Boosting of HIV envelope CD4 binding site antibodies with long variable heavy third complementarity determining region in the randomized double blind RV305 HIV-1 vaccine trial

Abstract

The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is antigenic for the unmutated common ancestor of V1V2 broadly neutralizing antibody (bnAbs), no plasma bnAb activity was induced. The RV305 (NCT01435135) HIV-1 clinical trial was a placebo-controlled randomized double-blinded study that assessed the safety and efficacy of vaccine boosting on B cell repertoires. HIV-1-uninfected RV144 vaccine recipients were reimmunized 6-8 years later with AIDSVAX B/E gp120 alone, ALVAC-HIV alone, or a combination of ALVAC-HIV and AIDSVAX B/E gp120 in the RV305 trial. Env-specific post-RV144 and RV305 boost memory B cell VH mutation frequencies increased from 2.9% post-RV144 to 6.7% post-RV305. The vaccine was well tolerated with no adverse events reports. While post-boost plasma did not have bnAb activity, the vaccine boosts expanded a pool of envelope CD4 binding site (bs)-reactive memory B cells with long third heavy chain complementarity determining regions (HCDR3) whose germline precursors and affinity matured B cell clonal lineage members neutralized the HIV-1 CRF01 AE tier 2 (difficult to neutralize) primary isolate, CNE8. Electron microscopy of two of these antibodies bound with near-native gp140 trimers showed that they recognized an open conformation of the Env trimer. Although late boosting of RV144 vaccinees expanded a novel pool of neutralizing B cell clonal lineages, we hypothesize that boosts with stably closed trimers would be necessary to elicit antibodies with greater breadth of tier 2 HIV-1 strains.

Trial registration: ClinicalTrials.gov NCT01435135.

Fig 1. Boosting of RV144 vaccinees increased V_H chain gene mutation frequency and caused a repertoire shift, increasing the frequency of antibodies with Heavy Chain Complementary Determinant Region 3 (HCDR3) ≥ 22 amino acids.

PBMCs from four vaccinees post-RV144 (A&B) and post-RV305 (C&D) were antigen-specific single-cell sorted with fluorophore labeled conjugates. The V_H/V_L chain genes were PCR-amplified and screened for Env-reactivity by ELISA. The V_H chain gene mutation frequency and HCDR3 lengths of 145 Env-reactive antibodies from RV135/144 and 242 Env-reactive antibodies from RV305 were analyzed with Cloanalyst[56]. (A&C) Error bars represent the standard error of the mean.

Fig 2. The long Heavy Chain Determinant Region 3 (HCDR3) antibodies that neutralize virus bind the CD4 binding site (CD4 bs).

Purified recombinant monoclonal antibodies (mAbs) were assayed by ELISA for (A) blocking the binding of soluble CD4 to AE.A244gp120, and (B) sensitivity to the CD4 binding site mutations Δ371I/P363N, D368R, N276A, and T278A in the AE.A244gp120 protein. (C) Epitope mapping of mAbs on yeast displayed YU2gp120 with point mutations within the inner domain, outer domain and CD4 binding site. (D) Assaying the long HCDR3 CD4 bs antibodies for HIV-1 neutralization in the TZM-bl neutralization assay.

Fig 3. Neutralization of the tier 1 HIV-1 isolate AE.92TH023 and the tier 2 HIV-1 isolate CRF_01 AE.CNE8 by the DH576 Unmutated Common Ancestor (UCA) and DH576 lineage members.

The DH576 clonal lineage was inferred with Cloanalyst[56]. The DH576 clonal lineage was assayed for neutralization of the tier 1 isolate AE.92TH023 and the tier 2 isolate CRF_01 AE.CNE8 in the TZM-bl neutralization assay. Affinity maturation of the DH576 clonal lineage improved neutralization for the tier 1 isolate AE.92TH023 by > 3 logs and for the tier 2 isolate CRF_01 AE.CNE8 by approximately 1 log.

Fig 4. Negative stained electron microscopy of DH576 in complex with CH505 SOSIP.664.

(A) 2D class averages of one, two, or three DH576 Fabs bound to the trimer and (B) Side and top views of the 3D EM reconstruction with the indicated Ab or sCD4 superimposed on the gp120 subunit for comparison with DH576 binding. Arrows indicate the angle at which the indicated antibody approaches the trimer.