch, Rebecca M; Alam, S Munir; Moody, M Anthony; Ferrari, Guido; Berrong, Mark; Kelsoe, Garnett; Shaw, George M; Hahn, Beatrice H; Montefiori, David C; Kamanga, Gift; Cohen, Myron S; Hraber, Peter; Kwong, Peter D; Korber, Bette T; Mascola, John R; Kepler, Thomas B; Haynes, Barton F

    2014-07-31

    Development of strategies for induction of HIV-1 broadly neutralizing antibodies (bnAbs) by vaccines is a priority. Determining the steps of bnAb induction in HIV-1-infected individuals who make bnAbs is a key strategy for immunogen design. Here, we study the B cell response in a bnAb-producing individual and report cooperation between two B cell lineages to drive bnAb development. We isolated a virus-neutralizing antibody lineage that targeted an envelope region (loop D) and selected virus escape mutants that resulted in both enhanced bnAb lineage envelope binding and escape mutant neutralization-traits associated with increased B cell antigen drive. Thus, in this individual, two B cell lineages cooperated to induce the development of bnAbs. Design of vaccine immunogens that simultaneously drive both helper and broadly neutralizing B cell lineages may be important for vaccine-induced recapitulation of events that transpire during the maturation of neutralizing antibodies in HIV-1-infected individuals.

  15. Sequential Immunization Elicits Broadly Neutralizing Anti-HIV-1 Antibodies in Ig Knockin Mice.

    PubMed

    Escolano, Amelia; Steichen, Jon M; Dosenovic, Pia; Kulp, Daniel W; Golijanin, Jovana; Sok, Devin; Freund, Natalia T; Gitlin, Alexander D; Oliveira, Thiago; Araki, Tatsuya; Lowe, Sarina; Chen, Spencer T; Heinemann, Jennifer; Yao, Kai-Hui; Georgeson, Erik; Saye-Francisco, Karen L; Gazumyan, Anna; Adachi, Yumiko; Kubitz, Michael; Burton, Dennis R; Schief, William R; Nussenzweig, Michel C

    2016-09-08

    A vaccine that elicits broadly neutralizing antibodies (bNAbs) against HIV-1 is likely to be protective, but this has not been achieved. To explore immunization regimens that might elicit bNAbs, we produced and immunized mice expressing the predicted germline PGT121, a bNAb specific for the V3-loop and surrounding glycans on the HIV-1 spike. Priming with an epitope-modified immunogen designed to activate germline antibody-expressing B cells, followed by ELISA-guided boosting with a sequence of directional immunogens, native-like trimers with decreasing epitope modification, elicited heterologous tier-2-neutralizing responses. In contrast, repeated immunization with the priming immunogen did not. Antibody cloning confirmed elicitation of high levels of somatic mutation and tier-2-neutralizing antibodies resembling the authentic human bNAb. Our data establish that sequential immunization with specifically designed immunogens can induce high levels of somatic mutation and shepherd antibody maturation to produce bNAbs from their inferred germline precursors.

  16. Ontogeny-based immunogens for the induction of V2-directed HIV broadly neutralizing antibodies.

    PubMed

    Moore, Penny L; Gorman, Jason; Doria-Rose, Nicole A; Morris, Lynn

    2017-01-01

    The development of a preventative HIV vaccine able to elicit broadly neutralizing antibodies (bNAbs) remains a major challenge. Antibodies that recognize the V2 region at the apex of the HIV envelope trimer are among the most common bNAb specificities during chronic infection and many exhibit remarkable breadth and potency. Understanding the developmental pathway of these antibodies has provided insights into their precursors, and the viral strains that engage them, as well as defined how such antibodies mature to acquire breadth. V2-apex bNAbs are derived from rare precursors with long anionic CDR H3s that are often deleted in the B cell repertoire. However, longitudinal studies suggest that once engaged, these precursors contain many of the structural elements required for neutralization, and can rapidly acquire breadth through moderate levels of somatic hypermutation in response to emerging viral variants. These commonalities in the precursors and mechanism of neutralization have enabled the identification of viral strains that show enhanced reactivity for V2 precursors from multiple donors, and may form the basis of germline targeting approaches. In parallel, new structural insights into the HIV trimer, the target of these quaternary antibodies, has created invaluable new opportunities for ontogeny-based immunogens designed to select for rare V2-bNAb precursors, and drive them toward breadth.

  17. Characterization and neutralization of Nemopilema nomurai (Scyphozoa: Rhizostomeae) jellyfish venom using polyclonal antibody.

    PubMed

    Kang, Changkeun; Han, Dae-Yong; Park, Kwang-Il; Pyo, Min-Jung; Heo, Yunwi; Lee, Hyunkyoung; Kim, Gon Sup; Kim, Euikyung

    2014-08-01

    Jellyfish stings have often caused serious health concerns for sea bathers especially in tropical waters. In the coastal areas of Korea, China and Japan, the blooming and stinging accidents of poisonous jellyfish species have recently increased, including Nemopilema nomurai. We have generated a polyclonal antibody against N. nomurai jellyfish venom (NnV) by the immunization of white rabbits with NnV antigen. In the present study, the antibody has been characterized for its neutralizing effect against NnV. At first, the presence of NnV polyclonal antibody has been confirmed from the immunized rabbit serum by Enzyme linked immunosorbent assay (ELISA). Then, the neutralizing activities of the polyclonal antibody have been investigated using cell-based toxicity test, hemolysis assay, and mice lethality test. When the polyclonal antibody was preincubated with NnV, it shows a high effectiveness in neutralizing the NnV toxicities in a concentration-dependent manner. Moreover, we explored proteomic analyses using 2-D SDS-PAGE and MALDI-TOF mass spectrometry to illustrate the molecular identities of the jellyfish venom. From this, 18 different protein families have been identified as jellyfish venom-derived proteins; the main findings of which are matrix metalloproteinase-14, astacin-like metalloprotease toxin 3 precursor. It is expected that the present results would have contributed to our understandings of the envenomation by N. nomurai, their treatment and some valuable knowledge on the pathological processes of the jellyfish stinging.

  18. HIV-1 VACCINES. HIV-1 neutralizing antibodies induced by native-like envelope trimers.

    PubMed

    Sanders, Rogier W; van Gils, Marit J; Derking, Ronald; Sok, Devin; Ketas, Thomas J; Burger, Judith A; Ozorowski, Gabriel; Cupo, Albert; Simonich, Cassandra; Goo, Leslie; Arendt, Heather; Kim, Helen J; Lee, Jeong Hyun; Pugach, Pavel; Williams, Melissa; Debnath, Gargi; Moldt, Brian; van Breemen, Mariëlle J; Isik, Gözde; Medina-Ramírez, Max; Back, Jaap Willem; Koff, Wayne C; Julien, Jean-Philippe; Rakasz, Eva G; Seaman, Michael S; Guttman, Miklos; Lee, Kelly K; Klasse, Per Johan; LaBranche, Celia; Schief, William R; Wilson, Ian A; Overbaugh, Julie; Burton, Dennis R; Ward, Andrew B; Montefiori, David C; Dean, Hansi; Moore, John P

    2015-07-10

    A challenge for HIV-1 immunogen design is the difficulty of inducing neutralizing antibodies (NAbs) against neutralization-resistant (tier 2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation, BG505 SOSIP.664, induced NAbs potently against the sequence-matched tier 2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (tier 1) viruses. Tier 2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas tier 1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous tier 2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for the development of HIV-1 vaccines aimed at inducing bNAbs.

  19. Neutralizing antibody responses to foot-and-mouth disease quadrivalent (type O, A, C and Asia 1) vaccines in growing calves with pre-existing maternal antibodies.

    PubMed

    Patil, Prasanna K; Sajjanar, Channabasavaraj M; Natarajan, Chitattor; Bayry, Jagadeesh

    2014-03-14

    The presence of maternal antibodies is a major obstacle for eliciting protective immune responses to foot-and-mouth disease (FMD) vaccines in young, growing animals. In this report, we analyzed the ability of inactivated quadrivalent oil emulsified and aluminium hydroxide adjuvanted FMD vaccines to elicit neutralizing antibody responses in growing calves that had maternal antibodies. Our results demonstrate that oil emulsified vaccines but not aluminium hydroxide adjuvanted FMD vaccines could surmount maternal antibodies to elicit strong and significant levels of neutralizing antibody responses in growing claves.

  20. A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield

    SciTech Connect

    Pejchal, Robert; Doores, Katie J.; Walker, Laura M.; Khayat, Reza; Huang, Po-Ssu; Wang, Sheng-Kai; Stanfield, Robyn L.; Julien, Jean-Philippe; Ramos, Alejandra; Crispin, Max; Depetris, Rafael; Katpally, Umesh; Marozsan, Andre; Cupo, Albert; Maloveste, Sebastien; Liu, Yan; McBride, Ryan; Ito, Yukishige; Sanders, Rogier W.; Ogohara, Cassandra; Paulson, James C.; Feizi, Ten; Scanlan, Christopher N.; Wong, Chi-Huey; Moore, John P.; Olson, William C.; Ward, Andrew B.; Poignard, Pascal; Schief, William R.; Burton, Dennis R.; Wilson, Ian A.

    2015-10-15

    The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man{sub 9} at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short {beta}-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

  1. Broadly Neutralizing Antibodies Against HIV: New Insights to Inform Vaccine Design.

    PubMed

    Sadanand, Saheli; Suscovich, Todd J; Alter, Galit

    2016-01-01

    HIV-1 poses immense immunological challenges to the humoral immune response because of its ability to shield itself and replicate and evolve rapidly. Although most currently licensed vaccines provide protection via the induction of antibodies (Abs) that can directly block infection ( 1 ), 30 years of HIV-1 vaccine research has failed to successfully elicit such Abs against globally relevant HIV strains. However, mounting evidence suggests that these broadly neutralizing antibodies (bNAbs) do emerge naturally in a significant fraction of infected subjects, albeit after years of infection, indicating that these responses can be selected naturally by the immune response but take long periods of time to evolve. We review the basic structural characteristics of broadly neutralizing antibodies and how they recognize the virus, and we discuss new vaccination strategies that aim to mimic natural evolution to guide B cells to produce protective Abs against HIV-1.

  2. A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield.

    PubMed

    Pejchal, Robert; Doores, Katie J; Walker, Laura M; Khayat, Reza; Huang, Po-Ssu; Wang, Sheng-Kai; Stanfield, Robyn L; Julien, Jean-Philippe; Ramos, Alejandra; Crispin, Max; Depetris, Rafael; Katpally, Umesh; Marozsan, Andre; Cupo, Albert; Maloveste, Sebastien; Liu, Yan; McBride, Ryan; Ito, Yukishige; Sanders, Rogier W; Ogohara, Cassandra; Paulson, James C; Feizi, Ten; Scanlan, Christopher N; Wong, Chi-Huey; Moore, John P; Olson, William C; Ward, Andrew B; Poignard, Pascal; Schief, William R; Burton, Dennis R; Wilson, Ian A

    2011-11-25

    The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man(9) at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificity. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

  3. Ricin-Holotoxin-Based Vaccines: Induction of Potent Ricin-Neutralizing Antibodies.

    PubMed

    Sabo, Tamar; Kronman, Chanoch; Mazor, Ohad

    2016-01-01

    Ricin is one of the most potent and lethal toxins known to which there is no available antidote. Currently, the most promising therapy is based on neutralizing antibodies elicited by active vaccination or given passively. Here, detailed protocols are provided for the production of two ricin holotoxin-based vaccines: monomerized subunit-based vaccine, and a formaldehyde-based ricin toxoid vaccine. Both vaccines were found to be stable with no toxic activity reversion even after long-term storage while eliciting high anti-ricin antibody titers possessing a potent neutralizing activity. The use of these vaccines is highly suitable for both the production of sera that can be used in passive protection experiments and immunization aimed to isolate potent anti-ricin monoclonal antibodies.

  4. A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield

    PubMed Central

    Pejchal, Robert; Doores, Katie J.; Walker, Laura M.; Khayat, Reza; Huang, Po-Ssu; Wang, Sheng-Kai; Stanfield, Robyn L.; Julien, Jean-Philippe; Ramos, Alejandra; Crispin, Max; Depetris, Rafael; Katpally, Umesh; Marozsan, Andre; Cupo, Albert; Maloveste, Sebastien; Liu, Yan; McBride, Ryan; Ito, Yukishige; Sanders, Rogier W.; Ogohara, Cassandra; Paulson, James C.; Feizi, Ten; Scanlan, Christopher N.; Wong, Chi-Huey; Moore, John P.; Olson, William C.; Ward, Andrew B.; Poignard, Pascal; Schief, William R.; Burton, Dennis R.; Wilson, Ian A.

    2012-01-01

    The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of Fabs PGT 127 and 128 with Man9 at 1.65 and 1.29 Å resolution, respectively, and glycan binding data delineate a specific high mannose binding site. Fab PGT 128 complexed with a fully-glycosylated gp120 outer domain at 3.25 Å reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 IgGs may be mediated by cross-linking Env trimers on the viral surface. PMID:21998254

  5. Neutralizing antibodies respond to a bivalent dengue DNA vaccine or/and a recombinant bivalent antigen.

    PubMed

    Zhang, Zhi-Shan; Weng, Yu-Wei; Huang, Hai-Long; Zhang, Jian-Ming; Yan, Yan-Sheng

    2015-02-01

    There is currently no effective vaccine to prevent dengue infection, despite the existence of multiple studies on potential methods of immunization. The aim of the present study was to explore the effect of DNA and/or recombinant protein on levels of neutralizing antibodies. For this purpose, envelope domain IIIs of dengue serotypes 1 and 2 (DEN-1/2)were spliced by a linker (Gly‑Gly‑Ser‑Gly‑Ser)3 and cloned into the prokaryotic expression plasmid pET30a (+) and eukaryotic vector pcDNA3.1 (+). The chimeric bivalent protein was expressed in Escherichia coli, and one‑step purification by high‑performance liquid chromatography was conducted. Protein expression levels of the DNA plasmid were tested in BHK‑21 cells by indirect immunofluorescent assay. In order to explore a more effective immunization strategy and to develop neutralizing antibodies against the two serotypes, mice were inoculated with recombinant bivalent protein, the DNA vaccine, or the two given simultaneously. Presence of the specific antibodies was tested by ELISA and the presence of the neutralizing antibodies was determined by plaque reduction neutralization test. Results of the analysis indicated that the use of a combination of DNA and protein induced significantly higher titers of neutralizing antibodies against either DEN‑1 or DEN‑2 (1:64.0 and 1:76.1, respectively) compared with the DNA (1:24.7 and 1:26.9, DEN‑1 and DEN‑2, respectively) or the recombinant protein (1:34.9 and 1:45.3 in DEN‑1 and DEN‑2, respectively). The present study demonstrated that the combination of recombinant protein and DNA as an immunization strategy may be an effective method for the development of a vaccine to prevent dengue virus infection.

  6. Detection of neutralizing antibodies against alpha-toxin of different Clostridium septicum strains in cell culture.

    PubMed

    Roth, F; Jansen, K; Petzke, S

    1999-07-01

    Clostridium septicum, a ubiquitious organism, is the pathogen which causes the classical malignant edema after injuries. Because of its strong cytotoxic alpha-toxin, infections are often lethal. To prevent losses in animals, vaccination with alpha-toxoid vaccines is carried out. Quality control of the vaccines is done by a neutralization test in mice. A cytotoxin test and as an alternative method to detect neutralizing antibodies, a cytotoxin inhibition test was standardized. In the studies, alpha-toxin of the C. septicum reference strain (NC 547) from the National Collection of Type Cultures was compared with alpha-toxin of a field strain from an outbreak in Germany. Sera from five heterologous polyvalent and three monovalent vaccines from eight rabbit groups were available. Each vaccination had been carried out according to the procedure of the German Pharmacopoeia. In three out of the five sera of the groups vaccinated with the heterologous polyvalent vaccine, cytotoxin neutralizing antibodies were detected. High antibody titers were observed in sera of rabbits vaccinated with a vaccine of strain NC 547, lower titers in the sera of rabbits vaccinated with a vaccine of the field strain. No cytotoxin neutralizing antibodies could be found in the sera of rabbits vaccinated with the monovalent C. chauvoei vaccine. The toxins of all strains showed the same ranking of the vaccines. Vaccines which caused high antibody titers in the animals were detected by all toxins as such, as well as vaccines which had medium or low antibody inducing capacity. The results were independent of the C. septicum strain used for the production of alpha-toxin.

  7. Levels of homotypic neutralizing antibody in human poliomyelitis three years after infection.

    PubMed

    WINSSER, J; SABIN, A B

    1952-11-01

    Quantitative neutralization tests in monkeys were carried out on sera obtained from 7 patients, 3 months, and 3 years after an attack of poliomyelitis. The serum specimens were tested against 100 to 1000 PD(50) of the patient's own strain of virus, recovered during the acute phase of the illness; all the strains were Type 1. The 6 patients, aged 6 months to 13 years, who had a paralytic attack of the disease, all exhibited very high levels of neutralizing antibody at 3 years as well as at 3 months after onset. The 50 per cent serum dilution titers ranged from about 1:180 to at least 1:860. Since the maximum titers were not established, it is not known to what extent, if any, the level of antibody may have dropped over the 3 year period. One of the patients, with a diagnosis of non-paralytic poliomyelitis, had a negligible or questionable antibody response during convalescence and no demonstrable antibody at 3 years; there is justifiable doubt as to whether the Type 1 poliomyelitis virus recovered from this patient had actually caused infection. Tests for Lansing neutralizing antibody indicated that the 5 patients who had no evidence of previous infection with Type 2 poliomyelitis virus had not become infected with it during the 3 year period. This suggested that these patients did not live in an environment in which infection with poliomyelitis virus is frequent. It is concluded, therefore, that in human beings, paralytic infections due to Type 1 poliomyelitis virus produce large amounts of homotypic neutralizing antibody, which persists at high levels for a period of at least 3 years.

  8. Mapping Polyclonal HIV-1 Antibody Responses via Next-Generation Neutralization Fingerprinting

    PubMed Central

    Doria-Rose, Nicole A.; Altae-Tran, Han R.; Roark, Ryan S.; Schmidt, Stephen D.; Sutton, Matthew S.; Louder, Mark K.; Chuang, Gwo-Yu; Bailer, Robert T.; McKee, Krisha; O’Dell, Sijy; Wang, Felicia; Binley, James M.; Connors, Mark; Haynes, Barton F.; Montefiori, David C.; Morris, Lynn; Overbaugh, Julie; Kwong, Peter D.; Mascola, John R.; Georgiev, Ivelin S.

    2017-01-01

    Computational neutralization fingerprinting, NFP, is an efficient and accurate method for predicting the epitope specificities of polyclonal antibody responses to HIV-1 infection. Here, we present next-generation NFP algorithms that substantially improve prediction accuracy for individual donors and enable serologic analysis for entire cohorts. Specifically, we developed algorithms for: (a) selection of optimized virus neutralization panels for NFP analysis, (b) estimation of NFP prediction confidence for each serum sample, and (c) identification of sera with potentially novel epitope specificities. At the individual donor level, the next-generation NFP algorithms particularly improved the ability to detect multiple epitope specificities in a sample, as confirmed both for computationally simulated polyclonal sera and for samples from HIV-infected donors. Specifically, the next-generation NFP algorithms detected multiple specificities in twice as many samples of simulated sera. Further, unlike the first-generation NFP, the new algorithms were able to detect both of the previously confirmed antibody specificities, VRC01-like and PG9-like, in donor CHAVI 0219. At the cohort level, analysis of ~150 broadly neutralizing HIV-infected donor samples suggested a potential connection between clade of infection and types of elicited epitope specificities. Most notably, while 10E8-like antibodies were observed in infections from different clades, an enrichment of such antibodies was predicted for clade B samples. Ultimately, such large-scale analyses of antibody responses to HIV-1 infection can help guide the design of epitope-specific vaccines that are tailored to take into account the prevalence of infecting clades within a specific geographic region. Overall, the next-generation NFP technology will be an important tool for the analysis of broadly neutralizing polyclonal antibody responses against HIV-1. PMID:28052137

  9. Neutralization of diverse human immunodeficiency virus type 1 variants by an anti-V3 human monoclonal antibody.

    PubMed Central

    Gorny, M K; Conley, A J; Karwowska, S; Buchbinder, A; Xu, J Y; Emini, E A; Koenig, S; Zolla-Pazner, S

    1992-01-01

    The third variable region (V3) of the HIV-1 gp120 envelope glycoprotein is thought to induce potent neutralizing antibodies which are generally defined as type specific and reactive with individual viral isolates. In contrast, the CD4-binding domain is thought to induce neutralizing antibodies that are group specific and capable of neutralizing all isolates of HIV-1. However, in this study, we used a panel of human monoclonal antibodies to these regions of gp120 which displays specificities and neutralizing activities that challenge these tenets. In particular, we used a human monoclonal antibody to the V3 domain with exceptionally potent and broad neutralizing activity against many diverse HIV-1 isolates. The anti-CD4-binding domain antibodies, on the other hand, showed a more restricted pattern of activity. PMID:1433529

  10. New connections: Cell to cell HIV-1 transmission, resistance to broadly neutralizing antibodies, and an envelope sorting motif.

    PubMed

    Smith, S Abigail; Derdeyn, Cynthia A

    2017-03-01

    HIV-1 infection from cell to cell may provide an efficient mode of viral spread in vivo and could therefore present a significant challenge for preventative or therapeutic strategies based on broadly neutralizing antibodies. Indeed, Li et al show that the potency and magnitude of multiple HIV-1 broadly neutralizing antibody classes are decreased during cell to cell infection in a context dependent manner. A functional motif in gp41 appears to contribute to this differential susceptibility by modulating exposure of neutralization epitopes.

  11. Lipophilicity is a key factor to increase the antiviral activity of HIV neutralizing antibodies.

    PubMed

    Augusto, Marcelo T; Hollmann, Axel; Troise, Fulvia; Veiga, Ana S; Pessi, Antonello; Santos, Nuno C

    2017-04-01

    The HIV broadly neutralizing antibody 2F5 targets the transiently exposed epitope in the membrane proximal external region (MPER) of HIV-1 gp41, by a two-step mechanism involving the viral membrane and this viral glycoprotein. It was recently shown that 2F5 conjugation with a cholesterol moiety outside of the antibody paratope substantially increases its antiviral activity. Additionally, the antiviral activity of D5, a human antibody that binds to the N-terminal heptad repeat (NHR) of gp41 and lacks membrane binding, was boosted by the same cholesterol conjugation. In this work, we evaluated the membrane affinity of both antibodies towards membranes of different compositions, using surface plasmon resonance. A correlation was found between membrane affinity and antiviral activity against HIV-1. We propose that the conjugation of cholesterol to 2F5 or D5 allows a higher degree of antibody pre-concentration at the viral membrane. This way, the antibodies become more available to bind efficiently to the gp41 epitope, blocking viral fusion faster than the unconjugated antibody. These results set up a relevant strategy to improve the rational design of therapeutic antibodies against HIV.

  12. Ockelbo virus (Togaviridae: Alphavirus) neutralizing antibodies in experimentally infected Swedish birds.

    PubMed

    Lundström, J O; Niklasson, B

    1996-01-01

    The ability of native Swedish birds to produce and maintain production of Ockelbo virus neutralizing (Nt) antibodies were evaluated experimentally between 6 June 1990 and 27 July 1991. After experimental infection of 57 birds of the orders Anseriformes and Passeriformes with Ockelbo virus, these birds were examined for Ockelbo virus Nt antibodies at 5 days, and at 1, 3, 6, 9, and 12 mo after inoculation. One month after inoculation, Nt antibodies were more prevalent in birds with a detectable viremia (100%, n = 22) than in non-viremic birds (65%, n = 26). The Nt antibody prevalence varied over time among taxa; detectable antibodies occurred earlier after inoculation and for a longer time in Anseriformes than in Passeriformes. By 5 days after inoculation, antibodies could be detected in 22 (71%) of 31 Anseriformes but in none of 16 Passeriformes. However, at 1 mo the antibody prevalences at 1 mo were similar: 84% among the Anseriformes and 73% among the Passeriformes; at 3 mo the prevalences were 50% in Anseriformes and 15% in Passeriformes. Forty-two percent of the Anseriformes had detectable antibodies even 12 mo after inoculation.

  13. Structural Constraints of Vaccine-Induced Tier-2 Autologous HIV Neutralizing Antibodies Targeting the Receptor-Binding Site.

    PubMed

    Bradley, Todd; Fera, Daniela; Bhiman, Jinal; Eslamizar, Leila; Lu, Xiaozhi; Anasti, Kara; Zhang, Ruijung; Sutherland, Laura L; Scearce, Richard M; Bowman, Cindy M; Stolarchuk, Christina; Lloyd, Krissey E; Parks, Robert; Eaton, Amanda; Foulger, Andrew; Nie, Xiaoyan; Karim, Salim S Abdool; Barnett, Susan; Kelsoe, Garnett; Kepler, Thomas B; Alam, S Munir; Montefiori, David C; Moody, M Anthony; Liao, Hua-Xin; Morris, Lynn; Santra, Sampa; Harrison, Stephen C; Haynes, Barton F

    2016-01-05

    Antibodies that neutralize autologous transmitted/founder (TF) HIV occur in most HIV-infected individuals and can evolve to neutralization breadth. Autologous neutralizing antibodies (nAbs) against neutralization-resistant (Tier-2) viruses are rarely induced by vaccination. Whereas broadly neutralizing antibody (bnAb)-HIV-Envelope structures have been defined, the structures of autologous nAbs have not. Here, we show that immunization with TF mutant Envs gp140 oligomers induced high-titer, V5-dependent plasma neutralization for a Tier-2 autologous TF evolved mutant virus. Structural analysis of autologous nAb DH427 revealed binding to V5, demonstrating the source of narrow nAb specificity and explaining the failure to acquire breadth. Thus, oligomeric TF Envs can elicit autologous nAbs to Tier-2 HIVs, but induction of bnAbs will require targeting of precursors of B cell lineages that can mature to heterologous neutralization.

  14. CD4 binding site broadly neutralizing antibody selection of HIV-1 escape mutants.

    PubMed

    Dreja, Hanna; Pade, Corinna; Chen, Lei; McKnight, Áine

    2015-07-01

    All human immunodeficiency virus type-1 (HIV-1) viruses use CD4 to enter cells. Consequently, the viral envelope CD4-binding site (CD4bs) is relatively conserved, making it a logical neutralizing antibody target. It is important to understand how CD4-binding site variation allows for escape from neutralizing antibodies. Alanine scanning mutagenesis identifies residues in antigenic sites, whereas escape mutant selection identifies viable mutants. We selected HIV-1 to escape CD4bs neutralizing mAbs b12, A12 and HJ16. Viruses that escape from A12 and b12 remained susceptible to HJ16, VRC01 and J3, whilst six different viruses that escape HJ16 remained sensitive to A12, b12 and J3. In contrast, their sensitivity to VRC01 was variable. Triple HJ16/A12/b12-resistant virus proved that HIV-1 could escape multiple broadly neutralizing monoclonal antibodies, but still retain sensitivity to VRC01 and the llama-derived J3 nanobody. This antigenic variability may reflect that occurring in circulating viruses, so studies like this can predict immunologically relevant antigenic forms of the CD4bs for inclusion in HIV-1 vaccines.

  15. Detection of HPV types and neutralizing antibodies in women with genital warts in Tianjin City, China.

    PubMed

    Wu, Xue-ling; Zhang, Chun-tao; Zhu, Xiao-ke; Wang, You-chun

    2010-02-01

    The serum samples and corresponding cervical swabs were collected from 50 women with genital warts from Tianjin city, China. The neutralizing antibodies against HPV-16, -18, -58, -45, -6 and -11 in serum samples were tested by using pseudovirus-based neutralization assays and HPV DNAs in cervical swabs were also tested by using a typing kit that can detect 21 types of HPV. The results revealed that 36% (18/50) of sera were positive for type-specific neutralizing antibodies with a titer range of 160-2560, of which 22%(11/50), 12%(6/50), 10%(5/50), 4%(2/50), 4%(2/50) and 2%(1/50) were against HPVs -6, -16, -18, -58, -45 and -11, respectively. Additionally, 60% (30/50) of samples were HPV DNA-positive, in which the most common types detected were HPV-68(18%), HPV-16(14%), HPV-58(12%), HPV-33(8%) and HPV-6, HPV-11, HPV-18 and HPV-52 (6% each). The concordance between HPV DNA and corresponding neutralizing antibodies was 56% (28/50) with a significant difference (P<0.05). The full-length sequences of five HPV types (HPV -42, -52, -53, -58 and -68) were determined and exhibited 98%-100% identities with their reported genomes. The present data may have utility for investigating the natural history of HPV infection and promote the development of HPV vaccines.

  16. Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody

    SciTech Connect

    Bonsignori, Mattia; Zhou, Tongqing; Sheng, Zizhang; Chen, Lei; Gao, Feng; Joyce, M.  Gordon; Ozorowski, Gabriel; Chuang, Gwo-Yu; Schramm, Chaim A.; Wiehe, Kevin; Alam, S.  Munir; Bradley, Todd; Gladden, Morgan A.; Hwang, Kwan-Ki; Iyengar, Sheelah; Kumar, Amit; Lu, Xiaozhi; Luo, Kan; Mangiapani, Michael C.; Parks, Robert J.; Song, Hongshuo; Acharya, Priyamvada; Bailer, Robert T.; Cao, Allen; Druz, Aliaksandr; Georgiev, Ivelin S.; Kwon, Young D.; Louder, Mark K.; Zhang, Baoshan; Zheng, Anqi; Hill, Brenna J.; Kong, Rui; Soto, Cinque; Mullikin, James C.; Douek, Daniel C.; Montefiori, David C.; Moody, Michael A.; Shaw, George M.; Hahn, Beatrice H.; Kelsoe, Garnett; Hraber, Peter T.; Korber, Bette T.; Boyd, Scott D.; Fire, Andrew Z.; Kepler, Thomas B.; Shapiro, Lawrence; Ward, Andrew B.; Mascola, John R.; Liao, Hua-Xin; Kwong, Peter D.; Haynes, Barton F.

    2016-04-01

    Here, we report that antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. We define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. Lastly, we integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.

  17. Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody

    DOE PAGES

    Bonsignori, Mattia; Zhou, Tongqing; Sheng, Zizhang; ...

    2016-04-01

    Here, we report that antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. We define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level ofmore » somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. Lastly, we integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.« less

  18. Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody.

    PubMed

    Bonsignori, Mattia; Zhou, Tongqing; Sheng, Zizhang; Chen, Lei; Gao, Feng; Joyce, M Gordon; Ozorowski, Gabriel; Chuang, Gwo-Yu; Schramm, Chaim A; Wiehe, Kevin; Alam, S Munir; Bradley, Todd; Gladden, Morgan A; Hwang, Kwan-Ki; Iyengar, Sheelah; Kumar, Amit; Lu, Xiaozhi; Luo, Kan; Mangiapani, Michael C; Parks, Robert J; Song, Hongshuo; Acharya, Priyamvada; Bailer, Robert T; Cao, Allen; Druz, Aliaksandr; Georgiev, Ivelin S; Kwon, Young D; Louder, Mark K; Zhang, Baoshan; Zheng, Anqi; Hill, Brenna J; Kong, Rui; Soto, Cinque; Mullikin, James C; Douek, Daniel C; Montefiori, David C; Moody, Michael A; Shaw, George M; Hahn, Beatrice H; Kelsoe, Garnett; Hraber, Peter T; Korber, Bette T; Boyd, Scott D; Fire, Andrew Z; Kepler, Thomas B; Shapiro, Lawrence; Ward, Andrew B; Mascola, John R; Liao, Hua-Xin; Kwong, Peter D; Haynes, Barton F

    2016-04-07

    Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.

  19. De Novo Sequencing and Resurrection of a Human Astrovirus-Neutralizing Antibody

    PubMed Central

    2016-01-01

    Monoclonal antibody (mAb) therapeutics targeting cancer, autoimmune diseases, inflammatory diseases, and infectious diseases are growing exponentially. Although numerous panels of mAbs targeting infectious disease agents have been developed, their progression into clinically useful mAbs is often hindered by the lack of sequence information and/or loss of hybridoma cells that produce them. Here we combine the power of crystallography and mass spectrometry to determine the amino acid sequence and glycosylation modification of the Fab fragment of a potent human astrovirus-neutralizing mAb. We used this information to engineer a recombinant antibody single-chain variable fragment that has the same specificity as the parent monoclonal antibody to bind to the astrovirus capsid protein. This antibody can now potentially be developed as a therapeutic and diagnostic agent. PMID:27213181

  20. Structural insights into the neutralization mechanism of monoclonal antibody 6C2 against ricin.

    PubMed

    Zhu, Yuwei; Dai, Jianxin; Zhang, Tiancheng; Li, Xu; Fang, Pengfei; Wang, Huajing; Jiang, Yongliang; Yu, Xiaojie; Xia, Tian; Niu, Liwen; Guo, Yajun; Teng, Maikun

    2013-08-30

    Ricin belongs to the type II ribosome-inactivating proteins that depurinate the universally conserved α-sarcin loop of rRNA. The RNA N-glycosidase activity of ricin also largely depends on the ribosomal proteins that play an important role during the process of rRNA depurination. Therefore, the study of the interaction between ricin and the ribosomal elements will be better to understand the catalysis mechanism of ricin. The antibody 6C2 is a mouse monoclonal antibody exhibiting unusually potent neutralizing ability against ricin, but the neutralization mechanism remains unknown. Here, we report the 2.8 Å crystal structure of 6C2 Fab in complex with the A-chain of ricin (RTA), which reveals an extensive antigen-antibody interface that contains both hydrogen bonds and van der Waals contacts. The complementarity-determining region loops H1, H2, H3, and L3 form a pocket to accommodate the epitope on the RTA (residues Asp(96)-Thr(116)). ELISA results show that Gln(98), Glu(99), Glu(102), and Thr(105) (RTA) are the key residues that play an important role in recognizing 6C2. With the perturbation of the 6C2 Fab-RTA interface, 6C2 loses its neutralization ability, measured based on the inhibition of protein synthesis in a cell-free system. Finally, we propose that the neutralization mechanism of 6C2 against ricin is that the binding of 6C2 hinders the interaction between RTA and the ribosome and the surface plasmon resonance and pulldown results confirm our hypothesis. In short, our data explain the neutralization mechanism of mAb 6C2 against ricin and provide a structural basis for the development of improved antibody drugs with better specificity and higher affinity.

  1. Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization

    SciTech Connect

    Guan, Jian; Bywaters, Stephanie M.; Brendle, Sarah A.; Lee, Hyunwook; Ashley, Robert E.; Makhov, Alexander M.; Conway, James F.; Christensen, Neil D.; Hafenstein, Susan

    2015-09-15

    Cryo-electron microscopy (cryo-EM) was used to solve the structures of human papillomavirus type 16 (HPV16) complexed with fragments of antibody (Fab) from three different neutralizing monoclonals (mAbs): H16.1A, H16.14J, and H263.A2. The structure-function analysis revealed predominantly monovalent binding of each Fab with capsid interactions that involved multiple loops from symmetry related copies of the major capsid protein. The residues identified in each Fab-virus interface map to a conformational groove on the surface of the capsomer. In addition to the known involvement of the FG and HI loops, the DE loop was also found to constitute the core of each epitope. Surprisingly, the epitope mapping also identified minor contributions by EF and BC loops. Complementary immunological assays included mAb and Fab neutralization. The specific binding characteristics of mAbs correlated with different neutralizing behaviors in pre- and post-attachment neutralization assays. - Highlights: • We present HPV16-Fab complexes from neutralizing mAbs: H16.1A, H16.14J, and H263.A2. • The structure-function analysis revealed predominantly monovalent binding of each mAb. • Capsid–Fab interactions involved multiple loops from symmetry related L1 proteins. • Besides the known FG and HI loops, epitope mapping also identified DE, EF, and BC loops. • Neutralizing assays complement the structures to show multiple neutralization mechanisms.

  2. Commercially available immunoglobulins contain virus neutralizing antibodies against all major genotypes of polyomavirus BK.

    PubMed

    Randhawa, P; Pastrana, D V; Zeng, G; Huang, Y; Shapiro, R; Sood, P; Puttarajappa, C; Berger, M; Hariharan, S; Buck, C B

    2015-04-01

    Neutralizing antibodies (NAbs) form the basis of immunotherapeutic strategies against many important human viral infections. Accordingly, we studied the prevalence, titer, genotype-specificity, and mechanism of action of anti-polyomavirus BK (BKV) NAbs in commercially available human immune globulin (IG) preparations designed for intravenous (IV) use. Pseudovirions (PsV) of genotypes Ia, Ib2, Ic, II, III, and IV were generated by co-transfecting a reporter plasmid encoding luciferase and expression plasmids containing synthetic codon-modified VP1, VP2, and VP3 capsid protein genes into 293TT cells. NAbs were measured using luminometry. All IG preparations neutralized all BKV genotypes, with mean EC50 titers as high as 254 899 for genotype Ia and 6,666 for genotype IV. Neutralizing titers against genotypes II and III were higher than expected, adding to growing evidence that infections with these genotypes are more common than currently appreciated. Batch to batch variation in different lots of IG was within the limits of experimental error. Antibody mediated virus neutralizing was dose dependent, modestly enhanced by complement, genotype-specific, and achieved without effect on viral aggregation, capsid morphology, elution, or host cell release. IG contains potent NAbs capable of neutralizing all major BKV genotypes. Clinical trials based on sound pharmacokinetic principles are needed to explore prophylactic and therapeutic applications of these anti-viral effects, until effective small molecule inhibitors of BKV replication can be developed.

  3. Virological features associated with the development of broadly neutralizing antibodies to HIV-1.

    PubMed

    Moore, Penny L; Williamson, Carolyn; Morris, Lynn

    2015-04-01

    The development of a preventative HIV-1 vaccine remains a global public health priority. This will likely require the elicitation of broadly neutralizing antibodies (bNAbs) able to block infection by diverse viral strains from across the world. Understanding the pathway to neutralization breadth in HIV-1 infected humans will provide insights into how bNAb lineages arise, a process that probably involves a combination of host and viral factors. Here, we focus on the role of viral characteristics and evolution in shaping bNAbs during HIV-1 infection, and describe how these findings may be translated into novel vaccine strategies.

  4. Neutralizing antibodies in mucosal secretions: IgG or IgA?

    PubMed

    Alexander, Rashada; Mestecky, Jiri

    2007-11-01

    The mucosal immune response to HIV weighs in heavily on the battle against it, as the majority of infections occur via the mucosal route. The antibody response in the mucosae, specifically the genital tract, is characterized by binding and, in some studies, neutralizing HIV-specific IgG and IgA antibodies. Ample evidence, however, points to discrepancies and difficulties in the detection of HIV-specific IgA in HIV-positive subjects, and an even more pronounced divide surfaces in studies done with individuals exposed to HIV, but uninfected. Reports in the literature detail HIV-specific (in some cases, neutralizing) IgA antibodies, in the absence of specific IgG, in the serum and mucosal secretions of virus-exposed, seronegative subjects; this has given rise to speculation that HIV-specific IgA provides a protective immune response to the virus in high-risk individuals who remain seronegative. Contradictory results, however, describe the absence of both IgA and IgG HIV antibodies in the mucosal secretions of similar cohorts. Considering the importance of the antibody response to ascertaining the correlates of HIV immunity, as well as on vaccine research and development, this review addresses the relevant studies and their implications.

  5. Epitope Mapping of Anti-Interleukin-13 Neutralizing Antibody CNTO607

    SciTech Connect

    Teplyakov, Alexey; Obmolova, Galina; Wu, Sheng-Jiun; Luo, Jinquan; Kang, James; O'Neil, Karyn; Gilliland, Gary L.

    2009-06-24

    CNTO607 is a neutralizing anti-interleukin-13 (IL-13) human monoclonal antibody obtained from a phage display library. To determine how this antibody inhibits the biological effect of IL-13, we determined the binding epitope by X-ray crystallography. The crystal structure of the complex between CNTO607 Fab and IL-13 reveals the antibody epitope at the surface formed by helices A and D of IL-13. This epitope overlaps with the IL-4Ralpha/IL-13Ralpha1 receptor-binding site, which explains the neutralizing effect of CNTO607. The extensive antibody interface covers an area of 1000 A(2), which is consistent with the high binding affinity. The key features of the interface are the charge and shape complementarity of the molecules that include two hydrophobic pockets on IL-13 that accommodate Phe32 [complementarity-determining region (CDR) L2] and Trp100a (CDR H3) and a number of salt bridges between basic residues of IL-13 and acidic residues of the antibody. Comparison with the structure of the free Fab shows that the CDR residues do not change their conformation upon complex formation, with the exception of two residues in CDR H3, Trp100a and Asp100b, which change rotamer conformations. To evaluate the relative contribution of the epitope residues to CNTO607 binding, we performed alanine-scanning mutagenesis of the A-D region of IL-13. This study confirmed the primary role of electrostatic interactions for antigen recognition.

  6. Stapled HIV-1 Peptides Recapitulate Antigenic Structures and Engage Broadly Neutralizing Antibodies

    PubMed Central

    Bird, Gregory H.; Irimia, Adriana; Ofek, Gilad; Kwong, Peter D.; Wilson, Ian A.; Walensky, Loren D.

    2014-01-01

    Hydrocarbon stapling can restore bioactive, α-helical structure to natural peptides, yielding research tools and prototype therapeutics to dissect and target protein interactions. Here, we explore the capacity of peptide stapling to generate high fidelity, protease-resistant mimics of antigenic structures for vaccine development. HIV-1 has been refractory to vaccine technologies thus far, although select human antibodies can broadly neutralize HIV-1 by targeting sequences of the gp41 juxtamembrane fusion apparatus. To develop candidate HIV-1 immunogens, we generated and characterized stabilized α-helices of the membrane proximal external region (SAH-MPER) of gp41. SAH-MPER peptides were remarkably protease-resistant and bound to the broadly neutralizing 4E10 and 10E8 antibodies with high affinity, recapitulating the structure of the MPER epitope when differentially engaged by the two anti-HIV Fabs. Thus, stapled peptides may provide a new opportunity to develop chemically-stabilized antigens for vaccination. PMID:25420104

  7. Stapled HIV-1 peptides recapitulate antigenic structures and engage broadly neutralizing antibodies.

    PubMed

    Bird, Gregory H; Irimia, Adriana; Ofek, Gilad; Kwong, Peter D; Wilson, Ian A; Walensky, Loren D

    2014-12-01

    Hydrocarbon stapling can restore bioactive α-helical structure to natural peptides, yielding research tools and prototype therapeutics to dissect and target protein interactions. Here we explore the capacity of peptide stapling to generate high-fidelity, protease-resistant mimics of antigenic structures for vaccine development. HIV-1 has been refractory to vaccine technologies thus far, although select human antibodies can broadly neutralize HIV-1 by targeting sequences of the gp41 juxtamembrane fusion apparatus. To develop candidate HIV-1 immunogens, we generated and characterized stabilized α-helices of the membrane-proximal external region (SAH-MPER) of gp41. SAH-MPER peptides were remarkably protease resistant and bound to the broadly neutralizing 4E10 and 10E8 antibodies with high affinity, recapitulating the structure of the MPER epitope when differentially engaged by the two anti-HIV Fabs. Thus, stapled peptides may provide a new opportunity to develop chemically stabilized antigens for vaccination.

  8. Peptide Paratope Mimics of the Broadly Neutralizing HIV-1 Antibody b12.

    PubMed

    Haußner, Christina; Damm, Dominik; Nirschl, Sandra; Rohrhofer, Anette; Schmidt, Barbara; Eichler, Jutta

    2017-01-26

    The broadly neutralizing HIV-1 antibody b12 recognizes the CD4 binding site of the HIV-1 envelope glycoprotein gp120 and efficiently neutralizes HIV-1 infections in vitro and in vivo. Based on the 3D structure of a b12⋅gp120 complex, we have designed an assembled peptide (b12-M) that presents the parts of the three heavy-chain complementarity-determining regions (CDRs) of b12, which contain the contact sites of the antibody for gp120. This b12-mimetic peptide, as well as a truncated peptide presenting only two of the three heavy-chain CDRs of b12, were shown to recognize gp120 in a similar manner to b12, as well as to inhibit HIV-1 infection, demonstrating functional mimicry of b12 by the paratope mimetic peptides.

  9. Glycan clustering stabilizes the mannose patch of HIV-1 and preserves vulnerability to broadly neutralizing antibodies

    PubMed Central

    Pritchard, Laura K.; Spencer, Daniel I. R.; Royle, Louise; Bonomelli, Camille; Seabright, Gemma E.; Behrens, Anna-Janina; Kulp, Dan; Menis, Sergey; Krumm, Stefanie A.; Dunlop, D. Cameron; Crispin, Daniel J.; Bowden, Thomas A.; Ward, Andrew B.; Schief, William R.; Doores, Katie J.; Crispin, Max

    2015-01-01

    The envelope spike of HIV-1 employs a ‘glycan shield’ to protect itself from antibody-mediated neutralization. Paradoxically, however, potent broadly neutralizing antibodies (bnAbs) have been isolated which target this shield. The unusually high glycan density on the gp120 subunit limits processing during biosynthesis, leaving a region of under-processed oligomannose-type structures which is a primary target of these bnAbs. Here we investigate the contribution of individual glycosylation sites to formation of this so-called intrinsic mannose patch. Deletion of individual sites has a limited effect on the overall size of the intrinsic mannose patch but leads to changes in the processing of neighboring glycans. These structural changes are largely tolerated by a panel of glycan-dependent bnAbs targeting these regions, indicating a degree of plasticity in their recognition. These results support the intrinsic mannose patch as a stable target for vaccine design. PMID:26105115

  10. Neutralizing antibodies to botulinum neurotoxin type A in aesthetic medicine: five case reports

    PubMed Central

    Torres, Sebastian; Hamilton, Mark; Sanches, Elena; Starovatova, Polina; Gubanova, Elena; Reshetnikova, Tatiana

    2014-01-01

    Botulinum neurotoxin injections are a valuable treatment modality for many therapeutic indications as well as in the aesthetic field for facial rejuvenation. As successful treatment requires repeated injections over a long period of time, secondary resistance to botulinum toxin preparations after repeated injections is an ongoing concern. We report five case studies in which neutralizing antibodies to botulinum toxin type A developed after injection for aesthetic use and resulted in secondary treatment failure. These results add to the growing number of reports in the literature for secondary treatment failure associated with high titers of neutralizing antibodies in the aesthetic field. Clinicians should be aware of this risk and implement injection protocols that minimize resistance development. PMID:24379687

  11. Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody

    SciTech Connect

    Kong, Rui; Xu, Kai; Zhou, Tongqing; Acharya, Priyamvada; Lemmin, Thomas; Liu, Kevin; Ozorowski, Gabriel; Taft, Justin D.; Bailer, Robert T.; Cale, Evan M.; Chen, Lei; Choi, Chang W.; Chuang, Gwo-Yu; Doria-Rose, Nicole A.; Druz, Aliaksandr; Georgiev, Ivelin S.; Gorman, Jason; Huang, Jinghe; Joyce, M. Gordon; Louder, Mark K.; Ma, Xiaochu; McKee, Krisha; O'Dell, Sijy; Pancera, Marie; Yang, Yongping; Blanchard, Scott C.; Mothes, Walther; Burton, Dennis R.; Koff, Wayne C.; Connors, Mark; Ward, Andrew B.; Mascola, John R.

    2016-05-13

    The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. In this paper, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showed that the N-terminal portion of the fusion peptide can be solvent-exposed. Finally, these results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.

  12. Reduced potency and incomplete neutralization of broadly neutralizing antibodies against cell-to-cell transmission of HIV-1 with transmitted founder Envs.

    PubMed

    Li, Hongru; Zony, Chati; Chen, Ping; Chen, Benjamin K

    2017-02-01

    Broadly neutralizing antibodies (bNAbs) have been isolated from HIV-1 patients and can potently block infection of a wide spectrum of HIV-1 subtypes. These antibodies define common epitopes shared by many viral isolates. While bNAbs potently antagonize infection with cell-free virus, inhibition of HIV-1 transmission from infected to uninfected CD4(+) T cells through virological synapses (VS), has been found to require greater amounts of antibody. In this study, we examined two well-studied molecular clones and two transmitted founder (T/F) viruses for their sensitivities to a panel of bNAbs in cell-free and cell-to-cell infection assays. We observed a relative resistance of cell-to-cell transmission to antibody neutralization that is reflected not only by reductions of antibody potency, but also by decreases in maximum neutralization capacity relative to cell-free infections. BNAbs targeting different epitopes exhibited incomplete neutralization against cell-associated virus with T/F Envs, which was not observed with cell-free form of the same virus. We further identified the membrane proximal internal tyrosine-based sorting motif as a determinant that can affect the incomplete neutralization of these T/F clones in cell-to-cell infection. These findings indicate that the signal that affects surface expression and/or internalization of Env from the plasma membrane can modulate the presentation of neutralizing epitopes on infected cells. These findings highlight that a fraction of virus can escape from high concentrations of antibody through cell-to-cell infection while maintaining sensitivity to neutralization in cell-free infection. The ability to fully inhibit cell-to-cell transmission may represent an important consideration in development of antibodies for treatment or prophylaxis.

  13. A Glycoprotein Subunit Vaccine Elicits a Strong Rift Valley Fever Virus Neutralizing Antibody Response in Sheep

    PubMed Central

    Lebedev, Maxim; McVey, D. Scott; Wilson, William; Morozov, Igor; Young, Alan

    2014-01-01

    Abstract Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, is a mosquito-borne zoonotic pathogen that causes serious morbidity and mortality in livestock and humans. The recent spread of the virus beyond its traditional endemic boundaries in Africa to the Arabian Peninsula coupled with the presence of susceptible vectors in nonendemic countries has created increased interest in RVF vaccines. Subunit vaccines composed of specific virus proteins expressed in eukaryotic or prokaryotic expression systems are shown to elicit neutralizing antibodies in susceptible hosts. RVFV structural proteins, amino-terminus glycoprotein (Gn), and carboxyl-terminus glycoprotein (Gc), were expressed using a recombinant baculovirus expression system. The recombinant proteins were reconstituted as a GnGc subunit vaccine formulation and evaluated for immunogenicity in a target species, sheep. Six sheep were each immunized with a primary dose of 50 μg of each vaccine immunogen with the adjuvant montanide ISA25; at day 21, postvaccination, each animal received a second dose of the same vaccine. The vaccine induced a strong antibody response in all animals as determined by indirect enzyme-linked immunosorbent assay (ELISA). A plaque reduction neutralization test (PRNT80) showed the primary dose of the vaccine was sufficient to elicit potentially protective virus neutralizing antibody titers ranging from 40 to 160, and the second vaccine dose boosted the titer to more than 1280. Furthermore, all animals tested positive for neutralizing antibodies at day 328 postvaccination. ELISA analysis using the recombinant nucleocapsid protein as a negative marker antigen indicated that the vaccine candidate is DIVA (differentiating infected from vaccinated animals) compatible and represents a promising vaccine platform for RVFV infection in susceptible species. PMID:25325319

  14. A glycoprotein subunit vaccine elicits a strong Rift Valley fever virus neutralizing antibody response in sheep.

    PubMed

    Faburay, Bonto; Lebedev, Maxim; McVey, D Scott; Wilson, William; Morozov, Igor; Young, Alan; Richt, Juergen A

    2014-10-01

    Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, is a mosquito-borne zoonotic pathogen that causes serious morbidity and mortality in livestock and humans. The recent spread of the virus beyond its traditional endemic boundaries in Africa to the Arabian Peninsula coupled with the presence of susceptible vectors in nonendemic countries has created increased interest in RVF vaccines. Subunit vaccines composed of specific virus proteins expressed in eukaryotic or prokaryotic expression systems are shown to elicit neutralizing antibodies in susceptible hosts. RVFV structural proteins, amino-terminus glycoprotein (Gn), and carboxyl-terminus glycoprotein (Gc), were expressed using a recombinant baculovirus expression system. The recombinant proteins were reconstituted as a GnGc subunit vaccine formulation and evaluated for immunogenicity in a target species, sheep. Six sheep were each immunized with a primary dose of 50 μg of each vaccine immunogen with the adjuvant montanide ISA25; at day 21, postvaccination, each animal received a second dose of the same vaccine. The vaccine induced a strong antibody response in all animals as determined by indirect enzyme-linked immunosorbent assay (ELISA). A plaque reduction neutralization test (PRNT80) showed the primary dose of the vaccine was sufficient to elicit potentially protective virus neutralizing antibody titers ranging from 40 to 160, and the second vaccine dose boosted the titer to more than 1280. Furthermore, all animals tested positive for neutralizing antibodies at day 328 postvaccination. ELISA analysis using the recombinant nucleocapsid protein as a negative marker antigen indicated that the vaccine candidate is DIVA (differentiating infected from vaccinated animals) compatible and represents a promising vaccine platform for RVFV infection in susceptible species.

  15. Mechanism of HIV-1 neutralization by antibodies targeting a membrane-proximal region of gp41.

    PubMed

    Chen, Jia; Frey, Gary; Peng, Hanqin; Rits-Volloch, Sophia; Garrity, Jetta; Seaman, Michael S; Chen, Bing

    2014-01-01

    Induction of broadly neutralizing antibodies (bNAbs) is an important goal for HIV-1 vaccine development. Two autoreactive bNAbs, 2F5 and 4E10, recognize a conserved region on the HIV-1 envelope glycoprotein gp41 adjacent to the viral membrane known as the membrane-proximal external region (MPER). They block viral infection by targeting a fusion-intermediate conformation of gp41, assisted by an additional interaction with the viral membrane. Another MPER-specific antibody, 10E8, has recently been reported to neutralize HIV-1 with potency and breadth much greater than those of 2F5 or 4E10, but it appeared not to bind phospholipids and might target the untriggered envelope spikes, raising the hope that the MPER could be harnessed for vaccine design without major immunological concerns. Here, we show by three independent approaches that 10E8 indeed binds lipid bilayers through two hydrophobic residues in its CDR H3 (third heavy-chain complementarity-determining region). Its weak affinity for membranes in general and preference for cholesterol-rich membranes may account for its great neutralization potency, as it is less likely than other MPER-specific antibodies to bind cellular membranes nonspecifically. 10E8 binds with high affinity to a construct mimicking the fusion intermediate of gp41 but fails to recognize the envelope trimers representing the untriggered conformation. Moreover, we can improve the potency of 4E10 without affecting its binding to gp41 by a modification of its lipid-interacting CDR H3. These results reveal a general mechanism of HIV-1 neutralization by MPER-specific antibodies that involves interactions with viral lipids.

  16. Toward Effective HIV Vaccination INDUCTION OF BINARY EPITOPE REACTIVE ANTIBODIES WITH BROAD HIV NEUTRALIZING ACTIVITY

    SciTech Connect

    Nishiyama, Yasuhiro; Planque, Stephanie; Mitsuda, Yukie; Nitti, Giovanni; Taguchi, Hiroaki; Jin, Lei; Symersky, Jindrich; Boivin, Stephane; Sienczyk, Marcin; Salas, Maria; Hanson, Carl V.; Paul, Sudhir

    2009-11-23

    We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421-433 and 288-306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (VH) domain framework (FR) residues. Substitution of the FR cavity VH Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and VH FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from VH1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421-433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.

  17. Quantification of Lyssavirus-Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles

    PubMed Central

    Moeschler, Sarah; Locher, Samira; Conzelmann, Karl-Klaus; Krämer, Beate; Zimmer, Gert

    2016-01-01

    Rabies is a highly fatal zoonotic disease which is primarily caused by rabies virus (RABV) although other members of the genus Lyssavirus can cause rabies as well. As yet, 14 serologically and genetically diverse lyssaviruses have been identified, mostly in bats. To assess the quality of rabies vaccines and immunoglobulin preparations, virus neutralization tests with live RABV are performed in accordance with enhanced biosafety standards. In the present work, a novel neutralization test is presented which takes advantage of a modified vesicular stomatitis virus (VSV) from which the glycoprotein G gene has been deleted and replaced by reporter genes. This single-cycle virus was trans-complemented with RABV envelope glycoprotein. Neutralization of this pseudotype virus with RABV reference serum or immune sera from vaccinated mice showed a strong correlation with the rapid fluorescent focus inhibition test (RFFIT). Importantly, pseudotype viruses containing the envelope glycoproteins of other lyssaviruses were neutralized by reference serum to a significantly lesser extent or were not neutralized at all. Taken together, a pseudotype virus system has been successfully developed which allows the safe, fast, and sensitive detection of neutralizing antibodies directed against different lyssaviruses. PMID:27649230

  18. Age-specific incidence of neutralization antibodies of Herpes simplex virus.

    PubMed Central

    Terzin, A. L.; Masic, M. G.

    1976-01-01

    Sera of 1255 individuals from Novi Sad, varying in age from less than 1 month to 69 years, have been tested for neutralization antibodies to Herpes implex virus type 1. The eight newborns tested and 97% of the 507 adults were positive, with titres ranging from 1/4 to 1/256. The titres in newborns were significantly lower than the titres in adults. After birth the maternal antibodies declined rapidly and 94% of infants at the age of greater than 6 months and less than 2 years were negative. After the first year infants in Novi Sad start to acquire herpes-neutralizing antibodies actively, reaching a 50% incidence of positives between the 2nd and 3rd year of age. Age-specific incidence rates of herpes positives found in Novi Sad have been compared with those reported from Edinburgh, Freiburg i. Br. and Louisiana. Possible influences of several circumstances upon the incidence rate of positives detected by the neutralization test are discussed. PMID:185287

  19. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin

    SciTech Connect

    Whittle, James R.R.; Zhang, Ruijun; Khurana, Surender; King, Lisa R.; Manischewitz, Jody; Golding, Hana; Dormitzer, Philip R.; Haynes, Barton F.; Walter, Emmanuel B.; Moody, M. Anthony; Kepler, Thomas B.; Liao, Hua-Xin; Harrison, Stephen C.

    2011-09-20

    Seasonal antigenic drift of circulating influenza virus leads to a requirement for frequent changes in vaccine composition, because exposure or vaccination elicits human antibodies with limited cross-neutralization of drifted strains. We describe a human monoclonal antibody, CH65, obtained by isolating rearranged heavy- and light-chain genes from sorted single plasma cells, coming from a subject immunized with the 2007 trivalent influenza vaccine. The crystal structure of a complex of the hemagglutinin (HA) from H1N1 strain A/Solomon Islands/3/2006 with the Fab of CH65 shows that the tip of the CH65 heavy-chain complementarity determining region 3 (CDR3) inserts into the receptor binding pocket on HA1, mimicking in many respects the interaction of the physiological receptor, sialic acid. CH65 neutralizes infectivity of 30 out of 36 H1N1 strains tested. The resistant strains have a single-residue insertion near the rim of the sialic-acid pocket. We conclude that broad neutralization of influenza virus can be achieved by antibodies with contacts that mimic those of the receptor.

  20. Influenza A HA's conserved epitopes and broadly neutralizing antibodies: a prediction method.

    PubMed

    Ren, Jing; Ellis, John; Li, Jinyan

    2014-10-01

    A conserved epitope is an epitope retained by multiple strains of influenza as the key target of a broadly neutralizing antibody. Identification of conserved epitopes is of strong interest to help design broad-spectrum vaccines against influenza. Conservation score measures the evolutionary conservation of an amino acid position in a protein based on the phylogenetic relationships observed amongst homologous sequences. Here, Average Amino Acid Conservation Score (AAACS) is proposed as a method to identify HA's conserved epitopes. Our analysis shows that there is a clear distinction between conserved epitopes and nonconserved epitopes in terms of AAACS. This method also provides an excellent classification performance on an independent dataset. In contrast, alignment-based comparison methods do not work well for this problem, because conserved epitopes to the same broadly neutralizing antibody are usually not identical or similar. Location-based methods are not successful either, because conserved epitopes are located at both the less-conserved globular head (HA1) and the more-conserved stem (HA2). As a case study, two conserved epitopes on HA are predicted for the influenza A virus H7N9: One should match the broadly neutralizing antibodies CR9114 or FI6v3, while the other is new and requires validation by wet-lab experiments.

  1. Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody

    PubMed Central

    Halstead, SB; O’Rourke, EJ

    1977-01-01

    Cultured mononuclear peripheral blood leukocytes (PBL) from nonimmune human beings and monkeys are nonpermissive to dengue 2 virus (D2V) infection at multiplicities of infection of 0.001-0.1, but become permissive when non-neutralizing dengue antibody is added to medium. D2V infection occurred in PBL prepared from anti-coagulated but not from defibrinated plasma. Infection enhancement was produced by multiple lots of heterotypic anti-dengue raised in several mammalian species. Homotypic anti-dengue neutralized D2V at high concentrations but enhanced at low concentrations; enhancement end point in one serum was 1:320,000. The infection-enhancing factor was a noncytophilic antibody of the IgG class. D2V infection occurred in the absence of heat-labile complement components but did not occur when complexes were prepared with anti- dengue F(ab)(2). Treatment of PBL with several proteases increased permissiveness to D2V infection by immune complexes but not by virus alone. Two rhesus monkey serums collected 14 days after D2V infection contained an IgG antibody with high-titered enhancing activity but with no hemagglutination-inhibition or neutralizing activity. Virus-antibody complexes are irreversibly attached to PBL within 15 min and completely internalized in 60 min. There was considerable variation in cellular infection in different experiments, however, maximum virus yields usually exceeded 1,000 plaque-forming units per 1 x 10(6) PBL occurring between 2 and 4 days in culture. In vitro antibody-dependent infection of PBL provides a possible model for study of pathogenetic mechanisms in infants with dengue shock syndrome who passively acquire maternal anti-dengue IgG. PMID:406347

  2. Germlining of the HIV-1 broadly neutralizing antibody domain m36

    PubMed Central

    Chen, Weizao; Li, Wei; Ying, Tianlei; Wang, Yanping; Feng, Yang; Dimitrov, Dimiter S.

    2015-01-01

    Engineered antibody domains (eAds) have emerged as a novel class of HIV-1 inhibitors and are currently under preclinical testing as promising drug candidates for prevention and therapy of HIV-1 infection. Reverse mutation of antibodies to germline sequences (germlining) could not only identify less mutated variants with lower probability of immunogenicity and other improved properties but also help elucidate their mechanisms of action. In this study, we sequentially reverted the framework (FRs) and complementary determining regions (CDRs) of m36, a human antibody heavy chain variable domain-based eAd targeting the coreceptor binding site of the viral envelope glycoprotein gp120, back to germline sequences. Two types of amino acid mutations and one region in the antibody V segment were identified that are critical for HIV-1 neutralization. These include four mutations to acidic acid residues distributed in the CDR1 and CDR2, two mutations to hydrophobic residues in the FR3 and CDR3, and partial FR2 and FR3 sequences flanking the CDR2 that are derived from a different gene family. An m36 variant with all five mutations in the FRs reverted back to germline showed slightly increased neutralizing activity against two HIV-1 isolates tested. Another variant with seven of twelve mutations in the V segment reverted retained potency within three-fold of that of the mature antibody. These results, together with an analysis of m36-gp120-CD4 docking structures, could have implications for the further development of m36 as candidate therapeutics and elucidation of its mechanism of potent and broad HIV-1 neutralization. PMID:25676867

  3. EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies.

    PubMed

    Lindsey, Changhong Y; Brown, J Edward; Torabazar, Nahid R; Smith, Leonard A

    2013-01-01

    A recombinant ricin toxin A-chain 1-33/44-198 vaccine (RVEc), developed at the United States Army Medical Research Institute of Infectious Diseases as a vaccine candidate, is under investigation in a phase 1 clinical study. To effectively evaluate the immunogenicity of this ricin vaccine and to eliminate the use of radioactive material, an EL4 cell-based colorimetric toxin neutralization activity (TNA) assay using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent has been developed, optimized, and applied in the vaccine efficacy studies. The TNA assay measures the protective neutralizing anti-ricin antibodies in animal sera by determining the cell viability after ricin exposure in the assay system and comparing it to a purified mouse polyclonal antiricin IgG standard curve. The standard curve of the anti-ricin TNA assay closely fits a four-parameter logistic regression model. The unknown test sample concentration was expressed as microg/mL, but not the 50% effective concentration (EC50), which was determined by most TNA assays. The neutralizing endpoint titers, not the 50% effective dilution (ED50), of human specimens were measured with the TNA assay in support of the clinical study of the RVEc vaccine. The optimal amount of ricin toxin, EL4 cells, and concentration of standards used in the assay system was established to minimize false-negative and false-positive results of serum specimens from the nonclinical and clinical studies of RVEc. The testing conditions were adjusted to optimize assay performance. The colorimetric TNA assay replaced a radioactive TNA assay previously used in the ricin vaccine studies.

  4. Persistence of Neutralizing Antibody Against Dengue Virus 2 After 70 Years from Infection in Nagasaki

    PubMed Central

    Ngwe Tun, Mya Myat; Muta, Yoshihito; Inoue, Shingo; Morita, Kouichi

    2016-01-01

    Abstract This study aimed to investigate the duration of humoral immune responses to dengue virus (DENV) infection in Japanese who experienced acute febrile illness with hemorrhagic manifestations 70 years ago, when an epidemic of dengue occurred in Nagasaki, Japan, from 1942 to 1944. A Japanese volunteer requested serological diagnosis of DENV infection in 2014 and donated blood sample to measure the antibody titer against DENV by antiflavi IgG indirect ELISA, focus reduction neutralization test, and plaque reduction neutralization test. The serum sample of the volunteer was positive in flavi IgG ELISA and it indicated primary infection. In the neutralization test, the highest neutralizing titer was ≥218 for DENV-2. We report here the existence of DENV-specific antibodies in the serum of a person after 70 years from infection. Published reports indicated that DENV-1 was responsible for the 1942–1944 outbreak in Nagasaki. However, our data suggested that DENV-2 also played a role in this Nagasaki dengue epidemic. PMID:27493841

  5. PEG conjugation moderately protects adeno-associated viral vectors against antibody neutralization.

    PubMed

    Lee, Gary K; Maheshri, Narendra; Kaspar, Brian; Schaffer, David V

    2005-10-05

    AAV gene therapy vectors have significant clinical promise, but serum neutralization poses a challenge that must be overcome. We have examined the potential of conjugating the AAV surface with activated polyethylene glycol chains to protect the vector from neutralizing antibodies. Two key parameters were investigated: the polymer chain size and the PEG:lysine conjugation ratio. Transduction data revealed that the vector is fully infectious until a critical PEG conjugation reaction ratio was exceeded, and this critical level was found to vary with polymer chain size. At this key conjugation ratio, however, particles were moderately protected from serum neutralization, 2.3-fold over unmodified vector, demonstrating that there is a small window of PEGylation for which particles are still fully infective and benefit from antibody protection. TEM results and structural analysis indicate that the drop of infectivity as the PEG concentration is increased beyond the critical conjugation ratio may be due to a combination of steric interference with viral regions necessary for infection as well as reaction at important lysine residues. However, this first study analyzing the potential of PEG to protect AAV from serum neutralization shows that the approach has promise, which can be further enhanced if the locations of PEG attachment can be more finely controlled.

  6. Neutralization of toxic shock syndrome toxin-1 by monoclonal antibodies in vitro and in vivo.

    PubMed Central

    Bonventre, P F; Thompson, M R; Adinolfi, L E; Gillis, Z A; Parsonnet, J

    1988-01-01

    Sixteen monoclonal antibodies (MAbs) directed against toxic shock syndrome toxin-1 (TSST-1) were generated by immunization of mice with purified TSST-1 and subsequent fusion of spleen cells with myeloma cells. Antibody-producing clones, identified by an enzyme-linked immunosorbent assay, were maintained as ascites tumors, and MAbs were purified by protein A chromatography. High-titered clones were further characterized and tested for the ability to neutralize several biological activities of TSST-1. The MAbs, which are of several immunoglobulin subtypes, reacted specifically with purified TSST-1 and TSST-1 present in Staphylococcus aureus culture supernatants. Three MAbs neutralized TSST-1-induced mitogenesis in a dose-dependent manner. Three of eight MAbs tested were able to neutralize induction by TSST-1 of interleukin-1 production by human monocytes. One neutralizing MAb, 8-5-7, was tested for the ability to protect rabbits from a constant infusion of TSST-1. Rabbits given the MAb had an attenuated clinical illness and were protected from the hypocalcemia, lipemia, and hepatic and renal insufficiency seen in control rabbits. Six of seven control rabbits died, compared with only one of seven rabbits treated with MAb 8-5-7. These experiments suggest that MAb 8-5-7 is directed against an antigenic determinant critical to the toxicity of TSST-1 and that the MAbs should be useful as probes in structure-function analyses of the TSST-1 molecule. Images PMID:3257201

  7. Lack of protection following passive transfer of polyclonal highly functional low-dose non-neutralizing antibodies.

    PubMed

    Dugast, Anne-Sophie; Chan, Ying; Hoffner, Michelle; Licht, Anna; Nkolola, Joseph; Li, Hualin; Streeck, Hendrik; Suscovich, Todd J; Ghebremichael, Musie; Ackerman, Margaret E; Barouch, Dan H; Alter, Galit

    2014-01-01

    Recent immune correlates analysis from the RV144 vaccine trial has renewed interest in the role of non-neutralizing antibodies in mediating protection from infection. While neutralizing antibodies have proven difficult to induce through vaccination, extra-neutralizing antibodies, such as those that mediate antibody-dependent cellular cytotoxicity (ADCC), are associated with long-term control of infection. However, while several non-neutralizing monoclonal antibodies have been tested for their protective efficacy in vivo, no studies to date have tested the protective activity of naturally produced polyclonal antibodies from individuals harboring potent ADCC activity. Because ADCC-inducing antibodies are highly enriched in elite controllers (EC), we passively transferred highly functional non-neutralizing polyclonal antibodies, purified from an EC, to assess the potential impact of polyclonal non-neutralizing antibodies on a stringent SHIV-SF162P3 challenge in rhesus monkeys. Passive transfer of a low-dose of ADCC inducing antibodies did not protect from infection following SHIV-SF162P3 challenge. Passively administered antibody titers and gp120-specific, but not gp41-specific, ADCC and antibody induced phagocytosis (ADCP) were detected in the majority of the monkeys, but did not correlate with post infection viral control. Thus these data raise the possibility that gp120-specific ADCC activity alone may not be sufficient to control viremia post infection but that other specificities or Fc-effector profiles, alone or in combination, may have an impact on viral control and should be tested in future passive transfer experiments.

  8. Novel replication-incompetent vector derived from adenovirus type 11 (Ad11) for vaccination and gene therapy: low seroprevalence and non-cross-reactivity with Ad5.

    PubMed

    Holterman, Lennart; Vogels, Ronald; van der Vlugt, Remko; Sieuwerts, Martijn; Grimbergen, Jos; Kaspers, Jorn; Geelen, Eric; van der Helm, Esmeralda; Lemckert, Angelique; Gillissen, Gert; Verhaagh, Sandra; Custers, Jerome; Zuijdgeest, David; Berkhout, Ben; Bakker, Margreet; Quax, Paul; Goudsmit, Jaap; Havenga, Menzo

    2004-12-01

    A novel plasmid-based adenovirus vector system that enables manufacturing of replication-incompetent (DeltaE1) adenovirus type 11 (Ad11)-based vectors is described. Ad11 vectors are produced on PER.C6/55K cells yielding high-titer vector batches after purification. Ad11 seroprevalence proves to be significantly lower than that of Ad5, and neutralizing antibody titers against Ad11 are low. Ad11 seroprevalence among human immunodeficiency virus-positive (HIV(+)) individuals is as low as that among HIV(-) individuals, independent of the level of immune suppression. The low level of coinciding seroprevalence between Ad11 and Ad35 in addition to a lack of correlation between high neutralizing antibody titers towards either adenovirus strongly suggest that the limited humoral cross-reactive immunity between these two highly related B viruses appears not to preclude the use of both vectors in the same individual. Ad11 transduces primary cells including smooth muscle cells, synoviocytes, and dendritic cells and cardiovascular tissues with higher efficiency than Ad5. Ad11 and Ad35 appear to have a similar tropism as judged by green fluorescent protein expression levels determined by using a panel of cancer cell lines. In addition, Ad5 preimmunization did not significantly affect Ad11-mediated transduction in C57BL/6 mice. We therefore conclude that the Ad11-based vector represents a novel and useful candidate gene transfer vehicle for vaccination and gene therapy.

  9. Somatic Hypermutation-Induced Changes in the Structure and Dynamics of HIV-1 Broadly Neutralizing Antibodies.

    PubMed

    Davenport, Thaddeus M; Gorman, Jason; Joyce, M Gordon; Zhou, Tongqing; Soto, Cinque; Guttman, Miklos; Moquin, Stephanie; Yang, Yongping; Zhang, Baoshan; Doria-Rose, Nicole A; Hu, Shiu-Lok; Mascola, John R; Kwong, Peter D; Lee, Kelly K

    2016-07-20

    Antibody somatic hypermutation (SHM) and affinity maturation enhance antigen recognition by modifying antibody paratope structure to improve its complementarity with the target epitope. SHM-induced changes in paratope dynamics may also contribute to antibody maturation, but direct evidence of this is limited. Here, we examine two classes of HIV-1 broadly neutralizing antibodies (bNAbs) for SHM-induced changes in structure and dynamics, and delineate the effects of these changes on interactions with the HIV-1 envelope glycoprotein (Env). In combination with new and existing structures of unmutated and affinity matured antibody Fab fragments, we used hydrogen/deuterium exchange with mass spectrometry to directly measure Fab structural dynamics. Changes in antibody structure and dynamics were positioned to improve complementarity with Env, with changes in dynamics primarily observed at the paratope peripheries. We conclude that SHM optimizes paratope complementarity to conserved HIV-1 epitopes and restricts the mobility of paratope-peripheral residues to minimize clashes with variable features on HIV-1 Env.

  10. Autoreactivity of primary human immunoglobulins ancestral to hypermutated human antibodies that neutralize HCMV.

    PubMed

    McLean, Gary R; Cho, Chin-wen; Schrader, John W

    2006-05-01

    The human antibody response to the AD-2S1 epitope of glycoprotein B (gB) of human cytomegalovirus (HCMV) is dominated by a family of closely related somatically mutated antibodies. These antibodies neutralize viral infectivity and the genes encoding them are derived from two commonly used germ-line variable (V) region genes, IGHV3-30 and IGKV3-11. Recombination of these V genes with the appropriate junctional diversity generates genes that encode primary immunoglobulins that bind to AD-2S1. To further understand the initial primary immunoglobulin response to AD-2S1 we synthesized the germ-line-based ancestor of one such family of antibodies and showed that it bound gB at the AD-2S1 epitope. Here we show that the germ-line ancestor of a second family of antibodies likewise binds to gB. We further show that one of the ancestral primary immunoglobulins, but not the other, also recognized autoantigens. In contrast, the hypermutated derivatives did not demonstrate autoreactivity and minor structural changes in the primary immunoglobulin were sufficient to generate or abolish autoreactivity or to change specificity. Thus, our demonstration that the ancestor of a highly mutated, non-autoreactive antiviral IgG antibody binds nuclear and cell-surface autoantigens indicates for the first time that self-reactivity is not necessarily a barrier to development into a follicular B lymphocyte that undergoes antigen-initiated affinity maturation.

  11. CROSS-REACTIVE AND POTENT NEUTRALIZING ANTIBODY RESPONSES IN HUMAN SURVIVORS OF NATURAL EBOLAVIRUS INFECTION

    PubMed Central

    Flyak, Andrew I.; Shen, Xiaoli; Murin, Charles D.; Turner, Hannah L.; David, Joshua A.; Fusco, Marnie L.; Lampley, Rebecca; Kose, Nurgun; Ilinykh, Philipp A.; Kuzmina, Natalia; Branchizio, Andre; King, Hannah; Brown, Leland; Bryan, Christopher; Davidson, Edgar; Doranz, Benjamin J.; Slaughter, James C.; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G.; Saphire, Erica Ollmann; Ward, Andrew B.; Bukreyev, Alexander; Crowe, James E.

    2015-01-01

    Summary Recent studies have suggested that antibody-mediated protection against the Ebolaviruses may be achievable, but little is known about whether or not antibodies can confer cross-reactive protection against viruses belonging to diverse Ebolavirus species, such as Ebola virus (EBOV), Sudan virus (SUDV) and Bundibugyo virus (BDBV). We isolated a large panel of human monoclonal antibodies (mAbs) against BDBV glycoprotein (GP) using peripheral blood B cells from survivors of the 2007 BDBV outbreak in Uganda. We determined that a large proportion of mAbs with potent neutralizing activity against BDBV bind to the glycan cap and recognize diverse epitopes within this major antigenic site. We identified several glycan cap-specific mAbs that neutralized multiple ebolaviruses including SUDV, and a cross-reactive mAb that completely protected guinea pigs from the lethal challenge with heterologous EBOV. Our results provide a roadmap to develop a single antibody-based treatment effective against multiple Ebolavirus infections. PMID:26806128

  12. Protective monotherapy against lethal Ebola virus infection by a potently neutralizing antibody.

    PubMed

    Corti, Davide; Misasi, John; Mulangu, Sabue; Stanley, Daphne A; Kanekiyo, Masaru; Wollen, Suzanne; Ploquin, Aurélie; Doria-Rose, Nicole A; Staupe, Ryan P; Bailey, Michael; Shi, Wei; Choe, Misook; Marcus, Hadar; Thompson, Emily A; Cagigi, Alberto; Silacci, Chiara; Fernandez-Rodriguez, Blanca; Perez, Laurent; Sallusto, Federica; Vanzetta, Fabrizia; Agatic, Gloria; Cameroni, Elisabetta; Kisalu, Neville; Gordon, Ingelise; Ledgerwood, Julie E; Mascola, John R; Graham, Barney S; Muyembe-Tamfun, Jean-Jacques; Trefry, John C; Lanzavecchia, Antonio; Sullivan, Nancy J

    2016-03-18

    Ebola virus disease in humans is highly lethal, with case fatality rates ranging from 25 to 90%. There is no licensed treatment or vaccine against the virus, underscoring the need for efficacious countermeasures. We ascertained that a human survivor of the 1995 Kikwit Ebola virus disease outbreak maintained circulating antibodies against the Ebola virus surface glycoprotein for more than a decade after infection. From this survivor we isolated monoclonal antibodies (mAbs) that neutralize recent and previous outbreak variants of Ebola virus and mediate antibody-dependent cell-mediated cytotoxicity in vitro. Strikingly, monotherapy with mAb114 protected macaques when given as late as 5 days after challenge. Treatment with a single human mAb suggests that a simplified therapeutic strategy for human Ebola infection may be possible.

  13. Distribution of neutralizing antibodies to California and Bunyamwera serogroup viruses in horses and rodents in California.

    PubMed

    Campbell, G L; Reeves, W C; Hardy, J L; Eldridge, B F

    1990-03-01

    Neutralization tests were done on sera from 141 horses from high elevation regions of California. Antibody prevalences to Jamestown Canyon, snowshoe hare, and California encephalitis viruses in the California serogroup and Northway virus in the Bunyamwera serogroup were 55%, 43%, 18%, and 46%, respectively. In 51 horses from rural low elevation regions, seroprevalences were 31%, 35%, 35%, and 37%, respectively. Twenty-four horses from a suburban lowland area were seronegative, except for a single horse with a low titer to snowshoe hare virus. Seroprevalence to Jamestown Canyon and snowshoe hare viruses was associated with increasing age. Only 2 of 177 rodents from the Sierra Nevada had antibodies to Northway virus; none had antibodies to Jamestown Canyon or snowshoe hare viruses.

  14. Broadly Neutralizing Antibody Responses in a Large Longitudinal Sub-Saharan HIV Primary Infection Cohort.

    PubMed

    Landais, Elise; Huang, Xiayu; Havenar-Daughton, Colin; Murrell, Ben; Price, Matt A; Wickramasinghe, Lalinda; Ramos, Alejandra; Bian, Charoan B; Simek, Melissa; Allen, Susan; Karita, Etienne; Kilembe, William; Lakhi, Shabir; Inambao, Mubiana; Kamali, Anatoli; Sanders, Eduard J; Anzala, Omu; Edward, Vinodh; Bekker, Linda-Gail; Tang, Jianming; Gilmour, Jill; Kosakovsky-Pond, Sergei L; Phung, Pham; Wrin, Terri; Crotty, Shane; Godzik, Adam; Poignard, Pascal

    2016-01-01

    Broadly neutralizing antibodies (bnAbs) are thought to be a critical component of a protective HIV vaccine. However, designing vaccines immunogens able to elicit bnAbs has proven unsuccessful to date. Understanding the correlates and immunological mechanisms leading to the development of bnAb responses during natural HIV infection is thus critical to the design of a protective vaccine. The IAVI Protocol C program investigates a large longitudinal cohort of primary HIV-1 infection in Eastern and South Africa. Development of neutralization was evaluated in 439 donors using a 6 cross-clade pseudo-virus panel predictive of neutralization breadth on larger panels. About 15% of individuals developed bnAb responses, essentially between year 2 and year 4 of infection. Statistical analyses revealed no influence of gender, age or geographical origin on the development of neutralization breadth. However, cross-clade neutralization strongly correlated with high viral load as well as with low CD4 T cell counts, subtype-C infection and HLA-A*03(-) genotype. A correlation with high overall plasma IgG levels and anti-Env IgG binding titers was also found. The latter appeared not associated with higher affinity, suggesting a greater diversity of the anti-Env responses in broad neutralizers. Broadly neutralizing activity targeting glycan-dependent epitopes, largely the N332-glycan epitope region, was detected in nearly half of the broad neutralizers while CD4bs and gp41-MPER bnAb responses were only detected in very few individuals. Together the findings suggest that both viral and host factors are critical for the development of bnAbs and that the HIV Env N332-glycan supersite may be a favorable target for vaccine design.

  15. Broadly Neutralizing Antibody Responses in a Large Longitudinal Sub-Saharan HIV Primary Infection Cohort

    PubMed Central

    Landais, Elise; Huang, Xiayu; Havenar-Daughton, Colin; Murrell, Ben; Price, Matt A.; Wickramasinghe, Lalinda; Ramos, Alejandra; Bian, Charoan B.; Simek, Melissa; Allen, Susan; Karita, Etienne; Kilembe, William; Lakhi, Shabir; Inambao, Mubiana; Kamali, Anatoli; Sanders, Eduard J.; Anzala, Omu; Edward, Vinodh; Bekker, Linda-Gail; Tang, Jianming; Gilmour, Jill; Kosakovsky-Pond, Sergei L.; Phung, Pham; Wrin, Terri; Crotty, Shane; Godzik, Adam; Poignard, Pascal

    2016-01-01

    Broadly neutralizing antibodies (bnAbs) are thought to be a critical component of a protective HIV vaccine. However, designing vaccines immunogens able to elicit bnAbs has proven unsuccessful to date. Understanding the correlates and immunological mechanisms leading to the development of bnAb responses during natural HIV infection is thus critical to the design of a protective vaccine. The IAVI Protocol C program investigates a large longitudinal cohort of primary HIV-1 infection in Eastern and South Africa. Development of neutralization was evaluated in 439 donors using a 6 cross-clade pseudo-virus panel predictive of neutralization breadth on larger panels. About 15% of individuals developed bnAb responses, essentially between year 2 and year 4 of infection. Statistical analyses revealed no influence of gender, age or geographical origin on the development of neutralization breadth. However, cross-clade neutralization strongly correlated with high viral load as well as with low CD4 T cell counts, subtype-C infection and HLA-A*03(-) genotype. A correlation with high overall plasma IgG levels and anti-Env IgG binding titers was also found. The latter appeared not associated with higher affinity, suggesting a greater diversity of the anti-Env responses in broad neutralizers. Broadly neutralizing activity targeting glycan-dependent epitopes, largely the N332-glycan epitope region, was detected in nearly half of the broad neutralizers while CD4bs and gp41-MPER bnAb responses were only detected in very few individuals. Together the findings suggest that both viral and host factors are critical for the development of bnAbs and that the HIV Env N332-glycan supersite may be a favorable target for vaccine design. PMID:26766578

  16. Increased frequencies of CD8+CD57+ T cells are associated with antibody neutralization breadth against HIV in viraemic controllers

    PubMed Central

    Palmer, Christine D; Romero-Tejeda, Marisol; Scully, Eileen P; Lockhart, Ainsley; Seaman, Michael S; Goldenthal, Ariel; Piechocka-Trocha, Alicja; Walker, Bruce D; Chibnik, Lori B; Jost, Stephanie; Porichis, Filippos

    2016-01-01

    Introduction An effective prophylactic vaccine against HIV will need to elicit antibody responses capable of recognizing and neutralizing rapidly evolving antigenic regions. The immunologic milieu associated with development of neutralizing antibody breadth remains to be fully defined. In this study, we sought to identify immunological signatures associated with neutralization breadth in HIV controllers. We applied an immune monitoring approach to analyze markers of T cell and myeloid cell activation by flow cytometry, comparing broad neutralizers with low- and non-neutralizers using multivariate and univariate analyses. Methods Antibody neutralization breadth was determined, and cryopreserved peripheral blood mononuclear cells were stained for T cell and myeloid cell activation markers. Subjects were grouped according to neutralization breadth, and T cell and myeloid cell activation was analyzed by partial least squares discriminant analysis to determine immune signatures associated with high neutralization breadth. Results We show that neutralization breadth in HIV viraemic controllers (VC) was strongly associated with increased frequencies of CD8+CD57+ T cells and that this association was independent of viral load, CD4 count and time since HIV diagnosis. Conclusions Our data show elevated frequencies of CD8+CD57+ T cells in VC who develop neutralization breadth against HIV. This immune signature could serve as a potential biomarker of neutralization breadth and should be further investigated in other HIV-positive cohorts and in HIV vaccine trials. PMID:27938646

  17. Neutralizing anti-interleukin-1β antibodies modulate fetal blood-brain barrier function after ischemia.

    PubMed

    Chen, Xiaodi; Sadowska, Grazyna B; Zhang, Jiyong; Kim, Jeong-Eun; Cummings, Erin E; Bodge, Courtney A; Lim, Yow-Pin; Makeyev, Oleksandr; Besio, Walter G; Gaitanis, John; Threlkeld, Steven W; Banks, William A; Stonestreet, Barbara S

    2015-01-01

    We have previously shown that increases in blood-brain barrier permeability represent an important component of ischemia-reperfusion related brain injury in the fetus. Pro-inflammatory cytokines could contribute to these abnormalities in blood-brain barrier function. We have generated pharmacological quantities of mouse anti-ovine interleukin-1β monoclonal antibody and shown that this antibody has very high sensitivity and specificity for interleukin-1β protein. This antibody also neutralizes the effects of interleukin-1β protein in vitro. In the current study, we hypothesized that the neutralizing anti-interleukin-1β monoclonal antibody attenuates ischemia-reperfusion related fetal blood-brain barrier dysfunction. Instrumented ovine fetuses at 127 days of gestation were studied after 30 min of carotid occlusion and 24h of reperfusion. Groups were sham operated placebo-control- (n=5), ischemia-placebo- (n=6), ischemia-anti-IL-1β antibody- (n=7), and sham-control antibody- (n=2) treated animals. Systemic infusions of placebo (0.154M NaCl) or anti-interleukin-1β monoclonal antibody (5.1±0.6 mg/kg) were given intravenously to the same sham or ischemic group of fetuses at 15 min and 4h after ischemia. Concentrations of interleukin-1β protein and anti-interleukin-1β monoclonal antibody were measured by ELISA in fetal plasma, cerebrospinal fluid, and parietal cerebral cortex. Blood-brain barrier permeability was quantified using the blood-to-brain transfer constant (Ki) with α-aminoisobutyric acid in multiple brain regions. Interleukin-1β protein was also measured in parietal cerebral cortices and tight junction proteins in multiple brain regions by Western immunoblot. Cerebral cortical interleukin-1β protein increased (P<0.001) after ischemia-reperfusion. After anti-interleukin-1β monoclonal antibody infusions, plasma anti-interleukin-1β monoclonal antibody was elevated (P<0.001), brain anti-interleukin-1β monoclonal antibody levels were higher (P<0

  18. Analysis of cross-reactive neutralizing antibodies in human HFMD serum with an EV71 pseudovirus-based assay.

    PubMed

    Zhang, Huafei; An, Dong; Liu, Wei; Mao, Qunying; Jin, Jun; Xu, Lin; Sun, Shiyang; Jiang, Liping; Li, Xiaojun; Shao, Jie; Ma, Hongxia; Huang, Xueyong; Guo, Shijie; Chen, Haiying; Cheng, Tong; Yang, Lisheng; Su, Weiheng; Kong, Wei; Liang, Zhenglun; Jiang, Chunlai

    2014-01-01

    Hand, foot and mouth disease, associated with enterovirus 71 (EV71) infections, has recently become an important public health issue throughout the world. Serum neutralizing antibodies are major indicators of EV71 infection and protective immunity. However, the potential for cross-reactivity of neutralizing antibodies for different EV71 genotypes and subgenotypes is unclear. Here we measured the cross-reactive neutralizing antibody titers against EV71 of different genotypes or subgenotypes in sera collected from EV71-infected children and vaccine-inoculated children in a phase III clinical trial (ClinicalTrials.gov Identifier: NCT01636245) using a new pseudovirus-based neutralization assay. Antibodies induced by EV71-C4a were cross-reactive for different EV71 genotypes, demonstrating that C4a is a good candidate strain for an EV71 vaccine. Our study also demonstrated that this new assay is practical for analyses of clinical samples from epidemiological and vaccine studies.

  19. Developmental Pathway of the MPER-Directed HIV-1-Neutralizing Antibody 10E8

    PubMed Central

    Zhang, Baoshan; McKee, Krisha; Longo, Nancy S.; Yang, Yongping; Huang, Jinghe; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E.; Alam, S. Munir; Haynes, Barton F.; Mullikin, James C.; Connors, Mark; Mascola, John R.; Shapiro, Lawrence; Kwong, Peter D.

    2016-01-01

    Antibody 10E8 targets the membrane-proximal external region (MPER) of HIV-1 gp41, neutralizes >97% of HIV-1 isolates, and lacks the auto-reactivity often associated with MPER-directed antibodies. The developmental pathway of 10E8 might therefore serve as a promising template for vaccine design, but samples from time-of-infection—often used to infer the B cell record—are unavailable. In this study, we used crystallography, next-generation sequencing (NGS), and functional assessments to infer the 10E8 developmental pathway from a single time point. Mutational analysis indicated somatic hypermutation of the 2nd-heavy chain-complementarity determining region (CDR H2) to be critical for neutralization, and structures of 10E8 variants with V-gene regions reverted to genomic origin for heavy-and-light chains or heavy chain-only showed structural differences >2 Å relative to mature 10E8 in the CDR H2 and H3. To understand these developmental changes, we used bioinformatic sieving, maximum likelihood, and parsimony analyses of immunoglobulin transcripts to identify 10E8-lineage members, to infer the 10E8-unmutated common ancestor (UCA), and to calculate 10E8-developmental intermediates. We were assisted in this analysis by the preservation of a critical D-gene segment, which was unmutated in most 10E8-lineage sequences. UCA and early intermediates weakly bound a 26-residue-MPER peptide, whereas HIV-1 neutralization and epitope recognition in liposomes were only observed with late intermediates. Antibody 10E8 thus develops from a UCA with weak MPER affinity and substantial differences in CDR H2 and H3 from the mature 10E8; only after extensive somatic hypermutation do 10E8-lineage members gain recognition in the context of membrane and HIV-1 neutralization. PMID:27299673

  20. H7N9 influenza virus neutralizing antibodies that possess few somatic mutations

    PubMed Central

    Thornburg, Natalie J.; Zhang, Heng; Bangaru, Sandhya; Kose, Nurgun; Lampley, Rebecca M.; Bombardi, Robin G.; Yu, Yingchun; Graham, Stephen; Branchizio, Andre; Yoder, Sandra M.; Rock, Michael T.; Creech, C. Buddy; Edwards, Kathryn M.; Lee, David; Li, Sheng; Wilson, Ian A.; García-Sastre, Adolfo; Albrecht, Randy A.; Crowe, James E.

    2016-01-01

    Avian H7N9 influenza viruses are group 2 influenza A viruses that have been identified as the etiologic agent for a current major outbreak that began in China in 2013 and may pose a pandemic threat. Here, we examined the human H7-reactive antibody response in 75 recipients of a monovalent inactivated A/Shanghai/02/2013 H7N9 vaccine. After 2 doses of vaccine, the majority of donors had memory B cells that secreted IgGs specific for H7 HA, with dominant responses against single HA subtypes, although frequencies of H7-reactive B cells ranged widely between donors. We isolated 12 naturally occurring mAbs with low half-maximal effective concentrations for binding, 5 of which possessed neutralizing and HA-inhibiting activities. The 5 neutralizing mAbs exhibited narrow breadth of reactivity with influenza H7 strains. Epitope-mapping studies using neutralization escape mutant analysis, deuterium exchange mass spectrometry, and x-ray crystallography revealed that these neutralizing mAbs bind near the receptor-binding pocket on HA. All 5 neutralizing mAbs possessed low numbers of somatic mutations, suggesting the clones arose from naive B cells. The most potent mAb, H7.167, was tested as a prophylactic treatment in a mouse intranasal virus challenge study, and systemic administration of the mAb markedly reduced viral lung titers. PMID:26950424

  1. HIV-1 neutralizing antibody response and viral genetic diversity characterized with next generation sequencing

    PubMed Central

    Carter, Christoph C.; Wagner, Gabriel A.; Hightower, George K.; Caballero, Gemma; Phung, Pham; Richman, Douglas D.; Kosakovsky Pond, Sergei L.; Smith, Davey M.

    2014-01-01

    To better understand the dynamics of HIV-specific neutralizing antibody (NAb), we examined associations between viral genetic diversity and the NAb response against a multi-subtype panel of heterologous viruses in a well-characterized, therapy-naïve primary infection cohort. Using next generation sequencing (NGS), we computed sequence-based measures of diversity within HIV-1 env, gag and pol, and compared them to NAb breadth and potency as calculated by a neutralization score. Contemporaneous env diversity and the neutralization score were positively correlated (p=0.0033), as were the neutralization score and estimated duration of infection (EDI) (p=0.0038), and env diversity and EDI (p=0.0005). Neither early env diversity nor baseline viral load correlated with future NAb breadth and potency (p>0.05). Taken together, it is unlikely that neutralizing capability in our cohort was conditioned on viral diversity, but rather that env evolution was driven by the level of NAb selective pressure. PMID:25463602

  2. Neutralizing monoclonal antibody to edema toxin and its effect on murine anthrax.

    PubMed

    Winterroth, Lisa; Rivera, Johanna; Nakouzi, Antonio S; Dadachova, Ekaterina; Casadevall, Arturo

    2010-06-01

    Edema factor (EF) is a component of an anthrax toxin that functions as an adenylate cyclase. Numerous monoclonal antibodies (MAbs) have been reported for the other Bacillus anthracis toxin components, but relatively few to EF have been studied. We report the generation of six murine hybridoma lines producing two IgM and four IgG1 MAbs to EF. Of the six MAbs, only one IgM neutralized EF, as assayed by an increase in cyclic AMP (cAMP) production by Chinese hamster ovary (CHO) cells. Analysis of the variable gene elements revealed that the single neutralizing MAb had a different binding site than the others. There was no competition between the neutralizing IgM and the nonneutralizing IgG MAbs indicative of different specificity. MAb-based capture enzyme-linked immunosorbent assay (ELISA) detected EF in liver lysates from mice infected with B. anthracis Sterne 34F2. Administration of the neutralizing IgM MAb to A/JCr mice lethally infected with B. anthracis strain Sterne had no significant effect on median time to death, but mice treated with the MAb were more likely to survive infection. Combining the neutralizing IgM to EF with a subprotective dose of a neutralizing MAb to protective antigen (PA) prolonged mean time to death of infected mice, suggesting that neutralization of EF and PA could produce synergistic beneficial effects. In summary, the results from our study and literature observations suggest that the majority of Abs to EF are nonneutralizing, but the toxin has some epitopes that can be targeted by the humoral response to generate useful Abs that may contribute to defense against anthrax.

  3. Structural Basis for the Binding of the Neutralizing Antibody, 7D11, to the Poxvirus L1 Protein

    DTIC Science & Technology

    2007-08-01

    enveloped particles, such as the humanized chimpanzee B5 antibodies (Chen et al., 2006), may serve as a replacement for vaccinia immune globulin and...Svitel, J., Schuck, P., Satterfield, W., Moss, B., Purcell, R., 2006. Chimpanzee / human mAbs to vaccinia virus B5 protein neutralize vaccinia and...frame, is the target of neutralizing antibodies and has been successfully used as a component of both protein subunit and DNA vaccines. L1-specific

  4. Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117.

    PubMed

    Caskey, Marina; Klein, Florian; Lorenzi, Julio C C; Seaman, Michael S; West, Anthony P; Buckley, Noreen; Kremer, Gisela; Nogueira, Lilian; Braunschweig, Malte; Scheid, Johannes F; Horwitz, Joshua A; Shimeliovich, Irina; Ben-Avraham, Sivan; Witmer-Pack, Maggi; Platten, Martin; Lehmann, Clara; Burke, Leah A; Hawthorne, Thomas; Gorelick, Robert J; Walker, Bruce D; Keler, Tibor; Gulick, Roy M; Fätkenheuer, Gerd; Schlesinger, Sarah J; Nussenzweig, Michel C

    2015-06-25

    HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned. However, recently developed single-cell-based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies to HIV-1 (refs 4, 5). These antibodies can prevent infection and suppress viraemia in humanized mice and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favourable pharmacokinetics. A single 30 mg kg(-1) infusion of 3BNC117 reduced the viral load in HIV-1-infected individuals by 0.8-2.5 log10 and viraemia remained significantly reduced for 28 days. Emergence of resistant viral strains was variable, with some individuals remaining sensitive to 3BNC117 for a period of 28 days. We conclude that, as a single agent, 3BNC117 is safe and effective in reducing HIV-1 viraemia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy and cure.

  5. 3BNC117 a Broadly Neutralizing Antibody Suppresses Viremia in HIV-1-Infected Humans

    PubMed Central

    Caskey, Marina; Klein, Florian; Lorenzi, Julio C. C.; Seaman, Michael S.; West, Anthony P.; Buckley, Noreen; Kremer, Gisela; Nogueira, Lilian; Braunschweig, Malte; Scheid, Johannes F.; Horwitz, Joshua A.; Shimeliovich, Irina; Ben Avraham-Shulman, Sivan; Witmer-Pack, Maggi; Platten, Martin; Lehmann, Clara; Burke, Leah A.; Hawthorne, Thomas; Gorelick, Robert J.; Walker, Bruce D.; Keler, Tibor; Gulick, Roy M.; Fätkenheuer, Gerd; Schlesinger, Sarah J.; Nussenzweig, Michel C.

    2016-01-01

    HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned1–3. However, recently developed single cell based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies (bNAbs) to HIV-14,5. These antibodies can prevent infection and suppress viremia in humanized mice (hu-mice) and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated6–10. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody11, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favorable pharmacokinetics. A single 30 mg/kg infusion of 3BNC117 reduced the viral load (VL) in HIV-1-infected individuals by 0.8 – 2.5 log10 and viremia remained significantly reduced for 28 days. Emergence of resistant viral strains was variable, with some individuals remaining sensitive to 3BNC117 for a period of 28 days. We conclude that as a single agent 3BNC117 is safe and effective in reducing HIV-1 viremia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy, and cure. PMID:25855300

  6. Detection of canine distemper virus serum neutralizing antibodies in captive U.S. phocids.

    PubMed

    Clancy, Meredith M; Gamble, Kathryn C; Travis, Dominic A

    2013-03-01

    Antibodies to morbilliviruses have been documented in free-ranging pinnipeds throughout populations in the Atlantic and Arctic Oceans, but not from the Pacific Ocean. As a symbolic geographic barrier between the exposed Atlantic and naive Pacific populations, the captive phocid population in North America had undocumented serologic status. In this study, canine distemper virus (CDV) serum neutralization assays were used to assess the prevalence of antibodies in this population with participation of 25 U.S. institutions from grey seals (Halichoerus grypus, n = 6) and harbor seals (Phoca vitulina, n = 108). Historic and environmental risk factors associated with the epidemiology of distemper virus were collected by survey. Based on antibodies to canine distemper virus, the prevalence of exposure in this population was 25.5%, with 28 seals (grey, n = 2; harbor, n = 26) demonstrating antibody titers > or = 1:16, and positive titers ranged from 1:4 to 1:1,536. By survey analysis, strong associations with seropositive status were identified for captive origin (P = 0.013) and movement among institutions (P = 0.024). Size of population has positive correlation with likelihood of seropositive seals at an institution (P = 0.020). However, no major husbandry or enclosure-based risk factors were identified in institutions with seropositive seals, and no interaction between individual or institutional risk factors was identified. Previously undocumented prior to this study, CDV antibodies were measured in harbor seals (n = 2) recently stranded from the Pacific coast.

  7. A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

    PubMed

    Scalley-Kim, Michelle L; Hess, Bruce W; Kelly, Ryan L; Krostag, Anne-Rachel F; Lustig, Kurt H; Marken, John S; Ovendale, Pamela J; Posey, Aaron R; Smolak, Pamela J; Taylor, Janelle D L; Wood, C L; Bienvenue, David L; Probst, Peter; Salmon, Ruth A; Allison, Daniel S; Foy, Teresa M; Raport, Carol J

    2012-01-01

    Chemokines play a key role in leukocyte recruitment during inflammation and are implicated in the pathogenesis of a number of autoimmune diseases. As such, inhibiting chemokine signaling has been of keen interest for the development of therapeutic agents. This endeavor, however, has been hampered due to complexities in the chemokine system. Many chemokines have been shown to signal through multiple receptors and, conversely, most chemokine receptors bind to more than one chemokine. One approach to overcoming this complexity is to develop a single therapeutic agent that binds and inactivates multiple chemokines, similar to an immune evasion strategy utilized by a number of viruses. Here, we describe the development and characterization of a novel therapeutic antibody that targets a subset of human CC chemokines, specifically CCL3, CCL4, and CCL5, involved in chronic inflammatory diseases. Using a sequential immunization approach, followed by humanization and phage display affinity maturation, a therapeutic antibody was developed that displays high binding affinity towards the three targeted chemokines. In vitro, this antibody potently inhibits chemotaxis and chemokine-mediated signaling through CCR1 and CCR5, primary chemokine receptors for the targeted chemokines. Furthermore, we have demonstrated in vivo efficacy of the antibody in a SCID-hu mouse model of skin leukocyte migration, thus confirming its potential as a novel therapeutic chemokine antagonist. We anticipate that this antibody will have broad therapeutic utility in the treatment of a number of autoimmune diseases due to its ability to simultaneously neutralize multiple chemokines implicated in disease pathogenesis.

  8. Opportunities to exploit non-neutralizing HIV-specific antibody activity

    PubMed Central

    Ackerman, Margaret E.; Alter, Galit

    2016-01-01

    Antibodies act as a nexus between innate and adaptive immunity: they provide a means to engage a spectrum of innate immune effector cells in order to clear viral particles and infected cells, and prime antigen presentation. This functional landscape is remarkably complex, and depends on antibody isotype, subclass, and glycosylation; the expression levels and patterns of a suite of Fc receptors with both complementary and opposing activities; and a host of innate immune cells capable of differential responses to opsonized particles and present at different sites. In vivo, even neutralizing antibodies rely on their ability to act as molecular beacons and recruit innate immune effector cells in order to provide protection, and results from both human and macaque studies have implicated these effector functions in vaccine-mediated protection. Thus, while enhancing effector function is a tractable handle for potentiating antibody-mediated protection from HIV infection, success will depend critically on leveraging understanding of the means by which antibodies with specific functional profiles could be elicited, which effector functions could provide optimal protection, and perhaps most critically, how to efficiently recruit the innate effector cells present at sites of infection. PMID:24191934

  9. Autologous and heterologous neutralizing antibody responses following initial seroconversion in human immunodeficiency virus type 1-infected individuals.

    PubMed Central

    Moog, C; Fleury, H J; Pellegrin, I; Kirn, A; Aubertin, A M

    1997-01-01

    In the course of human immunodeficiency virus type 1 (HIV-1) infection, patients develop a strong and persistent immune response characterized by the production of HIV-specific antibodies. The aim of our study was to analyze the appearance of autologous and heterologous neutralizing antibodies in the sera of HIV-infected individuals. For this purpose, primary strains have been isolated from 18 HIV-1-infected subjects prior to seroconversion (in one case) or within 1 to 8 months after seroconversion. Sera, collected at the same time as the virus was isolated and at various times after isolation, have been analyzed for their ability to neutralize the autologous primary strains isolated early after infection, heterologous primary isolates, and cell-line adapted strains. Our neutralization assay, which combines serial dilutions of virus and serial dilutions of sera, is based on the determination of the serum dilution at which a fixed reduction in virus titer (90%) occurs. We have shown that (i) we could not detect autologous neutralizing antibodies in sera collected at the same time as we isolated viruses; (ii) we detected neutralizing antibodies against the autologous strains about 1 year after seroconversion, occasionally after 8 months, but sera were not always available to exclude the presence of neutralizing antibodies at earlier times; (iii) after 1 year, the neutralization response was highly specific to virus present during the early phase of HIV infection; and (iv) heterologous neutralization of primary isolates was detected later (after about 2 years). These results reveal the enormous diversity of neutralization determinants on primary isolates as well as a temporal evolution of the humoral response generating cross-reactive neutralizing antibodies. PMID:9094648

  10. Development of a high-throughput β-Gal-based neutralization assay for quantitation of herpes simplex virus-neutralizing antibodies in human samples.

    PubMed

    Baccari, Amy; Cooney, Michael; Blevins, Tamara P; Morrison, Lynda A; Larson, Shane; Skoberne, Mojca; Belshe, Robert B; Flechtner, Jessica B; Long, Deborah

    2016-07-19

    Measurement of neutralizing antibodies against herpes simplex virus (HSV) is important for evaluation of candidate vaccines. The established plaque-reduction neutralization assay is time consuming, labor intensive, and difficult to validate and transfer. Here, we describe the characterization of a HSV-neutralization assay based on the expression of a reporter gene, β-galactosidase (β-Gal). Using previously constructed HSV-β-Gal recombinant viruses, HSV-2/Gal and HSV-1/tk12, we developed a colorimetric β-Gal-based neutralization assay that is sensitive and highly reproducible, and performed in less than 48h. HSV-1 and HSV-2 neutralizing titers measured by the β-Gal-based neutralization assay were equivalent to those obtained by a plaque reduction neutralization assay. Intra- and inter-assay precision studies demonstrated that the β-Gal-based assay was repeatable and yielded low and acceptable variation. In addition, comparison of HSV-2 neutralizing antibody (NAb) titers measured in two independent laboratories by two unique β-Gal-based assays showed a highly significant correlation (r=0.9499, p<0.0001) between the two assays. The new assay will serve as an important tool both for preclinical and clinical trials of new HSV vaccines.

  11. Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B

    PubMed Central

    Rasetti-Escargueil, Christine; Avril, Arnaud; Chahboun, Siham; Tierney, Rob; Bak, Nicola; Miethe, Sebastian; Mazuet, Christelle; Popoff, Michel R; Thullier, Philippe; Hust, Michael; Pelat, Thibaut; Sesardic, Dorothea

    2015-01-01

    Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2–7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2–7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2–7 and BLC3) are close to the human germline sequences

  12. Antibody Conjugation Approach Enhances Breadth and Potency of Neutralization of Anti-HIV-1 Antibodies and CD4-IgG

    PubMed Central

    Gavrilyuk, Julia; Ban, Hitoshi; Uehara, Hisatoshi; Sirk, Shannon J.; Saye-Francisco, Karen; Cuevas, Angelica; Zablowsky, Elise; Oza, Avinash; Seaman, Michael S.; Burton, Dennis R.

    2013-01-01

    Broadly neutralizing antibodies PG9 and PG16 effectively neutralize 70 to 80% of circulating HIV-1 isolates. In this study, the neutralization abilities of PG9 and PG16 were further enhanced by bioconjugation with aplaviroc, a small-molecule inhibitor of virus entry into host cells. A novel air-stable diazonium hexafluorophosphate reagent that allows for rapid, tyrosine-selective functionalization of proteins and antibodies under mild conditions was used to prepare a series of aplaviroc-conjugated antibodies, including b12, 2G12, PG9, PG16, and CD4-IgG. The conjugated antibodies blocked HIV-1 entry through two mechanisms: by binding to the virus itself and by blocking the CCR5 receptor on host cells. Chemical modification did not significantly alter the potency of the parent antibodies against nonresistant HIV-1 strains. Conjugation did not alter the pharmacokinetics of a model IgG in blood. The PG9-aplaviroc conjugate was tested against a panel of 117 HIV-1 strains and was found to neutralize 100% of the viruses. PG9-aplaviroc conjugate IC50s were lower than those of PG9 in neutralization studies of 36 of the 117 HIV-1 strains. These results support this new approach to bispecific antibodies and offer a potential new strategy for combining HIV-1 therapies. PMID:23427154

  13. Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus.

    PubMed

    Giang, Erick; Dorner, Marcus; Prentoe, Jannick C; Dreux, Marlène; Evans, Matthew J; Bukh, Jens; Rice, Charles M; Ploss, Alexander; Burton, Dennis R; Law, Mansun

    2012-04-17

    Hepatitis C virus (HCV) infects ∼2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human mAbs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies to HCV.

  14. Recombinant Encephalomyocarditis Viruses Elicit Neutralizing Antibodies against PRRSV and CSFV in Mice

    PubMed Central

    Zhu, Shu; Guo, Xin; Keyes, Lisa R.; Yang, Hanchun; Ge, Xinna

    2015-01-01

    Encephalomyocarditis virus (EMCV) is capable of infecting a wide range of species and the infection can cause myocarditis and reproductive failure in pigs as well as febrile illness in human beings. In this study, we introduced the entire ORF5 of the porcine reproductive and respiratory syndrome virus (PRRSV) or the neutralization epitope regions in the E2 gene of the classical swine fever virus (CSFV), into the genome of a stably attenuated EMCV strain, T1100I. The resultant viable recombinant viruses, CvBJC3m/I-ΔGP5 and CvBJC3m/I-E2, respectively expressed partial PRRSV envelope protein GP5 or CSFV neutralization epitope A1A2 along with EMCV proteins. These heterologous proteins fused to the N-terminal of the nonstructural leader protein could be recognized by anti-GP5 or anti-E2 antibody. We also tested the immunogenicity of these fusion proteins by immunizing BALB/c mice with the recombinant viruses. The immunized animals elicited neutralizing antibodies against PRRSV and CSFV. Our results suggest that EMCV can be engineered as an expression vector and serve as a tool in the development of novel live vaccines in various animal species. PMID:26076449

  15. Broadly neutralizing alphavirus antibodies bind an epitope on E2 and inhibit entry and egress

    PubMed Central

    Fox, Julie M.; Long, Feng; Edeling, Melissa A.; Lin, Hueylie; van Duijl-Richter, Mareike K.S.; Fong, Rachel H.; Kahle, Kristen M.; Smit, Jolanda M.; Jin, Jing; Simmons, Graham; Doranz, Benjamin J.; Crowe, James E.; Fremont, Daved H.; Rossmann, Michael G.; Diamond, Michael S.

    2015-01-01

    SUMMARY We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O’nyong’nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern. PMID:26553503

  16. Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress.

    PubMed

    Fox, Julie M; Long, Feng; Edeling, Melissa A; Lin, Hueylie; van Duijl-Richter, Mareike K S; Fong, Rachel H; Kahle, Kristen M; Smit, Jolanda M; Jin, Jing; Simmons, Graham; Doranz, Benjamin J; Crowe, James E; Fremont, Daved H; Rossmann, Michael G; Diamond, Michael S

    2015-11-19

    We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.

  17. Human Antibody Neutralizes Severe Fever with Thrombocytopenia Syndrome Virus, an Emerging Hemorrhagic Fever Virus

    PubMed Central

    Guo, Xiling; Zhang, Li; Zhang, Wenshuai; Chi, Ying; Zeng, Xiaoyan; Li, Xian; Qi, Xian; Jin, Qiu; Zhang, Xiao; Huang, Mingming; Wang, Hua; Chen, Yin; Bao, Changjun; Hu, Jianli; Liang, Shuyi; Bao, Lin; Wu, Tao

    2013-01-01

    Severe fever with thrombocytopenia syndrome virus (SFTSV), a newly discovered member of the Bunyaviridae family, is the causative agent of an emerging hemorrhagic fever, SFTS, in China. Currently, there are no vaccines or effective therapies against SFTS. In this study, a combinatorial human antibody library was constructed from the peripheral lymphocytes of 5 patients who had recovered from SFTS. The library was screened against purified virions for the production of single-chain variable-region fragments (ScFv). Of the 6 positive clones, one clone (monoclonal antibody [MAb] 4-5) showed neutralizing activity against SFTSV infection in Vero cells. MAb 4-5 was found to effectively neutralize all of the clinical isolates of SFTSV tested, which were isolated from patients in China from 2010 to 2012. MAb 4-5 was found to bind a linear epitope in the ectodomain of glycoprotein Gn. Its neutralizing activity is attributed to blockage of the interactions between the Gn protein and the cellular receptor, indicating that inhibition of virus-cell attachment is its main mechanism. These data suggest that MAb 4-5 can be used as a promising candidate molecule for immunotherapy against SFTSV infection. PMID:23863504

  18. Meta-analysis of neutralizing antibody conversion with onabotulinumtoxinA (BOTOX®) across multiple indications.

    PubMed

    Naumann, Markus; Carruthers, Alastair; Carruthers, Jean; Aurora, Sheena K; Zafonte, Ross; Abu-Shakra, Susan; Boodhoo, Terry; Miller-Messana, Mary Ann; Demos, George; James, Lynn; Beddingfield, Frederick; VanDenburgh, Amanda; Chapman, Mary Ann; Brin, Mitchell F

    2010-10-15

    This meta-analysis evaluated the frequency of neutralizing antibody (nAb) conversion with onabotulinumtoxinA (BOTOX®; Allergan) across five studied indications. The analysis was based on large, controlled or prospective, open-label trials (durations 4 months to ≥2 years). Serum samples were analyzed for nAbs using the Mouse Protection Assay. Subjects who were antibody negative at baseline and had at least one analyzable postbaseline antibody assay result were included. The 16 clinical studies included 3,006 subjects; of these, 2,240 met the inclusion criteria for this analysis. Subjects received 1-15 treatments (mean 3.8 treatments) with onabotulinumtoxinA. Total doses per treatment cycle ranged from 10 or 20 units in glabellar lines to 20-500 units in cervical dystonia. The numbers of subjects who converted from an antibody-negative status at baseline to antibody-positive status at any post-treatment time point were: cervical dystonia 4/312 (1.28%), glabellar lines 2/718 (0.28%), overactive bladder 0/22 (0%), post-stroke spasticity 1/317 (0.32%), and primary axillary hyperhidrosis 4/871 (0.46%). Across all indications, 11/2,240 subjects (0.49%) converted from antibody negative at baseline to positive at one or more post-treatment time points, but only three subjects became clinically unresponsive to onabotulinumtoxinA at some point following a positive assay. Based on these large trials, the frequency of antibody conversion after onabotulinumtoxinA treatment is very low, and infrequently leads to loss of efficacy. © 2010 Movement Disorder Society.

  19. Hypervariable antigenic region 1 of classical swine fever virus E2 protein impacts antibody neutralization.

    PubMed

    Liao, Xun; Wang, Zuohuan; Cao, Tong; Tong, Chao; Geng, Shichao; Gu, Yuanxing; Zhou, Yingshan; Li, Xiaoliang; Fang, Weihuan

    2016-07-19

    Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies and confers protection against CSFV infection. There are three hypervariable antigenic regions (HAR1, HAR2 and HAR3) of E2 that are different between the group 1 vaccine C-strain and group 2 clinical isolates. This study was aimed to characterize the antigenic epitope region recognized by monoclonal antibody 4F4 (mAb-4F4) that is present in the group 2 field isolate HZ1-08, but not in the C-strain, and examine its impact on neutralization titers when antisera from different recombinant viruses were cross-examined. Indirect ELISA with C-strain E2-based chimeric proteins carrying the three HAR regions showed that the mAb-4F4 bound to HAR1 from HZ1-08 E2, but not to HAR2 or HAR3, indicating that the specific epitope is located in the HAR1 region. Of the 6 major residues differences between C-strain and field isolates, Glu713 in the HAR1 region of strain HZ1-08 is critical for mAb-4F4 binding either at the recombinant protein level or using intact recombinant viruses carrying single mutations. C-strain-based recombinant viruses carrying the most antigenic part of E2 or HAR1 from strain HZ1-08 remained non-pathogenic to pigs and induced good antibody responses. By cross-neutralization assay, we observed that the anti-C-strain serum lost most of its neutralization capacity to RecC-HZ-E2 and QZ-14 (subgroup 2.1d field isolate in 2014), and vice versa. More importantly, the RecC-HAR1 virus remained competent in neutralizing ReC-HZ-E2 and QZ-14 strains without compromising the neutralization capability to the recombinant C-strain. Thus, we propose that chimeric C-strain carrying the HAR1 region of field isolates is a good vaccine candidate for classical swine fever.

  20. Efficient neutralizing activity of cocktailed recombinant human antibodies against hepatitis A virus infection in vitro and in vivo.

    PubMed

    Cao, Jingyuan; Meng, Shufang; Li, Chuan; Ji, Yan; Meng, Qingling; Zhang, Quanfu; Liu, Feng; Li, Jiandong; Bi, Shengli; Li, Dexin; Liang, Mifang

    2008-07-01

    Hepatitis A virus (HAV) is the major pathogen responsible for acute infectious hepatitis A, a disease that is prevalent worldwide. Although HAV immunization effectively prevents infection, primary immunizations must be administered at least 2 weeks prior to HAV exposure. In contrast, passive immunization with pooled human immunoglobulin (Ig) can provide immediate and rapid protection from HAV infection. Because the use of human sera-derived Igs carries the risk of contamination, we sought to develop recombinant HAV-neutralizing human antibodies. We prepared a combinatorial phage display library of recombinant human anti-HAV antibodies from RNA extracted from the blood lymphocytes of a convalescent hepatitis A patient. Two recombinant human IgG antibodies, HAIgG16 and HAIgG78, were screened from the antibody library by their ability to bind with high affinity to purified, inactivated HAV virions. These antibodies recognized different epitopes of the HAV virion capsid, and competed with both patient sera and well-characterized neutralizing mouse monoclonal antibodies. A cocktailed mixture of HAIgG16 and HAIgG78 at a 3:1 ratio was prepared to compare its combined biological activity with that conferred by each antibody individually. The cocktailed antibodies displayed a stronger neutralizing activity in vitro than that observed with either HAIgG16 and HAIgG78 alone. To determine the in vivo neutralizing abilities of these antibodies, rhesus monkeys were inoculated with cocktailed antibodies and challenged with HAV. Whereas control animals developed hepatitis A and seroconverted to the HAV antibody, animals receiving cocktailed antibodies were protected either from viral infection or from developing clinical hepatitis. These results demonstrate that recombinant human antibody preparations could be used to prevent or treat early-stage HAV infection.

  1. Synthetic glycopeptides reveal the glycan specificity of HIV-neutralizing antibodies

    PubMed Central

    Amin, Mohammed N.; McLellan, Jason S.; Huang, Wei; Orwenyo, Jared; Burton, Dennis R.; Koff, Wayne C.; Kwong, Peter D.

    2013-01-01

    A new class of glycan-reactive HIV-neutralizing antibodies, including PG9 and PG16, has been recently discovered that appear to recognize novel glycopeptide epitopes on HIV-1 gp120. However, further characterization and reconstitution of the precise neutralizing epitopes are complicated by the heterogeneity of glycosylation. We report here the design, synthesis, and antigenic evaluation of novel cyclic V1V2 glycopeptides carrying defined N-linked glycans at the conserved glycosylation sites (N160 and N156/N173) derived from gp120 of two HIV-1 isolates. Antibody binding studies confirmed the necessity of a Man5GlcNAc2 glycan at N160 for recognition by PG9 and PG16, and further revealed a critical role of a sialylated N-glycan at the secondary site (N156/N173) in the context of glycopeptides for antibody binding. In addition to defining the glycan specificities of PG9 and PG16, the identified synthetic glycopeptides provide a valuable template for HIV-1 vaccine design. PMID:23831758

  2. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike

    PubMed Central

    Lee, Jeong Hyun; Leaman, Daniel P.; Kim, Arthur S.; Torrents de la Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W.; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R.; Nussenzweig, Michel C.; Poignard, Pascal; Moore, John P.; Klasse, Per Johan; Sanders, Rogier W.; Zwick, Michael B.; Wilson, Ian A.; Ward, Andrew B.

    2015-01-01

    The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120–gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3 Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120–gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes. PMID:26404402

  3. Broadly Neutralizing Antibody 8ANC195 Recognizes Closed and Open States of HIV-1 Env.

    PubMed

    Scharf, Louise; Wang, Haoqing; Gao, Han; Chen, Songye; McDowall, Alasdair W; Bjorkman, Pamela J

    2015-09-10

    The HIV-1 envelope (Env) spike contains limited epitopes for broadly neutralizing antibodies (bNAbs); thus, most neutralizing antibodies are strain specific. The 8ANC195 epitope, defined by crystal and electron microscopy (EM) structures of bNAb 8ANC195 complexed with monomeric gp120 and trimeric Env, respectively, spans the gp120 and gp41 Env subunits. To investigate 8ANC195's gp41 epitope at higher resolution, we solved a 3.58 Å crystal structure of 8ANC195 complexed with fully glycosylated Env trimer, revealing 8ANC195 insertion into a glycan shield gap to contact gp120 and gp41 glycans and protein residues. To determine whether 8ANC195 recognizes the CD4-bound open Env conformation that leads to co-receptor binding and fusion, one of several known conformations of virion-associated Env, we solved EM structures of an Env/CD4/CD4-induced antibody/8ANC195 complex. 8ANC195 binding partially closed the CD4-bound trimer, confirming structural plasticity of Env by revealing a previously unseen conformation. 8ANC195's ability to bind different Env conformations suggests advantages for potential therapeutic applications.

  4. Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

    SciTech Connect

    Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki; Oshita, Masatoshi; Ideno, Shoji; Yunoki, Mikihiro; Kuhara, Motoki; Yamamoto, Naomasa; Okuno, Yoshinobu; Ikuta, Kazuyoshi

    2009-09-11

    Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

  5. Protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of Ebola hemorrhagic fever.

    PubMed

    Marzi, Andrea; Yoshida, Reiko; Miyamoto, Hiroko; Ishijima, Mari; Suzuki, Yasuhiko; Higuchi, Megumi; Matsuyama, Yukie; Igarashi, Manabu; Nakayama, Eri; Kuroda, Makoto; Saijo, Masayuki; Feldmann, Friederike; Brining, Douglas; Feldmann, Heinz; Takada, Ayato

    2012-01-01

    Ebola virus (EBOV) is the causative agent of severe hemorrhagic fever in primates, with human case fatality rates up to 90%. Today, there is neither a licensed vaccine nor a treatment available for Ebola hemorrhagic fever (EHF). Single monoclonal antibodies (MAbs) specific for Zaire ebolavirus (ZEBOV) have been successfully used in passive immunization experiments in rodent models, but have failed to protect nonhuman primates from lethal disease. In this study, we used two clones of human-mouse chimeric MAbs (ch133 and ch226) with strong neutralizing activity against ZEBOV and evaluated their protective potential in a rhesus macaque model of EHF. Reduced viral loads and partial protection were observed in animals given MAbs ch133 and ch226 combined intravenously at 24 hours before and 24 and 72 hours after challenge. MAbs circulated in the blood of a surviving animal until virus-induced IgG responses were detected. In contrast, serum MAb concentrations decreased to undetectable levels at terminal stages of disease in animals that succumbed to infection, indicating substantial consumption of these antibodies due to virus replication. Accordingly, the rapid decrease of serum MAbs was clearly associated with increased viremia in non-survivors. Our results indicate that EBOV neutralizing antibodies, particularly in combination with other therapeutic strategies, might be beneficial in reducing viral loads and prolonging disease progression during EHF.

  6. Neutralizing antibody immune response in children with primary and secondary rotavirus infections.

    PubMed Central

    Arias, C F; López, S; Mascarenhas, J D; Romero, P; Cano, P; Gabbay, Y B; de Freitas, R B; Linhares, A C

    1994-01-01

    We have characterized the neutralizing antibody immune response to six human rotavirus serotypes (G1 to G4, G8, and G9) in Brazilian children with primary and secondary rotavirus infections and correlated the response with the G serotype of the infecting rotavirus strain. Twenty-five children were studied: 17 had a single rotavirus infection, 4 were reinfected once, and 4 experienced three infections. Two of the reinfections were by non-group A rotaviruses. Among the 25 primary infections, we observed homotypic as well as heterotypic responses; the serotype G1 viruses, which accounted for 13 of these infections, induced mostly a homotypic response, while infections by serotype G2 and G4 viruses induced, in addition to the homotypic, a heterotypic response directed primarily to serotype G1. Two of the primary infections induced heterotypic antibodies to 69M, a serotype G8 virus that by RNA electrophoresis analysis was found not to circulate in the population during the time of the study. The specificity of the neutralizing antibody immune response induced by a virus of a given serotype was the same in primary as well as secondary infections. These results indicate that the heterotypic immune response induced in a primary rotavirus infection is an intrinsic property of the virus strain, and although there seem to be general patterns of serotype-specific seroconversion, these may vary from serotype to serotype and from strain to strain within a serotype. PMID:7496929

  7. Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire

    PubMed Central

    Yeung, Yik Andy; Foletti, Davide; Deng, Xiaodi; Abdiche, Yasmina; Strop, Pavel; Glanville, Jacob; Pitts, Steven; Lindquist, Kevin; Sundar, Purnima D.; Sirota, Marina; Hasa-Moreno, Adela; Pham, Amber; Melton Witt, Jody; Ni, Irene; Pons, Jaume; Shelton, David; Rajpal, Arvind; Chaparro-Riggers, Javier

    2016-01-01

    Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition. PMID:27857134

  8. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike.

    PubMed

    Lee, Jeong Hyun; Leaman, Daniel P; Kim, Arthur S; Torrents de la Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R; Nussenzweig, Michel C; Poignard, Pascal; Moore, John P; Klasse, Per Johan; Sanders, Rogier W; Zwick, Michael B; Wilson, Ian A; Ward, Andrew B

    2015-09-25

    The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120-gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3 Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120-gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes.

  9. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike

    NASA Astrophysics Data System (ADS)

    Lee, Jeong Hyun; Leaman, Daniel P.; Kim, Arthur S.; Torrents de La Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W.; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R.; Nussenzweig, Michel C.; Poignard, Pascal; Moore, John P.; Klasse, Per Johan; Sanders, Rogier W.; Zwick, Michael B.; Wilson, Ian A.; Ward, Andrew B.

    2015-09-01

    The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120-gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3 Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120-gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes.

  10. Enhanced humanization and affinity maturation of neutralizing anti-hepatitis B virus preS1 antibody based on antigen-antibody complex structure.

    PubMed

    Kim, Jin Hong; Gripon, Philippe; Bouezzedine, Fidaa; Jeong, Mun Sik; Chi, Seung-Wook; Ryu, Seong-Eon; Hong, Hyo Jeong

    2015-01-16

    To improve a previously constructed broadly neutralizing hepatitis B virus (HBV)-specific preS1 humanized antibody (HzKR127), we further humanized it through specificity-determining residue (SDR) grafting. Moreover, we improved affinity by mutating two residues in heavy-chain complementarity-determining regions (CDR), on the basis of the crystal structure of the antigen-antibody complex. HzKR127-3.2 exhibited 2.5-fold higher affinity and enhanced virus-neutralizing activity compared to the original KR127 antibody and showed less immunogenic potential than HzKR127. Enhanced virus-neutralizing activity was achieved by the increased association rate, providing insights into engineering potent antibody therapeutics for HBV immunoprophylaxis. HzKR127-3.2 may be a good candidate for HBV immunoprophylaxis.

  11. Evaluation of three different formats of a neutralizing single chain human antibody against toxin Cn2: neutralization capacity versus thermodynamic stability.

    PubMed

    Quintero-Hernández, Veronica; Del Pozo-Yauner, Luis; Pedraza-Escalona, Martha; Juárez-González, Victor R; Alcántara-Recillas, Israel; Possani, Lourival D; Becerril, Baltazar

    2012-04-30

    The single-chain antibody fragment (scFv) 6009F, obtained by directed evolution, neutralizes the effects of the Cn2 toxin, which is the major toxic component of Centruroides noxius scorpion venom. In this work we compared the neutralization capacity and the thermodynamic stability of scFv 6009F with those of two other derived formats: Fab 6009F and diabody 6009F. Additionally, the affinity constants to Cn2 toxin of the three recombinant antibody fragments were determined by means of BIAcore. We found a correlation between the thermodynamic stability of these antibody fragments with their neutralization capacity. The order of thermodynamic stability determined was Fab≫scFv>diabody. The Fab and scFv were capable of neutralizing the toxic effects of Cn2 and whole venom but the diabody was unable to fully neutralize intoxication. In silico analysis of the diabody format indicates that the reduction of stability and neutralization capacity could be explained by a less cooperative interface between the heavy and the light variable domains.

  12. Protection in Macaques Immunized with HIV-1 Candidate Vaccines Can Be Predicted Using the Kinetics of Their Neutralizing Antibodies

    PubMed Central

    Davis, David; Koornstra, Wim; Mortier, Daniella; Fagrouch, Zahra; Verschoor, Ernst J.; Heeney, Jonathan L.; Bogers, Willy M. J. M.

    2011-01-01

    Background A vaccine is needed to control the spread of human immunodeficiency virus type 1 (HIV-1). An in vitro assay that can predict the protection induced by a vaccine would facilitate the development of such a vaccine. A potential candidate would be an assay to quantify neutralization of HIV-1. Methods and Findings We have used sera from rhesus macaques that have been immunized with HIV candidate vaccines and subsequently challenged with simian human immunodeficiency virus (SHIV). We compared neutralization assays with different formats. In experiments with the standardized and validated TZMbl assay, neutralizing antibody titers against homologous SHIVSF162P4 pseudovirus gave a variable correlation with reductions in plasma viremia levels. The target cells used in the assays are not just passive indicators of virus infection but are actively involved in the neutralization process. When replicating virus was used with GHOST cell assays, events during the absorption phase, as well as the incubation phase, determine the level of neutralization. Sera that are associated with protection have properties that are closest to the traditional concept of neutralization: the concentration of antibody present during the absorption phase has no effect on the inactivation rate. In GHOST assays, events during the absorption phase may inactivate a fixed number, rather than a proportion, of virus so that while complete neutralization can be obtained, it can only be found at low doses particularly with isolates that are relatively resistant to neutralization. Conclusions Two scenarios have the potential to predict protection by neutralizing antibodies at concentrations that can be induced by vaccination: antibodies that have properties close to the traditional concept of neutralization may protect against a range of challenge doses of neutralization sensitive HIV isolates; a window of opportunity also exists for protection against isolates that are more resistant to

  13. Studies on the role of neutralizing antibodies against envelope genes in resolving HCV pseudo-particles infection.

    PubMed

    Rafique, Shazia; Idrees, Muhammad; Ali, Amjad; Iqbal, Muhammad

    2014-06-01

    Characterization of antibodies targeting the attachment and entry of the viral particles into host cells is important for studding antibody mediated neutralization. Antibodies against the envelope glycoproteins (EGP) have neutralizing capacity and can prevent HCV infections. System based on HCV pseudo typed-particles (HCVpp) stably expressing EGP can be used for screening of HCV anti envelope neutralizing antibodies in the serum of patients with acute and chronic HCV infections. The aim of the current study was to check HCVpp as a useful tool for the detection of anti-HCV envelope antibodies in the serum of HCV infected patients and to test the binding potential of these antiviral molecules to EGP of HCV 3a. Previously developed HCVpp harboring unmodified glycoproteins from local isolates in 293T cell line were used in this study. HCVpp were pre incubated with different concentrations of anti E1 antibody and different E2 antibodies to check antiviral activity. Further we used serum samples with low/medium (≤800,000 IU/mL), and high (>800,000 IU/mL) viral titer from chronic HCV male and female patients. Infection was done in Huh-7 cells for 1 h at 37 oC. Infectivity was checked through Luciferase assay. Considerable decrease in HCVpp infectivity with anti-envelope antibodies was observed in dose dependent manner. Maximum inhibition was seen when 5 µg/ml of monoclonal anti E1 antibody used. Further increase in concentration exhibited no decrease in infectivity which suggests that other factors are also involved in causing infection. Various well characterized E2-specific monoclonal antibodies (mAbs) have been screened for their capability to reduce infection in Huh-7 cells. Three of the four mAbs specific for the E2 had no effect on the infectivity of HCVpp. Confirmation sensitive antibody H53 showed maximum inhibition of infectivity. HCV ELISA positive samples from both male and female patients were used to neutralize the HCVpp. The neutralizing antibody response

  14. Optimal combinations of broadly neutralizing antibodies for prevention and treatments of HIV-1 clade C infection

    DOE PAGES

    Wagh, Kshitij; Bhattacharya, Tanmoy; Williamson, Carolyn; ...

    2016-03-30

    In this study, the identification of a new generation of potent broadly neutralizing HIV-1 antibodies (bnAbs) has generated substantial interest in their potential use for the prevention and/or treatment of HIV-1 infection. While combinations of bnAbs targeting distinct epitopes on the viral envelope (Env) will likely be required to overcome the extraordinary diversity of HIV-1, a key outstanding question is which bnAbs, and how many, will be needed to achieve optimal clinical benefit. We assessed the neutralizing activity of 15 bnAbs targeting four distinct epitopes of Env, including the CD4-binding site (CD4bs), the V1/V2-glycan region, the V3-glycan region, and themore » gp41 membrane proximal external region (MPER), against a panel of 200 acute/early clade C HIV-1 Env pseudoviruses. A mathematical model was developed that predicted neutralization by a subset of experimentally evaluated bnAb combinations with high accuracy. Using this model, we performed a comprehensive and systematic comparison of the predicted neutralizing activity of over 1,600 possible double, triple, and quadruple bnAb combinations. The most promising bnAb combinations were identified based not only on breadth and potency of neutralization, but also other relevant measures, such as the extent of complete neutralization and instantaneous inhibitory potential (IIP). By this set of criteria, triple and quadruple combinations of bnAbs were identified that were significantly more effective than the best double combinations, and further improved the probability of having multiple bnAbs simultaneously active against a given virus, a requirement that may be critical for countering escape in vivo. These results provide a rationale for advancing bnAb combinations with the best in vitro predictors of success into clinical trials for both the prevention and treatment of HIV-1 infection.« less

  15. Optimal Combinations of Broadly Neutralizing Antibodies for Prevention and Treatment of HIV-1 Clade C Infection.

    PubMed

    Wagh, Kshitij; Bhattacharya, Tanmoy; Williamson, Carolyn; Robles, Alex; Bayne, Madeleine; Garrity, Jetta; Rist, Michael; Rademeyer, Cecilia; Yoon, Hyejin; Lapedes, Alan; Gao, Hongmei; Greene, Kelli; Louder, Mark K; Kong, Rui; Karim, Salim Abdool; Burton, Dennis R; Barouch, Dan H; Nussenzweig, Michel C; Mascola, John R; Morris, Lynn; Montefiori, David C; Korber, Bette; Seaman, Michael S

    2016-03-01

    The identification of a new generation of potent broadly neutralizing HIV-1 antibodies (bnAbs) has generated substantial interest in their potential use for the prevention and/or treatment of HIV-1 infection. While combinations of bnAbs targeting distinct epitopes on the viral envelope (Env) will likely be required to overcome the extraordinary diversity of HIV-1, a key outstanding question is which bnAbs, and how many, will be needed to achieve optimal clinical benefit. We assessed the neutralizing activity of 15 bnAbs targeting four distinct epitopes of Env, including the CD4-binding site (CD4bs), the V1/V2-glycan region, the V3-glycan region, and the gp41 membrane proximal external region (MPER), against a panel of 200 acute/early clade C HIV-1 Env pseudoviruses. A mathematical model was developed that predicted neutralization by a subset of experimentally evaluated bnAb combinations with high accuracy. Using this model, we performed a comprehensive and systematic comparison of the predicted neutralizing activity of over 1,600 possible double, triple, and quadruple bnAb combinations. The most promising bnAb combinations were identified based not only on breadth and potency of neutralization, but also other relevant measures, such as the extent of complete neutralization and instantaneous inhibitory potential (IIP). By this set of criteria, triple and quadruple combinations of bnAbs were identified that were significantly more effective than the best double combinations, and further improved the probability of having multiple bnAbs simultaneously active against a given virus, a requirement that may be critical for countering escape in vivo. These results provide a rationale for advancing bnAb combinations with the best in vitro predictors of success into clinical trials for both the prevention and treatment of HIV-1 infection.

  16. Optimal combinations of broadly neutralizing antibodies for prevention and treatments of HIV-1 clade C infection

    SciTech Connect

    Wagh, Kshitij; Bhattacharya, Tanmoy; Williamson, Carolyn; Robles, Alex; Bayne, Madeleine; Garrity, Jetta; Rist, Michael; Rademeyer, Cecilia; Yoon, Hyejin; Lapedes, Alan Scott; Gao, Hongmei; Greene, Kelli; Louder, Mark K.; Kong, Rui; Karim, Salim Abdool; Burton, Dennis R.; Barouch, Dan H.; Nussenzweig, Michael C.; Mascola, John R.; Morris, Lynn; Montefiori, David; Korber, Bette Tina; Seamon, Michael S.

    2016-03-30

    In this study, the identification of a new generation of potent broadly neutralizing HIV-1 antibodies (bnAbs) has generated substantial interest in their potential use for the prevention and/or treatment of HIV-1 infection. While combinations of bnAbs targeting distinct epitopes on the viral envelope (Env) will likely be required to overcome the extraordinary diversity of HIV-1, a key outstanding question is which bnAbs, and how many, will be needed to achieve optimal clinical benefit. We assessed the neutralizing activity of 15 bnAbs targeting four distinct epitopes of Env, including the CD4-binding site (CD4bs), the V1/V2-glycan region, the V3-glycan region, and the gp41 membrane proximal external region (MPER), against a panel of 200 acute/early clade C HIV-1 Env pseudoviruses. A mathematical model was developed that predicted neutralization by a subset of experimentally evaluated bnAb combinations with high accuracy. Using this model, we performed a comprehensive and systematic comparison of the predicted neutralizing activity of over 1,600 possible double, triple, and quadruple bnAb combinations. The most promising bnAb combinations were identified based not only on breadth and potency of neutralization, but also other relevant measures, such as the extent of complete neutralization and instantaneous inhibitory potential (IIP). By this set of criteria, triple and quadruple combinations of bnAbs were identified that were significantly more effective than the best double combinations, and further improved the probability of having multiple bnAbs simultaneously active against a given virus, a requirement that may be critical for countering escape in vivo. These results provide a rationale for advancing bnAb combinations with the best in vitro predictors of success into clinical trials for both the prevention and treatment of HIV-1 infection.

  17. Pharmacokinetics and immunogenicity of broadly neutralizing HIV monoclonal antibodies in macaques.

    PubMed

    Rosenberg, Yvonne; Sack, Markus; Montefiori, David; Labranche, Celia; Lewis, Mark; Urban, Lori; Mao, Lingjun; Fischer, Rainer; Jiang, Xiaoming

    2015-01-01

    The identification of highly potent broadly neutralizing antibodies (bnAbs) against HIV-1, and success in preventing SHIV infection following their passive administration, have increased the likelihood that immunotherapeutic strategies can be adopted to prevent and treat HIV-1 infection. However, while broad and potent neutralizing activity is an essential prerequisite, in vivo properties such as good circulatory stability and non-immunogenicity are equally critical for developing a human treatment. In the present study, glycoforms of the bnAbs 10-1074, NIH45-46G54W, 10E8, PGT121, PGT128, PGT145, PGT135, PG9, PG16, VRC01 and b12 were produced by Agrobacterium-mediated transient transfection of Nicotiana benthamiana and assessed following administration in rhesus macaques. The results indicate that (i) N-glycans within the VL domain impair plasma stability of plant-derived bnAbs and (ii) while PGT121 and b12 exhibit no immunogenicity in rhesus macaques after multiple injections, VRC01, 10-1074 and NIH45-46G54W elicit high titer anti-idiotypic antibodies following a second injection. These anti-idiotypic antibodies specifically bind the administered bnAb or a close family member, and inhibit the bnAb in neutralization assays. These findings suggest that specific mutations in certain bnAbs contribute to their immunogenicity and call attention to the prospect that these mutated bnAbs will be immunogenic in humans, potentially compromising their value for prophylaxis and therapy of HIV-1.

  18. Recognition of influenza H3N2 variant virus by human neutralizing antibodies

    PubMed Central

    Bangaru, Sandhya; Nieusma, Travis; Kose, Nurgun; Thornburg, Natalie J.; Kaplan, Bryan S.; King, Hannah G.; Singh, Vidisha; Lampley, Rebecca M.; Cisneros, Alberto; Edwards, Kathryn M.; Edupuganti, Srilatha; Lai, Lilin; Richt, Juergen A.; Webby, Richard J.; Ward, Andrew B.; Crowe, James E.

    2016-01-01

    Since 2011, over 300 human cases of infection, especially in exposed children, with the influenza A H3N2 variant (H3N2v) virus that circulates in swine in the US have been reported. The structural and genetic basis for the lack of protection against H3N2v induced by vaccines containing seasonal H3N2 antigens is poorly understood. We isolated 17 human monoclonal antibodies (mAbs) that neutralized H3N2v virus from subjects experimentally immunized with an H3N2v candidate vaccine. Six mAbs exhibited very potent neutralizing activity (IC50 < 200 ng/ml) against the H3N2v virus but not against current human H3N2 circulating strains. Fine epitope mapping and structural characterization of antigen-antibody complexes revealed that H3N2v specificity was attributable to amino acid polymorphisms in the 150-loop and the 190-helix antigenic sites on the hemagglutinin protein. H3N2v-specific antibodies also neutralized human H3N2 influenza strains naturally circulating between 1995 and 2005. These results reveal a high level of antigenic relatedness between the swine H3N2v virus and previously circulating human strains, consistent with the fact that early human H3 seasonal strains entered the porcine population in the 1990s and reentered the human population, where they had not been circulating, as H3N2v about a decade later. The data also explain the increased susceptibility to H3N2v viruses in young children, who lack prior exposure to human seasonal strains from the 1990s. PMID:27482543

  19. Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to Guide Reductionist Vaccine Design

    PubMed Central

    Sarkar, Anita; Liang, Chi-Hui; Scherer, Erin A.; Henry Dunand, Carole J.; Adachi, Yumiko; Diwanji, Devan; Jones, Meaghan; Kalyuzhniy, Oleksandr; Kubitz, Michael; Spencer, Skye; Pauthner, Matthias; Saye-Francisco, Karen L.; Sesterhenn, Fabian; Wilson, Patrick C.; Galloway, Denise M.; Stanfield, Robyn L.; Wilson, Ian A.; Burton, Dennis R.; Schief, William R.

    2016-01-01

    An optimal HIV vaccine should induce broadly neutralizing antibodies (bnAbs) that neutralize diverse viral strains and subtypes. However, potent bnAbs develop in only a small fraction of HIV-infected individuals, all contain rare features such as extensive mutation, insertions, deletions, and/or long complementarity-determining regions, and some are polyreactive, casting doubt on whether bnAbs to HIV can be reliably induced by vaccination. We engineered two potent VRC01-class bnAbs that minimized rare features. According to a quantitative features frequency analysis, the set of features for one of these minimally mutated bnAbs compared favorably with all 68 HIV bnAbs analyzed and was similar to antibodies elicited by common vaccines. This same minimally mutated bnAb lacked polyreactivity in four different assays. We then divided the minimal mutations into spatial clusters and dissected the epitope components interacting with those clusters, by mutational and crystallographic analyses coupled with neutralization assays. Finally, by synthesizing available data, we developed a working-concept boosting strategy to select the mutation clusters in a logical order following a germline-targeting prime. We have thus developed potent HIV bnAbs that may be more tractable vaccine goals compared to existing bnAbs, and we have proposed a strategy to elicit them. This reductionist approach to vaccine design, guided by antibody and antigen structure, could be applied to design candidate vaccines for other HIV bnAbs or protective Abs against other pathogens. PMID:27560183

  20. Characterization of the glycoprotein of infectious hematopoietic necrosis virus using neutralizing monoclonal antibodies

    USGS Publications Warehouse

    Huang, Chienjin; Chien, Maw-Sheng; Landolt, Marsha; Winton, James

    1994-01-01

    To study the antigenic nature of the glycoprotein (G protein) of infectious hematopoietic necrosis virus (IHNV), 31 neutralizing monoclonal antibodies (MAbs) were produced against a reference isolate of the virus. The MAbs were compared using a neutralization assay, an enzyme-linked immunosorbent assay (ELISA), and by immunoblotting of the G protein in the native, reduced, and deglycosylated forms. Hybridoma culture fluids of the various MAbs could be diluted from 1:2 to 1:512 and still completely neutralize 1 X 104 plaque-forming units of IHNV. Similarly, the end point dilutions that produced optical density readings of 0.1 or greater in the ELISA were 1:40 to 1:10240. Western blotting showed that all of the MAbs reacted with the G protein in the unreduced (i.e. native) conformation; however, only 9 nine of the MAbs were able to react with the G protein following reduction by 2-mercaptoethanol. Deglycosylation of the protein did not influence the binding ability of any of the MAbs. These data indicate that all the MAbs recognized amino acid sequences on the protein itself and that the IHNV glycoprotein contains linear as well as conformation-dependent neutralizing epitopes. When rainbow trout Oncorhynchus mykiss fingerlings were passively immunized with MAbs against either a linear or a conformation-dependent epitope, the fish were protected against challenge with wild-type IHNV.

  1. Eliciting Neutralizing Antibodies with gp120 Outer Domain Constructs Based on M-Group Consensus Sequence

    PubMed Central

    Qin, Yali; Banasik, Marisa; Kim, SoonJeung; Penn-Nicholson, Adam; Habte, Habtom H; Labranche, Celia; Montefiori, David C; Wang, Chong; Cho, Michael W

    2014-01-01

    One strategy being evaluated for HIV-1 vaccine development is focusing immune responses towards neutralizing epitopes on the gp120 outer domain (OD) by removing the immunodominant, but non-neutralizing, inner domain. Previous OD constructs have not elicited strong neutralizing antibodies (nAbs). We constructed two immunogens, a monomeric gp120-OD and a trimeric gp120-OD×3, based on an M group consensus sequence (MCON6). Their biochemical and immunological properties were compared with intact gp120. Results indicated better preservation of critical neutralizing epitopes on gp120-OD×3. In contrast to previous studies, our immunogens induced potent, cross-reactive nAbs in rabbits. Although nAbs primarily targeted Tier 1 viruses, they exhibited significant breadth. Epitope mapping analyses indicated that nAbs primarily targeted conserved V3 loop elements. Although the potency and breadth of nAbs were similar for all three immunogens, nAb induction kinetics indicated that gp120-OD×3 was superior to gp120-OD, suggesting that gp120-OD×3 is a promising prototype for further gp120 OD-based immunogen development. PMID:25046154

  2. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits.

    PubMed

    Robinson, James E; Hastie, Kathryn M; Cross, Robert W; Yenni, Rachael E; Elliott, Deborah H; Rouelle, Julie A; Kannadka, Chandrika B; Smira, Ashley A; Garry, Courtney E; Bradley, Benjamin T; Yu, Haini; Shaffer, Jeffrey G; Boisen, Matt L; Hartnett, Jessica N; Zandonatti, Michelle A; Rowland, Megan M; Heinrich, Megan L; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C; Andersen, Kristian G; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J; Fonnie, Richard; Jalloh, Simbirie C; Kargbo, Brima; Vandi, Mohamed A; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A; Okokhere, Peter O; Follarin, Onikepe A; Schieffelin, John S; Pitts, Kelly R; Geisbert, Joan B; Kulakoski, Peter C; Wilson, Russell B; Happi, Christian T; Sabeti, Pardis C; Gevao, Sahr M; Khan, S Humarr; Grant, Donald S; Geisbert, Thomas W; Saphire, Erica Ollmann; Branco, Luis M; Garry, Robert F

    2016-05-10

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design.

  3. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

    PubMed Central

    Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  4. Structure-guided alterations of the gp41-directed HIV-1 broadly neutralizing antibody 2F5 reveal new properties regarding its neutralizing function.

    PubMed

    Guenaga, Javier; Wyatt, Richard T

    2012-01-01

    The broadly neutralizing HIV-1 antibody 2F5 recognizes an epitope in the gp41 membrane proximal external region (MPER). The MPER adopts a helical conformation as free peptide, as post-fusogenic forms of gp41, and when bound to the 4E10 monoclonal antibody (Mab). However, when bound to 2F5, the epitope is an extended-loop. The antibody-peptide structure reveals binding between the heavy and light chains with most the long, hydrophobic CDRH3 not contacting peptide. However, mutagenesis identifies this loop as critical for binding, neutralization and for putative hydrophobic membrane interactions. Here, we examined length requirements of the 2F5 CDRH3 and plasticity regarding binding and neutralization. We generated 2F5 variants possessing either longer or shorter CDRH3s and assessed function. The CDRH3 tolerated elongations and reductions up to four residues, displaying a range of binding affinities and retaining some neutralizing capacity. 2F5 antibody variants selective recognition of conformationally distinctive MPER probes suggests a new role for the CDRH3 loop in destabilizing the helical MPER. Binding and neutralization were enhanced by targeted tryptophan substitutions recapitulating fully the activities of the wild-type 2F5 antibody in a shorter CDRH3 variant. MPER alanine scanning revealed binding contacts of this variant downstream of the 2F5 core epitope, into the 4E10 epitope region. This variant displayed increased reactivity to cardiolipin-beta-2-glycoprotein. Tyrosine replacements maintained neutralization while eliminating cardiolipin-beta-2-glycoprotein interaction. The data suggest a new mechanism of action, important for vaccine design, in which the 2F5 CDRH3 contacts and destabilizes the MPER helix downstream of its core epitope to allow induction of the extended-loop conformation.

  5. Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins

    SciTech Connect

    Lok, Shee-Mei; Kostyuchenko, Victor; Nybakken, Grant E.; Holdaway, Heather A.; Battisti, Anthony J.; Sukupolvi-Petty, Soila; Sedlak, Dagmar; Fremont, Daved H.; Chipman, Paul R.; Roehrig, John T.; Diamond, Michael S.; Kuhn, Richard J.; Rossmann, Michael G.

    2008-04-02

    The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 degrees C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization of the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.

  6. Structural and Molecular Basis for Ebola Virus Neutralization by Protective Human Antibodies

    PubMed Central

    Misasi, John; Gilman, Morgan S.A.; Kanekiyo, Masaru; Gui, Miao; Cagigi, Alberto; Mulangu, Sabue; Corti, Davide; Ledgerwood, Julie E.; Lanzavecchia, Antonio; Cunningham, James; Muyembe-Tamfun, Jean Jacques; Baxa, Ulrich; Graham, Barney S.; Xiang, Ye; Sullivan, Nancy J.; McLellan, Jason S.

    2017-01-01

    Ebola virus causes hemorrhagic fever with a high mortality rate and for which there is no approved therapy. Two human monoclonal antibodies, mAb100 and mAb114, in combination protect non-human primates against all signs of Ebola virus disease, including viremia. Here, we demonstrate that mAb100 recognizes the base of the Ebola virus glycoprotein (GP) trimer, occludes access to the cathepsin-cleavage loop, and prevents the proteolytic cleavage of GP that is required for virus entry. We show that mAb114 interacts with the glycan cap and inner chalice of GP, remains associated following proteolytic removal of the glycan cap, and inhibits binding of cleaved GP to its receptor. These results define the basis of neutralization for two protective antibodies and may facilitate development of therapies and vaccines. PMID:26917592

  7. Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies

    PubMed Central

    Andersen, Kasper Krogh; Strokappe, Nika M.; Hultberg, Anna; Truusalu, Kai; Smidt, Imbi; Mikelsaar, Raik-Hiio; Mikelsaar, Marika; Verrips, Theo; Hammarström, Lennart

    2015-01-01

    Clostridium difficile is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of C. difficile are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered Lactobacillus strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB in vitro were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in Lactobacillus paracasei BL23, they were able to neutralize the cytotoxic effect of the toxin in an in vitro cell-based assay. Prophylactic treatment with a combination of two strains of engineered L. paracasei BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA− TcdB+ strain of C. difficile (P < 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with C. difficile for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients. PMID:26573738

  8. Evaluating the Synergistic Neutralizing Effect of Anti-Botulinum Oligoclonal Antibody Preparations

    PubMed Central

    Diamant, Eran; Lachmi, Bat-El; Keren, Adi; Barnea, Ada; Marcus, Hadar; Cohen, Shoshana; David, Alon Ben; Zichel, Ran

    2014-01-01

    Botulinum neurotoxins (BoNT) are considered some of the most lethal known substances. There are seven botulinum serotypes, of which types A, B and E cause most human botulism cases. Anti-botulinum polyclonal antibodies (PAbs) are currently used for both detection and treatment of the disease. However, significant improvements in immunoassay specificity and treatment safety may be made using monoclonal antibodies (MAbs). In this study, we present an approach for the simultaneous generation of highly specific and neutralizing MAbs against botulinum serotypes A, B, and E in a single process. The approach relies on immunization of mice with a trivalent mixture of recombinant C-terminal fragment (Hc) of each of the three neurotoxins, followed by a parallel differential robotic hybridoma screening. This strategy enabled the cloning of seven to nine MAbs against each serotype. The majority of the MAbs possessed higher anti-botulinum ELISA titers than anti-botulinum PAbs and had up to five orders of magnitude greater specificity. When tested for their potency in mice, neutralizing MAbs were obtained for all three serotypes and protected against toxin doses of 10 MsLD50–500 MsLD50. A strong synergistic effect of up to 400-fold enhancement in the neutralizing activity was observed when serotype-specific MAbs were combined. Furthermore, the highly protective oligoclonal combinations were as potent as a horse-derived PAb pharmaceutical preparation. Interestingly, MAbs that failed to demonstrate individual neutralizing activity were observed to make a significant contribution to the synergistic effect in the oligoclonal preparation. Together, the trivalent immunization strategy and differential screening approach enabled us to generate highly specific MAbs against each of the A, B, and E BoNTs. These new MAbs may possess diagnostic and therapeutic potential. PMID:24475231

  9. Antiviral Monoclonal Antibodies: Can They Be More Than Simple Neutralizing Agents?

    PubMed

    Pelegrin, Mireia; Naranjo-Gomez, Mar; Piechaczyk, Marc

    2015-10-01

    Monoclonal antibodies (mAbs) are increasingly being considered as agents to fight severe viral diseases. So far, they have essentially been selected and used on the basis of their virus-neutralizing activity and/or cell-killing activity to blunt viral propagation via direct mechanisms. There is, however, accumulating evidence that they can also induce long-lasting protective antiviral immunity by recruiting the endogenous immune system of infected individuals during the period of immunotherapy. Exploiting this property may revolutionize antiviral mAb-based immunotherapies, with benefits for both patients and healthcare systems.

  10. The amino acid residues at 102 and 104 in GP5 of porcine reproductive and respiratory syndrome virus regulate viral neutralization susceptibility to the porcine serum neutralizing antibody.

    PubMed

    Fan, Baochao; Liu, Xing; Bai, Juan; Zhang, Tingjie; Zhang, Qiaoya; Jiang, Ping

    2015-06-02

    Porcine reproductive and respiratory syndrome virus (PRRSV) is mainly responsible for the heavy economic losses in pig industry in the world. A number of neutralizing epitopes have been identified in the viral structural proteins GP3, GP4, GP5 and M. In this study, the important amino acid (aa) residues of HP-PRRSV strain BB affecting neutralization susceptibility of antibody were examined using resistant strains generated under neutralizing antibody (NAb) pressure in MARC-145 cells, reverse genetic technique and virus neutralization assay. HP-PRRSV strain BB was passaged under the pressure of porcine NAb serum in vitro. A resistant strain BB34s with 102 and 104 aa substitutions in GP5, which have been predicted to be the positive sites for pressure selection (Delisle et al., 2012), was cloned and identified. To determine the effect of the two aa residues on neutralization, eight recombinant PRRSV strains were generated, and neutralization assay results confirmed that the aa residues 102 and 104 in GP5 played an important role in NAbs against HP-PRRSV in MARC-145 cells and porcine alveolar macrophages. Alignment of GP5 sequences revealed that the variant aa residues at 102 and 104 were frequent among type 2 PRRSV strains. It may be helpful for understanding the mechanism regulating the neutralization susceptibility of PRRSV to the NAbs and monitoring the antigen variant strains in the field.

  11. The heptide repeat 2 and upstream region of TGEV induces potent cross-neutralizing antibodies against group I coronaviruses.

    PubMed

    Shi, Huiling; Wu, Nannan; Wang, Xiaoming; Wang, Tianhou

    2012-10-01

    The coronavirus heptide repeat (HR) region in the spike protein induces neutralizing antibodies that block the postfusion core formation and inhibit virus entry into target cells. The HR2 regions for coronaviruses of the same serogroup share high homology. We found that polyclonal antibodies derived from transmissible gastroenteritis coronavirus HR2 and upstream region were cross-reactive with the S proteins of the same serogroup in western blotting. The polyclonal antibodies also potently cross-neutralized viruses from the same serogroup. This study provides new insight for designing vaccine and therapeutic reagents against coronavirus infections.

  12. Impact of Clade, Geography, and Age of the Epidemic on HIV-1 Neutralization by Antibodies

    PubMed Central

    Hraber, Peter; Korber, Bette T.; Lapedes, Alan S.; Bailer, Robert T.; Seaman, Michael S.; Gao, Hongmei; Greene, Kelli M.; McCutchan, Francine; Williamson, Carolyn; Kim, Jerome H.; Tovanabutra, Sodsai; Hahn, Beatrice H.; Swanstrom, Ronald; Thomson, Michael M.; Gao, Feng; Harris, Linda; Giorgi, Elena; Hengartner, Nicholas; Bhattacharya, Tanmoy; Mascola, John R.

    2014-01-01

    ABSTRACT Neutralizing antibodies (nAbs) are a high priority for vaccines that aim to prevent the acquisition of HIV-1 infection. Vaccine effectiveness will depend on the extent to which induced antibodies neutralize the global diversity of circulating HIV-1 variants. Using large panels of genetically and geographically diverse HIV-1 Env-pseudotyped viruses and chronic infection plasma samples, we unambiguously show that cross-clade nAb responses are commonly induced in response to infection by any virus clade. Nonetheless, neutralization was significantly greater when the plasma clade matched the clade of the virus being tested. This within-clade advantage was diminished in older, more-diverse epidemics in southern Africa, the United States, and Europe compared to more recent epidemics in Asia. It was most pronounced for circulating recombinant form (CRF) 07_BC, which is common in China and is the least-divergent lineage studied; this was followed by the slightly more diverse Asian CRF01_AE. We found no evidence that transmitted/founder viruses are generally more susceptible to neutralization and are therefore easier targets for vaccination than chronic viruses. Features of the gp120 V1V2 loop, in particular, length, net charge, and number of N-linked glycans, were associated with Env susceptibility and plasma neutralization potency in a manner consistent with neutralization escape being a force that drives viral diversification and plasma neutralization breadth. The overall susceptibility of Envs and potencies of plasma samples were highly predictive of the neutralization outcome of any single virus-plasma combination. These findings highlight important considerations for the design and testing of candidate HIV-1 vaccines that aim to elicit effective nAbs. IMPORTANCE An effective HIV-1 vaccine will need to overcome the extraordinary variability of the virus, which is most pronounced in the envelope glycoproteins (Env), which are the sole targets for neutralizing

  13. Neutralizing antibodies obtained in a persistent immune response are effective against deleterious effects induced by the Thalassophryne nattereri fish venom.

    PubMed

    Piran-Soares, Ana Amélia; Komegae, Evilin Naname; Souza, Valdênia Maria Oliveira; Fonseca, Luiz Alberto; Lima, Carla; Lopes-Ferreira, Mônica

    2007-06-01

    Thalassophryne nattereri envenoming represents a great cost to North and Northeast Brazilian communities in terms of public healths, leisure and tourism. Victims rapidally develop symptoms as pain, local swelling, erythema followed by intense necrosis that persist for long days. The aim of this work was tested the immune competence of neutralizing antibodies in pre-immunized mice against principal toxic activities induced by venom. During the primary antibody response in mice, an elevation of IgG antibody levels was only observed on day 28. After boosting, high antibody levels were detected between days 49 and 70, with a 12-fold increase in IgG level over control values at day 49. We confirmed the in vitro neutralizing capacity of T. nattereri anti-venom against toxic effects and thereafter we show that neutralizing antibodies obtained in a persistent immune response are more effective, inclusive against edematous reaction. After boosting during the secondary response mice with high antibody levels do not present any alterations in venule or arteriole after topical application of venom on cremaster muscle. In addition, CK activity diminished in these mice with high neutralizing antibody levels corroborating the attenuation of the myonecrotic effect by venom. In addition, we determined the presence of high IgG antibodies levels in patients 6 months after injury by T. nattereri. In conclusion, the presence of neutralizing antibodies against to T. nattereri venom in the serum of pre-immunized mice could change the outcome of lesion at site of posterior envenoming. Antigen-specific antibodies of high affinity in consequence to specific immune response, dependent of T lymphocyte activation, could minimize the symptoms of intense and immediate inflammatory reaction caused by T. nattereri venom. These finding prompt us to the possibility of development of immune therapeutic strategies using specific anti-venom as an efficient intervention for protecting human victims.

  14. Atypical and classical memory B cells produce Plasmodium falciparum neutralizing antibodies

    PubMed Central

    Muellenbeck, Matthias F.; Ueberheide, Beatrix; Amulic, Borko; Epp, Alexandra; Fenyo, David; Busse, Christian E.; Esen, Meral; Theisen, Michael; Mordmüller, Benjamin

    2013-01-01

    Antibodies can protect from Plasmodium falciparum (Pf) infection and clinical malaria disease. However, in the absence of constant reexposure, serum immunoglobulin (Ig) levels rapidly decline and full protection from clinical symptoms is lost, suggesting that B cell memory is functionally impaired. We show at the single cell level that natural Pf infection induces the development of classical memory B cells (CM) and atypical memory B cells (AtM) that produce broadly neutralizing antibodies against blood stage Pf parasites. CM and AtM contribute to anti-Pf serum IgG production, but only AtM show signs of active antibody secretion. AtM and CM were also different in their IgG gene repertoire, suggesting that they develop from different precursors. The findings provide direct evidence that natural Pf infection leads to the development of protective memory B cell antibody responses and suggest that constant immune activation rather than impaired memory function leads to the accumulation of AtM in malaria. Understanding the memory B cell response to natural Pf infection may be key to the development of a malaria vaccine that induces long-lived protection. PMID:23319701

  15. Early Low-Titer Neutralizing Antibodies Impede HIV-1 Replication and Select for Virus Escape

    PubMed Central

    Bar, Katharine J.; Tsao, Chun-yen; Iyer, Shilpa S.; Decker, Julie M.; Yang, Yongping; Bonsignori, Mattia; Chen, Xi; Hwang, Kwan-Ki; Montefiori, David C.; Liao, Hua-Xin; Hraber, Peter; Fischer, William; Li, Hui; Wang, Shuyi; Sterrett, Sarah; Keele, Brandon F.; Ganusov, Vitaly V.; Perelson, Alan S.; Korber, Bette T.; Georgiev, Ivelin; McLellan, Jason S.; Pavlicek, Jeffrey W.; Gao, Feng; Haynes, Barton F.; Hahn, Beatrice H.; Kwong, Peter D.; Shaw, George M.

    2012-01-01

    Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses in vivo. By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab) responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC50) selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1–V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically

  16. Neutralizing human recombinant antibodies against herpes simplex virus type 1 glycoproteins B from a phage-displayed scFv antibody library.

    PubMed

    Bagheri, Vahid; Nejatollahi, Foroogh; Esmaeili, Seyed Alireza; Momtazi, Amir Abbas; Motamedifar, Mohamad; Sahebkar, Amirhossein

    2017-01-15

    The HSV-1 envelope glycoprotein B (gB) plays a critical role in virus entry into host cells. Neutralizing antibodies can therefore potentially prevent virus entry into target cells and cell-to-cell spread of infection. Our present study focused on the selection of neutralizing single-chain Fv (scFv) antibodies of a phage-displayed nonimmune human scFv antibody library against gB of HSV-1. To enrich specific scFvs, two phage antibodies were isolated against amino acid residues 31-43 derived from the N-terminal part of gB using panning technique. Two scFvs, scFv-gB1 and scFv-gB2, with frequencies of 45% and 20% were obtained from scFv clones after performing PCR and MvaI fingerprinting. In phage ELISA analysis, both gB1 and gB2 scFvs demonstrated high reactivity with the gB peptide. In the neutralization assay, scFv-gB1 and scFv-gB2 represented neutralizing effects of 55% and 59%, respectively. Upon further enhancement of the neutralizing effects of these antibodies, they can be considered as new potential alternatives in the treatment and prophylaxis of HSV-1 infections.

  17. Passive antibody therapy of Lassa fever in cynomolgus monkeys: importance of neutralizing antibody and Lassa virus strain.

    PubMed

    Jahrling, P B; Peters, C J

    1984-05-01

    Lassa virus-infected cynomolgus monkeys were passively immunized with immune plasma of primate or human origin to gain insight into criteria for plasma selection and administration to human Lassa fever patients. Protective efficacy was correlated with neutralizing antibody concentrations, expressed as a log10 neutralization index (LNI). Convalescent Lassa-immune monkey plasma was titrated for protective efficacy in monkeys by intravenous inoculation with dilutions of plasma on the day of subcutaneous Lassa virus inoculation (day 0) and again on days 3 and 6. Monkeys that received undiluted plasma (LNI = 4.1) (1 ml/kg per treatment) survived a lethal viral dose, whereas those given a 1:3 dilution (LNI = 2.6) of this same plasma (1 ml/kg per treatment) died. Protection was restored when the volume of the 1:3 plasma dilution was increased to 3 ml/kg per treatment. Plasma diluted 1:9 or more (LNI = 1.5 or less) delayed onset and suppressed the magnitude of viremia but failed to confer protection at 3 ml/kg per treatment. Immunological enhancement, defined as increased viremia or accelerated death, did not occur following inadequate treatment. Human convalescent plasma also protected recipient monkeys; reductions in mortality and viremia were accurately predicted by the LNI of the plasma. Plasma of Liberian origin neutralized a Liberian Lassa strain more effectively than a Sierra Leone strain in vitro (LNI = 2.8 and 1.6, respectively) and protected monkeys more effectively against the Liberian strain. Geographic origin is thus a factor in the selection of optimal plasma for treatment of human Lassa fever, since geographically matched plasma is more likely to contain adequate LNI titers against homologous Lassa virus strains.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Global Shape and Ligand Binding Efficiency of the HIV-1-neutralizing Antibodies Differ from Those of Antibodies That Cannot Neutralize HIV-1

    SciTech Connect

    Solanki, Ashish K.; Rathore, Yogendra S.; Badmalia, Maulik D.; Dhoke, Reema R.; Nath, Samir K.; Nihalani, Deepak

    2014-10-20

    Asymmetric disposition of Fab arms in the structures solved for the broadly neutralizing monoclonal antibody (nmAb) IgG1 b12 raised the question of whether the unusual shape observed for b12 is common for all IgG1 mAbs or if there is a difference in the overall shape of nmAbs versus non-nmAbs. In this paper, we compared small angle x-ray scattering (SAXS) data-based models and limited proteolysis profiles of some IgG1 mAbs known to be having and lacking HIV-1 neutralizing potency. In non-nmAbs, the Fab arms were found to be symmetrically disposed in space relative to central Fc, but in most nmAbs, the Fab arms were asymmetrically disposed, as seen for IgG1 b12. The only exceptions were 2G12 and 4E10, where both Fab arms were closed above Fc, suggesting some Fab-Fc and/or Fab-Fab interaction in the nmAbs that constrained extension of the Fab-Fc linker. Interestingly, these observations were correlated with differential proteolysis profiles of the mAbs by papain. Under conditions when papain could cut both Fab arms of non-nmAbs, only one Fab arm could be removed from neutralizing ones (except for 2G12 and 4E10). Chromatography and small angle x-ray scattering results of papain-digested products revealed that 1) the Fab-Fc or Fab-Fab interactions in unliganded mAbs are retained in digested products, and 2) whereas anti-gp120 non-nmAbs could bind two gp120 molecules, nmAbs could bind only one gp120. Finally, additional experiments showed that except for 2G12 and 4E10, unopen shapes of nmAbs remain uninfluenced by ionic strength but can be reversibly opened by low pH of buffer accompanied by loss of ligand binding ability.

  19. Global Shape and Ligand Binding Efficiency of the HIV-1-neutralizing Antibodies Differ from Those of Antibodies That Cannot Neutralize HIV-1

    DOE PAGES

    Solanki, Ashish K.; Rathore, Yogendra S.; Badmalia, Maulik D.; ...

    2014-10-20

    Asymmetric disposition of Fab arms in the structures solved for the broadly neutralizing monoclonal antibody (nmAb) IgG1 b12 raised the question of whether the unusual shape observed for b12 is common for all IgG1 mAbs or if there is a difference in the overall shape of nmAbs versus non-nmAbs. In this paper, we compared small angle x-ray scattering (SAXS) data-based models and limited proteolysis profiles of some IgG1 mAbs known to be having and lacking HIV-1 neutralizing potency. In non-nmAbs, the Fab arms were found to be symmetrically disposed in space relative to central Fc, but in most nmAbs, themore » Fab arms were asymmetrically disposed, as seen for IgG1 b12. The only exceptions were 2G12 and 4E10, where both Fab arms were closed above Fc, suggesting some Fab-Fc and/or Fab-Fab interaction in the nmAbs that constrained extension of the Fab-Fc linker. Interestingly, these observations were correlated with differential proteolysis profiles of the mAbs by papain. Under conditions when papain could cut both Fab arms of non-nmAbs, only one Fab arm could be removed from neutralizing ones (except for 2G12 and 4E10). Chromatography and small angle x-ray scattering results of papain-digested products revealed that 1) the Fab-Fc or Fab-Fab interactions in unliganded mAbs are retained in digested products, and 2) whereas anti-gp120 non-nmAbs could bind two gp120 molecules, nmAbs could bind only one gp120. Finally, additional experiments showed that except for 2G12 and 4E10, unopen shapes of nmAbs remain uninfluenced by ionic strength but can be reversibly opened by low pH of buffer accompanied by loss of ligand binding ability.« less

  20. Global Shape and Ligand Binding Efficiency of the HIV-1-neutralizing Antibodies Differ from Those of Antibodies That Cannot Neutralize HIV-1*

    PubMed Central

    Solanki, Ashish K.; Rathore, Yogendra S.; Badmalia, Maulik D.; Dhoke, Reema R.; Nath, Samir K.; Nihalani, Deepak; Ashish

    2014-01-01

    Asymmetric disposition of Fab arms in the structures solved for the broadly neutralizing monoclonal antibody (nmAb) IgG1 b12 raised the question of whether the unusual shape observed for b12 is common for all IgG1 mAbs or if there is a difference in the overall shape of nmAbs versus non-nmAbs. We compared small angle x-ray scattering (SAXS) data-based models and limited proteolysis profiles of some IgG1 mAbs known to be having and lacking HIV-1 neutralizing potency. In non-nmAbs, the Fab arms were found to be symmetrically disposed in space relative to central Fc, but in most nmAbs, the Fab arms were asymmetrically disposed, as seen for IgG1 b12. The only exceptions were 2G12 and 4E10, where both Fab arms were closed above Fc, suggesting some Fab-Fc and/or Fab-Fab interaction in the nmAbs that constrained extension of the Fab-Fc linker. Interestingly, these observations were correlated with differential proteolysis profiles of the mAbs by papain. Under conditions when papain could cut both Fab arms of non-nmAbs, only one Fab arm could be removed from neutralizing ones (except for 2G12 and 4E10). Chromatography and small angle x-ray scattering results of papain-digested products revealed that 1) the Fab-Fc or Fab-Fab interactions in unliganded mAbs are retained in digested products, and 2) whereas anti-gp120 non-nmAbs could bind two gp120 molecules, nmAbs could bind only one gp120. Additional experiments showed that except for 2G12 and 4E10, unopen shapes of nmAbs remain uninfluenced by ionic strength but can be reversibly opened by low pH of buffer accompanied by loss of ligand binding ability. PMID:25331945

  1. Differences in Glycoprotein Complex Receptor Binding Site Accessibility Prompt Poor Cross-Reactivity of Neutralizing Antibodies between Closely Related Arenaviruses.

    PubMed

    Brouillette, Rachel B; Phillips, Elisabeth K; Ayithan, Natarajan; Maury, Wendy

    2017-04-01

    The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide, GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related clade B viruses, Machupo virus (MACV) and Junín virus (JUNV), have nearly identical overall GPC architecture and share a host receptor, transferrin receptor 1 (TfR1). Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and the recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (MAbs) and mouse antisera against JUNV or MACV GPC. All six MAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these MAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block MAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV.IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses. A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple National

  2. Structural Basis for HIV-1 Neutralization by 2F5-Like Antibodies m66 and m66.6

    PubMed Central

    Zirkle, Brett; Yang, Yongping; Zhu, Zhongyu; McKee, Krisha; Zhang, Baoshan; Chuang, Gwo-Yu; Georgiev, Ivelin S.; O'Dell, Sijy; Doria-Rose, Nicole; Mascola, John R.; Dimitrov, Dimiter S.

    2014-01-01

    Antibodies m66.6 and 2F5 are the only effective human HIV-1-neutralizing antibodies reported thus far to recognize the N-terminal region of the membrane-proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein. Although 2F5 has been extensively characterized, much less is known about antibody m66.6 or antibody m66, a closely related light-chain variant. Here, we report the crystal structure of m66 in complex with its gp41 epitope, along with unbound structures of m66 and m66.6. We used mutational and binding analyses to decipher antibody elements critical for their recognition of gp41 and determined the molecular basis that underlies their neutralization of HIV-1. When bound by m66, the N-terminal region of the gp41 MPER adopts a conformation comprising a helix, followed by an extended loop. Comparison of gp41-bound m66 to unbound m66.6 identified three light-chain residues of m66.6 that were confirmed through mutagenesis to underlie the greater breadth of m66.6-mediated virus neutralization. Recognition of gp41 by m66 also revealed similarities to antibody 2F5 both in the conformation of crucial epitope residues as well as in the angle of antibody approach. Aromatic residues at the tip of the m66.6 heavy-chain third complementarity-determining region, as in the case of 2F5, were determined to be critical for virus neutralization in a manner that correlated with antibody recognition of the MPER in a lipid context. Antibodies m66, m66.6, and 2F5 thus utilize similar mechanistic elements to recognize a common gp41-MPER epitope and to neutralize HIV-1. PMID:24335316

  3. A Rapid Zika Diagnostic Assay to Measure Neutralizing Antibodies in Patients.

    PubMed

    Shan, Chao; Xie, Xuping; Ren, Ping; Loeffelholz, Michael J; Yang, Yujiao; Furuya, Andrea; Dupuis, Alan P; Kramer, Laura D; Wong, Susan J; Shi, Pei-Yong

    2017-03-01

    The potential association of microcephaly and other congenital abnormalities with Zika virus (ZIKV) infection during pregnancy underlines the critical need for a rapid and accurate diagnosis. Due to the short duration of ZIKV viremia in infected patients, a serologic assay that detects antibody responses to viral infection plays an essential role in diagnosing patient specimens. The current serologic diagnosis of ZIKV infection relies heavily on the labor-intensive Plaque Reduction Neutralization Test (PRNT) that requires more than one-week turnaround time and represents a major bottleneck for patient diagnosis. To overcome this limitation, we have developed a high-throughput assay for ZIKV and dengue virus (DENV) diagnosis that can attain the "gold standard" of the current PRNT assay. The new assay is homogeneous and utilizes luciferase viruses to quantify the neutralizing antibody titers in a 96-well format. Using 91 human specimens, we showed that the reporter diagnostic assay has a higher dynamic range and maintains the relative specificity of the traditional PRNT assay. Besides the improvement of assay throughput, the reporter virus technology has also shortened the turnaround time to less than two days. Collectively, our results suggest that, along with the viral RT-PCR assay, the reporter virus-based serologic assay could be potentially used as the first-line test for clinical diagnosis of ZIKV infection as well as for vaccine clinical trials.

  4. Seroprevalence of Neutralizing Antibodies Against Dengue Virus in Two Localities in the State of Morelos, Mexico

    PubMed Central

    Amaya-Larios, Irma Y.; Martínez-Vega, Ruth Aralí; Mayer, Sandra V.; Galeana-Hernández, Marisol; Comas-García, Andreu; Sepúlveda-Salinas, Karla J.; Falcón-Lezama, Jorge A.; Vasilakis, Nikos; Ramos-Castañeda, José

    2014-01-01

    Humoral immune response against dengue virus (DENV) is an important component in dengue-endemic transmission. We conducted a cross-sectional nested cohort study to determine the seroprevalence and frequency of neutralizing antibodies against DENV serotypes in two endemic localities in the state of Morelos, Mexico. The cohort participants (N = 1,196) were screened to determine previous exposure to DENV. Overall seroprevalence was 76.6% (95% confidence interval [95% CI] = 73.6–79.2), and prevalence of neutralizing antibodies in the 5- to 9-year-old group was 82.5% (95% CI = 67.2–92.7), 45% (95% CI = 29.3–61.5), and 65% (95% CI = 48.3–79.4) for DENV-1, DENV-2, and DENV-3, respectively. For participants older than 10 years, the observed seroprevalence was above 60% for each serotype, except DENV-4 in the 10- to 25-year-old group (42.9%); 81% of humoral responses were multitypic. The outcomes of our study contribute to understanding the immune component of dengue transmission and provide focal information for the evaluation of vaccine candidates under development. PMID:25294613

  5. Mutants of staphylococcal toxic shock syndrome toxin 1: mitogenicity and recognition by a neutralizing monoclonal antibody.

    PubMed Central

    Blanco, L; Choi, E M; Connolly, K; Thompson, M R; Bonventre, P F

    1990-01-01

    Toxic shock syndrome toxin 1 (TSST-1), a 22-kilodalton protein made by strains of Staphylococcus aureus harboring the chromosomal toxin gene, may elicit toxic shock syndrome in humans. In vitro, TSST-1 induces T cells to proliferate and macrophages to secrete interleukin-1. To conduct a structure-function analysis, point mutations on the TSST-1 gene were generated by site-directed mutagenesis to identify amino acids critical for activity of the toxin. Specific tyrosine and histidine residues were replaced by alanines. Wild-type and mutant TSST-1 gene constructs were expressed in Escherichia coli, and the products were tested for their mitogenic potential and reactivity with a TSST-1 neutralizing monoclonal antibody (MAb 8-5-7). Four of the mutants were similar to the wild type; i.e., the mutant toxins stimulated murine T cells and reacted with MAb 8-5-7 equally as well as the wild type. Two mutants exhibited a decrease in mitogenic activity, but one of these retained the capacity to bind with MAb 8-5-7 while the other was no longer recognized by the same antibody. One double mutant demonstrated minimal mitogenic activity and did not react in enzyme-linked immunosorbent and immunoblot assays with MAb 8-5-7. The data show that specific residues near the carboxy terminus of TSST-1 are essential for mitogenic activity and in forming the epitope recognized by neutralizing MAb 8-5-7. Images PMID:1696937

  6. Pediatric measles vaccine expressing a dengue tetravalent antigen elicits neutralizing antibodies against all four dengue viruses.

    PubMed

    Brandler, Samantha; Ruffie, Claude; Najburg, Valérie; Frenkiel, Marie-Pascale; Bedouelle, Hughes; Desprès, Philippe; Tangy, Frédéric

    2010-09-24

    Dengue disease is an increasing global health problem that threatens one-third of the world's population. To control this emerging arbovirus, an efficient preventive vaccine is still needed. Because four serotypes of dengue virus (DV) coexist and antibody-dependent enhanced infection may occur, most strategies developed so far rely on the administration of tetravalent formulations of four live attenuated or chimeric viruses. Here, we evaluated a new strategy based on the expression of a single minimal tetravalent DV antigen by a single replicating viral vector derived from pediatric live-attenuated measles vaccine (MV). We generated a recombinant MV vector expressing a DV construct composed of the four envelope domain III (EDIII) from the four DV serotypes fused with the ectodomain of the membrane protein (ectoM). After two injections in mice susceptible to MV infection, the recombinant vector induced neutralizing antibodies against the four serotypes of dengue virus. When immunized mice were further inoculated with live DV from each serotype, a strong memory neutralizing response was raised against all four serotypes. A combined measles-dengue vaccine might be attractive to immunize infants against both diseases where they co-exist.

  7. A peroxovanadium compound induces Xenopus oocyte maturation: inhibition by a neutralizing anti-insulin receptor antibody.

    PubMed

    Cummings, C; Zhu, L; Sorisky, A; Liu, X J

    1996-05-01

    Synthetic peroxovanadium compounds are a new class of potent inhibitors of protein phosphotyrosine phosphatases. These compounds exhibit insulin-like activity both in vitro and in experimental animals. However, the molecular mechanism by which these compounds exert their biological effect is not well defined. We demonstrate here that several of these compounds induce Xenopus oocyte maturation in vitro, as indicated by germinal vesicle breakdown. Using one of these compounds for further studies, we show that the induction is dose-dependent and is accompanied by activation of maturation promoting factor as well as activation of Xenopus MAP kinase. Like insulin, bpV(pic) causes an acute accumulation of PI(3,4,5)P3 (phosphotidylinositol-3,4,5-trisphosphate), a product of PI 3-kinase. More importantly, bpV(pic)-induced oocyte maturation was abolished by microinjection of a neutralizing monoclonal anti-insulin receptor antibody (17A3) into oocytes or preincubation of oocytes with a PI 3-kinase inhibitor (wortmannin). These results suggest that bpV(pic) acts upstream of the Xenopus IGF-1 receptor in the induction of meiotic maturation, presumably by neutralizing an inhibitory protein tyrosine phosphatase(s) that may regulate the receptor. Finally, using an oocyte-follicle cell complex that responded to human chorionic gonadotropin (hCG) to undergo GVBD, we showed that injection of 17A3 anti-insulin receptor antibody into oocytes did not affect hCG-induced oocyte maturation.

  8. Conformation-dependent high-affinity potent ricin-neutralizing monoclonal antibodies.

    PubMed

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M; Cherwonogrodzky, John W

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μ g, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes.

  9. Neutralizing Monoclonal Antibodies against Disparate Epitopes on Ricin Toxin's Enzymatic Subunit Interfere with Intracellular Toxin Transport.

    PubMed

    Yermakova, Anastasiya; Klokk, Tove Irene; O'Hara, Joanne M; Cole, Richard; Sandvig, Kirsten; Mantis, Nicholas J

    2016-03-07

    Ricin is a member of the A-B family of bacterial and plant toxins that exploit retrograde trafficking to the Golgi apparatus and endoplasmic reticulum (ER) as a means to deliver their cytotoxic enzymatic subunits into the cytoplasm of mammalian cells. In this study we demonstrate that R70 and SyH7, two well-characterized monoclonal antibodies (mAbs) directed against distinct epitopes on the surface of ricin's enzymatic subunit (RTA), interfere with toxin transport from the plasma membrane to the trans Golgi network. Toxin-mAb complexes formed on the cell surface delayed ricin's egress from EEA-1(+) and Rab7(+) vesicles and enhanced toxin accumulation in LAMP-1(+) vesicles, suggesting the complexes were destined for degradation in lysosomes. Three other RTA-specific neutralizing mAbs against different epitopes were similar to R70 and SyH7 in terms of their effects on ricin retrograde transport. We conclude that interference with toxin retrograde transport may be a hallmark of toxin-neutralizing antibodies directed against disparate epitopes on RTA.

  10. Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

    PubMed Central

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M.; Cherwonogrodzky, John W.

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes. PMID:23484120

  11. Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody

    DOE PAGES

    Kong, Rui; Xu, Kai; Zhou, Tongqing; ...

    2016-05-13

    The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. In this paper, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showedmore » that the N-terminal portion of the fusion peptide can be solvent-exposed. Finally, these results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.« less

  12. Neutralizing human monoclonal antibodies to conformational epitopes of human T-cell lymphotropic virus type 1 and 2 gp46.

    PubMed Central

    Hadlock, K G; Rowe, J; Perkins, S; Bradshaw, P; Song, G Y; Cheng, C; Yang, J; Gascon, R; Halmos, J; Rehman, S M; McGrath, M S; Foung, S K

    1997-01-01

    Ten human monoclonal antibodies derived from peripheral B cells of a patient with human T-cell lymphotropic virus (HTLV)-associated myelopathy are described. One monoclonal antibody recognized a linear epitope within the carboxy-terminal 43 amino acids of HTLV gp21, and two monoclonal antibodies recognized linear epitopes within HTLV type 1 (HTLV-1) gp46. The remaining seven monoclonal antibodies recognized denaturation-sensitive epitopes within HTLV-1 gp46 that were expressed on the surfaces of infected cells. Two of these antibodies also bound to viable HTLV-2 infected cells and immunoprecipitated HTLV-2 gp46. Virus neutralization was determined by syncytium inhibition assays. Eight monoclonal antibodies, including all seven that recognized denaturation-sensitive epitopes within HTLV-1 gp46, possessed significant virus neutralization activity. By competitive inhibition analysis it was determined that these antibodies recognized at least four distinct conformational epitopes within HTLV-1 gp46. These findings indicate the importance of conformational epitopes within HTLV-1 gp46 in mediating a neutralizing antibody response to HTLV infection. PMID:9223472

  13. Development of Germline-Humanized Antibodies Neutralizing Botulinum Neurotoxin A and B

    PubMed Central

    Liu, Yvonne; Tierney, Robert; Rasetti-Escargueil, Christine; Avril, Arnaud; Frenzel, André; Thullier, Philippe; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Sesardic, Dorothea; Hust, Michael; Popoff, Michel Robert

    2016-01-01

    Botulinum neurotoxins (BoNTs) are counted among the most toxic substances known and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. To date, 7 serologically distinct serotypes of BoNT (serotype A-G) are known. Due to the high toxicity of BoNTs the Centers for Disease Control and Prevention (CDC) have classified BoNTs as category A agent, including the six biological agents with the highest potential risk of use as bioweapons. Well tolerated antibodies neutralizing BoNTs are required to deal with the potential risk. In a previous work, we described the development of scFv and scFv-Fc (Yumab) from macaque origin (Macaca fascicularis) neutralizing BoNT/A and B by targeting the heavy and light chain of each serotype. In the present study, we humanized the macaque antibodies SEM120-IIIC1 (anti-BoNT/A light chain), A1HC38 (anti-BoNT/A heavy chain), BLC3 (anti-BoNT/B light chain) and B2-7 (anti-BoNT/B heavy chain) by germline-humanization to obtain a better potential immunotolerance in humans. We increased the Germinality Index (GI) of SEM120-IIIC1 to 94.5%, for A1HC38, to 95% for BLC3 and to 94.4% for B2-7. Furthermore, the neutralization efficacies of the germline-humanized antibodies were analyzed in lethal and non-lethal in vivo mouse assays as full IgG. The germline-humanized IgGs hu8SEM120-IIIC1, hu8A1HC38, hu8BLC3 and hu8B2-7 were protective in vivo, when anti-heavy and anti-light chain antibodies were combined. The synergistic effect and high humanness of the selected IgGs makes them promising lead candidates for further clinical development. PMID:27560688

  14. trans-Sialidase Neutralizing Antibody Detection in Trypanosoma cruzi-Infected Domestic Reservoirs ▿

    PubMed Central

    Sartor, Paula A.; Cardinal, Martha V.; Orozco, Marcela M.; Gürtler, Ricardo E.; Leguizamón, M. Susana

    2011-01-01

    The detection of Trypanosoma cruzi infection in domestic dogs and cats is relevant to evaluating human transmission risks and the effectiveness of insecticide spraying campaigns. However, the serological assays routinely used are associated with cross-reactivity in sera from mammals infected with Leishmania spp. We used a trans-sialidase inhibition assay (TIA) for T. cruzi diagnosis in serum samples from 199 dogs and 57 cats from areas where these types of infections are endemic. TIA is based on the antibody neutralization of recombinant trans-sialidase, an enzyme that is not detected in the coendemic Leishmania species or Trypanosoma rangeli parasites. T. cruzi infection was also evaluated by conventional serology (CS) (indirect immunofluorescence, indirect hemagglutination, enzyme-linked immunosorbent assay, and immunochromatographic dipstick test) and xenodiagnosis. Sera from 30 dogs and 15 cats from areas where these organisms are not endemic and 5 dogs with visceral leishmaniasis were found to be nonreactive by TIA and CS. Samples from dogs and cats demonstrated 91 and 95% copositivities between TIA and CS, whereas the conegativities were 98 and 97%, respectively. Sera from xenodiagnosis-positive dogs and cats also reacted by TIA (copositivities of 97 and 83%, respectively). TIA was reactive in three CS-negative samples and was able to resolve results in two cat serum samples that were CS inconclusive. Our study is the first to describe the development of trans-sialidase neutralizing antibodies in naturally infected dogs and cats. High CS conegativity and the absence of trans-sialidase neutralization in dog sera from areas where leishmaniasis is not endemic and from dogs with visceral leishmaniasis support TIA specificity. The TIA may be a useful tool for T. cruzi detection in the main domestic reservoirs. PMID:21471302

  15. A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes

    PubMed Central

    Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Zhai, Wenwu; Smith, Theresa J.; Smith, Leonard A.

    2017-01-01

    Human botulism is primarily caused by botulinum neurotoxin (BoNT) serotypes A, B and E, with around 1% caused by serotype F (BoNT/F). BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs) that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V) regions and yeast display to generate a panel of 33 lead single chain Fv (scFv) mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (KD) of 4.06 × 10−9 M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2×10−8 M to 1.47×10−12 pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism. PMID:28323873

  16. Characterization of Poliovirus Neutralization Escape Mutants of Single-Domain Antibody Fragments (VHHs)

    PubMed Central

    Schotte, Lise; Thys, Bert; Strauss, Mike; Filman, David J.; Rombaut, Bart

    2015-01-01

    To complete the eradication of poliovirus and to protect unvaccinated people subsequently, the development of one or more antiviral drugs will be necessary. A set of five single-domain antibody fragments (variable parts of the heavy chain of a heavy-chain antibody [VHHs]) with an in vitro neutralizing activity against poliovirus type 1 was developed previously (B. Thys, L. Schotte, S. Muyldermans, U. Wernery, G. Hassanzadeh-Ghassabeh, and B. Rombaut, Antiviral Res 87:257–264, 2010, http://dx.doi.org/10.1016/j.antiviral.2010.05.012), and their mechanisms of action have been studied (L. Schotte, M. Strauss, B. Thys, H. Halewyck, D. J. Filman, M. Bostina, J. M. Hogle, and B. Rombaut, J Virol 88:4403–4413, 2014, http://dx.doi.org/10.1128/JVI.03402-13). In this study, neutralization escape mutants were selected for each VHH. Sequencing of the P1 region of the genome showed that amino acid substitutions are found in the four viral proteins of the capsid and that they are located both in proximity to the binding sites of the VHHs and in regions further away from the canyon and hidden beneath the surface. Characterization of the mutants demonstrated that they have single-cycle replication kinetics that are similar to those of their parental strain and that they are all drug (VHH) independent. Their resistant phenotypes are stable, as they do not regain full susceptibility to the VHH after passage over HeLa cells in the absence of VHH. They are all at least as stable as the parental strain against heat inactivation at 44°C, and three of them are even significantly (P < 0.05) more resistant to heat inactivation. The resistant variants all still can be neutralized by at least two other VHHs and retain full susceptibility to pirodavir and 35-1F4. PMID:26014941

  17. Exploiting Cross-reactivity to Neutralize Two Different Scorpion Venoms with One Single Chain Antibody Fragment*

    PubMed Central

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D.; Becerril, Baltazar

    2011-01-01

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591–2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD50 of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity. PMID:21156801

  18. Exploiting cross-reactivity to neutralize two different scorpion venoms with one single chain antibody fragment.

    PubMed

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D; Becerril, Baltazar

    2011-02-25

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591-2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD(50) of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity.

  19. A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes.

    PubMed

    Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Zhai, Wenwu; Smith, Theresa J; Smith, Leonard A; Marks, James D

    2017-01-01

    Human botulism is primarily caused by botulinum neurotoxin (BoNT) serotypes A, B and E, with around 1% caused by serotype F (BoNT/F). BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs) that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V) regions and yeast display to generate a panel of 33 lead single chain Fv (scFv) mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (KD) of 4.06 × 10-9 M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2×10-8 M to 1.47×10-12 pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism.

  20. Targeting human vasohibin-2 by a neutralizing monoclonal antibody for anti-cancer treatment.

    PubMed

    Koyanagi, Takahiro; Suzuki, Yasuhiro; Komori, Kazuki; Saga, Yasushi; Matsubara, Shigeki; Fujiwara, Hiroyuki; Sato, Yasufumi

    2016-12-29

    There are 2 members of the vasohibin (VASH) family, VASH1 and VASH2. VASH1 is expressed mainly in endothelial cells to inhibit angiogenesis, whereas VASH2 is expressed mainly in cancer cells to stimulate tumor growth. The aim of the present study was to establish neutralizing monoclonal antibody (mAb) against human VASH2 and apply it as an anti-cancer treatment. We previously raised mAbs against several synthetic peptides of hVASH1, and found that one of them exhibited neutralizing activity against hVASH1. Because of the similarity in the amino acid sequences between VASH1 and VASH2, we hypothesized that they shared the bioactive center. When we mutated 4 amino acids within the region, the mutant VASH2 lost its pro-angiogenic activity. We therefore raised mAb against a synthetic peptide overlapping the mutated amino acids of hVASH2, and isolated one clone (1760) that almost completely inhibited the stimulatory effect of hVASH2 on the migration of and tube formation by ECs. When we used this clone 1760 antibody for cancer treatment, the peritoneal injection of it inhibited both tumor growth and angiogenesis in a mouse xenograft model of human cancer cells. In terms of anti-tumor activity, 25 mg/kg of clone 1760 was equivalent to 5 mg/kg of bevacizmab. From these results, we propose the targeting of human VASH2 with neutralizing mAb as a new strategy for cancer treatment. This article is protected by copyright. All rights reserved.

  1. Development and characterization of neutralizing monoclonal antibodies against canine distemper virus hemagglutinin protein.

    PubMed

    Bi, Zhenwei; Xia, Xingxia; Wang, Yongshan; Mei, Yongjie

    2015-04-01

    Canine distemper virus (CDV) causes a serious multisystemic disease in dogs and other carnivora. Hemagglutinin (H) protein-specific antibodies are mainly responsible for protective immunity against CDV infection. In the present study, six neutralizing MAbs to the H protein of CDV were newly obtained and characterized by immunizing BALB/c mice with a recent Chinese field isolate. Competitive binding inhibition assay revealed that they recognized four distinct antigenic regions of the H protein. Immunofluorescence assay and western blotting showed that all MAbs recognize the conformational rather than the linear epitopes of the H protein. Furthermore, in immunofluorescence and virus neutralization assays, two of the MAbs were found to react only with the recent Chinese field isolate and not with older CDV strains, including vaccine strain Onderstepoort, indicating there are neutralization-related antigenic variations between the recent Chinese field isolate and the older CDV strains examined in this study. The newly established MAbs are useful for differentiating the expanding CDV strains and could be used in immunotherapy and immunodiagnosis against infection with CDV.

  2. Radiometric cytolysis inhibition assay, a new rapid test for neutralizing antibodies to intact and trypsin-cleaved poliovirus

    SciTech Connect

    Hovi, T.; Roivainen, M.

    1989-04-01

    We have developed a new rapid test, the radiometric cytolysis inhibition assay (RACINA), for the determination of neutralizing poliovirus antibodies. HeLa cells prelabeled with /sup 51/Cr, (/sup 3/H)leucine, or, preferentially, with (/sup 3/H)uridine are used as sensitive quantitative indicators of residual infectious virus. Both suspensions and monolayer cultures of the indicator cells can be used. Neutralization of a fraction of a high-titer virus preparation can be scored after the first replication cycle at 8 to 10 h. By lowering the incubation temperature to 30/degree/C, the completion of the cytolysis due to the first replication cycle of poliovirus was delayed beyond 21 h. This makes it possible to use the RACINA, unlike the standard microneutralization assay, for measuring antibodies to trypsin-cleaved polioviruses. The RACINA was found to be as sensitive as and more reproducible than the standard microneutralization assay in the measurement of neutralizing poliovirus antibodies. The RACINA is a rapid and reliable test for neutralizing antibodies and in principle it may be applicable for quantitation of neutralizing antibodies to other cytolytic agents as well.

  3. Human Memory B Cells Producing Potent Cross-Neutralizing Antibodies against Human Parechovirus: Implications for Prevalence, Treatment, and Diagnosis

    PubMed Central

    Benschop, K. S. M.; Koen, G.; Claassen, Y. B.; Wagner, K.; Bakker, A. Q.; Wolthers, K. C.

    2015-01-01

    ABSTRACT The family Picornaviridae is a large and diverse group of positive-sense RNA viruses, including human enteroviruses (EVs) and human parechoviruses (HPeVs). The human immune response against EVs and HPeVs is thought to be mainly humoral, and an insufficient neutralizing antibody (Ab) response during infection is a risk factor and can ultimately be life threatening. The accessibility of different antigenic sites and observed cross-reactivity make HPeVs a good target for development of therapeutic human monoclonal antibodies (MAbs). In this study, we generated two different human MAbs specific for HPeV by screening culture supernatants of Ab-producing human B cell cultures for direct neutralization of HPeV1. Both MAbs showed HPeV1-specific neutralization as well as neutralization of HPeV2. One antibody, AM18, cross-neutralized HPeV4, -5, and -6 and coxsackievirus A9 (CV-A9). VP1 capsid protein-specific assays confirmed that AM18 bound VP1 of HPeV1, -2, and -4 with high affinity (11.5 pM). In contrast, the HPeV1-specific MAb AM28, which neutralized HPeV1 even more efficiently than did AM18, showed no cross-reactivity with HPeV3 to -6 or other EVs and did not bind any of the capsid proteins, suggesting that AM28 is specific for a conformation-dependent, nonlinear epitope on the virus. The discovery of MAbs that are cross-reactive between HPeVs may help development of HPeV treatment options with antibodies and vaccine design based on epitopes recognized by these antibodies. IMPORTANCE HPeV infections are widespread among young children and adults, causing a broad range of disease. Infections can be severe and life threatening, while no antiviral treatment is available. Given that the absence of neutralizing Abs is a risk factor for severe disease in infants, treatment of picornavirus infections with MAbs would be a therapeutic option. To study antibody neutralization of HPeV in more detail, we generated two different HPeV1-specific human MAbs. Both MAbs show HPe

  4. Oral iodine supplementation does not reduce neutralizing antibody responses to oral poliovirus vaccine.

    PubMed Central

    Taffs, R. E.; Enterline, J. C.; Rusmil, K.; Muhilal; Suwardi, S. S.; Rustama, D.; Djatnika; Cobra, C.; Semba, R. D.; Cohen, N.; Asher, D. M.

    1999-01-01

    Iodine deficiency is a major cause of impaired mental development, goitre, and cretinism in many parts of the world. Because existing immunization programmes can be used to deliver oral iodized oil (OIO) to infants at risk, it was important to know whether OIO could adversely affect the antibody response to vaccines, such as trivalent oral poliovirus vaccine (OPV). A randomized, double-blind, placebo-controlled clinical trial was conducted in Subang, West Java, Indonesia, in which 617 eight-week-old infants received either OIO or a placebo (poppy-seed oil) during a routine visit for their first dose of OPV as part of the Expanded Programme on Immunization (EPI). The infants received two boosters of OPV at 4-week intervals after the first dose, and were followed up when 6 months old. Neutralizing antibody titres to poliovirus serotypes 1, 2, and 3 were compared in serum samples that were taken from 478 of these infants just before the first dose of OPV and at 6 months. It was found that oral iodized oil did not reduce the antibody responses to any of the three serotypes of OPV. These results indicate that oral iodine may safely be delivered to infants at the same time as oral poliovirus vaccine according to current EPI immunization schedules. PMID:10427933

  5. Broadly Neutralizing Anti-HIV Antibodies Prevent HIV Infection of Mucosal Tissue Ex Vivo.

    PubMed

    Scott, Yanille M; Park, Seo Young; Dezzutti, Charlene S

    2016-02-01

    Broadly neutralizing monoclonal antibodies (nAbs) specific for HIV are being investigated for use in HIV prevention. Due to their ability to inhibit HIV attachment to and entry into target cells, nAbs may be suitable for use as topical HIV microbicides. As such, they would present an alternative intervention for individuals who may not benefit from using antiretroviral-based products for HIV prevention. We theorize that nAbs can inhibit viral transmission through mucosal tissue, thus reducing the incidence of HIV infection. The efficacy of the PG9, PG16, VRC01, and 4E10 antibodies was evaluated in an ex vivo human model of mucosal HIV transmission. nAbs reduced HIV transmission, causing 1.5- to 2-log10 reductions in HIV replication in ectocervical tissues and ≈3-log10 reductions in HIV replication in colonic tissues over 21 days. These antibodies demonstrated greater potency in colonic tissues, with a 50-fold higher dose being required to reduce transmission in ectocervical tissues. Importantly, nAbs retained their potency and reduced viral transmission in the presence of whole semen. No changes in tissue viability or immune activation were observed in colonic or ectocervical tissue after nAb exposure. Our data suggest that topically applied nAbs are safe and effective against HIV infection of mucosal tissue and support further development of nAbs as a topical microbicide that could be used for anal as well as vaginal protection.

  6. Broadly Neutralizing Anti-HIV Antibodies Prevent HIV Infection of Mucosal Tissue Ex Vivo

    PubMed Central

    Scott, Yanille M.; Park, Seo Young

    2015-01-01

    Broadly neutralizing monoclonal antibodies (nAbs) specific for HIV are being investigated for use in HIV prevention. Due to their ability to inhibit HIV attachment to and entry into target cells, nAbs may be suitable for use as topical HIV microbicides. As such, they would present an alternative intervention for individuals who may not benefit from using antiretroviral-based products for HIV prevention. We theorize that nAbs can inhibit viral transmission through mucosal tissue, thus reducing the incidence of HIV infection. The efficacy of the PG9, PG16, VRC01, and 4E10 antibodies was evaluated in an ex vivo human model of mucosal HIV transmission. nAbs reduced HIV transmission, causing 1.5- to 2-log10 reductions in HIV replication in ectocervical tissues and ≈3-log10 reductions in HIV replication in colonic tissues over 21 days. These antibodies demonstrated greater potency in colonic tissues, with a 50-fold higher dose being required to reduce transmission in ectocervical tissues. Importantly, nAbs retained their potency and reduced viral transmission in the presence of whole semen. No changes in tissue viability or immune activation were observed in colonic or ectocervical tissue after nAb exposure. Our data suggest that topically applied nAbs are safe and effective against HIV infection of mucosal tissue and support further development of nAbs as a topical microbicide that could be used for anal as well as vaginal protection. PMID:26596954

  7. Efficacy of broadly neutralizing monoclonal antibody PG16 in HIV-infected humanized mice.

    PubMed

    Stoddart, Cheryl A; Galkina, Sofiya A; Joshi, Pheroze; Kosikova, Galina; Long, Brian R; Maidji, Ekaterina; Moreno, Mary E; Rivera, Jose M; Sanford, Ukina R; Sloan, Barbara; Cieplak, Witold; Wrin, Terri; Chan-Hui, Po-Ying

    2014-08-01

    Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clades A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70-80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV inoculation. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier.

  8. A human/murine chimeric fab antibody neutralizes anthrax lethal toxin in vitro.

    PubMed

    Ding, Guipeng; Chen, Ximin; Zhu, Jin; Duesbery, Nicholas S; Cheng, Xunjia; Cao, Brian

    2013-01-01

    Human anthrax infection caused by exposure to Bacillus anthracis cannot always be treated by antibiotics. This is mostly because of the effect of the remaining anthrax toxin in the body. Lethal factor (LF) is a component of lethal toxin (LeTx), which is the major virulence of anthrax toxin. A murine IgG monoclonal antibody (mAb) against LF with blocking activity (coded LF8) was produced in a previous study. In this report, a human/murine chimeric Fab mAb (coded LF8-Fab) was developed from LF8 by inserting murine variable regions into human constant regions using antibody engineering to reduce the incompatibility of the murine antibody for human use. The LF8-Fab expressed in Escherichia coli could specifically identify LF with an affinity of 3.46 × 10(7) L/mol and could neutralize LeTx with an EC50 of 85  μ g/mL. Even after LeTx challenge at various time points, the LF8-Fab demonstrated protection of J774A.1 cells in vitro. The results suggest that the LF8-Fab might be further characterized and potentially be used for clinical applications against anthrax infection.

  9. Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B.

    PubMed

    Cheng, Luisa W; Henderson, Thomas D; Lam, Tina I; Stanker, Larry H

    2015-11-27

    Botulinum neurotoxins (BoNT) are some of nature's most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism.

  10. Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies.

    PubMed

    McGuire, Andrew T; Hoot, Sam; Dreyer, Anita M; Lippy, Adriana; Stuart, Andrew; Cohen, Kristen W; Jardine, Joseph; Menis, Sergey; Scheid, Johannes F; West, Anthony P; Schief, William R; Stamatatos, Leonidas

    2013-04-08

    Broadly neutralizing antibodies (bnAbs) against HIV are believed to be a critical component of the protective responses elicited by an effective HIV vaccine. Neutralizing antibodies against the evolutionarily conserved CD4-binding site (CD4-BS) on the HIV envelope glycoprotein (Env) are capable of inhibiting infection of diverse HIV strains, and have been isolated from HIV-infected individuals. Despite the presence of anti-CD4-BS broadly neutralizing antibody (bnAb) epitopes on recombinant Env, Env immunization has so far failed to elicit such antibodies. Here, we show that Env immunogens fail to engage the germline-reverted forms of known bnAbs that target the CD4-BS. However, we found that the elimination of a conserved glycosylation site located in Loop D and two glycosylation sites located in variable region 5 of Env allows Env-binding to, and activation of, B cells expressing the germline-reverted BCRs of two potent broadly neutralizing antibodies, VRC01 and NIH45-46. Our results offer a possible explanation as to why Env immunogens have been ineffective in stimulating the production of such bNAbs. Importantly, they provide key information as to how such immunogens can be engineered to initiate the process of antibody-affinity maturation against one of the most conserved Env regions.

  11. Characterization of a Large Panel of Rabbit Monoclonal Antibodies against HIV-1 gp120 and Isolation of Novel Neutralizing Antibodies against the V3 Loop

    PubMed Central

    Qin, Yali; Shi, Heliang; Banasik, Marisa; Lin, Feng; Rohl, Kari; LaBranche, Celia; Montefiori, David C.; Cho, Michael W.

    2015-01-01

    We recently reported the induction of potent, cross-clade neutralizing antibodies (nAbs) against Human Immunodeficiency Virus type-1 (HIV-1) in rabbits using gp120 based on an M-group consensus sequence. To better characterize these antibodies, 93 hybridomas were generated, which represent the largest panel of monoclonal antibodies (mAbs) ever generated from a vaccinated rabbit. The single most frequently recognized epitope of the isolated mAbs was at the very C-terminal end of the protein (APTKAKRRVVEREKR), followed by the V3 loop. A total of seven anti-V3 loop mAbs were isolated, two of which (10A3 and 10A37) exhibited neutralizing activity. In contrast to 10A3 and most other anti-V3 loop nAbs, 10A37 was atypical with its epitope positioned more towards the C-terminal half of the loop. To our knowledge, 10A37 is the most potent and broadly neutralizing anti-V3 loop mAb induced by vaccination. Interestingly, all seven anti-V3 loop mAbs competed with PGT121, suggesting a possibility that early induction of potent anti-V3 loop antibodies could prevent induction of more broadly neutralizing PGT121-like antibodies that target the conserved base of the V3 loop stem. PMID:26039641

  12. Enhanced Potency of a Broadly Neutralizing HIV-1 Antibody In Vitro Improves Protection against Lentiviral Infection In Vivo

    PubMed Central

    Rudicell, Rebecca S.; Kwon, Young Do; Ko, Sung-Youl; Pegu, Amarendra; Louder, Mark K.; Georgiev, Ivelin S.; Wu, Xueling; Zhu, Jiang; Boyington, Jeffrey C.; Chen, Xuejun; Shi, Wei; Yang, Zhi-yong; Doria-Rose, Nicole A.; McKee, Krisha; O'Dell, Sijy; Schmidt, Stephen D.; Chuang, Gwo-Yu; Druz, Aliaksandr; Soto, Cinque; Yang, Yongping; Zhang, Baoshan; Zhou, Tongqing; Todd, John-Paul; Lloyd, Krissey E.; Eudailey, Joshua; Roberts, Kyle E.; Donald, Bruce R.; Bailer, Robert T.; Ledgerwood, Julie; Mullikin, James C.; Shapiro, Lawrence; Koup, Richard A.; Graham, Barney S.; Nason, Martha C.; Connors, Mark; Haynes, Barton F.; Rao, Srinivas S.; Roederer, Mario; Kwong, Peter D.

    2014-01-01

    ABSTRACT Over the past 5 years, a new generation of highly potent and broadly neutralizing HIV-1 antibodies has been identified. These antibodies can protect against lentiviral infection in nonhuman primates (NHPs), suggesting that passive antibody transfer would prevent HIV-1 transmission in humans. To increase the protective efficacy of such monoclonal antibodies, we employed next-generation sequencing, computational bioinformatics, and structure-guided design to enhance the neutralization potency and breadth of VRC01, an antibody that targets the CD4 binding site of the HIV-1 envelope. One variant, VRC07-523, was 5- to 8-fold more potent than VRC01, neutralized 96% of viruses tested, and displayed minimal autoreactivity. To compare its protective efficacy to that of VRC01 in vivo, we performed a series of simian-human immunodeficiency virus (SHIV) challenge experiments in nonhuman primates and calculated the doses of VRC07-523 and VRC01 that provide 50% protection (EC50). VRC07-523 prevented infection in NHPs at a 5-fold lower concentration than VRC01. These results suggest that increased neutralization potency in vitro correlates with improved protection against infection in vivo, documenting the improved functional efficacy of VRC07-523 and its potential clinical relevance for protecting against HIV-1 infection in humans. IMPORTANCE In the absence of an effective HIV-1 vaccine, alternative strategies are needed to block HIV-1 transmission. Direct administration of HIV-1-neutralizing antibodies may be able to prevent HIV-1 infections in humans. This approach could be especially useful in individuals at high risk for contracting HIV-1 and could be used together with antiretroviral drugs to prevent infection. To optimize the chance of success, such antibodies can be modified to improve their potency, breadth, and in vivo half-life. Here, knowledge of the structure of a potent neutralizing antibody, VRC01, that targets the CD4-binding site of the HIV-1 envelope

  13. Complement requirement for virus neutralization by antibody and reduced serum complement levels associated with experimental equine herpesvirus 1 infection.

    PubMed Central

    Snyder, D B; Myrup, A C; Dutta, S K

    1981-01-01

    Pony foals, negative for detectable serum-neutralizing antibody to equine herpesvirus 1 by the standard tube-culture virus neutralization test, were experimentally infected with equine herpesvirus 1. Complement-requiring (CR) and non-complement-requiring (NCR) serum-neutralizing antibodies were evaluated in preinfection and postinfection sera by means of a complement-enhanced plaque reduction assay. Low levels of CR antibodies were found in the preinfection sera of only group II ponies. Upon infection, CR antibodies were detected by day 2 postinfection and reached peak titers between 7 and 14 days postinfection in the antisera of all ponies. NCR antibodies were detected later than CR antibodies and at levels approximately 40 to 150 times lower than the latter. CR/NCR ratios indicated that complement requirement was greatest early in the acute stages of disease and that this requirement decreased during the convalescent phase. Fractionation of 1-week and 2-week postinfection antisera of group I ponies indicated the CR antibody activity resided in both the 7S and 19S fractions. Total serum complement levels of the ponies were quantified throughout the infection with an equine anti-goat erythrocyte hemolytic system. In vivo, complement levels were depressed for all ponies during the first 2 weeks of infection. A decline in complement levels was seen as early as day 2, and they decreased to an average of 35% of preinfection levels on day 10 postinfection for all ponies. PMID:6260672

  14. Recombinant human monoclonal antibodies to human cytomegalovirus glycoprotein B neutralize virus in a complement-dependent manner.

    PubMed

    Ohta, Akane; Fujita, Ayano; Murayama, Tsugiya; Iba, Yoshitaka; Kurosawa, Yoshikazu; Yoshikawa, Tetsushi; Asano, Yoshizo

    2009-11-01

    Human antibodies specific for HCMV are currently considered as potential anti-HCMV therapeutic agents. In this study, we used a combinatorial human antibody library to isolate and characterize complete human monoclonal antibodies that effectively neutralize HCMV in a complement-dependent manner. One hundred and six clones were isolated in two independent screens using HCMV virions and recombinant glycoprotein B, gB654, as antigens. All of the clones recognized the same molecule gB and were classified into 14 groups based on the amino acid sequence of the V(H) region. Seven representative clones from these 14 groups had a strong gB654 binding affinity by surface plasmon resonance (SPR). A pairwise binding competition analysis suggested that there were three groups based on differences in the gB recognition sites. Although Fab fragments of the seven groups showed strong affinity for gB, none of the Fab fragments neutralized HCMV infectivity in vitro. In contrast, complete human IgG(1) antibodies of at least three groups neutralized HCMV in a complement-dependent manner. These data suggest that potent therapeutic antibodies can be obtained from a human antibody library, including most of the functional antibodies that mediate humoral immunity to the selected pathogen.

  15. Localization and synthesis of an antigenic determinant of herpes simplex virus glycoprotein D that stimulates the production of neutralizing antibody

    SciTech Connect

    Cohen, G.H.; Dietzschold, B.; Ponce de Leon, M.; Long, D.; Golub, E.; Varrichio, A.; Pereira, L.; Eisenberg, R.J.

    1984-01-01

    An antigenic determinant capable of inducing type-common herpes simplex virus (HSV)-neutralizing antibodies has been located on glycoprotein D (gD) of HSV type 1 (HSV-1). A peptide of 16 amino acids corresponding to residues 8 to 23 of the mature glycoprotein (residues 33 to 48 of the predicted gD-1 sequence) was synthesized. This peptide reacted with an anti-gD monoclonal antibody (group VII) previously shown to neutralize the infectivity of HSV-1 and HSV-2. The peptide was also recognized by polyclonal antibodies prepared against purified gD-1 but was less reactive with anti-gD2 sera. Sera from animals immunized with the synthetic peptide reacted with native gD and neutralized both HSV-1 and HSV-2.

  16. Passive transfer of modest titers of potent and broadly neutralizing anti-HIV monoclonal antibodies block SHIV infection in macaques

    PubMed Central

    Shingai, Masashi; Donau, Olivia K.; Plishka, Ronald J.; Buckler-White, Alicia; Mascola, John R.; Nabel, Gary J.; Nason, Martha C.; Montefiori, David; Moldt, Brian; Poignard, Pascal; Diskin, Ron; Bjorkman, Pamela J.; Eckhaus, Michael A.; Klein, Florian; Mouquet, Hugo; Cetrulo Lorenzi, Julio Cesar; Gazumyan, Anna; Burton, Dennis R.; Nussenzweig, Michel C.

    2014-01-01

    It is widely appreciated that effective human vaccines directed against viral pathogens elicit neutralizing antibodies (NAbs). The passive transfer of anti–HIV-1 NAbs conferring sterilizing immunity to macaques has been used to determine the plasma neutralization titers, which must be present at the time of exposure, to prevent acquisition of SIV/HIV chimeric virus (SHIV) infections. We administered five recently isolated potent and broadly acting anti-HIV neutralizing monoclonal antibodies (mAbs) to rhesus macaques and challenged them intrarectally 24 h later with either of two different R5-tropic SHIVs. By combining the results obtained from 60 challenged animals, we determined that the protective neutralization titer in plasma preventing virus infection in 50% of the exposed monkeys was relatively modest (∼1:100) and potentially achievable by vaccination. PMID:25155019

  17. Rapid High-Level Production of Functional HIV Broadly Neutralizing Monoclonal Antibodies in Transient Plant Expression Systems

    PubMed Central

    Rosenberg, Yvonne; Sack, Markus; Montefiori, David; Forthal, Donald; Mao, Lingjun; -Abanto, Segundo Hernandez; Urban, Lori; Landucci, Gary; Fischer, Rainer; Jiang, Xiaoming

    2013-01-01

    Passive immunotherapy using anti-HIV broadly neutralizing monoclonal antibodies (mAbs) has shown promise as an HIV treatment, reducing mother-to-child-transmission (MTCT) of simian/human immunodeficiency virus (SHIV) in non-human primates and decreasing viral rebound in patients who ceased receiving anti-viral drugs. In addition, a cocktail of potent mAbs may be useful as mucosal microbicides and provide an effective therapy for post-exposure prophylaxis. However, even highly neutralizing HIV mAbs used today may lose their effectiveness if resistance occurs, requiring the rapid production of new or engineered mAbs on an ongoing basis in order to counteract the viral resistance or the spread of a certain HIV-1 clade in a particular region or patient. Plant-based expression systems are fast, inexpensive and scalable and are becoming increasingly popular for the production of proteins and monoclonal antibodies. In the present study, Agrobacterium-mediated transient transfection of plants, utilizing two species of Nicotiana, have been tested to rapidly produce high levels of an HIV 89.6PΔ140env and several well-studied anti-HIV neutralizing monoclonal antibodies (b12, 2G12, 2F5, 4E10, m43, VRC01) or a single chain antibody construct (m9), for evaluation in cell-based viral inhibition assays. The protein-A purified plant-derived antibodies were intact, efficiently bound HIV envelope, and were equivalent to, or in one case better than, their counterparts produced in mammalian CHO or HEK-293 cells in both neutralization and antibody dependent viral inhibition assays. These data indicate that transient plant-based transient expression systems are very adaptable and could rapidly generate high levels of newly identified functional recombinant HIV neutralizing antibodies when required. In addition, they warrant detailed cost-benefit analysis of prolonged incubation in plants to further increase mAb production. PMID:23533588

  18. Structure of a Major Antigenic Site on the Respiratory Syncytial Virus Fusion Glycoprotein in Complex with Neutralizing Antibody 101F

    SciTech Connect

    McLellan, Jason S.; Chen, Man; Chang, Jung-San; Yang, Yongping; Kim, Albert; Graham, Barney S.; Kwong, Peter D.

    2010-11-19

    Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants and elderly people. Currently there is no effective vaccine against RSV, but passive prophylaxis with neutralizing antibodies reduces hospitalizations. To investigate the mechanism of antibody-mediated RSV neutralization, we undertook structure-function studies of monoclonal antibody 101F, which binds a linear epitope in the RSV fusion glycoprotein. Crystal structures of the 101F antigen-binding fragment in complex with peptides from the fusion glycoprotein defined both the extent of the linear epitope and the interactions of residues that are mutated in antibody escape variants. The structure allowed for modeling of 101F in complex with trimers of the fusion glycoprotein, and the resulting models suggested that 101F may contact additional surfaces located outside the linear epitope. This hypothesis was supported by surface plasmon resonance experiments that demonstrated 101F bound the peptide epitope {approx}16,000-fold more weakly than the fusion glycoprotein. The modeling also showed no substantial clashes between 101F and the fusion glycoprotein in either the pre- or postfusion state, and cell-based assays indicated that 101F neutralization was not associated with blocking virus attachment. Collectively, these results provide a structural basis for RSV neutralization by antibodies that target a major antigenic site on the fusion glycoprotein.

  19. Global Panel of HIV-1 Env Reference Strains for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies

    PubMed Central

    deCamp, Allan; Hraber, Peter; Bailer, Robert T.; Seaman, Michael S.; Ochsenbauer, Christina; Kappes, John; Gottardo, Raphael; Edlefsen, Paul; Self, Steve; Tang, Haili; Greene, Kelli; Gao, Hongmei; Daniell, Xiaoju; Sarzotti-Kelsoe, Marcella; Gorny, Miroslaw K.; Zolla-Pazner, Susan; LaBranche, Celia C.; Mascola, John R.; Korber, Bette T.

    2014-01-01

    ABSTRACT Standardized assessments of HIV-1 vaccine-elicited neutralizing antibody responses are complicated by the genetic and antigenic variability of the viral envelope glycoproteins (Envs). To address these issues, suitable reference strains are needed that are representative of the global epidemic. Several panels have been recommended previously, but no clear answers have been available on how many and which strains are best suited for this purpose. We used a statistical model selection method to identify a global panel of reference Env clones from among 219 Env-pseudotyped viruses assayed in TZM-bl cells with sera from 205 HIV-1-infected individuals. The Envs and sera were sampled globally from diverse geographic locations and represented all major genetic subtypes and circulating recombinant forms of the virus. Assays with a panel size of only nine viruses adequately represented the spectrum of HIV-1 serum neutralizing activity seen with the larger panel of 219 viruses. An optimal panel of nine viruses was selected and augmented with three additional viruses for greater genetic and antigenic coverage. The spectrum of HIV-1 serum neutralizing activity seen with the final 12-virus panel closely approximated the activity seen with subtype-matched viruses. Moreover, the final panel was highly sensitive for detection of many of the known broadly neutralizing antibodies. For broader assay applications, all 12 Env clones were converted to infectious molecular clones using a proviral backbone carrying a Renilla luciferase reporter gene (Env.IMC.LucR viruses). This global panel should facilitate highly standardized assessments of vaccine-elicited neutralizing antibodies across multiple HIV-1 vaccine platforms in different parts of the world. IMPORTANCE An effective HIV-1 vaccine will need to overcome the extraordinary genetic variability of the virus, where most variation occurs in the viral envelope glycoproteins that are the sole targets for neutralizing antibodies

  20. Production of Cytomegalovirus Dense Bodies by Scalable Bioprocess Methods Maintains Immunogenicity and Improves Neutralizing Antibody Titers.

    PubMed

    Schneider-Ohrum, Kirsten; Cayatte, Corinne; Liu, Yi; Wang, Zhaoti; Irrinki, Alivelu; Cataniag, Floro; Nguyen, Nga; Lambert, Stacie; Liu, Hui; Aslam, Shahin; Duke, Greg; McCarthy, Michael P; McCormick, Louise

    2016-11-15

    With the goal of developing a virus-like particle-based vaccine based on dense bodies (DB) produced by human cytomegalovirus (HCMV) infections, we evaluated scalable culture, isolation, and inactivation methods and applied technically advanced assays to determine the relative purity, composition, and immunogenicity of DB particles. Our results increase our understanding of the benefits and disadvantages of methods to recover immunogenic DB and inactivate contaminating viral particles. Our results indicate that (i) HCMV strain Towne replicates in MRC-5 fibroblasts grown on microcarriers, (ii) DB particles recovered from 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB)-treated cultures and purified by tangential flow filtration (TFF-DB) or glycerol tartrate gradient sedimentation (GT-DB) constitute 92% or 98%, respectively, of all particles in the final product, (iii) epithelial cell-tropic DB particles are recovered from a single round of coinfection by AD169 and Towne strain viruses, consistent with complementation between the UL130 and UL131A expressed by these strains and restoration of gH/gL/UL128-UL131A (gH pentamer), (iv) equivalent neutralizing antibody titers are induced in mice following immunization with epithelial cell-tropic DB or gH pentamer-deficient DB preparations, (v) UV-inactivated residual virus in GT-DB or TFF-DB preparations retained immunogenicity and induced neutralizing antibody, preventing viral entry into epithelial cells, and (vi) GT-DB and TFF-DB induced cellular immune responses to multiple HCMV peptides. Collectively, this work provides a foundation for future development of DB as an HCMV-based particle vaccine.

  1. AAV natural infection induces broad cross-neutralizing antibody responses to multiple AAV serotypes in chimpanzees.

    PubMed

    Calcedo, Roberto; Wilson, James M

    2016-06-01

    Cross-sectional studies of primates have revealed that natural neutralizing antibody (NAb) responses to adeno-associated viruses (AAV) span multiple serotypes. This differs from the phenotype of the NAb response to an AAV vector delivered to sero-negative nonhuman primates which is typically restricted to the administered AAV serotype. To better understand the mechanism by which natural AAV infections result in broad NAb responses, we conducted a longitudinal study spanning 10 years in which we evaluated serum-circulating AAV NAb levels in captive-housed chimpanzees. In a cohort of 25 chimpanzees we identified three distinct groups of animals: those which never sero-converted to AAV (naïve); those which were persistently seropositive (chronic); and those that seroconverted during the 10 year period (acute). For the chronic group we found a broad sero-response characterized by NAbs reacting to multiple AAV serotypes. A similar cross-neutralization pattern of NAbs was observed in the acute group. These data support our hypothesis that a single natural infection with AAV induces a broadly cross-reactive NAb response to multiple AAV serotypes.

  2. AAV Natural Infection Induces Broad Cross-Neutralizing Antibody Responses to Multiple AAV Serotypes in Chimpanzees.

    PubMed

    Calcedo, Roberto; Wilson, James M

    2016-06-01

    Cross-sectional studies of primates have revealed that natural neutralizing antibody (NAb) responses to adeno-associated viruses (AAV) span multiple serotypes. This differs from the phenotype of the NAb response to an AAV vector delivered to seronegative nonhuman primates that is typically restricted to the administered AAV serotype. To better understand the mechanism by which natural AAV infections result in broad NAb responses, we conducted a longitudinal study spanning 10 years in which we evaluated serum-circulating AAV NAb levels in captive-housed chimpanzees. In a cohort of 25 chimpanzees we identified 3 distinct groups of animals: those that never seroconverted to AAV (naïve), those that were persistently seropositive (chronic), and those that seroconverted during the 10-year period (acute). For the chronic group we found a broad seroresponse characterized by NAbs reacting to multiple AAV serotypes. A similar cross-neutralization pattern of NAbs was observed in the acute group. These data support our hypothesis that a single natural infection with AAV induces a broadly cross-reactive NAb response to multiple AAV serotypes.

  3. Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20

    SciTech Connect

    McCraw, Dustin M.; O'Donnell, Jason K.; Taylor, Kenneth A.; Stagg, Scott M.; Chapman, Michael S.

    2012-09-15

    The use of adeno-associated virus (AAV) as a gene therapy vector is limited by the host neutralizing immune response. The cryo-electron microscopy (EM) structure at 8.5 A resolution is determined for a complex of AAV-2 with the Fab' fragment of monoclonal antibody (MAb) A20, the most extensively characterized AAV MAb. The binding footprint is determined through fitting the cryo-EM reconstruction with a homology model following sequencing of the variable domain, and provides a structural basis for integrating diverse prior epitope mappings. The footprint extends from the previously implicated plateau to the side of the spike, and into the conserved canyon, covering a larger area than anticipated. Comparison with structures of binding and non-binding serotypes indicates that recognition depends on a combination of subtle serotype-specific features. Separation of the neutralizing epitope from the heparan sulfate cell attachment site encourages attempts to develop immune-resistant vectors that can still bind to target cells.

  4. Structural basis for the neutralization of hepatitis E virus by a cross-genotype antibody

    PubMed Central

    Gu, Ying; Tang, Xuhua; Zhang, Xiao; Song, Cuiling; Zheng, Minghua; Wang, Kaihang; Zhang, Jun; Ng, Mun-Hon; Hew, Choy-Leong; Li, Shaowei; Xia, Ningshao; Sivaraman, J.

    2015-01-01

    Hepatitis E virus (HEV), a non-enveloped, positive-sense, single-stranded RNA virus, is a major cause of enteric hepatitis. Classified into the family Hepeviridae, HEV comprises four genotypes (genotypes 1-4), which belong to a single serotype. We describe a monoclonal antibody (mAb), 8G12, which equally recognizes all four genotypes of HEV, with ∼2.53–3.45 nM binding affinity. The mAb 8G12 has a protective, neutralizing capacity, which can significantly block virus infection in host cells. Animal studies with genotypes 1, 3 and 4 confirmed the cross-genotype neutralizing capacity of 8G12 and its effective prevention of hepatitis E disease. The complex crystal structures of 8G12 with the HEV E2s domain (the most protruded region of the virus capsid) of the abundant genotypes 1 and 4 were determined at 4.0 and 2.3 Å resolution, respectively. These structures revealed that 8G12 recognizes both genotypes through the epitopes in the E2s dimerization region. Structure-based mutagenesis and cell-model assays with virus-like particles identified several conserved residues (Glu549, Lys554 and Gly591) that are essential for 8G12 neutralization. Moreover, the epitope of 8G12 is identified as a key epitope involved in virus-host interactions. These findings will help develop a common strategy for the prevention of the most abundant form of HEV infection. PMID:25793314

  5. A Novel Chimeric Anti-PA Neutralizing Antibody for Postexposure Prophylaxis and Treatment of Anthrax.

    PubMed

    Xiong, Siping; Tang, Qi; Liang, Xudong; Zhou, Tingting; Yang, Jin; Liu, Peng; Chen, Ya; Wang, Changjun; Feng, Zhenqing; Zhu, Jin

    2015-07-02

    Anthrax is a highly lethal infectious disease caused by the bacterium Bacillus anthracis, and the associated shock is closely related to the lethal toxin (LeTx) produced by the bacterium. The central role played by the 63 kDa protective antigen (PA63) region of LeTx in the pathophysiology of anthrax makes it an excellent therapeutic target. In the present study, a human/murine chimeric IgG mAb, hmPA6, was developed by inserting murine antibody variable regions into human constant regions using antibody engineering technology. hmPA6 expressed in 293F cells could neutralize LeTx both in vitro and in vivo. At a dose of 0.3 mg/kg, it could protect all tested rats from a lethal dose of LeTx. Even administration of 0.6 mg/kg hmPA6 48 h before LeTx challenge protected all tested rats. The results indicate that hmPA6 is a potential candidate for clinical application in anthrax treatment.

  6. Immunologic characteristics of HIV-infected individuals who make broadly neutralizing antibodies.

    PubMed

    Borrow, Persephone; Moody, M Anthony

    2017-01-01

    Induction of broadly neutralizing antibodies (bnAbs) capable of inhibiting infection with diverse variants of human immunodeficiency virus type 1 (HIV-1) is a key, as-yet-unachieved goal of prophylactic HIV-1 vaccine strategies. However, some HIV-infected individuals develop bnAbs after approximately 2-4 years of infection, enabling analysis of features of these antibodies and the immunological environment that enables their induction. Distinct subsets of CD4(+) T cells play opposing roles in the regulation of humoral responses: T follicular helper (Tfh) cells support germinal center formation and provide help for affinity maturation and the development of memory B cells and plasma cells, while regulatory CD4(+) (Treg) cells including T follicular regulatory (Tfr) cells inhibit the germinal center reaction to limit autoantibody production. BnAbs exhibit high somatic mutation frequencies, long third heavy-chain complementarity determining regions, and/or autoreactivity, suggesting that bnAb generation is likely to be highly dependent on the activity of CD4(+) Tfh cells, and may be constrained by host tolerance controls. This review discusses what is known about the immunological environment during HIV-1 infection, in particular alterations in CD4(+) Tfh, Treg, and Tfr populations and autoantibody generation, and how this is related to bnAb development, and considers the implications for HIV-1 vaccine design.

  7. Molecular basis for epitope recognition by non-neutralizing anti-gp41 antibody F240.

    PubMed

    Gohain, Neelakshi; Tolbert, William D; Orlandi, Chiara; Richard, Jonathan; Ding, Shilei; Chen, Xishan; Bonsor, Daniel A; Sundberg, Eric J; Lu, Wuyuan; Ray, Krishanu; Finzi, Andrés; Lewis, George K; Pazgier, Marzena

    2016-11-09

    Antibody-dependent cell-mediated cytotoxicity (ADCC) by non-neutralizing antibodies (nnAbs) specific to the HIV envelope (Env) glycoproteins present at the surface of virus sensitized or infected cells plays a role in the effective adaptive immune response to HIV. Here, we explore the molecular basis for the epitope at the disulfide loop region (DLR) of the principal immunodominant domain of gp41, recognized by the well-known nnAb F240. Our structural studies reveal details of the F240-gp41 interface and describe a structure of DLR that is distinct from known conformations of this region studied in the context of either CD4-unliganded Env trimer or the gp41 peptide in the unbound state. These data coupled with binding and functional analyses indicate that F240 recognizes non-trimeric Env forms which are significantly overexpressed on intact virions but poorly represented at surfaces of cells infected with infectious molecular clones and endogenously-infected CD4 T cells from HIV-1-infected individuals. Furthermore, although we detect ADCC activities of F240 against cells spinoculated with intact virions, our data suggest that these activities result from F240 recognition of gp41 stumps or misfolded Env variants present on virions rather than its ability to recognize functional gp41 transition structures emerging on trimeric Env post CD4 receptor engagement.

  8. The HIV glycan shield as a target for broadly neutralizing antibodies.

    PubMed

    Doores, Katie J

    2015-12-01

    The HIV envelope glycoprotein (Env) is the sole target for HIV broadly neutralizing antibodies (bnAbs). HIV Env is one of the most heavily glycosylated proteins known, with approximately half of its mass consisting of host-derived N-linked glycans. The high density of glycans creates a shield that impedes antibody recognition but, critically, some of the most potent and broadly active bnAbs have evolved to recognize epitopes formed by these glycans. Although the virus hijacks the host protein synthesis and glycosylation machinery to generate glycosylated HIV Env, studies have shown that HIV Env glycosylation diverges from that typically observed on host-derived glycoproteins. In particular, the high density of glycans leads to a nonself motif of underprocessed oligomannose-type glycans that forms the target of some of the most broad and potent HIV bnAbs. This review discusses the changing perception of the HIV glycan shield, and summarizes the protein-directed and cell-directed factors controlling HIV Env glycosylation that impact on HIV bnAb recognition and HIV vaccine design strategies.

  9. Molecular basis for epitope recognition by non-neutralizing anti-gp41 antibody F240

    PubMed Central

    Gohain, Neelakshi; Tolbert, William D.; Orlandi, Chiara; Richard, Jonathan; Ding, Shilei; Chen, Xishan; Bonsor, Daniel A.; Sundberg, Eric J.; Lu, Wuyuan; Ray, Krishanu; Finzi, Andrés; Lewis, George K.; Pazgier, Marzena

    2016-01-01

    Antibody-dependent cell-mediated cytotoxicity (ADCC) by non-neutralizing antibodies (nnAbs) specific to the HIV envelope (Env) glycoproteins present at the surface of virus sensitized or infected cells plays a role in the effective adaptive immune response to HIV. Here, we explore the molecular basis for the epitope at the disulfide loop region (DLR) of the principal immunodominant domain of gp41, recognized by the well-known nnAb F240. Our structural studies reveal details of the F240-gp41 interface and describe a structure of DLR that is distinct from known conformations of this region studied in the context of either CD4-unliganded Env trimer or the gp41 peptide in the unbound state. These data coupled with binding and functional analyses indicate that F240 recognizes non-trimeric Env forms which are significantly overexpressed on intact virions but poorly represented at surfaces of cells infected with infectious molecular clones and endogenously-infected CD4 T cells from HIV-1-infected individuals. Furthermore, although we detect ADCC activities of F240 against cells spinoculated with intact virions, our data suggest that these activities result from F240 recognition of gp41 stumps or misfolded Env variants present on virions rather than its ability to recognize functional gp41 transition structures emerging on trimeric Env post CD4 receptor engagement. PMID:27827447

  10. Mimicry of an HIV broadly neutralizing antibody epitope with a synthetic glycopeptide.

    PubMed

    Alam, S Munir; Aussedat, Baptiste; Vohra, Yusuf; Ryan Meyerhoff, R; Cale, Evan M; Walkowicz, William E; Radakovich, Nathan A; Anasti, Kara; Armand, Lawrence; Parks, Robert; Sutherland, Laura; Scearce, Richard; Joyce, M Gordon; Pancera, Marie; Druz, Aliaksandr; Georgiev, Ivelin S; Von Holle, Tarra; Eaton, Amanda; Fox, Christopher; Reed, Steven G; Louder, Mark; Bailer, Robert T; Morris, Lynn; Abdool-Karim, Salim S; Cohen, Myron; Liao, Hua-Xin; Montefiori, David C; Park, Peter K; Fernández-Tejada, Alberto; Wiehe, Kevin; Santra, Sampa; Kepler, Thomas B; Saunders, Kevin O; Sodroski, Joseph; Kwong, Peter D; Mascola, John R; Bonsignori, Mattia; Moody, M Anthony; Danishefsky, Samuel; Haynes, Barton F

    2017-03-15

    A goal for an HIV-1 vaccine is to overcome virus variability by inducing broadly neutralizing antibodies (bnAbs). One key target of bnAbs is the glycan-polypeptide at the base of the envelope (Env) third variable loop (V3). We have designed and synthesized a homogeneous minimal immunogen with high-mannose glycans reflective of a native Env V3-glycan bnAb epitope (Man9-V3). V3-glycan bnAbs bound to Man9-V3 glycopeptide and native-like gp140 trimers with similar affinities. Fluorophore-labeled Man9-V3 glycopeptides bound to bnAb memory B cells and were able to be used to isolate a V3-glycan bnAb from an HIV-1-infected individual. In rhesus macaques, immunization with Man9-V3 induced V3-glycan-targeted antibodies. Thus, the Man9-V3 glycopeptide closely mimics an HIV-1 V3-glycan bnAb epitope and can be used to isolate V3-glycan bnAbs.

  11. All eyes on the next generation of HIV vaccines: strategies for inducing a broadly neutralizing antibody response.

    PubMed

    Ahlers, Jeffrey D

    2014-04-01

    HIV-1 broadly neutralizing antibodies (BNAbs) develop after several years of infection through a recursive process of memory B cell adaptation and maturation against co-evolving virus quasispecies. Advances in single-cell sorting and memory B cell antibody cloning methods have identified many new HIV BNAbs targeting conserved epitopes on the HIV envelope (env) protein. 3D crystal structures and biophysical analyses of BNAbs bound to invariant virus structures expressed on monomeric gp120, epitope scaffolds, core structures, and native trimers have helped us to visualize unique binding interactions and paratope orientations that have been instrumental in guiding vaccine design. A paradigm shift in the approach to structure-based design of HIV-1 envelope immunogens came recently after several laboratories discovered that native viral envelopes or "env-structures" reverse-engineered to bind with high affinity to a handful of broadly neutralizing antibodies did not in fact bind the predicted germline precursors of these broadly neutralizing antibodies. A major challenge for HIV-1 B cell vaccine development moving forward is the design of new envelope immunogens that can trigger the selection and expansion of germline precursor and intermediate memory B cells to recapitulate B cell ontogenies associated with the maturation of a broadly neutralizing antibody response. Equally important for vaccine development is the identification of delivery systems, prime-boost strategies, and synergistic adjuvant combinations that can induce the magnitude and quality of antigen-specific T follicular helper (TFH) cell responses needed to drive somatic hypermutation (SHM) and B cell maturation against heterologous primary virus envelopes. Finding the combination of multi-protein envelope immunogens and immunization strategies that can evolve a potent broadly neutralizing antibody response portends to require a complex vaccine regimen that might be difficult to implement on any scale

  12. Novel neutralizing hedgehog antibody MEDI-5304 exhibits antitumor activity by inhibiting paracrine hedgehog signaling.

    PubMed

    Michaud, Neil R; Wang, Youzhen; McEachern, Kristen A; Jordan, Jerold J; Mazzola, Anne Marie; Hernandez, Axel; Jalla, Sanjoo; Chesebrough, Jon W; Hynes, Mark J; Belmonte, Matthew A; Wang, Lidong; Kang, Jaspal S; Jovanovic, Jelena; Laing, Naomi; Jenkins, David W; Hurt, Elaine; Liang, Meina; Frantz, Christopher; Hollingsworth, Robert E; Simeone, Diane M; Blakey, David C; Bedian, Vahe

    2014-02-01

    The hedgehog pathway has been implicated in the tumorigenesis, tumor progression, and metastasis of numerous human cancers. We generated the first fully human hedgehog antibody MEDI-5304 and characterized its antitumor activity and preclinical toxicology. MEDI-5304 bound sonic hedgehog (SHH) and Indian hedgehog (IHH) with low picomolar affinity and neutralized SHH and IHH activity in cellular mGLI1 reporter assays. The antibody inhibited transcription of hedgehog target genes and osteoblast differentiation of C3H10T1/2 cells. We evaluated the activity of MEDI-5304 in vivo in model systems that allowed us to evaluate two primary hypotheses of hedgehog function in human cancer, paracrine signaling between tumor and stromal cells and cancer stem cell (CSC) self-renewal. MEDI-5304 displayed robust pharmacodynamic effects in stromal cells that translated to antitumor efficacy as a single agent in an HT-29/MEF coimplantation model of paracrine hedgehog signaling. MEDI-5304 also improved responses to carboplatin in the HT-29/MEF model. The antibody, however, had no effect as a single agent or in combination with gemcitabine on the CSC frequency or growth of several primary pancreatic cancer explant models. These findings support the conclusion that hedgehog contributes to tumor biology via paracrine tumor-stromal signaling but not via CSC maintenance or propagation. Finally, the only safety study finding associated with MEDI-5304 was ondontodysplasia in rats. Thus, MEDI-5304 represents a potent dual hedgehog inhibitor suitable for continued development to evaluate efficacy and safety in human patients with tumors harboring elevated levels of SHH or IHH.

  13. Low frequency of broadly neutralizing HIV antibodies during chronic infection even in quaternary epitope targeting antibodies containing large numbers of somatic mutations.

    PubMed

    Hicar, Mark D; Chen, Xuemin; Kalams, Spyros A; Sojar, Hakimuddin; Landucci, Gary; Forthal, Donald N; Spearman, Paul; Crowe, James E

    2016-02-01

    Neutralizing antibodies (Abs) are thought to be a critical component of an appropriate HIV vaccine response. It has been proposed that Abs recognizing conformationally dependent quaternary epitopes on the HIV envelope (Env) trimer may be necessary to neutralize diverse HIV strains. A number of recently described broadly neutralizing monoclonal Abs (mAbs) recognize complex and quaternary epitopes. Generally, many such Abs exhibit extensive numbers of somatic mutations and unique structural characteristics. We sought to characterize the native antibody (Ab) response against circulating HIV focusing on such conformational responses, without a prior selection based on neutralization. Using a capture system based on VLPs incorporating cleaved envelope protein, we identified a selection of B cells that produce quaternary epitope targeting Abs (QtAbs). Similar to a number of broadly neutralizing Abs, the Ab genes encoding these QtAbs showed extensive numbers of somatic mutations. However, when expressed as recombinant molecules, these Abs failed to neutralize virus or mediate ADCVI activity. Molecular analysis showed unusually high numbers of mutations in the Ab heavy chain framework 3 region of the variable genes. The analysis suggests that large numbers of somatic mutations occur in Ab genes encoding HIV Abs in chronically infected individuals in a non-directed, stochastic, manner.

  14. Low frequency of broadly neutralizing HIV antibodies during chronic infection even in quaternary epitope targeting antibodies containing large numbers of somatic mutations

    PubMed Central

    Hicar, Mark D.; Chen, Xuemin; Kalams, Spyros A.; Sojar, Hakimuddin; Landucci, Gary; Forthal, Donald N.; Spearman, Paul; Crowe, James E.

    2016-01-01

    Neutralizing antibodies (Abs) are thought to be a critical component of an appropriate HIV vaccine response. It has been proposed that Abs recognizing conformationally dependent quaternary epitopes on the HIV envelope (Env) trimer may be necessary to neutralize diverse HIV strains. A number of recently described broadly neutralizing monoclonal Abs (mAbs) recognize complex and quaternary epitopes. Generally, many such Abs exhibit extensive numbers of somatic mutations and unique structural characteristics. We sought to characterize the native antibody (Ab) response against circulating HIV focusing on such conformational responses, without a prior selection based on neutralization. Using a capture system based on VLPs incorporating cleaved envelope protein, we identified a selection of B cells that produce quaternary epitope targeting Abs (QtAbs). Similar to a number of broadly neutralizing Abs, the Ab genes encoding these QtAbs showed extensive numbers of somatic mutations. However, when expressed as recombinant molecules, these Abs failed to neutralize virus or mediate ADCVI activity. Molecular analysis showed unusually high numbers of mutations in the Ab heavy chain framework 3 region of the variable genes. The analysis suggests that large numbers of somatic mutations occur in Ab genes encoding HIV Abs in chronically infected individuals in a non-directed, stochastic, manner. PMID:26748387

  15. Promiscuous glycan site recognition by antibodies to the high-mannose patch of gp120 broadens neutralization of HIV

    PubMed Central

    Sok, Devin; Doores, Katie J.; Briney, Bryan; Le, Khoa M.; Saye-Francisco, Karen F.; Ramos, Alejandra; Kulp, Daniel W.; Julien, Jean-Philippe; Menis, Sergey; Wickramasinghe, Lalinda; Seaman, Michael S.; Schief, William R.; Wilson, Ian A.; Poignard, Pascal; Burton, Dennis R.

    2014-01-01

    Broadly neutralizing monoclonal antibodies (bnMAbs) that target the high-mannose patch centered around the glycan at position 332 on HIV Env are promising vaccine leads and therapeutic candidates as they effectively protect against mucosal SHIV challenge and strongly suppress SHIV viraemia in established infection in macaque models. However, these antibodies demonstrate varying degrees of dependency on the N332 glycan site and the origins of their neutralization breadth are not always obvious. By measuring neutralization on an extended range of glycan site viral variants, we found that some bnMAbs can utilize alternate N-linked glycans in the absence of the N332 glycan site and therefore neutralize a substantial number of viruses lacking the site. Furthermore, many of the antibodies can neutralize viruses in which the N332 glycan site is shifted to the 334 position. Finally, we found that a combination of three antibody families that target the high-mannose patch can lead to 99% neutralization coverage of a large panel of viruses containing the N332/334 glycan site and up to 66% coverage for viruses that lack the N332/334 glycan site. The results indicate that a diverse response against the high-mannose patch may provide near equivalent coverage as a combination of bnMAbs targeting multiple epitopes. Additionally, the ability of some bnMAbs to utilize other N-linked glycan sites can help counter neutralization escape mediated by shifting of glycosylation sites. Overall, this work highlights the importance of promiscuous glycan binding properties in bnMAbs to the high-mannose patch for optimal anti-viral activity either in protective or therapeutic modalities. PMID:24828077

  16. Bispecific antibodies targeting tumor-associated antigens and neutralizing complement regulators increase the efficacy of antibody-based immunotherapy in mice.

    PubMed

    Macor, P; Secco, E; Mezzaroba, N; Zorzet, S; Durigutto, P; Gaiotto, T; De Maso, L; Biffi, S; Garrovo, C; Capolla, S; Tripodo, C; Gattei, V; Marzari, R; Tedesco, F; Sblattero, D

    2015-02-01

    The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, polymorphonuclear and natural killer cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications.

  17. Highly efficient neutralization by plasma antibodies from human immunodeficiency virus type-1 infected individuals on antiretroviral drug therapy.

    PubMed

    Andrabi, Raiees; Makhdoomi, M A; Kumar, Rajesh; Bala, Manju; Parray, Hilal; Gupta, Arjun; Kotnala, Ankita; Thirumurthy, Velpandian; Luthra, Kalpana

    2014-05-01

    Little is known about the neutralizing antibodies induced in HIV-1 patients on antiretroviral treatment, which constitute an interesting group of individuals with improved B cell profile. Plasma samples from 34 HIV-1 seropositive antiretroviral drug treated (ART) patients were tested for neutralization against a panel of 14 subtype-A, B and C tier 1 and tier 2 viruses in TZM-bl assay. Of the 34 plasma samples, remarkably all the plasma samples were able to neutralize at least one virus while 32 (94 %) were found to neutralize ≥50 % viruses tested. In terms of overall neutralization frequency, approximately 86 %, 68 % and 17 % of the virus/plasma combinations showed 50 % neutralizing activity at 1 > 60, 1 ≥ 200 and 1 ≥ 2000 dilutions respectively. The improvement in neutralizing activity was shown to be associated with ART in two follow up patients. The neutralization of viruses by two representative plasma samples, AIIMS221 and AIIMS265, was exclusively mediated by immunoglobulin G fractions independent of ART drugs and IgG retained cross-reactive binding to recombinant gp120 proteins. We observed a positive trend of neutralization with duration of ART (p = 0.06), however no such correlation was found with clinical and immunological variables like CD4 count (p = 0.35), viral load (p = 0.09) and plasma total IgG (p = 0.46). Our study suggests that the plasma antibodies from ART patients display high neutralizing activity most likely due to an improved B cell function induced by ART despite low antigenic stimulation.

  18. In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

    PubMed Central

    Richard, Gabrielle; Meyers, Ashley J.; McLean, Michael D.; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J. Christopher

    2013-01-01

    Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

  19. Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

    SciTech Connect

    Hashem, Anwar M.; Van Domselaar, Gary; Li, Changgui; Wang, Junzhi; She, Yi-Min; Cyr, Terry D.; Sui, Jianhua; He, Runtao; Marasco, Wayne A.; Li, Xuguang

    2010-12-10

    Research highlights: {yields} The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. {yields} Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. {yields} The universal antibodies cross-neutralize different influenza A subtypes. {yields} The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.

  20. Competitive detection of influenza neutralizing antibodies using a novel bivalent fluorescence-based microneutralization assay (BiFMA).

    PubMed

    Baker, Steven F; Nogales, Aitor; Santiago, Felix W; Topham, David J; Martínez-Sobrido, Luis

    2015-07-09

    Avian-derived influenza A zoonoses are closely monitored and may be an indication of virus strains with pandemic potential. Both successful vaccination and convalescence of influenza A virus in humans typically results in the induction of antibodies that can neutralize viral infection. To improve long-standing and new-generation methodologies for detection of neutralizing antibodies, we have employed a novel reporter-based approach that allows for multiple antigenic testing within a single sample. Central to this approach is a single-cycle infectious influenza A virus (sciIAV), where a functional hemagglutinin (HA) gene was changed to encode either the green or the monomeric red fluorescent protein (GFP and mRFP, respectively) and HA is complemented in trans by stable HA-expressing cell lines. By using fluorescent proteins with non-overlapping emission spectra, this novel bivalent fluorescence-based microneutralization assay (BiFMA) can be used to detect neutralizing antibodies against two distinct influenza isolates in a single reaction, doubling the speed of experimentation while halving the amount of sera required. Moreover, this approach can be used for the rapid identification of influenza broadly neutralizing antibodies. Importantly, this novel BiFMA can be used for any given influenza HA-pseudotyped virus under BSL-2 facilities, including highly pathogenic influenza HA isolates.

  1. Neutralizing Antibodies against IFN-[Beta] in Multiple Sclerosis: Antagonization of IFN-[Beta] Mediated Suppression of MMPs

    ERIC Educational Resources Information Center

    Gilli, Francesca; Bertolotto, Antonio; Sala, Arianna; Hoffmann, Francine; Capobianco, Marco; Malucchi, Simona; Glass, Tracy; Kappos, Ludwig; Lindberg, Raija L. P.; Leppert, David

    2004-01-01

    Neutralizing antibodies (NAb) against interferon-[Beta] (IFN-Beta) develop in about a third of treated multiple sclerosis patients and are believed to reduce therapeutic efficacy of IFN-[Beta] on clinical and MRI measures. The expression of the interferon acute-response protein, myxovirus resistance protein A (MxA) is a sensitive measure of the…

  2. A New Quaternary Structure Epitope on Dengue Virus Serotype 2 Is the Target of Durable Type-Specific Neutralizing Antibodies

    PubMed Central

    Gallichotte, E. N.; Widman, D. G.; Yount, B. L.; Wahala, W. M.; Durbin, A.; Whitehead, S.; Sariol, C. A.; Crowe, J. E.

    2015-01-01

    ABSTRACT Dengue virus serotype 2 (DENV2) is widespread and responsible for severe epidemics. While primary DENV2 infections stimulate serotype-specific protective responses, a leading vaccine failed to induce a similar protective response. Using human monoclonal antibodies (hMAbs) isolated from dengue cases and structure-guided design of a chimeric DENV, here we describe the major site on the DENV2 envelope (E) protein targeted by neutralizing antibodies. DENV2-specific neutralizing hMAb 2D22 binds to a quaternary structure epitope. We engineered and recovered a recombinant DENV4 that displayed the 2D22 epitope. DENV2 neutralizing antibodies in people exposed to infection or a live vaccine tracked with the 2D22 epitope on the DENV4/2 chimera. The chimera remained sensitive to DENV4 antibodies, indicating that the major neutralizing epitopes on DENV2 and -4 are at different sites. The ability to transplant a complex epitope between DENV serotypes demonstrates a hitherto underappreciated structural flexibility in flaviviruses, which could be harnessed to develop new vaccines and diagnostics. PMID:26463165

  3. The Neutralizing Capacity of Antibodies Elicited by Parainfluenza Virus Infection of African Green Monkeys is Dependent on Complement

    PubMed Central

    Mayer, Anne E.; Johnson, John B.; Parks, Griffith D.

    2014-01-01

    The African Green Monkey (AGM) model was used to analyze the role of complement in neutralization of parainfluenza virus. Parainfluenza virus 5 (PIV5) and human parainfluenza virus type 2 were effectively neutralized in vitro by naïve AGM sera, but neutralizing capacity was lost by heat-inactivation. The mechanism of neutralization involved formation of massive aggregates, with no evidence of virion lysis. Following inoculation of the respiratory tract with a PIV5 vector expressing HIV gp160, AGM produced high levels of serum and tracheal antibodies against gp120 and the viral F and HN proteins. However, in the absence of complement these anti-PIV5 antibodies had very poor neutralizing capacity. Virions showed extensive deposition of IgG and C1q with post- but not pre-immune sera. These results highlight the importance of complement in the initial antibody response to parainfluenza viruses, with implications for understanding infant immune responses and design of vaccine strategies for these pediatric pathogens. PMID:25010267

  4. Virus-neutralizing antibody response of mice to consecutive infection with human and avian influenza A viruses.

    PubMed

    Janulíková, J; Stropkovská, A; Bobišová, Z; Košík, I; Mucha, V; Kostolanský, F; Varečková, E

    2015-06-01

    In this work we simulated in a mouse model a naturally occurring situation of humans, who overcame an infection with epidemic strains of influenza A, and were subsequently exposed to avian influenza A viruses (IAV). The antibody response to avian IAV in mice previously infected with human IAV was analyzed. We used two avian IAV (A/Duck/Czechoslovakia/1956 (H4N6) and the attenuated virus rA/Viet Nam/1203-2004 (H5N1)) as well as two human IAV isolates (virus A/Mississippi/1/1985 (H3N2) of medium virulence and A/Puerto Rico/8/1934 (H1N1) of high virulence). Two repeated doses of IAV of H4 or of H5 virus elicited virus-specific neutralizing antibodies in mice. Exposure of animals previously infected with human IAV (of H3 or H1 subtype) to IAV of H4 subtype led to the production of antibodies neutralizing H4 virus in a level comparable with the level of antibodies against the human IAV used for primary infection. In contrast, no measurable levels of virus-neutralizing (VN) antibodies specific to H5 virus were detected in mice infected with H5 virus following a previous infection with human IAV. In both cases the secondary infection with avian IAV led to a significant increase of the titer of VN antibodies specific to the corresponding human virus used for primary infection. Moreover, cross-reactive HA2-specific antibodies were also induced by sequential infection. By virtue of these results we suggest that the differences in the ability of avian IAV to induce specific antibodies inhibiting virus replication after previous infection of mice with human viruses can have an impact on the interspecies transmission and spread of avian IAV in the human population.

  5. Neutralizing Antibodies Against Adeno-Associated Viral Capsids in Patients with mut Methylmalonic Acidemia.

    PubMed

    Harrington, Elizabeth A; Sloan, Jennifer L; Manoli, Irini; Chandler, Randy J; Schneider, Mark; McGuire, Peter J; Calcedo, Roberto; Wilson, James M; Venditti, Charles P

    2016-05-01

    Isolated methylmalonic acidemia (MMA), a group of autosomal recessive inborn errors of metabolism, is most commonly caused by complete (mut(0)) or partial (mut(-)) deficiency of the enzyme methylmalonyl-CoA mutase (MUT). The severe metabolic instability and increased mortality experienced by many affected individuals, especially those with mut(0) MMA, has led centers to use elective liver transplantation as a treatment for these patients. We have previously demonstrated the efficacy of systemic adeno-associated viral (AAV) gene delivery as a treatment for MMA in a murine model and therefore sought to survey AAV antibody titers against serotypes 2, 8, and 9 in a group of well-characterized MMA patients, accrued via a dedicated natural history study ( clinicaltrials.gov ID: NCT00078078). Plasma samples provided by 42 patients (8 mut(-) and 34 mut(0); 10 had received organ transplantation), who ranged in age between 2 and 31 years, were analyzed to examine AAV2 (n = 35), AAV8 (n = 41), and AAV9 (n = 42) antibody titers. In total, the seroprevalence of antibodies against AAV2, AAV8, or AAV9 was 20%, 22%, and 24%, respectively. We observed a lower-than-expected seropositivity rate (titers ≥1:20) in the pediatric MMA patients (2-18 years) for both AAV2 (p < 0.05) and AAV8 (p < 0.01) neutralizing antibodies (NAbs) compared with historical controls. Those with positive NAb titers were typically older than 18 years (p < 0.05 all serotypes) or had received solid organ transplantation (p < 0.01 AAV8, AAV9). The mut(0) patients who had not been transplanted (n = 24)-that is, the subset with the greatest need for improved treatments-represented the seronegative majority, with 21 out of 24 patients lacking Abs against all AAV capsids tested. The unexpected lack of NAbs against AAV in this patient population has encouraging implications for systemic gene delivery as a treatment for mut MMA.

  6. Neutralizing antibodies to HIV-1 envelope protect more effectively in vivo than those to the CD4 receptor

    PubMed Central

    Pegu, Amarendra; Yang, Zhi-yong; Boyington, Jeffrey C.; Wu, Lan; Ko, Sung-Youl; Schmidt, Stephen D.; McKee, Krisha; Kong, Wing-Pui; Shi, Wei; Chen, Xuejun; Todd, John-Paul; Huang, Jinghe; Nason, Martha C.; Hoxie, James A.; Kwong, Peter D.; Connors, Mark; Rao, Srinivas S.; Mascola, John R.; Nabel, Gary J.

    2015-01-01

    HIV-1 infection depends on effective viral entry mediated by the interaction of its envelope (Env) glycoprotein with specific cell surface receptors. Protective antiviral antibodies generated by passive or active immunization must prevent these interactions. Because the HIV-1 Env is highly variable, attention has also focused on blocking the HIV-1 primary cell receptor CD4. We therefore analyzed the in vivo protective efficacy of three potent neutralizing monoclonal antibodies (mAbs) to HIV-1 Env compared to an antibody against the CD4 receptor. Protection was assessed after mucosal challenge of rhesus macaques with simian/HIV (SHIV). Despite its comparable or greater neutralization potency in vitro, the anti-CD4 antibody did not provide effective protection in vivo, whereas the HIV-1–specific mAbs VRC01, 10E8, and PG9, targeting the CD4 binding site, membrane-proximal, and V1V2 glycan Env regions, respectively, conferred complete protection, albeit at different relative potencies. These findings demonstrate the protective efficacy of broadly neutralizing antibodies directed to the HIV-1 Env and suggest that targeting the HIV-1 Env is preferable to the cell surface receptor CD4 for the prevention of HIV-1 transmission. PMID:24990883

  7. Immunization routes in cattle impact the levels and neutralizing capacity of antibodies induced against S. aureus immune evasion proteins.

    PubMed

    Boerhout, Eveline; Vrieling, Manouk; Benedictus, Lindert; Daemen, Ineke; Ravesloot, Lars; Rutten, Victor; Nuijten, Piet; van Strijp, Jos; Koets, Ad; Eisenberg, Susanne

    2015-09-28

    Vaccines against S. aureus bovine mastitis are scarce and show limited protection only. All currently available vaccines are applied via the parenteral (usually intramuscular) route. It is unknown, however, whether this route is the most suitable to specifically increase intramammary immunity to combat S. aureus at the site of infection. Hence, in the present study, immunization via mucosal (intranasal; IN), intramuscular (triangle of the neck; IM), intramammary (IMM) and subcutaneous (suspensory ligament; SC) routes were analyzed for their effects on the quantity of the antibody responses in serum and milk as well as the neutralizing capacity of the antibodies within serum. The experimental vaccine comprised the recombinant S. aureus immune evasion proteins extracellular fibrinogen-binding protein (Efb) and the leukotoxin subunit LukM in an oil-in-water adjuvant combined with a hydrogel and alginate. The highest titer increases for both Efb and LukM specific IgG1 and IgG2 antibody levels in serum and milk were observed following SC/SC immunizations. Furthermore, the harmful effects of Efb and leukotoxin LukMF' on host-defense were neutralized by serum antibodies in a route-dependent manner. SC/SC immunization resulted in a significant increase in the neutralizing capacity of serum antibodies towards Efb and LukMF', shown by increased phagocytosis of S. aureus and increased viability of bovine leukocytes. Therefore, a SC immunization route should be considered when aiming to optimize humoral immunity against S. aureus mastitis in cattle.

  8. Induction of neutralizing antibodies by a tobacco chloroplast-derived vaccine based on a B cell epitope from canine parvovirus

    SciTech Connect

    Molina, Andrea; Veramendi, Jon . E-mail: jon@unavarra.es; Hervas-Stubbs, Sandra

    2005-11-25

    The 2L21 epitope of the VP2 protein from the canine parvovirus (CPV), fused to the cholera toxin B subunit (CTB-2L21), was expressed in transgenic tobacco chloroplasts. Mice and rabbits that received protein-enriched leaf extracts by parenteral route produced high titers of anti-2L21 antibodies able to recognize the VP2 protein. Rabbit sera were able to neutralize CPV in an in vitro infection assay with an efficacy similar to the anti-2L21 neutralizing monoclonal antibody 3C9. Anti-2L21 IgG and seric IgA antibodies were elicited when mice were gavaged with a suspension of pulverized tissues from CTB-2L21 transformed plants. Combined immunization (a single parenteral injection followed by oral boosters) shows that oral boosters help to maintain the anti-2L21 IgG response induced after a single injection, whereas parenteral administration of the antigen primes the subsequent oral boosters by promoting the induction of anti-2L21 seric IgA antibodies. Despite the induced humoral response, antibodies elicited by oral delivery did not show neutralizing capacity in the in vitro assay. The high yield of the fusion protein permits the preparation of a high number of vaccine doses from a single plant and makes feasible the oral vaccination using a small amount of crude plant material. However, a big effort has still to be done to enhance the protective efficacy of subunit vaccines by the oral route.

  9. Rapid assessment of antibody-induced ricin neutralization by employing a novel functional cell-based assay.

    PubMed

    Gal, Yoav; Alcalay, Ron; Sabo, Tamar; Noy-Porat, Tal; Epstein, Eyal; Kronman, Chanoch; Mazor, Ohad

    2015-09-01

    Ricin is one of the most potent and lethal toxins known against which there is no available antidote. Currently, the most promising countermeasures against the toxin are based on neutralizing antibodies elicited by active vaccination or administered passively. A cell-based assay is widely applied for the primary screening and evaluation of anti-ricin antibodies, yet such assays are usually time-consuming (18-72 h). Here, we report of a novel assay to monitor ricin activity, based on HeLa cells that stably express the rapidly-degraded ubiquitin-luciferase (Ub-FL, half-life of 2 min). Ricin-induced arrest of protein synthesis could be quantified within 3 to 6h post intoxication (IC90 of 300 and 100 ng/ml, respectively). Furthermore, by stabilizing the intracellular levels of Ub-FL in the last hour of the assay, a 3-fold increase in the assay sensitivity was attained. We applied this assay to monitor the efficacy of a ricin holotoxin-based vaccine by measuring the formation of neutralizing antibodies throughout the immunization course. The potency of anti-ricin monoclonal antibodies (directed to either subunit of the toxin) could also be easily and accurately measured in this assay format. Owing to its simplicity, this assay may be implemented for high-throughput screening of ricin-neutralizing antibodies and for identification of small-molecule inhibitors of the toxin, as well as other ribosome-inactivating toxins.

  10. Cooperativity Between CD8+ T Cells, Non-Neutralizing Antibodies, and Alveolar Macrophages Is Important for Heterosubtypic Influenza Virus Immunity

    PubMed Central

    Laidlaw, Brian J.; Decman, Vilma; Ali, Mohammed-Alkhatim A.; Abt, Michael C.; Wolf, Amaya I.; Monticelli, Laurel A.; Mozdzanowska, Krystyna; Angelosanto, Jill M.; Artis, David; Erikson, Jan; Wherry, E. John

    2013-01-01

    Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential “universal” vaccine. PMID:23516357

  11. Identification of a CD4-Binding-Site Antibody to HIV that Evolved Near-Pan Neutralization Breadth

    SciTech Connect

    Huang, Jinghe; Kang, Byong H.; Ishida, Elise; Zhou, Tongqing; Griesman, Trevor; Sheng, Zizhang; Wu, Fan; Doria-Rose, Nicole A.; Zhang, Baoshan; McKee, Krisha; O’Dell, Sijy; Chuang, Gwo-Yu; Druz, Aliaksandr; Georgiev, Ivelin S.; Schramm, Chaim A.; Zheng, Anqi; Joyce, M.  Gordon; Asokan, Mangaiarkarasi; Ransier, Amy; Darko, Sam; Migueles, Stephen A.; Bailer, Robert T.; Louder, Mark K.; Alam, S.  Munir; Parks, Robert; Kelsoe, Garnett; Von Holle, Tarra; Haynes, Barton F.; Douek, Daniel C.; Hirsch, Vanessa; Seaman, Michael S.; Shapiro, Lawrence; Mascola, John R.; Kwong, Peter D.; Connors, Mark

    2016-11-01

    Detailed studies of the broadly neutralizing antibodies (bNAbs) that underlie the best available examples of the humoral immune response to HIV are providing important information for the development of therapies and prophylaxis for HIV-1 infection. Here, we report a CD4-binding site (CD4bs) antibody, named N6, that potently neutralized 98% of HIV-1 isolates, including 16 of 20 that were resistant to other members of its class. N6 evolved a mode of recognition such that its binding was not impacted by the loss of individual contacts across the immunoglobulin heavy chain. In addition, structural analysis revealed that the orientation of N6 permitted it to avoid steric clashes with glycans, which is a common mechanism of resistance. Thus, an HIV-1-specific bNAb can achieve potent, near-pan neutralization of HIV-1, making it an attractive candidate for use in therapy and prophylaxis.

  12. Protective levels of canine distemper virus antibody in an urban dog population using plaque reduction neutralization test.

    PubMed

    Oyedele, O I; Oluwayelu, D O; Cadmus, S I B; Odemuyiwa, S O; Adu, F D

    2004-09-01

    Blood samples from 50 dogs were collected at three veterinary clinics in Ibadan and Abuja, Nigeria and the serum from each sample was evaluated serologically for neutralizing antibodies against canine distemper virus (CDV) by the highly sensitive plaque reduction (PRN) neutralization assay. Thirteen dogs had plaque reduction neutralization titres of 0-100, seven had titres of 100-1,000 while 30 had titres ranging from 1,000-6,000. The PRN titres of vaccinated dogs were found to be significantly higher than unvaccinated dogs. The widespread use of the highly reproducible PRN test for the evaluation of antibody response to CDV may be very important in the generation of international CDV positive serum standards that should help to improve pre-and post-vaccination testing of dogs worldwide.

  13. Neutralizing Antibodies Induced by Recombinant Virus-Like Particles of Enterovirus 71 Genotype C4 Inhibit Infection at Pre- and Post-attachment Steps

    PubMed Central

    Ku, Zhiqiang; Ye, Xiaohua; Huang, Xulin; Cai, Yicun; Liu, Qingwei; Li, Yan; Su, Zhiguo; Huang, Zhong

    2013-01-01

    Background Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia–Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear. Methods/Findings In the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs). Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC) located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment. Conclusions Collectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development. PMID:23451250

  14. Limited or no protection by weakly or nonneutralizing antibodies against vaginal SHIV challenge of macaques compared with a strongly neutralizing antibody.

    PubMed

    Burton, Dennis R; Hessell, Ann J; Keele, Brandon F; Klasse, Per Johan; Ketas, Thomas A; Moldt, Brian; Dunlop, D Cameron; Poignard, Pascal; Doyle, Lara A; Cavacini, Lisa; Veazey, Ronald S; Moore, John P

    2011-07-05

    To guide vaccine design, we assessed whether human monoclonal antibodies (MAbs) b12 and b6 against the CD4 binding site (CD4bs) on HIV-1 gp120 and F240 against an immundominant epitope on gp41 could prevent vaginal transmission of simian HIV (SHIV)-162P4 to macaques. The two anti-gp120 MAbs have similar monomeric gp120-binding properties, measured in vitro, but b12 is strongly neutralizing and b6 is not. F240 is nonneutralizing. Applied vaginally at a high dose, the strongly neutralizing MAb b12 provided sterilizing immunity in seven of seven animals, b6 in zero of five animals, and F240 in two of five animals. Compared with control animals, the protection by b12 achieved statistical significance, whereas that caused by F240 did not. For two of three unprotected F240-treated animals there was a trend toward lowered viremia. The potential protective effect of F240 may relate to the relatively strong ability of this antibody to capture infectious virions. Additional passive transfer experiments also indicated that the ability of the administered anti-gp120 MAbs to neutralize the challenge virus was a critical influence on protection. Furthermore, when data from all of the experiments were combined, there was a significant increase in the number of founder viruses establishing infection in animals receiving MAb b6, compared with other nonprotected macaques. Thus, a gp120-binding, weakly neutralizing MAb to the CD4bs was, at best, completely ineffective at protection. A nonneutralizing antibody to gp41 may have a limited capacity to protect, but the results suggest that the central focus of HIV-1 vaccine research should be on the induction of potently neutralizing antibodies.

  15. Prevalence of Neutralizing Antibodies to Japanese Encephalitis Virus among High-Risk Age Groups in South Korea, 2010.

    PubMed

    Lee, Eun Ju; Cha, Go-Woon; Ju, Young Ran; Han, Myung Guk; Lee, Won-Ja; Jeong, Young Eui

    2016-01-01

    After an extensive vaccination policy, Japanese encephalitis (JE) was nearly eliminated since the mid-1980s in South Korea. Vaccination in children shifted the affected age of JE patients from children to adults. However, an abrupt increase in JE cases occurred in 2010, and this trend has continued. The present study aimed to investigate the prevalence of neutralizing antibodies to the JE virus (JEV) among high-risk age groups (≥40 years) in South Korea. A plaque reduction neutralization test was conducted to evaluate the prevalence of neutralizing antibodies to JEV in 945 subjects within four age groups (30-39, 40-49, 50-59, and 60-69 years) in 10 provinces. Of the 945 enrolled subjects, 927 (98.1%) exhibited antibodies against JEV. No significant differences were found in the prevalence of neutralizing antibodies according to sex, age, or occupation. However, there were significant differences in the plaque reduction rate according to age and occupation; oldest age group had a higher reduction rate, and subjects who were employed in agriculture or forestry also had a higher value than the other occupations. We also found that three provinces (Gangwon, Jeonnam, and Gyeongnam) had a relatively lower plaque reduction rate than the other locations. In addition, enzyme-linked immunosorbent assays were conducted to determine recent viral infections and 12 (1.3%) subjects were found to have been recently infected by the virus [corrected]. In conclusion, the present study clearly indicated that the prevalence of neutralizing antibodies has been maintained at very high levels among adult age groups owing to vaccination or natural infections, or both. In the future, serosurveillance should be conducted periodically using more representative samples to better understand the population-level immunity to JE in South Korea.

  16. A neutralization test for specific detection of Nipah virus antibodies using pseudotyped vesicular stomatitis virus expressing green fluorescent protein.

    PubMed

    Kaku, Yoshihiro; Noguchi, Akira; Marsh, Glenn A; McEachern, Jennifer A; Okutani, Akiko; Hotta, Kozue; Bazartseren, Boldbaatar; Fukushi, Shuetsu; Broder, Christopher C; Yamada, Akio; Inoue, Satoshi; Wang, Lin-Fa

    2009-09-01

    Nipah virus (NiV) is a new zoonotic paramyxovirus that emerged in 1998 and is now classified in the genus Henipavirus along with the closely related Hendra virus (HeV). NiV is highly pathogenic in several vertebrate species including humans, and the lack of available vaccines or specific treatment restricts it to biosafety level 4 (BSL4) containment. A serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing green fluorescent protein (GFP) and bearing the F and G proteins of NiV (VSV-NiV-GFP). The neutralization titers were obtained by counting GFP-expressing cells or by measuring fluorescence. The performance of this new assay was compared against the conventional test using live NiV with panels of sera from several mammalian species, including sera from NiV outbreaks, experimental infections, as well as HeV-specific sera. The results obtained with the VSV-NiV-GFP based test correlated with those obtained using live NiV. Using a 50% reduction in VSV-NiV-GFP infected cells as the cut-off for neutralization, this new assay demonstrated its potential as an effective tool for detecting NiV neutralizing antibodies under BSL2 containment with greater speed, sensitivity and safety as compared to the conventional NiV serum neutralization test.

  17. Neutralizing monoclonal antibodies against ricin's enzymatic subunit interfere with protein disulfide isomerase-mediated reduction of ricin holotoxin in vitro.

    PubMed

    O'Hara, Joanne M; Mantis, Nicholas J

    2013-09-30

    The penultimate event in the intoxication of mammalian cells by ricin toxin is the reduction, in the endoplasmic reticulum (ER), of the intermolecular disulfide bond that links ricin's enzymatic (RTA) and binding (RTB) subunits. In this report we adapted an in vitro protein disulfide isomerase (PDI)-mediated reduction assay to test the hypothesis that the RTA-specific neutralizing monoclonal antibody (mAb) IB2 interferes with the liberation of RTA from RTB. IB2 recognizes an epitope located near the interface between RTA and RTB and, like a number of other RTA-specific neutralizing mAbs, is proposed to neutralize ricin intracellularly. In this study, we found that IB2 virtually eliminated the reduction of ricin holotoxin into RTA and RTB in vitro. Surprisingly, three other neutralizing mAbs (GD12, R70 and SyH7) that bind epitopes at considerable distance from ricin's disulfide bond were as effective (or nearly as effective) as IB2 in interfering with PDI-mediated liberation of RTA from RTB. By contrast, two non-neutralizing RTA-specific mAbs, FGA12 and SB1, did not affect PDI-mediated reduction of ricin. These data reveal a possible mechanism by which RTA-specific antibodies may neutralize ricin intracellularly, provided they are capable of trafficking in association with ricin from the cell surface to the ER.

  18. Anti-IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus.

    PubMed

    Zhang, Jiyong; Sadowska, Grazyna B; Chen, Xiaodi; Park, Seon Yeong; Kim, Jeong-Eun; Bodge, Courtney A; Cummings, Erin; Lim, Yow-Pin; Makeyev, Oleksandr; Besio, Walter G; Gaitanis, John; Banks, William A; Stonestreet, Barbara S

    2015-05-01

    Impaired blood-brain barrier function represents an important component of hypoxic-ischemic brain injury in the perinatal period. Proinflammatory cytokines could contribute to ischemia-related blood-brain barrier dysfunction. IL-6 increases vascular endothelial cell monolayer permeability in vitro. However, contributions of IL-6 to blood-brain barrier abnormalities have not been examined in the immature brain in vivo. We generated pharmacologic quantities of ovine-specific neutralizing anti-IL-6 mAbs and systemically infused mAbs into fetal sheep at 126 days of gestation after exposure to brain ischemia. Anti-IL-6 mAbs were measured by ELISA in fetal plasma, cerebral cortex, and cerebrospinal fluid, blood-brain barrier permeability was quantified using the blood-to-brain transfer constant in brain regions, and IL-6, tight junction proteins, and plasmalemma vesicle protein (PLVAP) were detected by Western immunoblot. Anti-IL-6 mAb infusions resulted in increases in mAb (P < 0.05) in plasma, brain parenchyma, and cerebrospinal fluid and decreases in brain IL-6 protein. Twenty-four hours after ischemia, anti-IL-6 mAb infusions attenuated ischemia-related increases in blood-brain barrier permeability and modulated tight junction and PLVAP protein expression in fetal brain. We conclude that inhibiting the effects of IL-6 protein with systemic infusions of neutralizing antibodies attenuates ischemia-related increases in blood-brain barrier permeability by inhibiting IL-6 and modulates tight junction proteins after ischemia.

  19. Epitope mapping of neutralizing monoclonal antibody in avian influenza A H5N1 virus hemagglutinin.

    PubMed

    Ohkura, Takashi; Kikuchi, Yuji; Kono, Naoko; Itamura, Shigeyuki; Komase, Katsuhiro; Momose, Fumitaka; Morikawa, Yuko

    2012-02-03

    The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.

  20. Structural basis for diverse N-glycan recognition by HIV-1-neutralizing V1-V2-directed antibody PG16

    SciTech Connect

    Pancera, Marie; Shahzad-ul-Hussan, Syed; Doria-Rose, Nicole A.; McLellan, Jason S.; Bailer, Robert T.; Dai, Kaifan; Loesgen, Sandra; Louder, Mark K.; Staupe, Ryan P.; Yang, Yongping; Zhang, Baoshan; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E.; Blinn, Julie; Alam, S. Munir; Haynes, Barton F.; Amin, Mohammed N.; Wang, Lai-Xi; Burton, Dennis R.; Koff, Wayne C.; Nabel, Gary J.; Mascola, John R.; Bewley, Carole A; Kwong, Peter D.

    2013-08-05

    HIV-1 uses a diverse N-linked-glycan shield to evade recognition by antibody. Select human antibodies, such as the clonally related PG9 and PG16, recognize glycopeptide epitopes in the HIV-1 V1–V2 region and penetrate this shield, but their ability to accommodate diverse glycans is unclear. Here we report the structure of antibody PG16 bound to a scaffolded V1–V2, showing an epitope comprising both high mannose–type and complex-type N-linked glycans. We combined structure, NMR and mutagenesis analyses to characterize glycan recognition by PG9 and PG16. Three PG16-specific residues, arginine, serine and histidine (RSH), were critical for binding sialic acid on complex-type glycans, and introduction of these residues into PG9 produced a chimeric antibody with enhanced HIV-1 neutralization. Although HIV-1–glycan diversity facilitates evasion, antibody somatic diversity can overcome this and can provide clues to guide the design of modified antibodies with enhanced neutralization.

  1. Neutralizing antibody titers against dengue virus correlate with protection from symptomatic infection in a longitudinal cohort

    PubMed Central

    Katzelnick, Leah C.; Montoya, Magelda; Gresh, Lionel; Balmaseda, Angel; Harris, Eva

    2016-01-01

    The four dengue virus serotypes (DENV1–4) are mosquito-borne flaviviruses that infect ∼390 million people annually; up to 100 million infections are symptomatic, and 500,000 cases progress to severe disease. Exposure to a heterologous DENV serotype, the specific infecting DENV strains, and the interval of time between infections, as well as age, ethnicity, genetic polymorphisms, and comorbidities of the host, are all risk factors for severe dengue. In contrast, neutralizing antibodies (NAbs) are thought to provide long-lived protection against symptomatic infection and severe dengue. The objective of dengue vaccines is to provide balanced protection against all DENV serotypes simultaneously. However, the association between homotypic and heterotypic NAb titers and protection against symptomatic infection remains poorly understood. Here, we demonstrate that the titer of preinfection cross-reactive NAbs correlates with reduced likelihood of symptomatic secondary infection in a longitudinal pediatric dengue cohort in Nicaragua. The protective effect of NAb titers on infection outcome remained significant when controlled for age, number of years between infections, and epidemic force, as well as with relaxed or more stringent criteria for defining inapparent DENV infections. Further, individuals with higher NAb titers immediately after primary infection had delayed symptomatic infections compared with those with lower titers. However, overall NAb titers increased modestly in magnitude and remained serotype cross-reactive in the years between infections, possibly due to reexposure. These findings establish that anti-DENV NAb titers correlate with reduced probability of symptomatic DENV infection and provide insights into longitudinal characteristics of antibody-mediated immunity to DENV in an endemic setting. PMID:26729879

  2. Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

    PubMed Central

    Nambiar, Jonathan; Clarke, Adam W; Shim, Doris; Mabon, David; Tian, Chen; Windloch, Karolina; Buhmann, Chris; Corazon, Beau; Lindgren, Matilda; Pollard, Matthew; Domagala, Teresa; Poulton, Lynn; Doyle, Anthony G

    2015-01-01

    CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (KD ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC50 typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical. PMID:25751125

  3. Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model.

    PubMed

    Nambiar, Jonathan; Clarke, Adam W; Shim, Doris; Mabon, David; Tian, Chen; Windloch, Karolina; Buhmann, Chris; Corazon, Beau; Lindgren, Matilda; Pollard, Matthew; Domagala, Teresa; Poulton, Lynn; Doyle, Anthony G

    2015-01-01

    CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (KD ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC50 typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.

  4. A murine cytomegalovirus-neutralizing monoclonal antibody exhibits autoreactivity and induces tissue damage in vivo.

    PubMed Central

    Chapman, A J; Farrell, H E; Thomas, J A; Papadimitriou, J M; Garlepp, M J; Scalzo, A A; Shellam, G R

    1994-01-01

    The autoreactivity of murine cytomegalovirus (MCMV)-neutralizing monoclonal antibody (mAb) AC1 was examined in vitro and in vivo. Both mAb AC1 and a human antiserum reactive with U1-small nuclear ribonucleoprotein (U1-snRNP) stained uninfected mouse embryo fibroblasts (MEF) in a speckled nuclear pattern and reacted with 70,000 molecular weight (MW) MEF nuclear antigens by immunoblotting, suggesting that mAb AC1 cross-reacted with the 70,000 MW component of U1-snRNP. However, only mAb AC1 cross-reacted with an additional epithelial cytoplasmic autoantigen present in cultured HEp2 cells. On tissue sections from uninfected mice, mAb AC1 predominantly reacted with a component of central and peripheral nervous systems, although cross-reactivity with the stratum spinosum of the skin and the outer sheath of hair follicles was also observed. Immunoblotting revealed that mAb AC1 reacted with phosphorylated epitopes present on a 98,000 MW MCMV structural protein and the 200,000 MW mouse neurofilament protein (NFP). Treatment of uninfected mice with mAb AC1 resulted in a severe interstitial pneumonia with greatly thickened and congested alveolar septa. Severe oedema of the hypodermis and a mild mesangial proliferative glomerulonephritis were also observed. These results demonstrate that a mAb reacting with a MCMV structural phosphoprotein which can protect mice against the dissemination of MCMV, can also promote the development of autoimmune disease. Therefore, the production of such cross-reactive antibodies may be an important mechanism in the development of autoimmunity following viral infection. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7515848

  5. Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies

    PubMed Central

    Yee, Michael; Konopka, Krystyna; Balzarini, Jan; Düzgüneş, Nejat

    2011-01-01

    Background: Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy. Results: Cell-cell fusion was inhibited completely by the carbohydrate binding proteins (CBPs), Hippeastrum hybrid (Amaryllis) agglutinin (HHA), and Galanthus nivalis (Snowdrop) agglutinin (GNA), and by the peptide, T-20, at relatively low concentrations. Anti-gp120 and anti-gp41 antibodies, at concentrations much higher than those required for neutralization, were not particularly effective in inhibiting fusion. Monoclonal antibodies b12, m14 IgG and 2G12 had moderate inhibitory activity; the IC50 of 2G12 was about 80 µg/ml. Antibodies 4E10 and 2F5 had no inhibitory activity at the concentrations tested. Conclusions: These observations raise concerns about the ability of neutralizing antibodies to inhibit the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. The interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion reactions, and may explain the differential effects of antibodies in these two systems. The fluorescence assay described here may be useful in high throughput screening of potential HIV fusion inhibitors. PMID:21660189

  6. Crystal Structures of Ricin Toxin’s Enzymatic Subunit (RTA) in Complex with Neutralizing and Non-neutralizing Single Chain Antibodies

    PubMed Central

    Rudolph, Michael J.; Vance, David J.; Cheung, Jonah; Franklin, Matthew C.; Burshteyn, Fiana; Cassidy, Michael S.; Gary, Ebony N.; Herrera, Cristina; Shoemaker, Charles B.; Mantis, Nicholas J.

    2014-01-01

    Ricin is a Select Agent Toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins (RIPs). In this study, we determined x-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA’s RNA N-glycosidase activity or induced notable allosteric changes in the toxin’s subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines. PMID:24907552

  7. Production of neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in carcinoma patients following GM-CSF combination therapy.

    PubMed

    Wadhwa, M; Bird, C; Fagerberg, J; Gaines-Das, R; Ragnhammar, P; Mellstedt, H; Thorpe, R

    1996-05-01

    In this study, the development of neutralizing and non-neutralizing GM-CSF antibodies and the clinical consequences related to the induction of these antibodies were analysed in 20 patients with metastatic colorectal carcinoma receiving a combination therapy of Escherichia coli-derived GM-CSF and a colon carcinoma-reactive MoAb in the absence of any concomitant chemotherapy. The recombinant human GM-CSF was administered subcutaneously for 10 days every month for 4 months. Following the first cycle of treatment, no GM-CSF antibodies were detected, but during subsequent therapy, 19 of the 20 patients studied developed GM-CSF binding antibodies. However, only a proportion (40%) of the 19 antibody-positive patients developed antibodies that neutralized the biological activity of GM-CSF in an in vitro bioassay. The presence of GM-CSF neutralizing antibodies was associated with a significant reduction in GM-CSF-induced expansion of leucocytes, neutrophils and eosinophils. Such clinical effects were not apparent in patients with non-neutralizing antibodies. Further characterization of sera from patients with neutralizing antibodies showed that, in most cases, the antibodies neutralized the biological activity of GM-CSF preparations derived using different expression systems (Chinese hamster ovary cells and yeast), suggesting that these antibodies may have the potential to cross-react with endogenously produced GM-CSF. These effects should be considered before therapeutic use of cytokines, particularly in patients who are not immunosuppressed, and therefore capable of mounting an effective immune response. Our results indicate that assessment of production of neutralizing antibodies induced during cytokine therapy can be used to predict diminished clinical response to further therapy.

  8. Comparison of agglutinating and neutralizing antibodies to serovar hardjo in sows immunized with two commercial whole culture polivalent anti-leptospira bacterins

    PubMed Central

    Soto, Francisco Rafael Martins; Pinheiro, Sônia Regina; Morais, Zenaide Maria; Gonçales, Amane Paldês; de Azevedo, Sérgio Santos; Bernardi, Fernanda; Camargo, Sebastião Rodrigues; Vasconcellos, Silvio Arruda

    2008-01-01

    It was performed the comparison of the intensity and duration of agglutinating and neutralizing antibodies to serovar Hardjo in swines vaccinated with two commercial anti-leptospira bacterins. Sows no reactive to 24 Leptospira sp serovars in the microscopic agglutination test (MAT) were divided in three groups: Group A (n=08): received two vaccine A doses with 30 days interval, Group B (n=08) two vaccine B doses with 30 days interval and Group C (n=08): control no vaccinated against leptospirosis.Blood samples were collected each 30 days during six months following the first vaccination. The sera were tested by MAT and growth inhibition test (GIT) to serovar Hardjo in order to evaluate respectively agglutinating and neutralizing antibodies. It was found that neutralizing antibodies persisted for a longer time than the agglutinating ones and that the absence of agglutinating antibodies does not means in the absence of the neutralizing. The peaks of agglutinating antibodies was obtained at least 30 days earlier than that produced by neutralizing. The duration of both kinds of antibodies measured differed between the two bacterines tested. The period for inducing neutralizing antibodies against serovar Hardjo indicated that gilts must be immunized with two doses of whole culture anti-leptospira bacterines applied 30 days each other at least 90 days before the first mating. For the maintenance of hight levels of neutralizing antibodies the revaccinations must be performed every six months after the first vaccination. PMID:24031250

  9. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers

    SciTech Connect

    Swanson, Kurt A.; Settembre, Ethan C.; Shaw, Christine A.; Dey, Antu K.; Rappuoli, Rino; Mandl, Christian W.; Dormitzer, Philip R.; Carfi, Andrea

    2012-02-07

    Respiratory syncytial virus (RSV), the main cause of infant bronchiolitis, remains a major unmet vaccine need despite more than 40 years of vaccine research. Vaccine candidates based on a chief RSV neutralization antigen, the fusion (F) glycoprotein, have foundered due to problems with stability, purity, reproducibility, and potency. Crystal structures of related parainfluenza F glycoproteins have revealed a large conformational change between the prefusion and postfusion states, suggesting that postfusion F antigens might not efficiently elicit neutralizing antibodies. We have generated a homogeneous, stable, and reproducible postfusion RSV F immunogen that elicits high titers of neutralizing antibodies in immunized animals. The 3.2-{angstrom} X-ray crystal structure of this substantially complete RSV F reveals important differences from homology-based structural models. Specifically, the RSV F crystal structure demonstrates the exposure of key neutralizing antibody binding sites on the surface of the postfusion RSV F trimer. This unanticipated structural feature explains the engineered RSV F antigen's efficiency as an immunogen. This work illustrates how structural-based antigen design can guide the rational optimization of candidate vaccine antigens.

  10. HIV-1 clade C escapes broadly neutralizing autologous antibodies with N332 glycan specificity by distinct mechanisms.

    PubMed

    Deshpande, Suprit; Patil, Shilpa; Kumar, Rajesh; Hermanus, Tandile; Murugavel, Kailapuri G; Srikrishnan, Aylur K; Solomon, Suniti; Morris, Lynn; Bhattacharya, Jayanta

    2016-08-30

    The glycan supersite centered on N332 in the V3 base of the HIV-1 envelope (Env) is a target for broadly neutralizing antibodies (bnAbs) such as PGT121 and PGT128. In this study, we examined the basis of resistance of HIV-1 clade C Envs obtained from broadly cross neutralizing (BCN) plasma of an Indian donor with N332 specificity. Pseudotyped viruses expressing autologous envs were found to be resistant to autologous BCN plasma as well as to PGT121 and PGT128 mAbs despite the majority of Envs containing an intact N332 residue. While resistance of one of the Envs to neutralization by autologous plasma antibodies with shorter V1 loop length was found to be correlated with a N332S mutation, resistance to neutralization of rest of the Envs was found to be associated with longer V1 loop length and acquisition of protective N-glycans. In summary, we show evidence of escape of circulating HIV-1 clade C in an individual from autologous BCN antibodies by three distinct mechanisms.

  11. Role of neutralizing antibodies and T-cells in pathogenesis of herpes simplex virus infection in congenitally athymic mice.

    PubMed

    Kapoor, A K; Buckmaster, A; Nash, A A; Field, H J; Wildy, P

    1982-11-01

    Congenitally athymic nude mice were infected with 10(4) p.f.u. herpes simplex type 1 (strain SC16). Following the passive transfer of neutralizing monoclonal antibodies (AP7, AP8 and AP12) it was observed that AP7 alone reduced the virus infectivity in the nervous system; AP8 and AP12 failed to protect mice probably due to poor in vivo binding to the neutralization site on the virus. Latent ganglionic infection could be established in nude mice following adoptive transfer of optimum number (2 x 10(7) cells/mouse) of immune lymph node cells from day 7 herpes virus-infected hairy immunocompetent donor mice. Moreover, in some of the immune lymph node cell protected nudes, latency could be maintained even in complete absence of neutralizing antibodies. Results of ear-ablation experiments revealed that removal of primary source of infection after day 5 of infection reduced the amount of virus in the ganglia and spinal cord. Acute neurological infection was not detected following transfer of protective anti-gp-D neutralizing antibody (LP2) in combination with removal of infected pinna. These data suggest that continuous seeding of virus occurs in related ganglia via the axonal route from infected ear pinna. It appears that local T-cell-mediated immune mechanisms are involved in maintenance of latency.

  12. Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in newborn macaques

    PubMed Central

    Hessell, Ann J.; Jaworski, J. Pablo; Epson, Erin; Matsuda, Kenta; Pandey, Shilpi; Kahl, Christoph; Reed, Jason; Sutton, William F.; Hammond, Katherine B.; Cheever, Tracy A.; Barnette, Philip T.; Legasse, Alfred W.; Planer, Shannon; Stanton, Jeffrey J.; Pegu, Amarendra; Chen, Xuejun; Wang, Keyun; Siess, Don; Burke, David; Park, Byung S.; Axthelm, Michael K.; Lewis, Anne; Hirsch, Vanessa M.; Graham, Barney S.; Mascola, John R.; Sacha, Jonah B.; Haigwood, Nancy L.

    2016-01-01

    Prevention of mother to child transmission (MTCT) of HIV remains a major objective where antenatal care is not readily accessible. We tested anti-HIV-1 human neutralizing monoclonal antibodies (NmAb) as post-exposure therapy in an infant macaque model for intrapartum MTCT. One-month-old rhesus macaques were inoculated orally with SHIVSF162P3. On days 1, 4, 7, and 10 after virus exposure, we injected animals subcutaneously with NmAbs and quantified systemic distribution of NmAbs in multiple tissues within 24 h following administration. Replicating virus was found in multiple tissues by day 1 in animals without treatment. All NmAb-treated macaques were free of virus in blood and tissues at 6 months post-exposure. We detected no anti-SHIV T cell responses in blood or tissues at necropsy, and no virus emerged following CD8+ T cell depletion. These results suggest early passive immunotherapy can eliminate early viral foci and thereby prevent the establishment of viral reservoirs. PMID:26998834

  13. HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen.

    PubMed

    Jardine, Joseph G; Kulp, Daniel W; Havenar-Daughton, Colin; Sarkar, Anita; Briney, Bryan; Sok, Devin; Sesterhenn, Fabian; Ereño-Orbea, June; Kalyuzhniy, Oleksandr; Deresa, Isaiah; Hu, Xiaozhen; Spencer, Skye; Jones, Meaghan; Georgeson, Erik; Adachi, Yumiko; Kubitz, Michael; deCamp, Allan C; Julien, Jean-Philippe; Wilson, Ian A; Burton, Dennis R; Crotty, Shane; Schief, William R

    2016-03-25

    Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.

  14. A broadly neutralizing human monoclonal antibody is effective against H7N9

    PubMed Central

    Tharakaraman, Kannan; Subramanian, Vidya; Viswanathan, Karthik; Sloan, Susan; Yen, Hui-Ling; Barnard, Dale L.; Leung, Y. H. Connie; Szretter, Kristy J.; Koch, Tyree J.; Delaney, James C.; Babcock, Gregory J.; Wogan, Gerald N.; Sasisekharan, Ram; Shriver, Zachary

    2015-01-01

    Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (−24 h) or double-dose (−12 h, +48 h) administration (P < 0.001). A single dose of VIS410 at 50 mg/kg (−12 h) combined with oseltamivir at 50 mg/kg (−12 h, twice daily for 7 d) in C57BL/6 mice infected with A/Shanghai 2/2013 (H7N9) resulted in significant decreased lung viral load (P = 0.002) and decreased lung cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains. PMID:26283346

  15. Immunoglobulin gene insertions and deletions in the affinity maturation of HIV-1 broadly reactive neutralizing antibodies.

    PubMed

    Kepler, Thomas B; Liao, Hua-Xin; Alam, S Munir; Bhaskarabhatla, Rekha; Zhang, Ruijun; Yandava, Chandri; Stewart, Shelley; Anasti, Kara; Kelsoe, Garnett; Parks, Robert; Lloyd, Krissey E; Stolarchuk, Christina; Pritchett, Jamie; Solomon, Erika; Friberg, Emma; Morris, Lynn; Karim, Salim S Abdool; Cohen, Myron S; Walter, Emmanuel; Moody, M Anthony; Wu, Xueling; Altae-Tran, Han R; Georgiev, Ivelin S; Kwong, Peter D; Boyd, Scott D; Fire, Andrew Z; Mascola, John R; Haynes, Barton F

    2014-09-10

    Induction of HIV-1 broad neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development but has remained challenging partially due to unusual traits of bnAbs, including high somatic hypermutation (SHM) frequencies and in-frame insertions and deletions (indels). Here we examined the propensity and functional requirement for indels within HIV-1 bnAbs. High-throughput sequencing of the immunoglobulin (Ig) VHDJH genes in HIV-1 infected and uninfected individuals revealed that the indel frequency was elevated among HIV-1-infected subjects, with no unique properties attributable to bnAb-producing individuals. This increased indel occurrence depended only on the frequency of SHM point mutations. Indel-encoded regions were generally proximal to antigen binding sites. Additionally, reconstruction of a HIV-1 CD4-binding site bnAb clonal lineage revealed that a large compound VHDJH indel was required for bnAb activity. Thus, vaccine development should focus on designing regimens targeted at sustained activation of bnAb lineages to achieve the required SHM and indel events.

  16. Detection of Infertility-related Neutralizing Antibodies with a Cell-free Microfluidic Method

    NASA Astrophysics Data System (ADS)

    Eyer, Klaus; Root, Katharina; Verboket, Pascal E.; Dittrich, Petra S.

    2015-11-01

    The unwanted emergence of neutralizing antibodies (nAbs) against an endogenous or a therapeutic protein can result in deficiency diseases or therapy failure. Here, we developed a cell-free microfluidic method for the sensitive detection and quantification of nAbs in human serum that are associated with infertility. We used cell-derived vesicles containing the luteinizing hormone (LH)/choriogonadotropin receptor (LHHCGR) to detect nAbs against LH. The method exploits the entire cellular signal amplification mechanism, and facilitates the detection of as little as 0.44 nM of LH-nAb (Kd 1.5 nM) in human serum matrix within only 15 minutes. In addition, dose-response curves can be generated in less than 2 hours to evaluate the nAB concentration and dissociation constant. The developed system is devoid of problems associated with cell-based assays and we believe that this simple effect-directed analysis can be used in clinical environments, and is adaptable to other hormones or cytokines and their respective nAbs.

  17. Dose-response curve slope helps predict therapeutic potency and breadth of HIV broadly neutralizing antibodies.

    PubMed

    Webb, Nicholas E; Montefiori, David C; Lee, Benhur

    2015-09-29

    A new generation of HIV broadly neutralizing antibodies (bnAbs) with remarkable potency, breadth and epitope diversity has rejuvenated interest in immunotherapeutic strategies. Potencies defined by in vitro IC50 and IC80 values (50 and 80% inhibitory concentrations) figure prominently into the selection of clinical candidates; however, much higher therapeutic levels will be required to reduce multiple logs of virus and impede escape. Here we predict bnAb potency at therapeutic levels by analysing dose-response curve slopes, and show that slope is independent of IC50/IC80 and specifically relates to bnAb epitope class. With few exceptions, CD4-binding site and V3-glycan bnAbs exhibit slopes >1, indicative of higher expected therapeutic effectiveness, whereas V2-glycan, gp41 membrane-proximal external region (MPER) and gp120-gp41 bnAbs exhibit less favourable slopes <1. Our results indicate that slope is one major predictor of both potency and breadth for bnAbs at clinically relevant concentrations, and may better coordinate the relationship between bnAb epitope structure and therapeutic expectations.

  18. Detection of Infertility-related Neutralizing Antibodies with a Cell-free Microfluidic Method

    PubMed Central

    Eyer, Klaus; Root, Katharina; Verboket, Pascal E.; Dittrich, Petra S.

    2015-01-01

    The unwanted emergence of neutralizing antibodies (nAbs) against an endogenous or a therapeutic protein can result in deficiency diseases or therapy failure. Here, we developed a cell-free microfluidic method for the sensitive detection and quantification of nAbs in human serum that are associated with infertility. We used cell-derived vesicles containing the luteinizing hormone (LH)/choriogonadotropin receptor (LHHCGR) to detect nAbs against LH. The method exploits the entire cellular signal amplification mechanism, and facilitates the detection of as little as 0.44 nM of LH-nAb (Kd 1.5 nM) in human serum matrix within only 15 minutes. In addition, dose-response curves can be generated in less than 2 hours to evaluate the nAB concentration and dissociation constant. The developed system is devoid of problems associated with cell-based assays and we believe that this simple effect-directed analysis can be used in clinical environments, and is adaptable to other hormones or cytokines and their respective nAbs. PMID:26585778

  19. Interferon and neutralizing antibody in sera of exercised mice with coxsackievirus B-3 myocarditis.

    PubMed

    Reyes, M P; Lerner, A M

    1976-02-01

    Weanling ICR albino Swiss mice were inoculated ip with 1.9 x 10(4) PFU of coxsackievirus B-3 (Nancy) and subsequently forced to swim vigorously daily in a preheated pool (33 degrees). Viremias and virus in hearts of exercised mice were respectively 75 x 1000 x greater than in infected, but not exercised mice. At 24 hr after inoculation, pooled serum from mice that had been swum had no circulating interferon, while infected but not swum mice had interferon activity at a dilution of 1:10. At 72 hr after infection, circulating interferon disappeared from infected (not swum) mice, but continued to be present in high titers through the sixth day in sucklings forced to swim. Interferon was first detected in the hearts of both groups at 48 hr. Quantities in both infected groups were generally similar. Neutralizing antibodies were found in these baby mice on the 13th day of infection and were 16 x greater in nurslings that were not exercised. Measures of corticosterone taken at 4 PM daily were similar in infected, infected-swum, and uninfected mice.

  20. HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen

    DOE PAGES

    Jardine, Joseph G.; Kulp, Daniel W.; Havenar-Daughton, Colin; ...

    2016-03-25

    Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. We employed deep mutational scanning and multi-target optimization to develop a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen asmore » a candidate human vaccine prime. Lastly, these methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.« less

  1. HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen

    SciTech Connect

    Jardine, Joseph G.; Kulp, Daniel W.; Havenar-Daughton, Colin; Sarkar, Anita; Briney, Bryan; Sok, Devin; Sesterhenn, Fabian; Ereno-Orbea, June; Kalyuzhniy, Oleksandr; Deresa, Isaiah; Hu, Xiaozhen; Spencer, Skye; Jones, Meaghan; Georgeson, Erik; Adachi, Yumiko; Kubitz, Michael; deCamp, Allan C.; Julien, Jean -Philippe; Wilson, Ian A.; Burton, Dennis R.; Crotty, Shane; Schief, William R.

    2016-03-25

    Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. We employed deep mutational scanning and multi-target optimization to develop a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. Lastly, these methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.

  2. Development of Patient-specific AAV Vectors After Neutralizing Antibody Selection for Enhanced Muscle Gene Transfer.

    PubMed

    Li, Chengwen; Wu, Shuqing; Albright, Blake; Hirsch, Matthew; Li, Wuping; Tseng, Yu-Shan; Agbandje-McKenna, Mavis; McPhee, Scott; Asokan, Aravind; Samulski, R Jude

    2016-02-01

    A major hindrance in gene therapy trials with adeno-associated virus (AAV) vectors is the presence of neutralizing antibodies (NAbs) that inhibit AAV transduction. In this study, we used directed evolution techniques in vitro and in mouse muscle to select novel NAb escape AAV chimeric capsid mutants in the presence of individual patient serum. AAV mutants isolated in vitro escaped broad patient-specific NAb activity but had poor transduction ability in vivo. AAV mutants isolated in vivo had enhanced NAb evasion from cognate serum and had high muscle transduction ability. More importantly, structural modeling identified a 100 amino acid motif from AAV6 in variable region (VR) III that confers this enhanced muscle tropism. In addition, a predominantly AAV8 capsid beta barrel template with a specific preference for AAV1/AAV9 in VR VII located at threefold symmetry axis facilitates NAb escape. Our data strongly support that chimeric AAV capsids composed of modular and nonoverlapping domains from various serotypes are capable of evading patient-specific NAbs and have enhanced muscle transduction.

  3. Crystal structure of the HIV neutralizing antibody 2G12 in complex with a bacterial oligosaccharide analog of mammalian oligomannose

    PubMed Central

    Stanfield, Robyn L; De Castro, Cristina; Marzaioli, Alberto M; Wilson, Ian A; Pantophlet, Ralph

    2015-01-01

    Human immunodeficiency virus-1 (HIV-1) is a major public health threat that continues to infect millions of people worldwide each year. A prophylactic vaccine remains the most cost-effective way of globally reducing and eliminating the spread of the virus. The HIV envelope spike, which is the target of many vaccine design efforts, is densely mantled with carbohydrate and several potent broadly neutralizing antibodies to HIV-1 recognize carbohydrate on the envelope spike as a major part of their epitope. However, immunizing with recombinant forms of the envelope glycoprotein does not typically elicit anti-carbohydrate antibodies. Thus, studies of alternative antigens that may serve as a starting point for carbohydrate-based immunogens are of interest. Here, we present the crystal structure of one such anti-carbohydrate HIV neutralizing antibody (2G12) in complex with the carbohydrate backbone of the lipooligosaccharide from Rhizobium radiobacter strain Rv3, which exhibits a chemical structure that naturally mimics the core high-mannose carbohydrate epitope of 2G12 on HIV-1 gp120. The structure described here provides molecular evidence of the structural homology between the Rv3 oligosaccharide and highly abundant carbohydrates on the surface of HIV-1 and raises the potential for the design of novel glycoconjugates that may find utility in efforts to develop immunogens for eliciting carbohydrate-specific neutralizing antibodies to HIV. PMID:25380763

  4. Prevalence and distribution of serum neutralizing antibodies to Tillamook (bovine) calicivirus in selected populations of marine mammals.

    PubMed

    Barlough, J E; Berry, E S; Smith, A W; Skilling, D E

    1987-01-01

    Neutralizing antibodies to Tillamook calicivirus (TCV) were found in sera collected from California sea lions (Zalophus c. californianus Lesson) in 1983 and 1984 and in sera collected from Steller sea lions (Eumetopias jubatus Schreber) in 1976 and 1985. The combined prevalence of antibodies for these two species was 10/228 = 4.38%. Titers ranged from 1:20 (five animals), to 1:40 (four animals), to 1:80 (one animal) by standard microtiter neutralization assay. The seropositive pinnipeds were dispersed widely along the margins of the eastern Pacific rim, from the Bering Sea to the Santa Barbara Channel. Antibodies to TCV were not found in sera collected from northern fur seals (Callorhinus ursinus L.), Pacific walruses (Odobenus rosmarus divergens Illiger), seals of the family Phocidae, or several cetacean species. Tillamook calicivirus was isolated originally in 1981 from dairy calves in Oregon; the finding of neutralizing antibodies in two widely distributed species of sea lions suggests the possibility of a marine origin for this agent.

  5. Neutralizing antibody and functional mapping of Bacillus anthracis protective antigen-The first step toward a rationally designed anthrax vaccine.

    PubMed

    McComb, Ryan C; Martchenko, Mikhail

    2016-01-02

    Anthrax is defined by the Centers for Disease Control and Prevention as a Category A pathogen for its potential use as a bioweapon. Current prevention treatments include Anthrax Vaccine Adsorbed (AVA). AVA is an undefined formulation of Bacillus anthracis culture supernatant adsorbed to aluminum hydroxide. It has an onerous vaccination schedule, is slow and cumbersome to produce and is slightly reactogenic. Next-generation vaccines are focused on producing recombinant forms of anthrax toxin in a well-defined formulation but these vaccines have been shown to lose potency as they are stored. In addition, studies have shown that a proportion of the antibody response against these vaccines is focused on non-functional, non-neutralizing regions of the anthrax toxin while some essential functional regions are shielded from eliciting an antibody response. Rational vaccinology is a developing field that focuses on designing vaccine antigens based on structural information provided by neutralizing antibody epitope mapping, crystal structure analysis, and functional mapping through amino acid mutations. This information provides an opportunity to design antigens that target only functionally important and conserved regions of a pathogen in order to make a more optimal vaccine product. This review provides an overview of the literature related to functional and neutralizing antibody epitope mapping of the Protective Antigen (PA) component of anthrax toxin.

  6. Capsid protein: evidences about the partial protective role of neutralizing antibody-independent immunity against dengue in monkeys.

    PubMed

    Gil, Lázaro; Izquierdo, Alienys; Lazo, Laura; Valdés, Iris; Ambala, Peris; Ochola, Lucy; Marcos, Ernesto; Suzarte, Edith; Kariuki, Thomas; Guzmán, Guadalupe; Guillén, Gerardo; Hermida, Lisset

    2014-05-01

    The role of cellular immune response in dengue virus infection is not yet fully understood. Only few studies in murine models propose that CD8(+) T-cells are associated with protection from infection and disease. At the light of recent reports about the protective role of CD8(+) T-cells in humans and the no correlation between neutralizing antibodies and protection observed in several studies, a vaccine based on cell-mediated immunity constitute an attractive approach. Our group has developed a capsid-based vaccine as nucleocpasid-like particles from dengue-2 virus, which induced a protective CD4(+) and CD8(+) cell-mediated immunity in mice, without the contribution of neutralizing antibodies. Herein we evaluated the immunogenicity and protective efficacy of this molecule in monkeys. Neither IgG antibodies against the whole virus nor neutralizing antibodies were elicited after the antigen inoculation. However, animals developed a cell-mediated immunity, measured by gamma interferon secretion and cytotoxic capacity. Although only one out of three vaccinated animals was fully protected against viral challenge, a viral load reduction was observed in this group compared with the placebo one, suggesting that capsid could be the base on an attractive vaccine against dengue.

  7. Crystal structure of the HIV neutralizing antibody 2G12 in complex with a bacterial oligosaccharide analog of mammalian oligomannose.

    PubMed

    Stanfield, Robyn L; De Castro, Cristina; Marzaioli, Alberto M; Wilson, Ian A; Pantophlet, Ralph

    2015-04-01

    Human immunodeficiency virus-1 (HIV-1) is a major public health threat that continues to infect millions of people worldwide each year. A prophylactic vaccine remains the most cost-effective way of globally reducing and eliminating the spread of the virus. The HIV envelope spike, which is the target of many vaccine design efforts, is densely mantled with carbohydrate and several potent broadly neutralizing antibodies to HIV-1 recognize carbohydrate on the envelope spike as a major part of their epitope. However, immunizing with recombinant forms of the envelope glycoprotein does not typically elicit anti-carbohydrate antibodies. Thus, studies of alternative antigens that may serve as a starting point for carbohydrate-based immunogens are of interest. Here, we present the crystal structure of one such anti-carbohydrate HIV neutralizing antibody (2G12) in complex with the carbohydrate backbone of the lipooligosaccharide from Rhizobium radiobacter strain Rv3, which exhibits a chemical structure that naturally mimics the core high-mannose carbohydrate epitope of 2G12 on HIV-1 gp120. The structure described here provides molecular evidence of the structural homology between the Rv3 oligosaccharide and highly abundant carbohydrates on the surface of HIV-1 and raises the potential for the design of novel glycoconjugates that may find utility in efforts to develop immunogens for eliciting carbohydrate-specific neutralizing antibodies to HIV.

  8. Enhanced clearance of HIV-1-infected cells by anti-HIV-1 broadly neutralizing antibodies in vivo

    PubMed Central

    Lu, Ching-Lan; Murakowski, Dariusz K.; Bournazos, Stylianos; Schoofs, Till; Sarkar, Debolina; Halper-Stromberg, Ariel; Horwitz, Joshua A.; Nogueira, Lilian; Golijanin, Jovana; Gazumyan, Anna; Ravetch, Jeffrey V.; Caskey, Marina; Chakraborty, Arup K.; Nussenzweig, Michel C.

    2016-01-01

    Anti-retroviral drugs and antibodies limit HIV-1 infection by interfering with the viral life-cycle. In addition, antibodies also have the potential to guide host immune effector cells to kill HIV-1 infected cells. Examination of the kinetics of HIV-1 suppression in infected individuals by passively administered 3BNC117, a broadly neutralizing antibody (bNAb), suggested that the effects of the antibody are not limited to free viral clearance and blocking new infection, but also include acceleration of infected cell clearance. Consistent with these observations, we find that bNAbs can target CD4+ T cells infected with patient viruses and decrease their in vivo half-lives by a mechanism that requires FcγR engagement in a humanized mouse model. The results indicate that passive immunotherapy can accelerate elimination of HIV-1 infected cells. PMID:27199430

  9. High Concentrations of Measles Neutralizing Antibodies and High-Avidity Measles IgG Accurately Identify Measles Reinfection Cases

    PubMed Central

    Rota, Jennifer S.; Hickman, Carole J.; Mercader, Sara; Redd, Susan; McNall, Rebecca J.; Williams, Nobia; McGrew, Marcia; Walls, M. Laura; Rota, Paul A.; Bellini, William J.

    2016-01-01

    In the United States, approximately 9% of the measles cases reported from 2012 to 2014 occurred in vaccinated individuals. Laboratory confirmation of measles in vaccinated individuals is challenging since IgM assays can give inconclusive results. Although a positive reverse transcription (RT)-PCR assay result from an appropriately timed specimen can provide confirmation, negative results may not rule out a highly suspicious case. Detection of high-avidity measles IgG in serum samples provides laboratory evidence of a past immunologic response to measles from natural infection or immunization. High concentrations of measles neutralizing antibody have been observed by plaque reduction neutralization (PRN) assays among confirmed measles cases with high-avidity IgG, referred to here as reinfection cases (RICs). In this study, we evaluated the utility of measuring levels of measles neutralizing antibody to distinguish RICs from noncases by receiver operating characteristic curve analysis. Single and paired serum samples with high-avidity measles IgG from suspected measles cases submitted to the CDC for routine surveillance were used for the analysis. The RICs were confirmed by a 4-fold rise in PRN titer or by RT-quantitative PCR (RT-qPCR) assay, while the noncases were negative by both assays. Discrimination accuracy was high with serum samples collected ≥3 days after rash onset (area under the curve, 0.953; 95% confidence interval [CI], 0.854 to 0.993). Measles neutralizing antibody concentrations of ≥40,000 mIU/ml identified RICs with 90% sensitivity (95% CI, 74 to 98%) and 100% specificity (95% CI, 82 to 100%). Therefore, when serological or RT-qPCR results are unavailable or inconclusive, suspected measles cases with high-avidity measles IgG can be confirmed as RICs by measles neutralizing antibody concentrations of ≥40,000 mIU/ml. PMID:27335386

  10. Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission

    PubMed Central

    Mangala Prasad, Vidya; Wang, Cheng-I; Akahata, Wataru; Ng, Lisa F. P.

    2015-01-01

    ABSTRACT Chikungunya virus is a positive-stranded RNA alphavirus. Structures of chikungunya virus-like particles in complex with strongly neutralizing antibody Fab fragments (8B10 and 5F10) were determined using cryo-electron microscopy and X-ray crystallography. By fitting the crystallographically determined structures of these Fab fragments into the cryo-electron density maps, we show that Fab fragments of antibody 8B10 extend radially from the viral surface and block receptor binding on the E2 glycoprotein. In contrast, Fab fragments of antibody 5F10 bind the tip of the E2 B domain and lie tangentially on the viral surface. Fab 5F10 fixes the B domain rigidly to the surface of the virus, blocking exposure of the fusion loop on glycoprotein E1 and therefore preventing the virus from becoming fusogenic. Although Fab 5F10 can neutralize the wild-type virus, it can also bind to a mutant virus without inhibiting fusion or attachment. Although the mutant virus is no longer able to propagate by extracellular budding, it can, however, enter the next cell by traveling through junctional complexes without being intercepted by a neutralizing antibody to the wild-type virus, thus clarifying how cell-to-cell transmission can occur. IMPORTANCE Alphaviral infections are transmitted mainly by mosquitoes. Chikungunya virus (CHIKV), which belongs to the Alphavirus genus, has a wide distribution in the Old World that has expanded in recent years into the Americas. There are currently no vaccines or drugs against alphaviral infections. Therefore, a better understanding of CHIKV and its associated neutralizing antibodies will aid in the development of effective treatments. PMID:26537684

  11. Relative contribution of dengue prM- and E-specific polyclonal antibodies to neutralization and enhancement.

    PubMed

    Rodpothong, P; Boonarkart, Ch; Ruangrung, K; Onsirisakul, N; Kanistanon, D; Auewarakul, P

    Viral surface proteins, premembrane protein (prM) and envelope (E) protein have been shown to induce a production of antibodies that are involved in both enhancement and neutralization. To explore the feasibility of modifying the relative immune responses to prM and E proteins, four DNA constructs were created and administered into groups of Balb/c mice; pPW01 contains prM and E genes of DENV1, pPW02 contains prM and E genes of DENV2, pPW03 contains DENV1 prM and DENV2 E, and pPW04 contains DENV2 prM and DENV1 E. Exchange of either prM or E from a heterologous serotype does not appear to have an effect on the immunogenicity of the proteins. We have proved that the chimeric pPW03 and pPW04 constructs can produce humoral response in mice. Immunized sera were subjected to neutralization and enhancement assays against DENV2. The results showed that only serotype-specific anti-E antibodies conferred protective function, while the cross-reactive anti-E and anti-prM enhanced infection. In addition, the enhancement of DENV2 infection exhibited a serotype-preference for anti-E antibodies while such response was not observed with anti-prM, reflecting a degree of structural conservation of prM. Taken together, neutralization and enhancement appeared to occur at the same time during the course of infection. Successful prevention of severe symptoms of DENV infection depends on the ability to induce high levels of neutralizing antibodies to subdue the effect of enhancing antibodies.

  12. Isolation and characterization of a neutralizing antibody specific to internalization domain of Clostridium botulinum neurotoxin type B.

    PubMed

    Yang, Gi-Hyeok; Kim, Kyu-Sik; Kim, Hak-Woo; Jeong, Sung Tae; Huh, Gyung Heng; Kim, Ji Cheon; Jung, Hyun Ho

    2004-07-01

    Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. In this study, a neutralizing mouse monoclonal antibody against botulinum neurotoxin serotype B (BoNT/B), named BTBH-N1, was developed from mice immunized with BoNT/B toxoid without non-toxic components, which are generally associated with the toxin. Western blot analysis, using recombinant toxin fragments containing light (L), N-terminal half of heavy (HN) and C-terminal half of heavy chains, indicated that BTBH-N1 recognizes linear epitopes located on the HN domain. An in vivo neutralization assay with mice, was conducted to characterize the neutralization capacity of the BTBH-N1. Only 10 microg of BTBH-N1 completely neutralized 20 units (1 unit = one 50% lethal dose) of BoNT/B. Even though the Mab (up to 100 microg) failed to protect mice challenged with 100 units, it significantly prolonged the time to death in a dose dependent manner. BTBH-N1, the first neutralizing antibody against BoNT/B, could be further developed as effective biological therapeutics for preventing and treating botulism, as well as other diseases caused by BoNT/B.

  13. Bivalent vaccine platform based on Japanese encephalitis virus (JEV) elicits neutralizing antibodies against JEV and hepatitis C virus.

    PubMed

    Saga, Ryohei; Fujimoto, Akira; Watanabe, Noriyuki; Matsuda, Mami; Hasegawa, Makoto; Watashi, Koichi; Aizaki, Hideki; Nakamura, Noriko; Tajima, Shigeru; Takasaki, Tomohiko; Konishi, Eiji; Kato, Takanobu; Kohara, Michinori; Takeyama, Haruko; Wakita, Takaji; Suzuki, Ryosuke

    2016-06-27

    Directly acting antivirals recently have become available for the treatment of hepatitis C virus (HCV) infection, but there is no prophylactic vaccine for HCV. In the present study, we took advantage of the properties of Japanese encephalitis virus (JEV) to develop antigens for use in a HCV vaccine. Notably, the surface-exposed JEV envelope protein is tolerant of inserted foreign epitopes, permitting display of novel antigens. We identified 3 positions that permitted insertion of the HCV E2 neutralization epitope recognized by HCV1 antibody. JEV subviral particles (SVP) containing HCV-neutralization epitope (SVP-E2) were purified from culture supernatant by gel chromatography. Sera from mice immunized with SVP-E2 inhibited infection by JEV and by trans-complemented HCV particles (HCVtcp) derived from multi-genotypic viruses, whereas sera from mice immunized with synthetic E2 peptides did not show any neutralizing activity. Furthermore, sera from mice immunized with SVP-E2 neutralized HCVtcp with N415K escape mutation in E2. As with the SVP-E2 epitope-displaying particles, JEV SVPs with HCV E1 epitope also elicited neutralizing antibodies against HCV. Thus, this novel platform harboring foreign epitopes on the surface of the particle may facilitate the development of a bivalent vaccine against JEV and other pathogens.

  14. Bivalent vaccine platform based on Japanese encephalitis virus (JEV) elicits neutralizing antibodies against JEV and hepatitis C virus

    PubMed Central

    Saga, Ryohei; Fujimoto, Akira; Watanabe, Noriyuki; Matsuda, Mami; Hasegawa, Makoto; Watashi, Koichi; Aizaki, Hideki; Nakamura, Noriko; Tajima, Shigeru; Takasaki, Tomohiko; Konishi, Eiji; Kato, Takanobu; Kohara, Michinori; Takeyama, Haruko; Wakita, Takaji; Suzuki, Ryosuke

    2016-01-01

    Directly acting antivirals recently have become available for the treatment of hepatitis C virus (HCV) infection, but there is no prophylactic vaccine for HCV. In the present study, we took advantage of the properties of Japanese encephalitis virus (JEV) to develop antigens for use in a HCV vaccine. Notably, the surface-exposed JEV envelope protein is tolerant of inserted foreign epitopes, permitting display of novel antigens. We identified 3 positions that permitted insertion of the HCV E2 neutralization epitope recognized by HCV1 antibody. JEV subviral particles (SVP) containing HCV-neutralization epitope (SVP-E2) were purified from culture supernatant by gel chromatography. Sera from mice immunized with SVP-E2 inhibited infection by JEV and by trans-complemented HCV particles (HCVtcp) derived from multi-genotypic viruses, whereas sera from mice immunized with synthetic E2 peptides did not show any neutralizing activity. Furthermore, sera from mice immunized with SVP-E2 neutralized HCVtcp with N415K escape mutation in E2. As with the SVP-E2 epitope-displaying particles, JEV SVPs with HCV E1 epitope also elicited neutralizing antibodies against HCV. Thus, this novel platform harboring foreign epitopes on the surface of the particle may facilitate the development of a bivalent vaccine against JEV and other pathogens. PMID:27345289

  15. Neutralizing Antibodies from the Sera of Human Immunodeficiency Virus Type 1-Infected Individuals Bind to Monomeric gp120 and Oligomeric gp140

    PubMed Central

    Stamatos, Nicholas M.; Mascola, John R.; Kalyanaraman, Vaniambadi S.; Louder, Mark K.; Frampton, Lynn M.; Birx, Deborah L.; VanCott, Thomas C.

    1998-01-01

    Antibodies that neutralize primary isolates of human immunodeficiency virus type 1 (HIV-1) appear during HIV-1 infection but are difficult to elicit by immunization with current vaccine products comprised of monomeric forms of HIV-1 envelope glycoprotein gp120. The limited neutralizing antibody response generated by gp120 vaccine products could be due to the absence or inaccessibility of the relevant epitopes. To determine whether neutralizing antibodies from HIV-1-infected patients bind to epitopes accessible on monomeric gp120 and/or oligomeric gp140 (ogp140), purified total immunoglobulin from the sera of two HIV-1-infected patients as well as pooled HIV immune globulin were selectively depleted of antibodies which bound to immobilized gp120 or ogp140. After passage of each immunoglobulin preparation through the respective columns, antibody titers against gp120 and ogp140 were specifically reduced at least 128-fold. The gp120- and gp140-depleted antibody fraction from each serum displayed reduced neutralization activity against three primary and two T-cell line-adapted (TCLA) HIV-1 isolates. Significant residual neutralizing activity, however, persisted in the depleted sera, indicating additional neutralizing antibody specificities. gp120- and ogp140-specific antibodies eluted from each column neutralized both primary and TCLA viruses. These data demonstrate the presence and accessibility of epitopes on both monomeric gp120 and ogp140 that are specific for antibodies that are capable of neutralizing primary isolates of HIV-1. Thus, the difficulties associated with eliciting neutralizing antibodies by using current monomeric gp120 subunit vaccines may be related less to improper protein structure and more to ineffective immunogen formulation and/or presentation. PMID:9811699

  16. Cervicovaginal neutralizing antibodies to herpes simplex virus (HSV) in women seropositive for HSV Types 1 and 2.

    PubMed

    Mbopi-Kéou, Francois-Xavier; Bélec, Laurent; Dalessio, Julie; Legoff, Jérôme; Grésenguet, Gérard; Mayaud, Philippe; Brown, David W G; Morrow, Rhoda Ashley

    2003-05-01

    Antibodies to herpes simplex virus type 1 (HSV-1) and HSV-2 of the immunoglobulin G (IgG) and IgA isotypes were detected in the cervicovaginal secretions (CVS) of 77 HSV-1- and HSV-2-seropositive but clinically asymptomatic African women by type-specific enhanced chemiluminescence Western blotting (ECL-WB). Of the 77 subjects, 34 were HIV negative, shedding HSV-2 DNA in their genital secretions; 20 were HIV positive, shedding HSV-2 DNA; and 23 were HIV negative, not shedding HSV-2 DNA. HSV-specific IgG was detected in CVS of nearly 70% of the women studied. HSV-specific IgA was found in CVS of 50% of the women studied. The distribution of CVS HSV-specific antibodies to each HSV type was highly heterogeneous, with a slight predominance of detectable IgG to HSV-1 (59%) over IgG to HSV-2 (41%), whereas the frequency of detectable IgA to HSV-1 (39%) was similar to that of IgA to HSV-2 (36%). The presence of detectable HSV-specific antibodies was inversely associated with HSV-2 DNA genital asymptomatic shedding but was not affected by HIV seropositivity. In addition, 13 of 77 (17%) CVS samples showed neutralizing activity against HSV-2, as assessed by an HSV-2 in vitro infectivity reduction assay. Neutralizing activity in CVS was associated with the presence of IgG and/or IgA antibodies to HSV-1 and/or to HSV-2 by ECL-WB. Among women whose CVS showed HSV-2-neutralizing activity, the specific activity of HSV-specific neutralizing antibodies was substantially (fivefold) higher in HSV-2 DNA shedders than in nonshedders. In conclusion, HSV-specific antibodies are frequently detected in CVS of asymptomatic African women seropositive for HSV-1 and HSV-2. A subset of these women had functional neutralizing activity against HSV-2 in their CVS. The origin of these antibodies and their role in HSV-2 disease of the female genital tract remain to be determined.

  17. Cervicovaginal Neutralizing Antibodies to Herpes Simplex Virus (HSV) in Women Seropositive for HSV Types 1 and 2

    PubMed Central

    Mbopi-Kéou, Francois-Xavier; Bélec, Laurent; Dalessio, Julie; Legoff, Jérôme; Grésenguet, Gérard; Mayaud, Philippe; Brown, David W. G.; Ashley Morrow, Rhoda

    2003-01-01

    Antibodies to herpes simplex virus type 1 (HSV-1) and HSV-2 of the immunoglobulin G (IgG) and IgA isotypes were detected in the cervicovaginal secretions (CVS) of 77 HSV-1- and HSV-2-seropositive but clinically asymptomatic African women by type-specific enhanced chemiluminescence Western blotting (ECL-WB). Of the 77 subjects, 34 were HIV negative, shedding HSV-2 DNA in their genital secretions; 20 were HIV positive, shedding HSV-2 DNA; and 23 were HIV negative, not shedding HSV-2 DNA. HSV-specific IgG was detected in CVS of nearly 70% of the women studied. HSV-specific IgA was found in CVS of 50% of the women studied. The distribution of CVS HSV-specific antibodies to each HSV type was highly heterogeneous, with a slight predominance of detectable IgG to HSV-1 (59%) over IgG to HSV-2 (41%), whereas the frequency of detectable IgA to HSV-1 (39%) was similar to that of IgA to HSV-2 (36%). The presence of detectable HSV-specific antibodies was inversely associated with HSV-2 DNA genital asymptomatic shedding but was not affected by HIV seropositivity. In addition, 13 of 77 (17%) CVS samples showed neutralizing activity against HSV-2, as assessed by an HSV-2 in vitro infectivity reduction assay. Neutralizing activity in CVS was associated with the presence of IgG and/or IgA antibodies to HSV-1 and/or to HSV-2 by ECL-WB. Among women whose CVS showed HSV-2-neutralizing activity, the specific activity of HSV-specific neutralizing antibodies was substantially (fivefold) higher in HSV-2 DNA shedders than in nonshedders. In conclusion, HSV-specific antibodies are frequently detected in CVS of asymptomatic African women seropositive for HSV-1 and HSV-2. A subset of these women had functional neutralizing activity against HSV-2 in their CVS. The origin of these antibodies and their role in HSV-2 disease of the female genital tract remain to be determined. PMID:12738636

  18. Protection of Macaques against Pathogenic Simian/Human Immunodeficiency Virus 89.6PD by Passive Transfer of Neutralizing Antibodies

    PubMed Central

    Mascola, John R.; Lewis, Mark G.; Stiegler, Gabriela; Harris, Dawn; VanCott, Thomas C.; Hayes, Deborah; Louder, Mark K.; Brown, Charles R.; Sapan, Christine V.; Frankel, Sarah S.; Lu, Yichen; Robb, Merlin L.; Katinger, Hermann; Birx, Deborah L.

    1999-01-01

    The role of antibody in protection against human immunodeficiency virus (HIV-1) has been difficult to study in animal models because most primary HIV-1 strains do not infect nonhuman primates. Using a chimeric simian/human immunodeficiency virus (SHIV) based on the envelope of a primary isolate (HIV-89.6), we performed passive-transfer experiments in rhesus macaques to study the role of anti-envelope antibodies in protection. Based on prior in vitro data showing neutralization synergy by antibody combinations, we evaluated HIV immune globulin (HIVIG), and human monoclonal antibodies (MAbs) 2F5 and 2G12 given alone, compared with the double combination 2F5/2G12 and the triple combination HIVIG/2F5/2G12. Antibodies were administered 24 h prior to intravenous challenge with the pathogenic SHIV-89.6PD. Six control monkeys displayed high plasma viremia, rapid CD4+-cell decline, and clinical AIDS within 14 weeks. Of six animals given HIVIG/2F5/2G12, three were completely protected; the remaining three animals became SHIV infected but displayed reduced plasma viremia and near normal CD4+-cell counts. One of three monkeys given 2F5/2G12 exhibited only transient evidence of infection; the other two had marked reductions in viral load. All monkeys that received HIVIG, 2F5, or 2G12 alone became infected and developed high-level plasma viremia. However, compared to controls, monkeys that received HIVIG or MAb 2G12 displayed a less profound drop in CD4+ T cells and a more benign clinical course. These data indicate a general correlation between in vitro neutralization and protection and suggest that a vaccine that elicits neutralizing antibody should have a protective effect against HIV-1 infection or disease. PMID:10196297

  19. Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies.

    PubMed

    Mascola, J R; Lewis, M G; Stiegler, G; Harris, D; VanCott, T C; Hayes, D; Louder, M K; Brown, C R; Sapan, C V; Frankel, S S; Lu, Y; Robb, M L; Katinger, H; Birx, D L

    1999-05-01

    The role of antibody in protection against human immunodeficiency virus (HIV-1) has been difficult to study in animal models because most primary HIV-1 strains do not infect nonhuman primates. Using a chimeric simian/human immunodeficiency virus (SHIV) based on the envelope of a primary isolate (HIV-89.6), we performed passive-transfer experiments in rhesus macaques to study the role of anti-envelope antibodies in protection. Based on prior in vitr