HIV-1 VACCINES. Diversion of HIV-1 vaccine-induced immunity by gp41-microbiota cross-reactive antibodies

Abstract

An HIV-1 DNA prime vaccine, with a recombinant adenovirus type 5 (rAd5) boost, failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1-reactive plasma Ab titers were higher to Env gp41 than to gp120, and repertoire analysis demonstrated that 93% of HIV-1-reactive Abs from memory B cells responded to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies were non-neutralizing and frequently polyreactive with host and environmental antigens, including intestinal microbiota (IM). Next-generation sequencing of an immunoglobulin heavy chain variable region repertoire before vaccination revealed an Env-IM cross-reactive Ab that was clonally related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy.

Figure 1. Characteristics of HIV-1-reactive antibodies (Abs) induced by DNA prime, rAd5 boost vaccine

A) Plasma Ab titers (AUC, area under the curve) to gp120 and gp41 proteins in a subset of 40 HVTN 505 vaccine recipients; red circles represent vaccine responders (percents) to the antigens tested, and blue represent non-responders. (B) Plasma binding Abs from 8 HVTN 082 and 204 trial participants (vaccine responders) were screened for gp120 and gp41 reactivity via binding Ab multiplex assay (BAMA). (C) HIV-1-reactive Abs were isolated from single memory B cells of the 8 HVTN 082 and 204 trial participants and screened for binding gp120 and gp41 proteins via ELISA. Statistics: (A) *** p < 0.0001, Wilcoxon signed rank test, B.MN gp41 vs. gp120 (maximum; which is the highest value of all gp120 Envs for each participant), *p < 0.05 McNemar’s test, B.MN gp41 vs. each gp120 (not shown); (B) *p < 0.05**p < 0.01, Wilcoxon signed rank test [Graphpad Prism].

Figure 2. Polyreactivity of vaccine-induced gp41-reactive monoclonal antibodies (mAbs)

Five representative polyreactive and gp41-reactive mAbs demonstrated binding with auto-antigens (ANA) (A), Hep-2 cells (immunofluorescence staining) (B), and anaerobe and aerobe whole cell lysates of intestinal microbiota (IM), bacterial (E. coli) RNA polymerase, MN gp41, and vaccine-strain VRC-A gp140 antigens (BAMA) (C). (B) All images were taken on an Olympus AX70 fluorescence microscope using a 40x objective with a SPOT Flex camera. All images were prepared using 50 µg/mL of mAb. Images were acquired for 5 sec (2F5, 17b) or 10 sec (DH432, DH438, DH440, DH443, DH444). Size bars are 25 µm. (A, B) Control antibodies: 4E10 (gp41), 2F5 (gp41), 17b (gp41), and palivizumab (RSV). (C) MAb binding was reported as MFI-background-blank, where background is plate specific background binding and blank represents non-specific sample binding to a negative control bead. Positivity cutoff for binding was 100 MFI as previously reported (2); AbCLL1324 as an IM-reactive control was used to validate IM binding and gp41 mAb 7B2 that does not react with IM was used as a negative control. Data shown were generated from the same commercially available kits (A, B), and a single BAMA experiment (C), which was in agreement with Ab-IM reactivity in western blots (Figure S10 or not shown).

Figure 3. Characteristics of clonally-related antibodies (Abs)

Pre- (DH477) and post- (DH476) vaccine clonally-related Abs found in vaccinee 082-003 had the same V_HDJ_H recombination. IGHV, IGHD and IGHJ Ab segments were statistically inferred (A, B); nucleotides in the 3’ end of the V-gene and 5’ end of J-gene shown with rearrangement junctions (gray-shaded nucleotides - NN1, NN2), and HCDR3 (underlined nucleotides) indicated. The lower case letters show nucleotides that have been removed from the germline gene during V_HDJ_H rearrangement. Pre- (DH477) and post- (DH476) vaccine heavy chain sequences were paired with the natural light chain (IGκV3–20*01, IGκJ1*01, 1.7% mutated nt, 10 aa CDR3) of the post-vaccine Ab DH476. (C) Ab binding to recombinant HIV-1 Env gp140, 5-Helix gp41 and MN gp41 proteins was determined by surface plasmon resonance (SPR) analysis, and (D) the antibody binding K_d determined by rate constants or steady-state analysis measurements. (E) Binding of DH477 to VRC-A gp140 expressed on the surface of 293i cells. Anti-V3 mAb (19B) and a representative gp41-reactive Ab (DH440) from this study, both of which bind recombinant VRC-A gp140, were used as positive controls, while palivizumab (RSV Ab) was used as a negative IgG1 control. (F) The cross-reactivity of pre-(DH477) and post-(DH476) vaccine Abs with intestinal microbiota (IM) whole cell lysates (WCLs) was determined via western blot and SPR as previously described (2). DH438 (gp41-reactive Ab) and palivizumab were used as positive and negative control Abs, respectively, for western blots, and the arrows indicate candidate IM antigens bound by DH476 and/or DH477. In SPR analysis, IM WCLs were diluted in PBS buffer as indicated and injected over mAbs captured on an anti-IgFc immobilized sensor surface. Specific binding of the lysate proteins to the mAbs in SPR were measured after subtraction of non-specific binding to anti-HA mAb CH65. Abbreviations - nt, nucleotides; aa, amino acid. Data shown (C–F) were representative of duplicate experiments, except SPR screen of DH477 and DH476 for binding rgp140 proteins that was performed once (C).