Immune Correlates Analysis Research Articles

Antibody responses induced to the C1 region of HIV-1 gp120 in acute HIV-1 infection and after vaccination

Abstract Based on the analysis of secondary immune correlates, the RV144 phase III trial demonstrated a reduced risk of infection in vaccine recipients with high levels of ADCC activity and low HIV-1 Env specific plasma IgA antibodies (Abs). Ab responses to the HIV-1 Env constant 1 (C1) region constituted the dominant ADCC Ab response. To further explore the binding Ab profiles and determine differences in the Ab responses to the C1 region (100 amino acids) induced by vaccine versus natural HIV-1 infection, plasma samples from an early acute HIV-1 infection Thai cohort (RV217) and a phase II clinical trial, RV135 (ALVAC prime and gp120 SF2/CM235 boost adjuvanted with MF59) were evaluated by Biacore. Based on the sequencing data from 16 RV217 individuals at 3 time points (week 1, weeks 4–6 and weeks 21–27 post infection), consensus 40-mer C1 peptides with 10-mer-overlapping amino acid sequences were initially screened for binding responses in RV217 and RV135 plasma samples. This was then followed with 16-mer peptides with 8-mer overlapping amino acid sequences to match the viral sequences in individual RV217 patients at these 3 time points. Vaccinees (RV135) induced Ab responses that recognized linear C1 peptides spanning amino acids 31-46, 55-70, 61-76, 77-92, and 85-100, whereas HIV-1 infected individuals (RV217) recognized linear C1 peptides spanning amino acids 1-16, 55-70, and 77-92. Future experiments will examine the functional role of these C1-specific Abs, in particular ADCC activity.

D-106 Cellular immune correlates analysis of an HIV-1 pre- exposure prophylaxis trial

D-106 Cellular immune correlates analysis of an HIV-1 pre- exposure prophylaxis trial

Erratum.

Journal of the International AIDS SocietyVolume 19, Issue 1 21252 ErratumOpen Access Erratum This article corrects the following: HIV Vaccine Awareness Day: sustaining the momentum Kathleen M MacQueen, Mitchell Warren, Volume 19Issue 1Journal of the International AIDS Society First Published online: May 18, 2016 First published: 10 June 2016 https://doi.org/10.7448/IAS.19.1.21252AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Regarding the paper ‘HIV Vaccine Awareness Day: sustaining the momentum’ by Kathleen M MacQueen, Mitchell Warren. Published in Journal of the International AIDS Society, Citation: MacQueen KM and Warren M. Journal of the International AIDS Society 2016, 19:21202. http://www.jiasociety.org/index.php/jias/article/view/21202 ∣ http://dx.doi.org/10.7448/IAS.19.1.21202 Reference 6 is missing in the reference list, and should be cited in the text according to the following: “This is no different than the requirements for advancing research on a cure for HIV [6], on new methods …”. The correct reference list is displayed below. References 1Snow B. Vaccine v. Cure: an HIV-positive perspective. In: B Snow, editor. HIV vaccine handbook: community perspectives on participating in research, advocacy, and progress. Washington, DC: AIDS Vaccine Advocacy Coalition; 1999. pp. 17– 20. Google Scholar 2Esparza J. What has 30 years of HIV vaccine research taught us? Vaccines. 2013; 1: 513– 26. doi: http://dx.doi.org/10.3390/vaccines1040513 Google Scholar 3Harmon TM, Fisher KA, McGlynn MG, Stover J, Warren MJ, Teng Y, et al. Exploring the potential health impact and cost-effectiveness of AIDS vaccine within a comprehensive HIV/AIDS response in low and middle-income countries. PLoS One. 2016; 11(1): e0146387. doi: http://dx.doi.org/10.1371/journal.pone.0146387 Google Scholar 4Rerks-Ngarm, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, et al. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med. 2009; 361: 2209– 20. doi: http://dx.doi.org/10.1056/NEJMoa0908492 Google Scholar 5Haynes BF, Gilbert PB, McElrath J, Zolla-Pazner S, Tomaras GD, Alam SM, et al. Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N Engl J Med. 2012; 366: 1275– 86. doi: http://dx.doi.org/10.1056/NEJMoa1113425 Google Scholar 6Meier BM, Gelpi A, Kavanagh MW, Forman L, Amon JJ. Employing human rights frameworks to realize access to an HIV cure. JIAS. 2015; 18: 20305. doi: http://dx.doi.org/10.7448/IAS.18.1.20305 Google Scholar Volume19, Issue1January 201621252 ReferencesRelatedInformation

Breakthrough Virus Neutralization Resistance as a Correlate of Protection in a Nonhuman Primate Heterologous Simian Immunodeficiency Virus Vaccine Challenge Study.

Comprehensive assessments of immune correlates of protection in human immunodeficiency virus (HIV) vaccine trials are essential to vaccine design. Neutralization sieve analysis compares the neutralization sensitivity of the breakthrough transmitted/founder (TF) viruses from vaccinated and control animals to infer the molecular mechanisms of vaccine protection. Here, we report a robust neutralization sieve effect in a nonhuman primate simian immunodeficiency virus (SIV) vaccine trial (DNA prime/recombinant adenovirus type 5 [rAd5] boost) (VRC-10-332) that demonstrated substantial protective efficacy and revealed a genetic signature of neutralization resistance in the C1 region of env. We found significant enrichment for neutralization resistance in the vaccine compared to control breakthrough TF viruses when tested with plasma from vaccinated study animals, plasma from chronically SIV-infected animals, and a panel of SIV-specific monoclonal antibodies targeting six discrete Env epitopes (P < 0.008 for all comparisons). Neutralization resistance was significantly associated with the previously identified genetic signature of resistance (P < 0.0001), and together, the results identify virus neutralization as a correlate of protection. These findings further demonstrate the in vivo relevance of our previous in vitro analyses of the SIVsmE660 challenge stock, which revealed a broad range of neutralization sensitivities of its component viruses. In sum, this report demonstrates proof-of-concept that phenotypic sieve analyses can elucidate mechanistic correlates of immune protection following vaccination and raises a cautionary note for SIV and SHIV (simian-human immunodeficiency virus) vaccine studies that employ challenge strains with envelope glycoproteins that fail to exhibit neutralization resistance profiles typical of TF viruses. With more than 2 million new infections annually, the development of an effective vaccine against HIV-1 is a global health priority. Understanding immunologic correlates of protection generated in vaccine trials is critical to advance vaccine development. Here, we assessed the role of vaccine-elicited neutralizing antibodies in a recent nonhuman primate study of a vaccine that showed significant protection against simian immunodeficiency virus (SIV) challenge and suggested a genetic signature of neutralization sensitivity. We found that breakthrough viruses able to establish infection in vaccinated animals were substantially more resistant to antibody-mediated neutralization than were viruses from controls. These findings suggest that vaccine-elicited neutralizing antibodies selectively blocked the transmission of more sensitive challenge viruses. Sieve analysis also corroborated a genetic signature of neutralization sensitivity and highlighted the impact of challenge swarm diversity. Our findings suggest an important role for neutralization sieve analyses as an informative component of comprehensive immune-correlates analyses.

Cellular immune correlates analysis of an HIV-1 preexposure prophylaxis trial

HIV-1-specific T-cell responses in exposed seronegative subjects suggest that a viral breach of the exposure site is more common than current transmission rates would suggest and that host immunity can extinguish subsequent infection foci. The Preexposure Prophylaxis Initiative (iPrEx) chemoprophylaxis trial provided an opportunity to rigorously investigate these responses in a case-control immunology study; 84 preinfection peripheral blood mononuclear cell samples from individuals enrolled in the iPrEx trial who later seroconverted were matched with 480 samples from enrolled subjects who remained seronegative from both the placebo and active treatment arms. T-cell responses to HIV-1 Gag, Protease, Integrase, Reverse Transcriptase, Vif, and Nef antigens were quantified for all subjects in an IFN-γ enzyme-linked immunospot (ELISpot) assay. IFN-γ responses varied in magnitude and frequency across subjects. A positive response was more prevalent in those who remained persistently HIV-1-negative for Gag (P = 0.007), Integrase (P < 0.001), Vif (P < 0.001), and Nef (P < 0.001). When correlated with outcomes in the iPrEx trial, Vif- and Integrase-specific T-cell responses were associated with reduced HIV-1 infection risk [hazard ratio (HR) = 0.36, 95% confidence interval (95% CI) = 0.19-0.66 and HR = 0.52, 95% CI = 0.28-0.96, respectively]. Antigen-specific responses were independent of emtricitabine/tenofovir disoproxil fumarate use. IFN-γ secretion in the ELISpot was confirmed using multiparametric flow cytometry and largely attributed to effector memory CD4+ or CD8+ T cells. Our results show that HIV-1-specific T-cell immunity can be detected in exposed but uninfected individuals and that these T-cell responses can differentiate individuals according to infection outcomes.

Immune-correlates analysis of an HIV-1 vaccine efficacy trial reveals an association of nonspecific interferon-γ secretion with increased HIV-1 infection risk: a cohort-based modeling study.

BackgroundElevated risk of HIV-1 infection among recipients of an adenovirus serotype 5 (Ad5)-vectored HIV-1 vaccine was previously reported in the Step HIV-1 vaccine efficacy trial. We assessed pre-infection cellular immune responses measured at 4 weeks after the second vaccination to determine their roles in HIV-1 infection susceptibility among Step study male participants.MethodsWe examined ex vivo interferon-γ (IFN-γ) secretion from peripheral blood mononuclear cells (PBMC) using an ELISpot assay in 112 HIV-infected and 962 uninfected participants. In addition, we performed flow cytometric assays to examine T-cell activation, and ex vivo IFN-γ and interleukin-2 secretion from CD4+ and CD8+ T cells. We accounted for the sub-sampling design in Cox proportional hazards models to estimate hazard ratios (HRs) of HIV-1 infection per 1-loge increase of the immune responses.FindingsWe found that HIV-specific immune responses were not associated with risk of HIV-1 infection. However, each 1-loge increase of mock responses measured by the ELISpot assay (i.e., IFN-γ secretion in the absence of antigen-specific stimulation) was associated with a 62% increase of HIV-1 infection risk among vaccine recipients (HR = 1.62, 95% CI: (1.28, 2.04), p<0.001). This association remains after accounting for CD4+ or CD8+ T-cell activation. We observed a moderate correlation between ELISpot mock responses and CD4+ T-cells secreting IFN-γ (ρ = 0.33, p = 0.007). In addition, the effect of the Step vaccine on infection risk appeared to vary with ELISpot mock response levels, especially among participants who had pre-existing anti-Ad5 antibodies (interaction p = 0.04).ConclusionsThe proportion of cells, likely CD4+ T-cells, producing IFN-γ without stimulation by exogenous antigen appears to carry information beyond T-cell activation and baseline characteristics that predict risk of HIV-1 infection. These results motivate additional investigation to understand the potential link between IFN-γ secretion and underlying causes of elevated HIV-1 infection risk among vaccine recipients in the Step study.

Lessons from the RV144 Thai phase III HIV-1 vaccine trial and the search for correlates of protection.

RV144 remains the only HIV-1 vaccine trial to demonstrate efficacy against HIV-1 acquisition. The prespecified analysis of immune correlates of risk showed that antibodies directed against the V1V2 region of gp120, in particular the IgG1 and IgG3 subclass mediating antibody-dependent cell-mediated cytotoxicity, seem to play a predominant role in protection against HIV-1 acquisition and that plasma envelope (Env)-specific IgA antibodies were directly correlated with risk. RV144 and recent nonhuman primate challenge studies suggest that Env is essential, and perhaps sufficient, to induce protective antibody responses against mucosal HIV-1 acquisition. Follow-up clinical trials are ongoing to further dissect the immune responses elicited by the RV144 ALVAC-HIV and AIDSVAX® B/E regimen. The study of gp120 Env immunogens and immune correlates of risk has resulted in the development of improved antigens. Whether the RV144 immune correlates of risk will generalize to other populations vaccinated with similar immunogens with different modes and intensity of transmission remains to be demonstrated. Efficacy trials are now planned in heterosexual populations in southern Africa and men who have sex with men in Thailand.

Sequence-conserved and antibody-accessible sites in the V1V2 domain of HIV-1 gp120 envelope protein.

The immune-correlates analysis of the RV144 trial suggested that epitopes targeted by protective antibodies (Abs) reside in the V1V2 domain of gp120. We mapped V1V2 positional sequence variation onto the conserved V1V2 structural fold and showed that while most of the solvent-accessible V1V2 amino acids vary between strains, there are two accessible molecular surface regions that are conserved and also naturally antigenic. These sites may contain epitopes targeted by broadly cross-reactive anti-V1V2 antibodies.

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

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.

Dissecting the Antibody Constant Region Protective Immune Parameters in HIV Infection

ABSTRACT: RV144 vaccine immune-correlates analysis has generated a renewed interest in understanding the potentially protective role of non-neutralizing antibodies in HIV infection and vaccine design. Antibodies consist of a variable region involved in antigen binding and a constant region. While both ends of the antibody collaborate to induce protective immunity, it is through the constant portion that an antibody provides instructions to the innate immune system on how the recognized antigen should be processed, contributing directly to antiviral immunity. Antibody constant regions, despite their name, are not uniform structures, but can vary both in protein sequence and glycosylation, together modulating antibody functionality via conformational changes that alter antibody affinity for Fc receptors, complement and so on. This review will focus on how the immune system naturally modulates the Fc domain of antibodies to achieve optimum protective Fc effector responses for vaccine and monoclonal therapeutic design efforts aimed at preventing or curing HIV infection.

Lessons Learned from HIV Vaccine Clinical Efficacy Trials

The past few years have witnessed many promising advances in HIV prevention strategies involving preexposure prophylaxis approaches. Some may now wonder whether an HIV vaccine is still needed, and whether developing one is even possible. The partial efficacy reported in the RV144 trial and the encouraging results of the accompanying immune correlates analysis suggest that an effective HIV vaccine is achievable. These successes have provided a large impetus and guidance for conducting more HIV vaccine trials. A key lesson learned from RV144 is that assessment of HIV acquisition is now a feasible and valuable primary objective for HIV preventive vaccine trials. In this article we review how RV144 and other HIV vaccine efficacy trials have instructed the field and highlight some of the HIV vaccine concepts in clinical development. After a long and significant investment, HIV vaccine clinical research is paying off in the form of valuable lessons that, if applied effectively, will accelerate the path toward a safe and effective vaccine. Together with other HIV prevention approaches, preventive and therapeutic HIV vaccines will be invaluable tools in bringing the epidemic to an end.

Toward an effective AIDS vaccine development

Toward an effective AIDS vaccine development

Retrospective proteomic analysis of cellular immune responses and protective correlates of p24 vaccination in an HIV elite controller

BackgroundHIV p24 is an extracellular HIV antigen that is involved in viral replication. Falling p24 antibody responses are associated with clinical disease progression, whereas their preservation with non-progression implies that stimulation of p24 antibody production by immunisation might delay progression. This was the basis of the discontinued p24 vaccine. We identified a therapy-naive man aged 61 years from Sydney, Australia, infected in 1988 through his partner. He received the HIV-p24-virus like particle (VLP) vaccine and a 3 week course of azidothymidin monotherapy in 1993, and continued to show vigorous p24 antigen responses with more than 4% p24-specific CD4+ T cells, coupled with undetectable plasma viraemia, until December, 2011. The patient has since remained therapy naive, with plasma viraemia below detectable levels. The patient is heterozygous for CCR5Δ32 and has an HLA type A1, 2; B8, 44; DR4, 15. In 2011, a transitory viral spike (163 HIV RNA copies per mL plasma) was recorded, which without antiretroviral drug treatment reverted to undetectable virus (<20 HIV copies per mL) in 3 weeks. We aimed to define the immune-protective correlates of p24 vaccination in this individual by retrospective analysis of cellular responses to p24 antigen in CD4+ and CD8+ T cells, and CD14+ monocytes, in terms of the concentrations of 270 cytokines and chemokines during viraemia and aviraemia. MethodsWe used whole peripheral blood mononuclear cells from the two phases, stimulated them with p24 antigen, and then separated them by magnetic beads as stimulated and unstimulated CD4+ and CD8+ T cells, and monocyte fractions. Cellular proteins were extracted, quantified, and analysed with a cytokine antibody array. Differentially expressed proteins were analysed with online bioinformatic tools, and statistics evaluated with the student t test and three k-way analyses. FindingsEncompassing all three cell types, we found statistically significant coordinated upregulation with high fold-changes of fractalkine, ITAC, IGFBP-2, cathepsin-S, and CCL14a in the aviraemic phase. TECK and TRAIL-R4 were downregulated in the viraemic phase and upregulated in the aviraemic phase. Fractalkine, which showed high fold-changes across cell types during the aviraemic phase and no expression during viraemia, plays a vital part in promotion of cell survival during homeostatic and inflammatory conditions. The upregulation of fractalkine in all three cell types (14-fold in CD4+ T cells, 12-fold in monocytes, and five-fold in CD8+ T cells) during aviraemia might be associated with a protective effect. Interestingly, downregulation of TRAIL-R3/R4 and TECK/CCL25 coincided with plasma viraemia and their upregulation with aviraemia, suggesting that dysfunction in antiapoptotic chemokines might be a mechanism contributing to loss of immune function during HIV infection, and that an effective T-cell-derived immune response can be mounted without T-cell exhaustion. Decrease in TECK expression is associated with increased apoptosis in lymphoid tissues in macaques infected with simian immunodeficiency virus. InterpretationBecause the p24 VLP vaccine failed to induce antibody titres, the possible beneficial effect of p24 vaccine in this individual could be attributed to broad and coordinated cellular responses mounted by all three cell types in tandem in modulating viraemic and aviraemic phases. This study highlights that induction of HIV-1-specific helper cells might be important in immunotherapeutic interventions and HIV vaccine development. FundingWe acknowledge the support of a National Health and Medical Research Council Development grant to NKS.

Progress in HIV-1 vaccine development.

In this review, examples of recent progress in HIV-1 vaccine research are discussed. New insights from the immune correlates analyses of the RV144 efficacy trial have accelerated vaccine development with leads to follow in nonhuman primate studies and improved vaccine designs. Several new vaccine vector approaches offer promise in the exquisite control of acute infection and in improving the breadth of T-cell responses. New targets of broadly neutralizing antibodies (BnAbs) have been elucidated, and improved understanding of how the human host controls BnAb development have emerged from BnAb knock-in mice and from analyses of BnAb maturation and virus evolution in individuals followed from the time of HIV-1 transmission to BnAb induction. Based on these observations, it is clear that the development of a successful HIV-1 vaccine will require new vaccine approaches and iterative testing of immunogens in well designed animal and human trials.

A Novel Protective MHC-I Haplotype Not Associated with Dominant Gag-Specific CD8+ T-Cell Responses in SIVmac239 Infection of Burmese Rhesus Macaques

Several major histocompatibility complex class I (MHC-I) alleles are associated with lower viral loads and slower disease progression in human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections. Immune-correlates analyses in these MHC-I-related HIV/SIV controllers would lead to elucidation of the mechanism for viral control. Viral control associated with some protective MHC-I alleles is attributed to CD8+ T-cell responses targeting Gag epitopes. We have been trying to know the mechanism of SIV control in multiple groups of Burmese rhesus macaques sharing MHC-I genotypes at the haplotype level. Here, we found a protective MHC-I haplotype, 90-010-Id (D), which is not associated with dominant Gag-specific CD8+ T-cell responses. Viral loads in five D+ animals became significantly lower than those in our previous cohorts after 6 months. Most D+ animals showed predominant Nef-specific but not Gag-specific CD8+ T-cell responses after SIV challenge. Further analyses suggested two Nef-epitope-specific CD8+ T-cell responses exerting strong suppressive pressure on SIV replication. Another set of five D+ animals that received a prophylactic vaccine using a Gag-expressing Sendai virus vector showed significantly reduced viral loads compared to unvaccinated D+ animals at 3 months, suggesting rapid SIV control by Gag-specific CD8+ T-cell responses in addition to Nef-specific ones. These results present a pattern of SIV control with involvement of non-Gag antigen-specific CD8+ T-cell responses.

Antigenicity and Immunogenicity of RV144 Vaccine AIDSVAX Clade E Envelope Immunogen Is Enhanced by a gp120 N-Terminal Deletion

An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity.

Molecular epidemiology of the protective RV144 V2 loop epitope

Background The immune correlate analysis of the RV144 trial identified antibodies (Abs) targeted at the V1-V2 domain as potentially protective vaccine responses. Reactivity of vaccinee serum Abs with a glycosylated gp70-V1-V2 fusion protein or with certain V2 loop peptides was significantly associated with a lower risk of viral infection. A V2 loop segment approximately comprising amino acid residues 165-178 of gp120 was also identified by pilot vaccinee studies as a key immunogenic region. Methods To identify whether the segment identified by pilot studies coincides with the reagents reactive in the case control study, I mapped the locations of the reagents associated with lower risk of viral infection onto the V1-V2 domain structure. With a single segment identified as associated both with immunogenicity in the pilot studies and protection in the case control study, I calculated the Dayhoff evolutionary sequence distance between the the sequences of this segment in the immunogens and in a panel of V2 loop based reagents used in the case control study, each with associated odds ratios (OR) for risk. OR was then plotted against distance. Results The same V2 loop segment from positions 165-178 that was specifically most immunogenic in the pilot studies appears to have been associated with protection in the immune correlate analysis. This segment corresponds to the “C” strand of the V1-V2 domain beta-sheet fold, exactly where the broadly neutralizing antibody PG9 binds. Plotting the evolutionary distance between this segment in the RV144 immunogens and synthetic V2 loop based antigens used in the case control study shows that lower risk (lower OR) correlates with greater evolutionary distance.

Antibody-dependent cellular cytotoxicity-mediating antibodies from an HIV-1 vaccine efficacy trial preferentially use the VH1 gene family

Background The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine efficacy trial showed an estimated efficacy of 31%. The immune correlates analysis raised the hypothesis that the observed protection in RV144 may be partially due to AntibodyDependent Cellular Cytotoxicity (ADCC)-mediating antibodies in the presence of low levels of Env IgA antibodies. In this study we analyzed the Ig VH family usage of vaccine-induced ADCC mAbs isolated from memory B cells of vaccinees. Methods From a total of 321,945 memory B-cells of 6 vaccinees we obtained 23 mAbs that mediated ADCC using IgG+ memory B-cell cultures (n=9) and Env-specific flow cytometric single memory B-cell sorting (n=14). ADCC activity was measured using both E.CM243 gp120-coated and E. CM235-infected target cells in a flow-based assay. Results ADCC-mediating mAbs displayed a disproportionate usage of VH1 family genes (17/23; 74%), in particular the VH1-2 gene segment (10/17; 59%), as recently observed for CD4bs broadly neutralizing antibodies (HAAD bNAbs). In contrast, only 17.1% of 111 heavy chains isolated from cultures that did not mediate ADCC used the VH1 gene. VH1 ADCC-mediating mAbs showed a high degree of V(D)J amino acid similarity to both the VH (68-84%) and VL (70-87%) HAAD motifs. V(D)J rearrangements displayed modest levels of affinity maturation (0.5-5.1% for heavy chains and 0.4-4.3% for light chains). While none of the VH1 ADCC-mediating mAbs was capable of mediating HIV-1 neutralization, the strength of their ADCC activity correlated with the levels of heavy chain somatic mutations (p=0.02). We produced the reverted unmutated ancestor antibodies of two VH1 ADCC-mediating mAbs: one bound to B.MN Env and both reacted against autoantigens. Conclusion ADCC-mediating antibodies induced by the ALVAC-HIV/ AIDSVAX-B/E vaccine underwent limited affinity maturation, and preferentially used VH1 gene segments which share the HAAD motif with CD4bs bNAbs. These observations raise the hypothesis that HIV-1 Env preferentially selects VH1 family usage for distinct subsets of antibodies with different functions.

Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies from an HIV-1 Vaccine Efficacy Trial Target Multiple Epitopes and Preferentially Use the VH1 Gene Family

The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs.

Optimization and qualification of a multiplex bead array to assess cytokine and chemokine production by vaccine-specific cells

The magnitude and functional phenotype (e.g. proliferation, immune stimulation) of vaccine-induced T-cell responses are likely to be critical in defining responses that can control pathogenic challenge. Current multi-parameter flow cytometric techniques may not be sufficient to measure all of these different functions, since characterizing T-cell responses by flow cytometry is presently limited to concurrent measurement of at most 10 cytokines/chemokines. Here, we describe extensive studies conducted using standardized GCLP procedures to optimize and qualitatively/quantitatively qualify a multiplex bead array (MBA) performed on supernatant collected from stimulated peripheral blood mononuclear cells (PBMC) to assess 12 cytokines and chemokines of interest. Our optimized MBA shows good precision (intra-assay, inter-day, inter-technician; coefficients of variation <30%) and linearity for most of the analytes studied. We also developed positivity criteria that allow us to define a response as positive or negative with a high degree of confidence. In conclusion, we provide a detailed description of the qualification of an MBA, which permits quantitative and qualitative evaluation of vaccine-induced immunogenicity and analysis of immune correlates of protection. This assay provides an excellent complement to the existing repertoire of assays for assessing immunogenicity in HIV vaccine clinical trials.