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

Abstract

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.

Fig 1

Vaccine-induced ADCC responses. To measure plasma ADCC activity induced by the ALVAC-HIV(vCP1521)/AIDSVAX B/E vaccine, plasma samples from 40 vaccine recipients and 10 placebo recipients were collected before immunization (week 0) and 2 weeks after the last boost (week 26). ADCC activity was measured using the ADCC-CM243 assay (A and B) and ADCC-92TH023 assay (C and D) as described in Materials and Methods. Results are reported as areas under the curve (AUC). Each dot represents one sample. The lines connect samples obtained from the same donor.

Fig 2

Recognition of the A32 epitope in plasma of ALVAC-HIV(vCP1521)/AIDSVAX B/E vaccine recipients. (A) Plasma samples collected at week 26 from 20 placebo recipients and 79 vaccine recipients were evaluated for the presence of Abs with A32-like binding specificities by competition ELISA. We defined plasma samples that inhibited >50% of A32 MAb binding as positive (red dots). While none of the placebo recipients displayed A32-like responses, the plasma of 76/79 vaccine recipients (96.2%) competed for A32 MAb binding to its cognate epitope. The whisker boxes show the average plasma ID₅₀ titer and the 95% confidence intervals for each test group. (B) Inhibition of plasma ADCC activity by epitope blocking with the MAb A32 Fab fragment was evaluated in the ADCC-CM243 assay (see Materials and Methods). Plasma samples were collected at week 26 from 30 vaccine recipients and were tested at dilutions corresponding to peak activity. Data are reported as maximum percent GzB activity detected using CM243-gp120-coated targets without pretreatment (no Fab pretreatment; left) or treated with 10 μg/ml MAb A32 Fab (center) or palivizumab Fab (negative control; right). Lines and error bars represent the mean percent GzB activity ± SD. The P values were obtained using repeated-measure analysis of variance. pos, positive; neg, negative; n.s., not significant.

Fig 3

ADCC activity of vaccine-induced MAbs. ADCC activity mediated by monoclonal antibodies isolated from memory B cells of ALVAC-HIV(vCP1521)/AIDSVAX B/E vaccine recipients. Twenty-three MAbs were isolated from six vaccine recipients. Each bar is color-coded by subject: T141485 (light blue), T141449 (red), T143859 (brown), 609107 (green), 210884 (orange), and 347759 (dark blue). MAb A32 (positive control) and palivizumab (negative control) are shown in black and white, respectively. (A) The plots show the maximum percent GzB with the threshold of positivity (5%) indicated by the black line. (B) The endpoint titer expressed in μg/ml for each MAb. The threshold of positivity was determined by averaging the results obtained by testing more than 70 MAbs with different binding capacities to gp120 and infected cells. Data shown refer to the results obtained with the ADCC-CM235 assay with the exception of MAb CH23, for which results of the ADCC-CM243 assay are shown. ADCC activity of all MAbs was confirmed in the ADCC-CM235 assay with a 6-h incubation (not shown; P = 0.001 by Spearman correlation analysis).

Fig 4

Monoclonal antibody competition of A32, 17B, and 19B Fab ADCC activity. The 20 ADCC-mediating MAbs that did not bind to linear epitopes were tested for their ability to inhibit ADCC mediated by Fab A32 (left), 17B (middle), and 19B (right) in the ADCC-E.CM235 assay. The y axis shows the average inhibition of ADCC activity in duplicate assays, and each bar is color-coded by subject as described for Fig. 3.

Fig 5

Monoclonal antibody competition of A32 MAb binding to HIV-1 AE.A244 gp120 envelope glycoprotein. The ADCC-mediating MAbs (with the exception of CH55 and CH80) were tested for their ability to compete for MAb A32 binding to AE.A244 gp120 envelope glycoprotein. The y axis shows the percentage of blocking of binding activity, and each bar is color-coded by subject as described for Fig. 3. The data shown are representative of duplicate independent experiments.

Fig 6

Cross-clade activity of RV144-induced, ADCC-mediating MAbs. Twenty-one MAbs isolated from six vaccine recipients were tested against CEM.NKR_CCR5 target cells infected with E.CM235 (black bar), B.BaL (red bar), C.DU422 (blue bar), and C.DU151 (green bar) using the GTL assay. The plot shows the average endpoint titer from duplicate values expressed in μg/ml for each MAb and calculated as previously described for Fig. 3.

Fig 7

VH gene segment usage of the ADCC-mediating monoclonal antibodies. The pie chart shows the distribution of VH gene segments and allele usage of the 23 ADCC-mediating MAbs. Each antibody is color-coded by subject of origin using the same color scheme as that described for Fig. 3. The yellow fill indicates all MAbs that used VH1.

Fig 8

Characteristics of antibodies that used VH1 gene segments. (A) Amino acid sequences of ADCC-mediating antibodies that used VH1 gene segments were aligned to the heavy- and light-chain consensus HAAD motifs previously identified for CD4bs bNAbs, which were described to preferentially use the VH1 gene, in particular the VH 1-2*02 and 1-46 segments (41). The consensus HAAD motifs of the heavy and light chains are 68 and 53 amino acids long, respectively. Data are plotted as the number of identical amino acids for heavy chain (x axis) and light chain (y axis). Black X, CD4bs bNAbs (41); red circles, VH1 ADCC mediating antibodies (range, 46- to 57/68-aa identity for heavy chain, 68 to 84%, and 37- to 46/53-aa identity for light chain, 70 to 87%); blue diamonds, influenza virus broadly neutralizing antibodies (49) (52- to 55/68-aa identity for heavy chain, 76 to 81%, and 31- to 32/53-aa identity for light chain, 58 to 60%). (B) Maximal percent GzB activity is correlated with HC mutation frequency (Spearman correlation, ρ = 0.56; P = 0.02). Antibodies that blocked sCD4 binding to gp120 are shown as red diamonds and were found throughout the range of mutation frequencies; those without blocking activity are shown as black circles.