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.
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.
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.
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.
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.
Multiplex bead array; Vaccine; HIV; Cytokine; Chemokine
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.
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.
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.
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.
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.
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.
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.
HIV Associated Dementia (HAD) is a complication of HIV infection in developed countries and is still poorly defined in resource-limited settings. In this study we investigated the expression of the monocyte phenotype CD14CD16HLADR and the inflammatory profiles in monocytes supernatants by surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry in a cohort of HAD and non-HAD Thai volunteers prior to the initiation of ARV. The CD14CD16HLADR phenotype was significantly increased in monocytes from HAD and non-HAD versus negative controls, but there was no difference in phenotype and in the secretion protein profiles between the two seropositive groups. In addition, monocytes supernatants from HAD and non-HAD did not induced apoptosis or cell death in brain aggregate culture. In conclusion it appears that HAD in Thai individuals has a different immunological profile then in North America cohorts.
HIV-1; Thailand; Monocyte markers; HAD
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.
Human; Vaccination; Viral; AIDS; HIV-1; T cells
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.
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.
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.
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.
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.
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.
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.
Background. A recombinant canarypox vector expressing human immunodeficiency virus type 1 (HIV-1) Gag, Pro, and membrane-linked gp120 (vCP1521), combined with a bivalent gp120 protein boost (AIDSVAX B/E), provided modest protection against HIV-1 infection in a community-based population in Thailand (RV144 trial). No protection was observed in Thai injection drug users who received AIDSVAX B/E alone (Vax003 trial). We compared the neutralizing antibody response in these 2 trials.
Methods. Neutralization was assessed with tier 1 and tier 2 strains of virus in TZM-bl and A3R5 cells.
Results. Neutralization of several tier 1 viruses was detected in both RV144 and Vax003. Peak titers were higher in Vax003 and waned rapidly in both trials. The response in RV144 was targeted in part to V3 of gp120.vCP1521 priming plus 2 boosts with gp120 protein was superior to 2 gp120 protein inoculations alone, confirming a priming effect for vCP1521. Sporadic weak neutralization of tier 2 viruses was detected only in Vax003 and A3R5 cells.
Conclusion. The results suggest either that weak neutralizing antibody responses can be partially protective against HIV-1 in low-risk heterosexual populations or that the modest efficacy seen in RV144 was mediated by other immune responses, either alone or in combination with neutralizing antibodies.
A number of human immunodeficiency virus type 1 (HIV-1) non-B-subtype products have been developed for present or future vaccine trials; in Thailand, several studies using subtype B and/or CRF01_AE vaccines have been conducted. To better characterize the biologic properties of these subtypes, 70 HIV-1 subtype B and E isolates were phenotyped as syncytium-inducing (SI) or non-syncytium-inducing (NSI) isolates and assessed for sensitivity to neutralizing antibody (NAb). A significantly higher number of NSI subtype E viruses were neutralization sensitive than SI subtype E viruses (P = 0.009), while no association between viral phenotype and sensitivity to NAb was observed for subtype B (P = 0.856), suggesting a difference in the neutralization patterns of subtypes B and E. Strikingly, concurrent CD4 T-cell numbers were significantly lower for subtype E-infected patients whose isolates were more resistant to NAb, both for the overall study group (P < 0.001) as well as for the 22 patients with NSI isolates (P = 0.013). Characterization of the evolution of biologic properties of both B and non-B HIV-1 subtypes will provide a clearer understanding of the repertoire of antibodies that must be elicited for a vaccine to be effective against all phenotypes and subtypes.
Characterization of the HIV epidemic in Thailand has benefited from the systematic testing of young men upon entry into the military. These data, which have shown that public health measures can reverse an HIV epidemic, have been reanalyzed with current geographic information systems methods. The resulting maps, thus far, are the best means of visualizing the geography of the dynamic HIV epidemic in Thailand.
human immunodeficiency virus; epidemiology; Thailand; geographic information systems; dispatch