A Highly Conserved Residue of the HIV-1 gp120 Inner Domain Is Important for Antibody-Dependent Cellular Cytotoxicity Responses Mediated by Anti-cluster A Antibodies

Abstract

Previous studies have shown that sera from HIV-1-infected individuals contain antibodies able to mediate antibody-dependent cellular cytotoxicity (ADCC). These antibodies preferentially recognize envelope glycoprotein (Env) epitopes induced upon CD4 binding. Here, we show that a highly conserved tryptophan at position 69 of the gp120 inner domain is important for ADCC mediated by anti-cluster A antibodies and sera from HIV-1-infected individuals.

FIG 1

Effect of Nef, Vpu, and Env-CD4 interaction on recognition of infected cells by HIV⁺ sera and a panel of monoclonal antibodies. CEM.NKr cells infected with a panel of vesicular stomatitis virus glycoprotein-pseudotyped NL4.3–GFP ADA viruses expressing wild type (WT) or a CD4-binding site (D368R) Env variant, lacking Nef (N⁻), Vpu (U⁻), or both Nef and Vpu (N⁻ U⁻), were stained at 48 h postinfection with an anti-CD4 antibody (OKT4) (A); 1 μg/ml of the CD4-independent outer domain recognizing 2G12 antibody (B); a 1/1,000 dilution of sera from 12 HIV-1-infected individuals (HIV⁺ sera) (C); or 1 μg/ml of anti-cluster A (A32 and N12-i3) (D), anti-CoRBS (17b and LF17) (E), anti-V3 (19b and GE2-JG8) (F), anti-gp120-gp41 interface (PGT151 and 8ANC195) (G), anti-CD4-binding site (VRC01 and b12) (H), and anti-gp41 (F240, N5-U1, N5-U3, 7B2, M785-U1, and N10-U1) (I) antibodies and then fluorescently labeled with an Alexa Fluor 647-conjugated anti-human IgG secondary Ab. Shown is the fold increase of staining relative to mock for all tested sera and antibodies. Data shown are the results of 3 different experiments, and error bars depict the standard errors of the means. Statistical significance was tested using paired one-way analyses of variance (**, P < 0.01; ***, P < 0.001). MFI, mean fluorescence intensity.

FIG 2

Recognition of HIV-1-infected cells by the anti-cluster A32 antibody. CEM.NKr cells infected with a panel of vesicular stomatitis virus glycoprotein-pseudotyped NL4.3–GFP ADA viruses expressing wild type (WT) or a CD4-binding site (D368R) Env variant, lacking Nef (N⁻), Vpu (U⁻), or both Nef and Vpu (N⁻ U⁻) (A), or with vesicular stomatitis virus glycoprotein-pseudotyped primary T/F CH77 infectious molecular clone (B) were stained at 48 h postinfection with the anti-cluster A A32 antibody (1 μg/ml) and then fluorescently labeled with an Alexa Fluor 647-conjugated anti-human IgG secondary Ab. Histograms depicting representative staining of infected (GFP⁺ [A] or p24⁺ [B]) cells are shown. Right panels show the percentages of infected cells, the percentages of infected (GFP⁺ [A] or p24⁺ [B]) cells that were recognized by A32, and the mean fluorescence intensity (MFI) of these cells. Mean fluorescence intensity of infected cells over that of mock-infected cells is shown in the last column.

FIG 3

Effect of Nef and Vpu on recognition of cells infected with a primary isolate by HIV⁺ sera and a panel of monoclonal antibodies. (A and B) CEM.NKr cells infected with vesicular stomatitis virus glycoprotein-pseudotyped T/F CH77 expressing wild type (WT) or lacking Nef and Vpu (N⁻ U⁻) were stained at 48 h postinfection with an anti-CD4 antibody (OKT4) (A) or a 1/1,000 dilution of sera from 12 HIV-1-infected individuals (HIV⁺ sera) (B). (C) CH77-infected cells were also stained with a panel of anti-gp120 and -gp41 antibodies (1 μg/ml) and then fluorescently labeled with an Alexa Fluor 647-conjugated anti-human IgG secondary Ab. Data shown are the results of 3 independent experiments, with medians ± interquartile ranges. Statistical significance was tested using paired one-way analyses of variance (***, P < 0.001).

FIG 4

Effect of Nef, Vpu, and Env-CD4 interaction on ADCC responses mediated by HIV⁺ sera and a panel of monoclonal antibodies. (A) CEM.NKr cells infected with a panel of vesicular stomatitis virus glycoprotein-pseudotyped NL4.3–GFP ADA viruses expressing wild type (WT) or a CD4-binding site (D368R) Env variant, lacking Nef (N⁻), Vpu (U⁻), or both Nef and Vpu (N⁻ U⁻), were used at 48 h postinfection as target cells in our FACS-based ADCC assay (5) to determine their susceptibility to ADCC mediated by a 1/1,000 dilution of sera from 12 HIV-1-infected individuals. (B) The susceptibility of vesicular stomatitis virus glycoprotein-pseudotyped T/F CH77-infected cells expressing wild type (WT) or lacking Nef and Vpu (N⁻ U⁻) to ADCC mediated by the same panel of HIV⁺ sera was also evaluated. (C and D) The susceptibility of pNL4.3-ADA-infected cells to ADCC mediated by 5 μg/ml of anti-gp120 (anti-cluster A antibodies A32 and N12-i3; anti-CD4-binding site antibodies VRC01 and b12; anti-CoRBS antibodies 17b and LF17; anti-V3 antibodies 19b and GE2-JG8; anti-outer domain antibody 2G12) or anti-gp120-gp41 (PGT151 and 8ANC195) (C) or anti-gp41 (F240, N5-U1, N5-U3, 7B2, M785-U1, and N10-U1) (D) antibodies was also evaluated. (E) Susceptibility of CH77-infected cells to anti-gp120 and anti-gp41 antibodies. Peripheral blood mononuclear cells from healthy donors were used as effector cells. Data shown are the results of 3 independent experiments, with medians ± interquartile ranges. Statistical significance was tested using paired one-way analyses of variance (***, P < 0.001).

FIG 5

Tryptophan 69 in layer 1 is important for ADCC responses mediated by anti-cluster A antibodies and some HIV⁺ sera. CEM.NKr cells infected with N⁻ U⁻ vesicular stomatitis virus glycoprotein-pseudotyped NL4-3–GFP ADA virus were used at 48 h postinfection for surface staining (A and B) or FACS-based ADCC assay (C and D) using 5 μg/ml of anti-cluster A antibodies (A32, N5-i5, N12-i3, N26-i1, and 2.2c) or a 1/1,000 dilution of sera from 12 HIV-1-infected individuals (HIV⁺ sera, different color or symbol for each serum sample) in the absence or presence of recombinant gp120/D368R (10 μg/μg of antibody), ΔV1V2V3V5/D368R (6 μg/μg of antibody), or ΔV1V2V3V5/D368R/W69A (6 μg/μg of antibody) for 30 min at room temperature. Data shown are representative of at least 3 independent experiments. Statistical significance was tested using paired one-way analyses of variance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).