Vaccine-Induced IgG Antibodies to V1V2 Regions of Multiple HIV-1 Subtypes Correlate with Decreased Risk of HIV-1 Infection

<div><p></p><p>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.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="http://clinicaltrials.gov/ct2/show/NCT00223080" target="_blank">NCT00223080</a></p></div

V2 sequences in V1V2-scaffold antigens tested with case-control specimens^*.

*<p>Sequence of V2 residues present in the designated V1V2-scaffold antigens. Residues in bold indicate the V2 epitope recognized by RV144 vaccinees’ plasma as mapped with peptides <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087572#pone.0087572-ZollaPazner1" target="_blank">[4]</a>. Underlined residues represent the putative α4β7 binding site.</p>‡<p>Sequence of V1V2 in ALVAC-HIV subtype E gp120 priming immunogen.</p

Response rates in Placebo and Vaccine recipients as measured by ELISA and BAMA.

*<p>For ELISA, the criterion for a positive response was a value of 3 SD above the mean of the Week 26 placebo group read-outs (n = 40) where read-outs are the natural logarithm-transformed averages of the OD readings across four replicates.</p>**<p>For BAMA, a positive response is defined as (a) MFI greater than 263; (b) read-outs at Week 26 divided by read-outs at Week 0 greater than 3 for both blank-subtracted and blank-unsubtracted read-outs, and (c) blank MFI less than 5,000.</p

Reactivity in ELISA of the 13 V1V2-scaffold antigens used in the Phase 2 study.

<p>Results are shown as box plots of reactivity with each of the 13 Phase 2 V1V2-scaffold antigens and the scaffold control antigen (gp70_WT) with 60 plasma samples from set V2L. Results shown are Week 26 read-outs at a 1∶100 dilution with the exception of tags.A(Q23)-V1V2 and tags.C(1086)-V1V2 which were run at a 1∶300, dilution and gp70.AE(92TH023)-V1V2 which was run at 1∶900. AP, LL and GN denote production of comparable reagents by Drs. Pinter, Liao, and Nabel.</p

Analysis of HIV Env-specific cytokine-producing T cells.

<p>(A) Env peptide-specific CD4⁺ T cell responses and (B) Env peptide-specific CD8⁺ T cell responses from PBMC samples taken at week 6 (2 weeks post NYVAC-KC administration for G1 and G2 only) (blue circles) and week 22 (2 weeks post the final αLOX-1.Env gp140 administration) (red circles). The data are based on total CD4⁺ and CD8⁺ T cells and show the sum of IFNγ⁺, IL-2⁺, and TNFα⁺ T cells specific to the Env peptide pools for individual NHP as filled circles. The grey horizontal bars are the median value for the group NHP (n = 6 for G1, G2; n = 4 for G3, G4) and the vertical grey bars are the IQR.</p

Study design for testing immunogenicity of αLOX-1.Env gp140 in NHP.

<p>Study design for testing immunogenicity of αLOX-1.Env gp140 in NHP.</p

Estimated vaccine efficacy (VE).

<p>Estimated VE is shown for vaccine recipients with Low, Medium and High V1V2-scaffold IgG Ab responses at Week 26 versus the placebo group as defined by (A) ELISA data, and (B) BAMA data. VE is estimated as one minus the odds of infection in vaccine recipients with Low/Medium/High responses divided by the odds of infection in the entire HIV-1 negative placebo group at week 24. Points show the estimated VE values, and vertical lines delineate 95% confidence intervals. Two-sided p-values are from Wald tests.</p

Analysis of HIV-specific T cell responses.

<p>The upper pie charts show the percentage of epitope specificities for (A) CD4⁺ T cell responses and (B) CD8⁺ T cell responses from PBMCs sampled at week 6 (2 weeks post NYVAC-KC administration, NHP groups G1 and G2 only) and week 22 (2 weeks post αLOX-1.Env gp140 administration) as defined by IFNγ⁺ T cells specific to each peptide pool. The lower pie charts show analysis of multifunctional HIV-specific T cell responses. ICS analysis of HIV-specific (C) CD4⁺ (left 6 pies) and (D) CD8⁺ (right 6 pies) T cells in PBMC samples taken at week 6 (2 weeks post NYVAC-KC administration, G1 Nkc2Lp3Nkc and G2 Nkc2Lg3Nkc only) and week 22 (2 weeks post αLOX-1.Env gp140 administration). The data show the breakdown of IFNγ⁺ (labeled as G), IL-2⁺ (labeled as 2) and TNFα⁺ (labeled as T) T cells specific to the combined HIV peptide pools (n = 6 NHP for G1, G2; n = 4 NHP for G3, G4). The lower linear presentation in (C) and (D) shows the same data as the pie charts, but includes data points for individual NHPs.</p

Data used for down-selection of V1V2-scaffold antigens to be used in the case-control studies.

*<p>Correlation: the Spearman correlation between the original gp70-V1V2 antigen read-out and the listed scaffold antigen.</p>§<p>Response, signal-to-noise, and P-values are defined in the Methods section.</p>‡<p>Antigens shown in bold were selected for use in case-control study.</p

Analysis of neutralizing antibody titers.

<p>Plasma samples taken at peak response week 22 (2 weeks post αLOX-1.Env gp140 administrations) and week 32 (2 weeks post NYVAC-KC boost) for groups 1–4 (G1-G4) were tested for neutralizing titers versus each indicated virus (MW965.26 or TH023.6). Log to base 10 of titers inhibiting replication by 50% for individual NHP (n = 6 for G1, G2; n = 4 for G3, G4) are shown. Boxes represent the middle 95^th percentile. Horizontal lines are the median. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153484#pone.0153484.s004" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153484#pone.0153484.s005" target="_blank">S3</a> Tables show similar tests against other viruses.</p