Co-receptor Binding Site Antibodies Enable CD4-Mimetics to Expose Conserved Anti-cluster A ADCC Epitopes on HIV-1 Envelope Glycoproteins

Abstract

Human immunodeficiency virus type 1 (HIV-1) has evolved a sophisticated strategy to conceal conserved epitopes of its envelope glycoproteins (Env) recognized by antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. These antibodies, which are present in the sera of most HIV-1-infected individuals, preferentially recognize Env in its CD4-bound conformation. Accordingly, recent studies showed that small CD4-mimetics (CD4mc) able to "push" Env into this conformation sensitize HIV-1-infected cells to ADCC mediated by HIV+ sera. Here we test whether CD4mc also expose epitopes recognized by anti-cluster A monoclonal antibodies such as A32, thought to be responsible for the majority of ADCC activity present in HIV+ sera and linked to decreased HIV-1 transmission in the RV144 trial. We made the surprising observation that CD4mc are unable to enhance recognition of HIV-1-infected cells by this family of antibodies in the absence of antibodies such as 17b, which binds a highly conserved CD4-induced epitope overlapping the co-receptor binding site (CoRBS). Our results indicate that CD4mc initially open the trimeric Env enough to allow the binding of CoRBS antibodies but not anti-cluster A antibodies. CoRBS antibody binding further opens the trimeric Env, allowing anti-cluster A antibody interaction and sensitization of infected cells to ADCC. Therefore, ADCC responses mediated by cluster A antibodies in HIV-positive sera involve a sequential opening of the Env trimer on the surface of HIV-1-infected cells. The understanding of the conformational changes required to expose these vulnerable Env epitopes might be important in the design of new strategies aimed at fighting HIV-1.

Keywords: ADCC; CD4; CD4-mimetics; Envelope glycoproteins; HIV-1; Non-neutralizing antibodies.

Graphical abstract

Fig. 1

CD4mc fail to enhance recognition of HIV-1-infected cells by anti-cluster A antibodies. Primary CD4 + T cells, which were either mock-infected or infected with NL4.3 GFP expressing the primary R5 ADA Env (NL4.3 ADA GFP), were stained with sera from 10 HIV-1-infected individuals followed by an anti-human Alexa-Fluor 647 (AF647)-conjugated secondary antibody (a, c) or with (b, d) AF647-conjugated anti-cluster A (b, d) A32, (e) N5-i5 or (f) C11, in the presence of CD4mc JP-III-48, BNM-III-170, M48U1 or DMSO (vehicle). Shown are histograms depicting representative staining obtained with (a) HIV + sera or (b) A32-AF647 and (c-f) the mean fluorescence intensities (MFI) obtained from at least three experiments. Error bars indicate mean ± SEM. Statistical significance was tested using a Kruskal-Wallis with a Dunn's post-test (**p < 0.01, ***p < 0.001, ns: non-significant).

Fig. 2

Sera from HIV-1-infected individuals enable recognition of HIV-1-infected cells by anti-cluster A antibodies upon CD4mc addition. Cell-surface staining of primary CD4 + T cells either mock-infected or infected with NL4.3 GFP expressing the primary R5 ADA Env with AF647-conjugated anti-cluster A antibodies (a–b) A32, (c) C11 or (d) N5-i5, in the presence or absence of HIV + sera and CD4mc JP-III-48, BNM-III-170, M48U1 or DMSO. Shown in (a) are histograms depicting representative staining obtained with A32-AF647 in the presence the CD4mc JP-III-48 or DMSO. Shown in (b–d) are the mean fluorescence intensities (MFI) obtained for staining obtained in at least 5 experiments with (b) A32-AF647, (c) C11-AF647 and (d) N5-i5-AF647 in the presence or absence of 10 different HIV + sera. Error bars indicate mean ± SEM. Statistical significance was tested using a Kruskal-Wallis with a Dunn's post-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: non-significant). See also Fig. S1.

Fig. 3

Co-receptor binding site Fab fragments reduce HIV + sera ability to enhance recognition of infected cells in the presence of CD4mc. (a–c) Cell-surface staining of primary CD4 + T cells either mock-infected or infected with CH58 T/F with AF647 anti-cluster A antibodies (a–b) A32 or (c) C11, in the presence or not of HIV + sera, with or without the Fab fragment of (a) 17b or (b) N12-i2 and CD4mc JP-III-48 or DMSO (vehicle). Shown in (a–c) are (left) histograms depicting representative staining and (right) the mean fluorescence intensities (MFI) obtained for staining obtained in at least 5 experiments in the presence of 10 different HIV + sera. (d) Cell-surface staining of primary CD4 + T cells isolated from 3 HIV-1-infected individuals after ex-vivo expansion with AF647-A32 in the presence or not of autologous (circle), an HIV + heterologous (square) sera, with or without the 17b Fab fragment and CD4mc JP-III-48 or DMSO. Left panels show histograms depicting representative staining with autologous sera. Right panels present the mean fluorescence intensities (MFI) obtained for multiple staining. Error bars indicate mean ± SEM. Statistical significance was tested using a Kruskal-Wallis with a Dunn's post-test (*p < 0.05, ***p < 0.001, ****p < 0.0001, ns: non-significant).

Fig. 4

Co-receptor binding site antibodies facilitate anti-cluster A antibody recognition of HIV-1-infected cells in the presence of CD4mc. (a–c) Cell-surface staining of primary CD4 + T cells either mock-infected or infected with NL4.3 GFP expressing the primary R5 ADA Env with AF647-conjugated (a–b) A32 or (c) C11, in the presence or not of co-receptor binding site (CoRBS) mAbs 17b, C2, N12-i2, 48d or 412d, CD4 binding site (CD4BS) mAbs VRC01 or b12, anti-V3 mAbs 19b, anti-V2 mAbs CH58 or human IgG, with CD4mc JP-III-48 or DMSO. Shown in (a) are histograms depicting representative A32-AF647 staining and (b–c) the mean fluorescence intensities (MFI) obtained for at least 7 different staining. (d) Cell-surface staining of primary CD4 + T cells either mock-infected or infected with CH58 T/F with AF647-conjugated A32 in the presence or not of the Fab fragment, the Fab′2 fragment or the full 17b with CD4mc JP-III-48 or DMSO. Shown in (d) are (left) histograms depicting representative staining and (right) the MFI for at least 9 different staining. Error bars indicate mean ± SEM. Statistical significance was tested using a Kruskal-Wallis with a Dunn's post-test (*p < 0.05, **p < 0.01, ****p < 0.0001, ns: non-significant). See also Fig. S2, S3 and S4.

Fig. 5

CD4-mimetic sensitize HIV-1-infected cells to ADCC-mediated by anti-cluster A antibodies in the presence of CoRBS antibodies. Primary CD4 T cells infected with CH58 T/F were used as target cells and autologous PBMC as effector cells in our FACS-based ADCC assay. Shown in (a) are the percentages of ADCC-mediated killing obtained with serial dilution of A32 (0.3125, 0.625, and 1.25 μg/ml) alone or in combination with 17b (5 μg/ml), in the presence of CD4mc JP-III-48 or equivalent volume of DMSO for 7 different experiments. Shown in (b) are the percentages of ADCC-mediated killing obtained with A32 (0.3125 μg/ml), 17b, 17b Fab, alone or in combination, or with sera from an HIV + individual, in the presence of JP-III-48 or equivalent volume of DMSO. Error bars indicate mean ± SEM. Statistical significance was tested using an Ordinary one-way ANOVA with a Holm-Sidak's post-test (***p < 0.001, ****p < 0.0001, ns: non-significant), See also Fig. S5 and S6.

Fig. 6

Sequential opening of Env required for anti-cluster A antibody binding. CD4mc induce a partial opening of trimeric Env allowing subsequent exposure of the co-receptor binding site. Binding of Env by CoRBS antibodies induce additional conformational changes in Env resulting in the exposure of highly-conserved epitopes recognized by anti-cluster A antibodies. Env exposing anti-cluster A epitopes become a suitable target for ADCC.