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1.  Identification of Immunodominant CD4-Restricted Epitopes Co-Located with Antibody Binding Sites in Individuals Vaccinated with ALVAC-HIV and AIDSVAX B/E 
PLoS ONE  2015;10(2):e0115582.
We performed fine epitope mapping of the CD4+ responses in the ALVAC-HIV-AIDSVAX B/E prime-boost regimen in the Thai Phase III trial (RV144). Non-transformed Env-specific T cell lines established from RV144 vaccinees were used to determine the fine epitope mapping of the V2 and C1 responses and the HLA class II restriction. Data showed that there are two CD4+ epitopes contained within the V2 loop: one encompassing the α4β7 integrin binding site (AA179-181) and the other nested between two previously described genetic sieve signatures (AA169, AA181). There was no correlation between the frequencies of CD4+ fine epitope responses and binding antibody.
doi:10.1371/journal.pone.0115582
PMCID: PMC4321833  PMID: 25665096
2.  Comprehensive Sieve Analysis of Breakthrough HIV-1 Sequences in the RV144 Vaccine Efficacy Trial 
PLoS Computational Biology  2015;11(2):e1003973.
The RV144 clinical trial showed the partial efficacy of a vaccine regimen with an estimated vaccine efficacy (VE) of 31% for protecting low-risk Thai volunteers against acquisition of HIV-1. The impact of vaccine-induced immune responses can be investigated through sieve analysis of HIV-1 breakthrough infections (infected vaccine and placebo recipients). A V1/V2-targeted comparison of the genomes of HIV-1 breakthrough viruses identified two V2 amino acid sites that differed between the vaccine and placebo groups. Here we extended the V1/V2 analysis to the entire HIV-1 genome using an array of methods based on individual sites, k-mers and genes/proteins. We identified 56 amino acid sites or “signatures” and 119 k-mers that differed between the vaccine and placebo groups. Of those, 19 sites and 38 k-mers were located in the regions comprising the RV144 vaccine (Env-gp120, Gag, and Pro). The nine signature sites in Env-gp120 were significantly enriched for known antibody-associated sites (p = 0.0021). In particular, site 317 in the third variable loop (V3) overlapped with a hotspot of antibody recognition, and sites 369 and 424 were linked to CD4 binding site neutralization. The identified signature sites significantly covaried with other sites across the genome (mean = 32.1) more than did non-signature sites (mean = 0.9) (p < 0.0001), suggesting functional and/or structural relevance of the signature sites. Since signature sites were not preferentially restricted to the vaccine immunogens and because most of the associations were insignificant following correction for multiple testing, we predict that few of the genetic differences are strongly linked to the RV144 vaccine-induced immune pressure. In addition to presenting results of the first complete-genome analysis of the breakthrough infections in the RV144 trial, this work describes a set of statistical methods and tools applicable to analysis of breakthrough infection genomes in general vaccine efficacy trials for diverse pathogens.
Author Summary
We present an analysis of the genomes of the HIV viruses that infected some participants of the RV144 Thai trial, which was the first study to show efficacy of a vaccine to prevent HIV infection. We analyzed the HIV genomes of infected vaccine recipients and infected placebo recipients, and found differences between them. These differences coincide with previously-studied genetic features that are relevant to the biology of HIV infection, including features involved in immune recognition of the virus. The findings presented here generate testable hypotheses about the mechanism of the partial protection seen in the Thai trial, and may ultimately lead to improved vaccines. The article also presents a toolkit of methods for computational analyses that can be applied to other vaccine efficacy trials.
doi:10.1371/journal.pcbi.1003973
PMCID: PMC4315437  PMID: 25646817
3.  Vaccine-Induced HIV-1 Envelope gp120 Constant Region 1-Specific Antibodies Expose a CD4-Inducible Epitope and Block the Interaction of HIV-1 gp140 with Galactosylceramide 
Journal of Virology  2014;88(16):9406-9417.
ABSTRACT
Mucosal epithelial cell surface galactosylceramide (Galcer) has been postulated to be a receptor for HIV-1 envelope (Env) interactions with mucosal epithelial cells. Disruption of the HIV-1 Env interaction with such alternate receptors could be one strategy to prevent HIV-1 entry through the mucosal barrier. To study antibody modulation of HIV-1 Env-Galcer interactions, we used Galcer-containing liposomes to assess whether natural- and vaccine-induced monoclonal antibodies can block HIV-1 Env binding to Galcer. HIV-1 Env gp140 proteins bound to Galcer liposomes with Kds (dissociation constants) in the nanomolar range. Several HIV-1 ALVAC/AIDSVAX vaccinee-derived monoclonal antibodies (MAbs) specific for the gp120 first constant (C1) region blocked Galcer binding of a transmitted/founder HIV-1 Env gp140. Among the C1-specific MAbs that showed Galcer blocking, the antibody-dependent cellular cytotoxicity-mediating CH38 IgG and its natural IgA isotype were the most potent blocking antibodies. C1-specific IgG monoclonal antibodies that blocked Env binding to Galcer induced upregulation of the gp120 CD4-inducible (CD4i) epitope bound by MAb 17B, demonstrating that a conformational change in gp120 may be required for Galcer blocking. However, the MAb 17B itself did not block Env-Galcer binding, suggesting that the C1 antibody-induced gp120 conformational changes resulted in alteration in a Galcer binding site distant from the CD4i 17B MAb binding site.
IMPORTANCE Galactosyl ceramide, a glycosphingolipid, has been postulated to be a receptor for the HIV-1 envelope glycoprotein (Env) interaction with mucosal epithelial cells. Here, we have mimicked this interaction by using an artificial membrane containing synthetic Galcer and recombinant HIV-1 Env proteins to identify antibodies that would block the HIV-1 Env-Galcer interaction. Our study revealed that a class of vaccine-induced human antibodies potently blocks HIV-1 Env-Galcer binding by perturbing the HIV-1 Env conformation.
doi:10.1128/JVI.01031-14
PMCID: PMC4136246  PMID: 24920809
4.  Issues in Women's Participation in a Phase III Community HIV Vaccine Trial in Thailand 
AIDS Research and Human Retroviruses  2013;29(11):1524-1534.
Abstract
To assess qualities and outcomes of women participating in a large, community-based HIV vaccine trial, the present study was conducted among female participants of the RV 144 prime-boost trial in Thailand from 2003 to 2009. Qualities of participation refer to complete vaccination, retention, and status change. Outcomes of participation refer to incident rate, adverse event, and participation impact event. A total of 6,334 (38.6%) women participated in the trial, of whom about 50% were classified as low risk and 11% as high risk. About 85% of participants completed four vaccinations and 76% were included in the per-protocol analysis of the on-time vaccination schedule. More women (88%) completed 42 months follow-up compared with men (85%). Women aged 21 and above had more adverse events compared to younger age groups. More women (5%) compared with men (3%) reported participation impact events (PIEs). High-risk women had more PIEs and a higher infection rate compared to the low-risk group. Complete vaccination and retention on last follow-up were more common in married women aged above 21, and being a housewife. Female volunteers showed the same qualities and outcomes of participation as males in the HIV vaccine trial. There was no statistically significant difference in vaccine efficacy between men and women, especially among the high-risk and married women. The study highlighted the important behavioral, social, and cultural issues that could be considered for future HIV vaccine trial designs.
doi:10.1089/aid.2012.0265
PMCID: PMC3809940  PMID: 23343395
5.  CD8 and CD4 Epitope Predictions in RV144: No Strong Evidence of a T-Cell Driven Sieve Effect in HIV-1 Breakthrough Sequences from Trial Participants 
PLoS ONE  2014;9(10):e111334.
The modest protection afforded by the RV144 vaccine offers an opportunity to evaluate its mechanisms of protection. Differences between HIV-1 breakthrough viruses from vaccine and placebo recipients can be attributed to the RV144 vaccine as this was a randomized and double-blinded trial. CD8 and CD4 T cell epitope repertoires were predicted in HIV-1 proteomes from 110 RV144 participants. Predicted Gag epitope repertoires were smaller in vaccine than in placebo recipients (p = 0.019). After comparing participant-derived epitopes to corresponding epitopes in the RV144 vaccine, the proportion of epitopes that could be matched differed depending on the protein conservation (only 36% of epitopes in Env vs 84–91% in Gag/Pol/Nef for CD8 predicted epitopes) or on vaccine insert subtype (55% against CRF01_AE vs 7% against subtype B). To compare predicted epitopes to the vaccine, we analyzed predicted binding affinity and evolutionary distance measurements. Comparisons between the vaccine and placebo arm did not reveal robust evidence for a T cell driven sieve effect, although some differences were noted in Env-V2 (0.022≤p-value≤0.231). The paucity of CD8 T cell responses identified following RV144 vaccination, with no evidence for V2 specificity, considered together both with the association of decreased infection risk in RV 144 participants with V-specific antibody responses and a V2 sieve effect, lead us to hypothesize that this sieve effect was not T cell specific. Overall, our results did not reveal a strong differential impact of vaccine-induced T cell responses among breakthrough infections in RV144 participants.
doi:10.1371/journal.pone.0111334
PMCID: PMC4211711  PMID: 25350851
6.  Aggregate complexes of HIV-1 induced by multimeric antibodies 
Retrovirology  2014;11(1):78.
Background
Antibody mediated viral aggregation may impede viral transfer across mucosal surfaces by hindering viral movement in mucus, preventing transcytosis, or reducing inter-cellular penetration of epithelia thereby limiting access to susceptible mucosal CD4 T cells and dendritic cells. These functions may work together to provide effective immune exclusion of virus from mucosal tissue; however little is known about the antibody characteristics required to induce HIV aggregation. Such knowledge may be critical to the design of successful immunization strategies to facilitate viral immune exclusion at the mucosal portals of entry.
Results
The potential of neutralizing and non-neutralizing IgG and IgA monoclonals (mAbs) to induce HIV-1 aggregation was assessed by Dynamic light scattering (DLS). Although neutralizing and non-neutralizing IgG mAbs and polyclonal HIV-Ig efficiently aggregated soluble Env trimers, they were not capable of forming viral aggregates. In contrast, dimeric (but not monomeric) IgA mAbs induced stable viral aggregate populations that could be separated from uncomplexed virions. Epitope specificity influenced both the degree of aggregation and formation of higher order complexes by dIgA. IgA purified from serum of uninfected RV144 vaccine trial responders were able to efficiently opsonize viral particles in the absence of significant aggregation, reflective of monomeric IgA.
Conclusions
These results collectively demonstrate that dIgA is capable of forming stable viral aggregates providing a plausible basis for testing the effectiveness of aggregation as a potential protection mechanism at the mucosal portals of viral entry.
Electronic supplementary material
The online version of this article (doi:10.1186/s12977-014-0078-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12977-014-0078-8
PMCID: PMC4193994  PMID: 25274446
HIV-1; Mucosal immunity; Immunoglobulin A; Aggregation
7.  Vaccine-Induced Env V1–V2 IgG3 Correlates with Lower HIV-1 Infection Risk and Declines Soon After Vaccination 
Science translational medicine  2014;6(228):228ra39.
HIV-1–specific immunoglobulin G (IgG) subclass antibodies bind to distinct cellular Fc receptors. Antibodies of the same epitope specificity but of a different subclass therefore can have different antibody effector functions. The study of IgG subclass profiles between different vaccine regimens used in clinical trials with divergent efficacy outcomes can provide information on the quality of the vaccine-induced B cell response. We show that HIV-1–specific IgG3 distinguished two HIV-1 vaccine efficacy studies (RV144 and VAX003 clinical trials) and correlated with decreased risk of HIV-1 infection in a blinded follow-up case-control study with the RV144 vaccine. HIV-1–specific IgG3 responses were not long-lived, which was consistent with the waning efficacy of the RV144 vaccine. These data suggest that specific vaccine-induced HIV-1 IgG3 should be tested in future studies of immune correlates in HIV-1 vaccine efficacy trials.
doi:10.1126/scitranslmed.3007730
PMCID: PMC4116665  PMID: 24648342
8.  FCGR2C polymorphisms associate with HIV-1 vaccine protection in RV144 trial 
The Journal of Clinical Investigation  2014;124(9):3879-3890.
The phase III RV144 HIV-1 vaccine trial estimated vaccine efficacy (VE) to be 31.2%. This trial demonstrated that the presence of HIV-1–specific IgG-binding Abs to envelope (Env) V1V2 inversely correlated with infection risk, while the presence of Env-specific plasma IgA Abs directly correlated with risk of HIV-1 infection. Moreover, Ab-dependent cellular cytotoxicity responses inversely correlated with risk of infection in vaccine recipients with low IgA; therefore, we hypothesized that vaccine-induced Fc receptor–mediated (FcR-mediated) Ab function is indicative of vaccine protection. We sequenced exons and surrounding areas of FcR-encoding genes and found one FCGR2C tag SNP (rs114945036) that associated with VE against HIV-1 subtype CRF01_AE, with lysine at position 169 (169K) in the V2 loop (CRF01_AE 169K). Individuals carrying CC in this SNP had an estimated VE of 15%, while individuals carrying CT or TT exhibited a VE of 91%. Furthermore, the rs114945036 SNP was highly associated with 3 other FCGR2C SNPs (rs138747765, rs78603008, and rs373013207). Env-specific IgG and IgG3 Abs, IgG avidity, and neutralizing Abs inversely correlated with CRF01_AE 169K HIV-1 infection risk in the CT- or TT-carrying vaccine recipients only. These data suggest a potent role of Fc-γ receptors and Fc-mediated Ab function in conferring protection from transmission risk in the RV144 VE trial.
doi:10.1172/JCI75539
PMCID: PMC4151214  PMID: 25105367
9.  Analysis of HLA A*02 Association with Vaccine Efficacy in the RV144 HIV-1 Vaccine Trial 
Journal of Virology  2014;88(15):8242-8255.
ABSTRACT
The RV144 HIV-1 vaccine trial demonstrated partial efficacy of 31% against HIV-1 infection. Studies into possible correlates of protection found that antibodies specific to the V1 and V2 (V1/V2) region of envelope correlated inversely with infection risk and that viruses isolated from trial participants contained genetic signatures of vaccine-induced pressure in the V1/V2 region. We explored the hypothesis that the genetic signatures in V1 and V2 could be partly attributed to selection by vaccine-primed T cells. We performed a T-cell-based sieve analysis of breakthrough viruses in the RV144 trial and found evidence of predicted HLA binding escape that was greater in vaccine versus placebo recipients. The predicted escape depended on class I HLA A*02- and A*11-restricted epitopes in the MN strain rgp120 vaccine immunogen. Though we hypothesized that this was indicative of postacquisition selection pressure, we also found that vaccine efficacy (VE) was greater in A*02-positive (A*02+) participants than in A*02− participants (VE = 54% versus 3%, P = 0.05). Vaccine efficacy against viruses with a lysine residue at site 169, important to antibody binding and implicated in vaccine-induced immune pressure, was also greater in A*02+ participants (VE = 74% versus 15%, P = 0.02). Additionally, a reanalysis of vaccine-induced immune responses that focused on those that were shown to correlate with infection risk suggested that the humoral responses may have differed in A*02+ participants. These exploratory and hypothesis-generating analyses indicate there may be an association between a class I HLA allele and vaccine efficacy, highlighting the importance of considering HLA alleles and host immune genetics in HIV vaccine trials.
IMPORTANCE The RV144 trial was the first to show efficacy against HIV-1 infection. Subsequently, much effort has been directed toward understanding the mechanisms of protection. Here, we conducted a T-cell-based sieve analysis, which compared the genetic sequences of viruses isolated from infected vaccine and placebo recipients. Though we hypothesized that the observed sieve effect indicated postacquisition T-cell selection, we also found that vaccine efficacy was greater for participants who expressed HLA A*02, an allele implicated in the sieve analysis. Though HLA alleles have been associated with disease progression and viral load in HIV-1 infection, these data are the first to suggest the association of a class I HLA allele and vaccine efficacy. While these statistical analyses do not provide mechanistic evidence of protection in RV144, they generate testable hypotheses for the HIV vaccine community and they highlight the importance of assessing the impact of host immune genetics in vaccine-induced immunity and protection. (This study has been registered at ClinicalTrials.gov under registration no. NCT00223080.)
doi:10.1128/JVI.01164-14
PMCID: PMC4135964  PMID: 24829343
10.  HIV-1 Vaccine-Induced C1 and V2 Env-Specific Antibodies Synergize for Increased Antiviral Activities 
Journal of Virology  2014;88(14):7715-7726.
ABSTRACT
The RV144 ALVAC/AIDSVax HIV-1 vaccine clinical trial showed an estimated vaccine efficacy of 31.2%. Viral genetic analysis identified a vaccine-induced site of immune pressure in the HIV-1 envelope (Env) variable region 2 (V2) focused on residue 169, which is included in the epitope recognized by vaccinee-derived V2 monoclonal antibodies. The ALVAC/AIDSVax vaccine induced antibody-dependent cellular cytotoxicity (ADCC) against the Env V2 and constant 1 (C1) regions. In the presence of low IgA Env antibody levels, plasma levels of ADCC activity correlated with lower risk of infection. In this study, we demonstrate that C1 and V2 monoclonal antibodies isolated from RV144 vaccinees synergized for neutralization, infectious virus capture, and ADCC. Importantly, synergy increased the HIV-1 ADCC activity of V2 monoclonal antibody CH58 at concentrations similar to that observed in plasma of RV144 vaccinees. These findings raise the hypothesis that synergy among vaccine-induced antibodies with different epitope specificities contributes to HIV-1 antiviral antibody responses and is important to induce for reduction in the risk of HIV-1 transmission.
IMPORTANCE The Thai RV144 ALVAC/AIDSVax prime-boost vaccine efficacy trial represents the only example of HIV-1 vaccine efficacy in humans to date. Studies aimed at identifying immune correlates involved in the modest vaccine-mediated protection identified HIV-1 envelope (Env) variable region 2-binding antibodies as inversely correlated with infection risk, and genetic analysis identified a site of immune pressure within the region recognized by these antibodies. Despite this evidence, the antiviral mechanisms by which variable region 2-specific antibodies may have contributed to lower rates of infection remain unclear. In this study, we demonstrate that vaccine-induced HIV-1 envelope variable region 2 and constant region 1 antibodies synergize for recognition of virus-infected cells, infectious virion capture, virus neutralization, and antibody-dependent cellular cytotoxicity. This is a major step in understanding how these types of antibodies may have cooperatively contributed to reducing infection risk and should be considered in the context of prospective vaccine design.
doi:10.1128/JVI.00156-14
PMCID: PMC4097802  PMID: 24807721
12.  Vaccine-Induced IgG Antibodies to V1V2 Regions of Multiple HIV-1 Subtypes Correlate with Decreased Risk of HIV-1 Infection 
PLoS ONE  2014;9(2):e87572.
In the RV144 HIV-1 vaccine efficacy trial, IgG antibody (Ab) binding levels to variable regions 1 and 2 (V1V2) of the HIV-1 envelope glycoprotein gp120 were an inverse correlate of risk of HIV-1 infection. To determine if V1V2-specific Abs cross-react with V1V2 from different HIV-1 subtypes, if the nature of the V1V2 antigen used to asses cross-reactivity influenced infection risk, and to identify immune assays for upcoming HIV-1 vaccine efficacy trials, new V1V2-scaffold antigens were designed and tested. Protein scaffold antigens carrying the V1V2 regions from HIV-1 subtypes A, B, C, D or CRF01_AE were assayed in pilot studies, and six were selected to assess cross-reactive Abs in the plasma from the original RV144 case-control cohort (41 infected vaccinees, 205 frequency-matched uninfected vaccinees, and 40 placebo recipients) using ELISA and a binding Ab multiplex assay. IgG levels to these antigens were assessed as correlates of risk in vaccine recipients using weighted logistic regression models. Levels of Abs reactive with subtype A, B, C and CRF01_AE V1V2-scaffold antigens were all significant inverse correlates of risk (p-values of 0.0008–0.05; estimated odds ratios of 0.53–0.68 per 1 standard deviation increase). Thus, levels of vaccine-induced IgG Abs recognizing V1V2 regions from multiple HIV-1 subtypes, and presented on different scaffolds, constitute inverse correlates of risk for HIV-1 infection in the RV144 vaccine trial. The V1V2 antigens provide a link between RV144 and upcoming HIV-1 vaccine trials, and identify reagents and methods for evaluating V1V2 Abs as possible correlates of protection against HIV-1 infection.
Trial Registration
ClinicalTrials.gov NCT00223080
doi:10.1371/journal.pone.0087572
PMCID: PMC3913641  PMID: 24504509
13.  Vaccine Induction of Antibodies Against a Structurally Heterogeneous Site of Immune Pressure within HIV-1 Envelope Protein Variable Regions 1 and 2 
Immunity  2013;38(1):176-186.
Summary
The RV144 HIV-1 trial of the canary pox vector (ALVAC-HIV) plus the gp120 AIDSVAX B/E vaccine demonstrated an estimated efficacy of 31%, that correlated directly with antibodies to HIV-1 envelope variable regions 1 and 2 (V1–V2). Genetic analysis of trial viruses revealed increased vaccine efficacy against viruses matching the vaccine strain at V2 residue 169. Here, we isolated four V2 monoclonal antibodies from RV144 vaccinees that recognize residue 169, neutralize laboratory-adapted HIV-1, and mediate killing of field isolate HIV-1-infected CD4+ T cells. Crystal structures of two of the V2 antibodies demonstrated residue 169 can exist within divergent helical and loop conformations, which contrasted dramatically with the beta strand conformation previously observed with a broadly neutralizing antibody PG9. Thus, RV144 vaccine-induced immune pressure appears to target a region that may be both sequence variable and structurally polymorphic. Variation may signal sites of HIV-1 envelope vulnerability, providing vaccine designers with new options.
doi:10.1016/j.immuni.2012.11.011
PMCID: PMC3569735  PMID: 23313589
14.  The Thai Phase III HIV Type 1 Vaccine Trial (RV144) Regimen Induces Antibodies That Target Conserved Regions Within the V2 Loop of gp120 
AIDS Research and Human Retroviruses  2012;28(11):1444-1457.
Abstract
The Thai Phase III clinical trial (RV144) showed modest efficacy in preventing HIV-1 acquisition. Plasma collected from HIV-1-uninfected trial participants completing all injections with ALVAC-HIV (vCP1521) prime and AIDSVAX B/E boost were tested for antibody responses against HIV-1 gp120 envelope (Env). Peptide microarray analysis from six HIV-1 subtypes and group M consensus showed that vaccination induced antibody responses to the second variable (V2) loop of gp120 of multiple subtypes. We further evaluated V2 responses by ELISA and surface plasmon resonance using cyclic (Cyc) and linear V2 loop peptides. Thirty-one of 32 vaccine recipients tested (97%) had antibody responses against Cyc V2 at 2 weeks postimmunization with a reciprocal geometric mean titer (GMT) of 1100 (range: 200–3200). The frequency of detecting plasma V2 antibodies declined to 19% at 28 weeks post-last injection (GMT: 110, range: 100–200). Antibody responses targeted the mid-region of the V2 loop that contains conserved epitopes and has the amino acid sequence KQKVHALFYKLDIVPI (HXB2 Numbering sequence 169–184). Valine at position 172 was critical for antibody binding. The frequency of V3 responses at 2 weeks postimmunization was modest (18/32, 56%) with a GMT of 185 (range: 100–800). In contrast, naturally infected HIV-1 individuals had a lower frequency of antibody responses to V2 (10/20, 50%; p=0.003) and a higher frequency of responses to V3 (19/20, 95%), with GMTs of 400 (range: 100–3200) and 3570 (range: 200–12,800), respectively. RV144 vaccination induced antibodies that targeted a region of the V2 loop that contains conserved epitopes. Early HIV-1 transmission events involve V2 loop interactions, raising the possibility that anti-V2 antibodies in RV144 may have contributed to viral inhibition.
doi:10.1089/aid.2012.0103
PMCID: PMC3484815  PMID: 23035746
15.  Plasma IgG to Linear Epitopes in the V2 and V3 Regions of HIV-1 gp120 Correlate with a Reduced Risk of Infection in the RV144 Vaccine Efficacy Trial 
PLoS ONE  2013;8(9):e75665.
Neutralizing and non-neutralizing antibodies to linear epitopes on HIV-1 envelope glycoproteins have potential to mediate antiviral effector functions that could be beneficial to vaccine-induced protection. Here, plasma IgG responses were assessed in three HIV-1 gp120 vaccine efficacy trials (RV144, Vax003, Vax004) and in HIV-1-infected individuals by using arrays of overlapping peptides spanning the entire consensus gp160 of all major genetic subtypes and circulating recombinant forms (CRFs) of the virus. In RV144, where 31.2% efficacy against HIV-1 infection was seen, dominant responses targeted the C1, V2, V3 and C5 regions of gp120. An analysis of RV144 case-control samples showed that IgG to V2 CRF01_AE significantly inversely correlated with infection risk (OR= 0.54, p=0.0042), as did the response to other V2 subtypes (OR=0.60-0.63, p=0.016-0.025). The response to V3 CRF01_AE also inversely correlated with infection risk but only in vaccine recipients who had lower levels of other antibodies, especially Env-specific plasma IgA (OR=0.49, p=0.007) and neutralizing antibodies (OR=0.5, p=0.008). Responses to C1 and C5 showed no significant correlation with infection risk. In Vax003 and Vax004, where no significant protection was seen, serum IgG responses targeted the same epitopes as in RV144 with the exception of an additional C1 reactivity in Vax003 and infrequent V2 reactivity in Vax004. In HIV-1 infected subjects, dominant responses targeted the V3 and C5 regions of gp120, as well as the immunodominant domain, heptad repeat 1 (HR-1) and membrane proximal external region (MPER) of gp41. These results highlight the presence of several dominant linear B cell epitopes on the HIV-1 envelope glycoproteins. They also generate the hypothesis that IgG to linear epitopes in the V2 and V3 regions of gp120 are part of a complex interplay of immune responses that contributed to protection in RV144.
doi:10.1371/journal.pone.0075665
PMCID: PMC3784573  PMID: 24086607
16.  Optimization and Qualification of a Multiplex Bead Array to Assess Cytokine and Chemokine Production by Vaccine-specific Cells 
Journal of Immunological Methods  2012;382(1-2):117-128.
The magnitude and functional phenotype (e.g. proliferation, immune stimulation) of the 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.
doi:10.1016/j.jim.2012.05.011
PMCID: PMC3406745  PMID: 22626638
Multiplex bead array; Vaccine; HIV; Cytokine; Chemokine
17.  Antigenicity and Immunogenicity of RV144 Vaccine AIDSVAX Clade E Envelope Immunogen Is Enhanced by a gp120 N-Terminal Deletion 
Journal of Virology  2013;87(3):1554-1568.
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.
doi:10.1128/JVI.00718-12
PMCID: PMC3554162  PMID: 23175357
18.  Infectious Virion Capture by HIV-1 gp120-Specific IgG from RV144 Vaccinees 
Journal of Virology  2013;87(14):7828-7836.
The detailed examination of the antibody repertoire from RV144 provides a unique template for understanding potentially protective antibody functions. Some potential immune correlates of protection were untested in the correlates analyses due to inherent assay limitations, as well as the need to keep the correlates analysis focused on a limited number of endpoints to achieve statistical power. In an RV144 pilot study, we determined that RV144 vaccination elicited antibodies that could bind infectious virions (including the vaccine strains HIV-1 CM244 and HIV-1 MN and an HIV-1 strain expressing transmitted/founder Env, B.WITO.c). Among vaccinees with the highest IgG binding antibody profile, the majority (78%) captured the infectious vaccine strain virus (CM244), while a smaller proportion of vaccinees (26%) captured HIV-1 transmitted/founder Env virus. We demonstrated that vaccine-elicited HIV-1 gp120 antibodies of multiple specificities (V3, V2, conformational C1, and gp120 conformational) mediated capture of infectious virions. Although capture of infectious HIV-1 correlated with other humoral immune responses, the extent of variation between these humoral responses and virion capture indicates that virion capture antibodies occupy unique immunological space.
doi:10.1128/JVI.02737-12
PMCID: PMC3700223  PMID: 23658446
19.  Ad hoc analysis of behavior and time as co-variates of the Thai phase III efficacy trial: RV 144 
The Lancet infectious diseases  2012;12(7):531-537.
Background
The Thai phase III HIV vaccine trial's modest efficacy (VE 31.2% 95% CI 1.1, 51.2) represents the first demonstration that a vaccine can protect against HIV acquisition. Baseline variables of age, gender, marital status, and risk did not modify vaccine efficacy (VE). Here we explore behavioral risk and efficacy at 6 monthly intervals following vaccination.
Methods
Behavioral risk was assessed with a self-administered questionnaire every 6 months during trial participation. Both the acquisition endpoint and the early viral load endpoint are examined for interactions with risk status over time and temporal effects following vaccination.
Finding
Risk for HIV acquisition is low in each risk group, but the majority of participants reported higher-risk behavior at least once during the study (N= 9187, 58%). In post-hoc analyses, comparing those participants categorized as high or rising risk at least once during study follow-up versus those who maintained low or medium risk behavior as a time-varying covariate, the interaction of risk status and acquisition efficacy is significant (P = 0.010) with greater benefit in the lower risk individuals. VE appears to peak early with an estimate of cumulative VE = 60% through 12 months after initial vaccination (95% CI 22 –80%), and declines quickly. Vaccination did not appear to affect viral load in either early or late infections.
Interpretation
Future HIV vaccine trials must recognize potential interactions between challenge intensity and risk heterogeneity in the population and treatment effects. The regimen tested in the Thai phase III trial may benefit from extended immunization schedules.
doi:10.1016/S1473-3099(12)70088-9
PMCID: PMC3530398  PMID: 22652344
20.  Molecular Evolution of the HIV-1 Thai Epidemic between the Time of RV144 Immunogen Selection to the Execution of the Vaccine Efficacy Trial 
Journal of Virology  2013;87(13):7265-7281.
The RV144 HIV-1 vaccine trial (Thailand, 2003 to 2009), using immunogens genetically matched to the regional epidemic, demonstrated the first evidence of efficacy for an HIV-1 vaccine. Here we studied the molecular evolution of the HIV-1 epidemic from the time of immunogen selection to the execution of the efficacy trial. We studied HIV-1 genetic diversity among 390 volunteers who were deferred from enrollment in RV144 due to preexisting HIV-1 infection using a multiregion hybridization assay, full-genome sequencing, and phylogenetic analyses. The subtype distribution was 91.7% CRF01_AE, 3.5% subtype B, 4.3% B/CRF01_AE recombinants, and 0.5% dual infections. CRF01_AE strains were 31% more diverse than the ones from the 1990s Thai epidemic. Sixty-nine percent of subtype B strains clustered with the cosmopolitan Western B strains. Ninety-three percent of B/CRF01_AE recombinants were unique; recombination breakpoint analysis showed that these strains were highly embedded within the larger network that integrates recombinants from East/Southeast Asia. Compared to Thai sequences from the early 1990s, the distance to the RV144 immunogens increased 52% to 68% for CRF01_AE Env immunogens and 12% to 29% for subtype B immunogens. Forty-three percent to 48% of CRF01_AE sequences differed from the sequence of the vaccine insert in Env variable region 2 positions 169 and 181, which were implicated in vaccine sieve effects in RV144. In conclusion, compared to the molecular picture at the early stages of vaccine development, our results show an overall increase in the genetic complexity of viruses in the Thai epidemic and in the distance to vaccine immunogens, which should be considered at the time of the analysis of the trial results.
doi:10.1128/JVI.03070-12
PMCID: PMC3700312  PMID: 23576510
21.  The Thai Phase III Trial (RV144) Vaccine Regimen Induces T Cell Responses that Preferentially Target Epitopes within the V2 Region of HIV-1 Envelope 
The Thai HIV phase III prime-boost trial (RV144) using ALVAC-HIV® (vCP1521) and AIDSVAX B/E® was, to our knowledge, the first to demonstrate acquisition efficacy. Vaccine-induced, cell-mediated immune responses were assessed. T cell epitope mapping studies using IFN-γ ELISPOT were performed on PBMC from HIV-1 uninfected vaccine (N=61) and placebo (N=10) recipients using HIV-1 Env peptides. Positive responses were measured in 25 (41%) vaccinees and were predominantly CD4+ T cell mediated. Responses were targeted within the HIV Env region, with 15/25 (60%) of vaccinees recognizing peptides derived from the V2 region of HIV-1 Env, which includes the α4β7 integrin binding site. Intracellular cytokine staining confirmed that Env responses predominated (19/30; 63% of vaccine recipients) and were mediated by polyfunctional effector memory CD4+ T cells, with the majority of responders producing both IL-2 and IFN-γ (12/19; 63%). HIV-Env Ab titers were higher in subjects with IL-2 compared to those without IL-2 secreting HIV-Env specific effector memory T cells. Proliferation assays revealed that HIV Ag-specific T cells were CD4+ with the majority (80%) expressing CD107a. HIV-specific T cell lines obtained from vaccine recipients confirmed V2 specificity, polyfunctionality and functional cytolytic capacity. While the RV144 T cell responses were modest in frequency compared to humoral immune responses, the CD4+ T cell response was directed to HIV-1 Env and more particularly the V2 region.
doi:10.4049/jimmunol.1102756
PMCID: PMC3383859  PMID: 22529301
Human; Vaccination; Viral; AIDS; HIV-1; T cells
22.  Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies from an HIV-1 Vaccine Efficacy Trial Target Multiple Epitopes and Preferentially Use the VH1 Gene Family 
Journal of Virology  2012;86(21):11521-11532.
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.
doi:10.1128/JVI.01023-12
PMCID: PMC3486290  PMID: 22896626
23.  Increased HIV-1 vaccine efficacy against viruses with genetic signatures in Env-V2 
Nature  2012;490(7420):417-420.
Summary
The RV144 trial demonstrated 31% vaccine efficacy (VE) at preventing HIV-1 infection1. Antibodies against the HIV-1 envelope variable loops 1 and 2 (V1/V2) domain correlated inversely with infection risk2. We hypothesized that vaccine-induced immune responses against V1/V2 would selectively impact, or sieve, HIV-1 breakthrough viruses. 936 HIV-1 genome sequences from 44 vaccine and 66 placebo recipients were examined. We show that vaccine-induced immune responses were associated with two signatures in V1/V2 at amino-acid positions 169 and 181. VE against viruses matching the vaccine at position 169 was 48% (CI: 18 to 66%; p=0.0036), whereas VE against viruses mismatching the vaccine at position 181 was 78% (CI: 35% to 93%; p=0.0028). Residue 169 is in a cationic glycosylated region recognized by broadly neutralizing and RV144-derived antibodies. The predicted distance between the two signatures sites (21±7 Å), and their match/mismatch dichotomy, suggest that multiple factors may be involved in the protection observed in RV144. Genetic signatures of RV144 vaccination in V2 complement the finding of an association between high V1/V2 binding antibodies and reduced risk of HIV-1 acquisition and provide evidence that vaccine-induced V2 responses plausibly played a role in the partial protection conferred by the RV144 regimen.
doi:10.1038/nature11519
PMCID: PMC3551291  PMID: 22960785
24.  Analysis of V2 Antibody Responses Induced in Vaccinees in the ALVAC/AIDSVAX HIV-1 Vaccine Efficacy Trial 
PLoS ONE  2013;8(1):e53629.
The RV144 clinical trial of a prime/boost immunizing regimen using recombinant canary pox (ALVAC-HIV) and two gp120 proteins (AIDSVAX B and E) was previously shown to have a 31.2% efficacy rate. Plasma specimens from vaccine and placebo recipients were used in an extensive set of assays to identify correlates of HIV-1 infection risk. Of six primary variables that were studied, only one displayed a significant inverse correlation with risk of infection: the antibody (Ab) response to a fusion protein containing the V1 and V2 regions of gp120 (gp70-V1V2). This finding prompted a thorough examination of the results generated with the complete panel of 13 assays measuring various V2 Abs in the stored plasma used in the initial pilot studies and those used in the subsequent case-control study. The studies revealed that the ALVAC-HIV/AIDSVAX vaccine induced V2-specific Abs that cross-react with multiple HIV-1 subgroups and recognize both conformational and linear epitopes. The conformational epitope was present on gp70-V1V2, while the predominant linear V2 epitope mapped to residues 165–178, immediately N-terminal to the putative α4β7 binding motif in the mid-loop region of V2. Odds ratios (ORs) were calculated to compare the risk of infection with data from 12 V2 assays, and in 11 of these, the ORs were ≤1, reaching statistical significance for two of the variables: Ab responses to gp70-V1V2 and to overlapping V2 linear peptides. It remains to be determined whether anti-V2 Ab responses were directly responsible for the reduced infection rate in RV144 and whether anti-V2 Abs will prove to be important with other candidate HIV vaccines that show efficacy, however, the results support continued dissection of Ab responses to the V2 region which may illuminate mechanisms of protection from HIV-1 infection and may facilitate the development of an effective HIV-1 vaccine.
doi:10.1371/journal.pone.0053629
PMCID: PMC3547933  PMID: 23349725
25.  Immune-Correlates Analysis of an HIV-1 Vaccine Efficacy Trial 
The New England Journal of Medicine  2012;366(14):1275-1286.
BACKGROUND
In the RV144 trial, the estimated efficacy of a vaccine regimen against human immunodeficiency virus type 1 (HIV-1) was 31.2%. We performed a case–control analysis to identify antibody and cellular immune correlates of infection risk.
METHODS
In pilot studies conducted with RV144 blood samples, 17 antibody or cellular assays met prespecified criteria, of which 6 were chosen for primary analysis to determine the roles of T-cell, IgG antibody, and IgA antibody responses in the modulation of infection risk. Assays were performed on samples from 41 vaccinees who became infected and 205 uninfected vaccinees, obtained 2 weeks after final immunization, to evaluate whether immune-response variables predicted HIV-1 infection through 42 months of follow-up.
RESULTS
Of six primary variables, two correlated significantly with infection risk: the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of HIV-1 envelope proteins (Env) correlated inversely with the rate of HIV-1 infection (estimated odds ratio, 0.57 per 1-SD increase; P = 0.02; q = 0.08), and the binding of plasma IgA antibodies to Env correlated directly with the rate of infection (estimated odds ratio, 1.54 per 1-SD increase; P = 0.03; q = 0.08). Neither low levels of V1V2 antibodies nor high levels of Env-specific IgA antibodies were associated with higher rates of infection than were found in the placebo group. Secondary analyses suggested that Env-specific IgA antibodies may mitigate the effects of potentially protective antibodies.
CONCLUSIONS
This immune-correlates study generated the hypotheses that V1V2 antibodies may have contributed to protection against HIV-1 infection, whereas high levels of Env-specific IgA antibodies may have mitigated the effects of protective antibodies. Vaccines that are designed to induce higher levels of V1V2 antibodies and lower levels of Env-specific IgA antibodies than are induced by the RV144 vaccine may have improved efficacy against HIV-1 infection.
doi:10.1056/NEJMoa1113425
PMCID: PMC3371689  PMID: 22475592

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