HIV-1 vaccine-induced C1 and V2 Env-specific antibodies synergize for increased antiviral activities

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.

FIG 1

Synergy of MAb binding to monomeric gp120 by SPR. (A) Schematic of the SPR assay utilized to test the presence of synergy between the V2 and C1 MAbs for binding to the recombinant AE.244Δ11 gp120 according to the procedure reported in Materials and Methods. (B) SPR of binding of the CH58 MAb in combination with negative-control MAb Palivizumab and the C1 MAbs A32, 16H3, CH57, CH54, and CH90. The y axis represents the response unit values, and the x axis represents the time in milliseconds. (C) Fold increase of the V2 MAb CH58 binding to the recombinant AE.A244Δ11 gp120. The data are reported as percentage of increase compared to the binding of the CH58 MAb to gp120 incubated with Palivizumab used as negative control. The results represent the average ± standard error of the mean (SEM) of the results obtained in three separate experiments.

FIG 2

Synergy of MAb for binding to the infected CD4 T cells. Primary CD4⁺ T cells were activated and infected with HIV-1 AE.92TH023 (A to C) and AE.CM235 (D and E) for 72 h. Cells were stained with viability dye and anti-p24 Ab to identify viable (live) infected (p24⁺) cells using the gating strategy shown by the dot plot in panel A. The CH58 MAb was conjugated with Alexa Fluor 488 fluoropohore. The other MAbs and MAb Fab fragments (Palivizumab [Neg], A32, CH54, CH57, and CH90) were used as nonconjugated reagents. The histogram in panel A is representative of the CH58 Alexa Fluor-488 staining of infected cells (p24⁺) observed with individual and combined MAbs. (B to E) The infected CD4⁺ T cells were stained with CH58 Alexa Fluor 488 in combination with the MAbs or Fab fragments indicated on the x axes at 10 μg/ml each. The y axes represent the percentage of increase of stained cells (B and D) and mean fluorescent intensity (MFI) (C and E) for each combination of MAb or Fab fragment relative to the staining of cells observed when the CH58 MAb was used alone. The results represent the average ± SEM of the results obtained in four experiments.

FIG 3

ADCC synergy with combinations of V2 and C1 MAbs. (A to E) ADCC synergy with equimolar combinations of V2 and C1 MAbs. Shown is the percentage of specific killing observed by V2 MAbs CH58 (A), CH59 (B), HG107 (C), and HG120 (D) alone and in combination with negative-control Palivizumab or C1 MAbs CH54, CH57, and CH90 at a 1:1 ratio over 5-fold serial dilutions in the luciferase ADCC assay with CM235-infected targets. Each MAb and the combinations were tested in duplicate wells. The results represent the average ± SEM of the results obtained in a minimum of two independent experiments. (E) ADCC synergy with nonequimolar combinations of V2 and C1 MAbs. The percentage of specific killing was detected by incubating individual MAbs and the combinations indicated with HIV-1 AE.CM235-infected CEM.NKR_CCR5 target cells for 3 h in the luciferase ADCC assay. The expected ADCC activities for an additive effect are represented by white bars. The actual observed activities are represented by filled bars. Results represent the mean and SEM of two experiments, each tested in duplicate. Significantly different results between the expected and observed mean activities by t test are indicated (*). (F) Synergy of CH58 anti-V2 IgG and CH90 anti-C1 F(ab′)₂ for ADCC. The graph represents the percentage of specific killing observed by V2 MAb CH58 and CH90 C1 F(ab′)₂ alone and in combination at a 1:1 ratio over 5-fold serial dilutions in the luciferase ADCC assay with CM235-infected targets. The graphs represent the average ± SEM of the results obtained in three experiments with each run in duplicate.

FIG 4

V2 MAbs synergize with C1 MAb for enhanced selective capture of infectious HIV-1 AE.92TH023 virions. IVCI (i.e., the fold change of the ratios of infectious HIV-1 AE.92TH023 virions to total virion particles) was used to quantify the selective capture of infectious versus noninfectious virions as described in Materials and Methods. (A) IVCI values of V2 MAbs CH58 and C59 and the positive-control gp41 MAb 7B2 when tested alone (black squares) or in combination with the C1 MAb A32 (red circles). (B) Fold increase of IVCI values observed in the presence of C1 MAb A32. The line represents the mean of the results obtained in three experiments.