Three families of CD4-induced antibodies are associated with the capacity of plasma from people living with HIV to mediate ADCC in the presence of CD4-mimetics

Abstract

CD4-mimetics (CD4mcs) are small molecule compounds that mimic the interaction of the CD4 receptor with HIV-1 envelope glycoproteins (Env). Env from primary viruses normally samples a "closed" conformation that occludes epitopes recognized by CD4-induced (CD4i) non-neutralizing antibodies (nnAbs). CD4mcs induce conformational changes on Env resulting in the exposure of these otherwise inaccessible epitopes. Here, we evaluated the capacity of plasma from a cohort of 50 people living with HIV to recognize HIV-1-infected cells and eliminate them by antibody-dependent cellular cytotoxicity (ADCC) in the presence of a potent indoline CD4mc. We observed a marked heterogeneity among plasma samples. By measuring the levels of different families of CD4i Abs, we found that the levels of anti-cluster A, anti-coreceptor binding site, and anti-gp41 cluster I antibodies are responsible for plasma-mediated ADCC in the presence of CD4mc.

Importance: There are several reasons that make it difficult to target the HIV reservoir. One of them is the capacity of infected cells to prevent the recognition of HIV-1 envelope glycoproteins (Env) by commonly elicited antibodies in people living with HIV. Small CD4-mimetic compounds expose otherwise occluded Env epitopes, thus enabling their recognition by non-neutralizing antibodies (nnAbs). A better understanding of the contribution of these antibodies to eliminate infected cells in the presence of CD4mc could lead to the development of therapeutic cure strategies.

Keywords: ADCC; CD4 mimetics; Env; HIV cure; HIV-1; cluster A region; co-receptor binding site; gp41 cluster I; non-neutralizing antibodies.

Fig 1

CJF-III-288 enhances recognition and ADCC-mediated elimination of HIV+ infected cells by plasma from PLWH. (A, D) HIV+ infected CD4+ T cells recognition by 50 plasma from PLWH in the presence of CJF-III-288 (50 µM) or equivalent volume of DMSO. Panel A shows the results obtained with the plasma from the 50 PLWH, and panel D shows the results depending on acquisition duration and treatment with antiretroviral therapy. (B, E) Plasma from PLWH-mediated ADCC in the presence of CJF-III-288 (50 µM) or equivalent volume of DMSO. Panel B shows the results obtained with plasma from the 50 PLWH, and panel E shows the results depending on acquisition duration and treatment with antiretroviral therapy of the individuals. (C) Spearman correlation between the capacity of the plasma to recognize infected cells and to mediate ADCC in the presence of DMSO (gray), CJF-III-288 (blue), or both conditions (black). Error bars indicate means ± SEM (**P < 0.01; ***P < 0.001; ****P < 0.0001). The data shows the mean of three independent experiments. Statistical significance was tested using paired t-tests or Wilcoxon matched-pairs signed rank test, based on statistical normality.

Fig 2

Correlations between the level of three families of non-neutralizing antibodies and plasma binding and ADCC activity. (A–B) Indirect ELISA was performed by incubating plasma samples from 50 PLWH to measure the levels of antibodies recognizing specifically the CoRBS, cluster A, or the gp41 cluster I epitopes (see Materials and Methods for more details). CD4i Abs were detected using horseradish peroxidase (HRP)-conjugated anti-human IgG. Relative light unit values obtained with bovine serum albumin (BSA) (negative control) were subtracted and further normalized to the signal obtained with the appropriate control mAb (17b for the CoRBS, A32 for the cluster A, and F240 for the gp41 cluster I ELISA) present in each plate. Panel A shows the results obtained with the plasma from the 50 PLWH, and panel B shows the results depending on acquisition duration and treatment with antiretroviral therapy of the individuals. (C–E) Spearman correlations between the capacity of plasma samples from 50 PLWH to recognize the infected cells in the presence of DMSO (gray) or CJF-III-288 at 50 µM (blue) and the level of anti-CoRBS (C), anti-cluster A (D), or anti-gp41 cluster I (E) antibodies present in these plasmas. (F–H) Spearman or Pearson correlations between the capacity of plasma samples to mediate ADCC in the presence of DMSO (gray) or CJF-III-288 at 50 µM (blue) and the level of anti-CoRBS (F), anti-cluster A (G), or anti-gp41 cluster I (H) antibodies present in these plasmas. The data shows the mean of three independent experiments. Error bars indicate means ± SEM (*P < 0.05; **P < 0.01; ****P < 0.0001; ns, non-significant). Statistical significance was tested using paired t-tests or Wilcoxon matched-pairs signed rank test, based on statistical normality.

Fig 3

Anti-CoRBS, anti-cluster A, and anti-gp41 cluster I CD4-induced Abs contribute to ADCC mediated by plasma from PLWH in the presence of a CD4mc. (A) HIV+ infected CD4+ T cells recognition and (B) ADCC activity of plasma from 20 PLWH (from the two groups of donors, untreated or ART-treated, collected 2–5 years after acquisition) in the presence of DMSO or CJF-III-288 (50 µM, blue) with or without pre-incubation with anti-cluster A A32 Fab, or anti-gp41 cluster I F240 Fab or anti-CoRBS 17b Fab or the three Fab fragments. Error bars indicate means ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant). Statistical significance was tested using paired t-tests, based on statistical normality.

Fig 4

ADCC activity of plasma from PLWH against autologous ex vivo expanded CD4+ T cells in the presence of CJF-III-288. (A) HIV+ infected CD4+ T cells isolated from PLWH were expanded ex vivo. (B) HIV-1-infected cells recognition at the surface of infected cells by autologous plasma in the presence of DMSO (gray), CJF-III-288 (blue), or CJF-III-288 with or without preincubation with the three Fab fragments (red). The graph shows the results obtained for a representative donor. (C) ADCC activity in the presence of DMSO (gray) or CJF-III-288 (50 µM, blue) with autologous plasma after or not pre-incubation with anti-cluster A A32 Fab, anti-gp41 cluster I F240 Fab, and anti-CoRBS 17b Fab fragments. Each symbol represents data from an individual donor. Error bars indicate means ± SEM (*P < 0.05; ns, non-significant). Statistical significance was tested using the Wilcoxon matched-pairs signed rank test, based on statistical normality.

Fig 5

Anti-cluster A, anti-CoRBS, and anti-gp41 cluster I Abs recognize highly conserved epitopes in Env. Env residues forming epitopes for cluster A, CoRBS, and gp41 cluster I region are highlighted in pink, yellow, and cyan, respectively. Epitope residues are defined as gp120 (cluster A and CoRBS) or gp41 (cluster I) residues contributing buried surface area to the Fab-Env antigen interface as calculated by PISA (http://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). Calculations were done based on available Env antigen complexes of cluster A Abs: A32 (PDB code: 4YC2 and 4YBL), N5-i5 (PDB code: 4H8W), 2.2c (PDB code: 4R4F), N60-i3 (PDB code: 4RFO), JR4 (PDB code: 4RFN), CH54 (PDB code: 6MG7), CH55 (PDB code: 6OFI), DH677.3 (PDB code: 6MFP), C11 (PDB code: 6OEJ), and N12-i3 (PDB code: 5W4L); CoRBS Abs: 17b (PDB code: 1GC1), 48d (PDB code: 4DVR), N12-i2 (PDB code: 6W4M), 412d (PDB code: 2QAD), X5 (PDB code: 2B4C); and cluster I Abs: F240 (PDB code: 7N05) and 7B2 (PDB code: 4YDV) Cluster I region includes also 246D epitope residues defined based on peptide mapping as in PMID: 1714520.