Identification of HIV gp41-specific antibodies that mediate killing of infected cells

Abstract

Antibodies that mediate killing of HIV-infected cells through antibody-dependent cellular cytotoxicity (ADCC) have been implicated in protection from HIV infection and disease progression. Despite these observations, these types of HIV antibodies are understudied compared to neutralizing antibodies. Here we describe four monoclonal antibodies (mAbs) obtained from one individual that target the HIV transmembrane protein, gp41, and mediate ADCC activity. These four mAbs arose from independent B cell lineages suggesting that in this individual, multiple B cell responses were induced by the gp41 antigen. Competition and phage peptide display mapping experiments suggested that two of the mAbs target epitopes in the cysteine loop that are highly conserved and a common target of HIV gp41-specific antibodies. The amino acid sequences that bind these mAbs are overlapping but distinct. The two other mAbs were competed by mAbs that target the C-terminal heptad repeat (CHR) and the fusion peptide proximal region (FPPR) and appear to both target a similar unique conformational epitope. These gp41-specific mAbs mediated killing of infected cells that express high levels of Env due to either pre-treatment with interferon or deletion of vpu to increase levels of BST-2/Tetherin. They also mediate killing of target cells coated with various forms of the gp41 protein, including full-length gp41, gp41 ectodomain or a mimetic of the gp41 stump. Unlike many ADCC mAbs that target HIV gp120, these gp41-mAbs are not dependent on Env structural changes associated with membrane-bound CD4 interaction. Overall, the characterization of these four new mAbs that target gp41 and mediate ADCC provides evidence for diverse gp41 B cell lineages with overlapping but distinct epitopes within an individual. Such antibodies that can target various forms of envelope protein could represent a common response to a relatively conserved HIV epitope for a vaccine.

Diversity and function of maternal HIV-1-specific antibodies at the time of vertical transmission

“…To map epitope specificities of each clonal nAb family, we used a combination of phage immunoprecipitation sequencing (PhIP-seq) (24) (Figures 1 and 2) and enzyme-linked immunosorbent assays (ELISA) (Figure 2). For PhIP-seq, we utilized a previously described phage library (24) that contains multiple HIV Env sequences: consensus sequences for clades A, B, C and D and specific sequences circulating in Kenya, including the transmitted BG505.W6.C2 virus.…”

“…To map epitope specificities of each clonal nAb family, we used a combination of phage immunoprecipitation sequencing (PhIP-seq) (24) (Figures 1 and 2) and enzyme-linked immunosorbent assays (ELISA) (Figure 2). For PhIP-seq, we utilized a previously described phage library (24) that contains multiple HIV Env sequences: consensus sequences for clades A, B, C and D and specific sequences circulating in Kenya, including the transmitted BG505.W6.C2 virus. For 9 of the 21 nAb families, phage-displayed peptides were significantly enriched within the V3 region of HIV Envelope (spanning positions 302-322, based on HXB2 numbering), suggesting that this region of HIV Env comprises a key part of the epitope of these isolated nAbs (Figure 3A).…”

“…Wells were washed four times with 300 μl wash buffer (1X PBS, 0.05% Tween-20) and blocked for 1 hour at room temperature (RT) in blocking buffer (1X PBS with 10% non-fat milk and 0.05% Tween-20). MG505 nAbs and control antibodies (gp120-specific VRC01 (39) and gp41-specific QA255.006 (24)) were applied at 10 μg ml −1 in blocking buffer and incubated at 37°C for 2 hours. Wells were washed and incubated with goat anti-human IgG-HRP (Sigma) diluted 1:2500 in blocking buffer for 1 hour at RT.…”

“…The RF-ADCC assay was performed as described (24, 40). In short, CEM-NKr cells (AIDS Research and Reference Reagent Program, NIAID, NIH from Dr. Alexandra Trkola) were double labeled with PKH-26-cell membrane dye (Sigma-Aldrich) and a cytoplasmic-staining dye (Vybrant CFDA SE Cell Tracer Kit, Life Technologies).…”

“…To precisely map the epitopes of antibodies in this study, we employed an approach that combines phage display, immunoprecipitation and highly-multiplexed sequencing, as previously described (24, 41). In brief, amplified phage (1 mL at 2×10 5 -fold representation of each phage clone) that display peptides from several Envelope and full-length HIV sequences was added to each well of a 96-deep-well plate (CoStar).…”