The HIV-1 gp120 CD4-bound conformation is preferentially targeted by antibody-dependent cellular cytotoxicity-mediating antibodies in sera from HIV-1-infected individuals

Abstract

Recent studies have linked antibody Fc-mediated effector functions with protection or control of human immunodeficiency type 1 (HIV-1) and simian immunodeficiency (SIV) infections. Interestingly, the presence of antibodies with potent antibody-dependent cellular cytotoxicity (ADCC) activity in the Thai RV144 vaccine trial was suggested to correlate with decreased HIV-1 acquisition risk. These antibodies recently were found to recognize HIV envelope (Env) epitopes exposed upon Env-CD4 interaction. CD4 downregulation by Nef and Vpu, as well as Vpu-mediated BST-2 antagonism, were reported to modulate exposure of those CD4-induced HIV-1 Env epitopes and were proposed to play a role in reducing the susceptibility of infected cells to ADCC mediated by this class of antibodies. Here, we report the high prevalence of antibodies recognizing CD4-induced HIV-1 Env epitopes in sera from HIV-1-infected individuals, which correlated with their ability to mediate ADCC responses against HIV-1-infected cells, exposing these Env epitopes at the cell surface. Furthermore, our results indicate that Env variable regions V1, V2, V3, and V5 do not represent a major determinant for ADCC responses mediated by sera from HIV-1-infected individuals. Altogether, these findings suggest that HIV-1 tightly controls the exposure of certain Env epitopes at the surface of infected cells in order to prevent elimination by Fc-effector functions.

Importance: Here, we identified a particular conformation of HIV-1 Env that is specifically targeted by ADCC-mediating antibodies present in sera from HIV-1-infected individuals. This observation suggests that HIV-1 developed sophisticated mechanisms to minimize the exposure of these epitopes at the surface of infected cells.

FIG 1

Env-CD4 interaction is required for efficient recognition of infected cells by sera from HIV-1-infected individuals. CEM.NKr cells infected with a panel of VSV-G-pseudotyped NL4.3 GFP ADA-Env-based viruses expressing the wild type (wt) or a CD4-binding site (D368R) Env variant, lacking Nef (Nef−) or expressing a Nef variant (L166A-L168A) defective for CD4 downregulation (34) (NefAA) or lacking Vpu (Vpu−) or both Nef and Vpu (Nef− Vpu−), were stained at 48 h postinfection with sera from 30 HIV-infected individuals and then fluorescently labeled with an Alexa Fluor 647-conjugated anti-human IgG secondary Ab. (A) Histograms depicting representative staining of infected (GFP⁺) cells by serum from one HIV⁻ and one HIV⁺ donor. (B) Fold increase of staining relative to mock infection for all tested sera. Data shown are the results from two different experiments, and error bars depict the standard errors of the means (SEM). Statistical significance was tested using paired one-way analyses of variance (ANOVA) (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 2

Env-CD4 interaction modulates the exposure of the Env ADCC-mediating A32 epitope at the surface of infected cells. CEM.NKr cells infected with a panel of VSV-G-pseudotyped NL4.3 GFP ADA-Env-based viruses expressing the wild type (wt) or a CD4-binding site (D368R) Env variant, lacking Nef (Nef−) or expressing a Nef variant (L166A-L168A) defective for CD4 downregulation (34) (NefAA) or lacking Vpu (Vpu−) or both Nef and Vpu (Nef− Vpu−), were stained at 48 h postinfection for surface CD4 levels (A) or Env A32 epitope exposure (B). Data shown are the results from at least three different experiments, and error bars depict SEM. Statistical significance was tested using paired one-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 3

Env-CD4 interaction modulates susceptibility of infected cells to ADCC killing mediated by sera from HIV-1-infected individuals. (A) CEM.NKr cells infected with a panel of VSV-G-pseudotyped NL4.3 GFP ADA-based viruses expressing the wild type (wt) or a CD4-binding site (D368R) Env variant, lacking Nef (Nef−) or expressing a Nef variant (L166A-L168A) defective for CD4 downregulation (34) (NefAA) or lacking Vpu (Vpu−) or both Nef and Vpu (Nef− Vpu−), were used at 48 h postinfection as target cells in our FACS-based ADCC assay (18, 33) to determine their susceptibility to sera from HIV-1-infected individuals to mediate cell lysis by PBMCs from healthy donors. Data shown are the results from three different experiments (median ± interquartile range). (B) A positive correlation was observed between the staining intensity of sera from HIV-1-infected individuals on cells infected with Nef- and Vpu-defective virus and their ability to mediate ADCC. (C) Paired values of ADCC mediated by sera from HIV-1-infected individuals and those of cells infected by HIV-1 viruses lacking both Nef and Vpu and encoding a wt or D368R Env variant. Statistical significance was tested using paired one-way ANOVA (A) or a paired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) (C).

FIG 4

A recombinant gp120 protein lacking V1V2V3 and V5 variable regions is sufficient to adsorb the majority of ADCC activity present in sera from HIV-infected individuals. CEM.NKr cells infected with wt or Nef- and Vpu-defective VSV-G-pseudotyped NL4.3 GFP ADA-based viruses were used at 48 h postinfection for surface staining (A) or FACS-based ADCC assay (B) using sera from HIV-1-infected individuals preincubated in the absence or presence of 83.3 pmol/μl ΔV1V2V3V5 D368R serum for 30 min at room temperature. Data shown are representative of at least two different experiments. Statistical significance was tested using paired t test (**, P < 0.01; ns, not significant).