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. 2014 Mar;88(5):2633-44.
doi: 10.1128/JVI.03230-13. Epub 2013 Dec 18.

Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by antibody-dependent cell-mediated cytotoxicity

Maxime Veillette  1 , Anik Désormeaux, Halima Medjahed, Nour-Elhouda Gharsallah, Mathieu Coutu, Joshua Baalwa, Yongjun Guan, George Lewis, Guido Ferrari, Beatrice H Hahn, Barton F Haynes, James E Robinson, Daniel E Kaufmann, Mattia Bonsignori, Joseph Sodroski, Andrés Finzi
Affiliations

Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by antibody-dependent cell-mediated cytotoxicity

Maxime Veillette et al. J Virol. 2014 Mar.

Abstract

Anti-HIV-1 envelope glycoprotein (Env) antibodies without broadly neutralizing activity correlated with protection in the RV144 clinical trial, stimulating interest in other protective mechanisms involving antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Env epitopes targeted by many antibodies effective at mediating ADCC are poorly exposed on the unliganded Env trimer. Here we investigated the mechanism of exposure of ADCC epitopes on Env and showed that binding of Env and CD4 within the same HIV-1-infected cell effectively exposes these epitopes. Env capacity to transit to the CD4-bound conformation is required for ADCC epitope exposure. Importantly, cell surface CD4 downregulation by Nef and Vpu accessory proteins and Vpu-mediated BST-2 antagonism modulate exposure of ADCC-mediating epitopes and reduce the susceptibility of infected cells to this effector function in vitro. Significantly, Env conformational changes induced by cell surface CD4 are conserved among Env from HIV-1 and HIV-2/SIVmac lineages. Altogether, our observations describe a highly conserved mechanism required to expose ADCC epitopes that might help explain the evolutionary advantage of downregulation of cell surface CD4 by the HIV-1 Vpu and Nef proteins.

Importance: HIV-1 envelope epitopes targeted by many antibodies effective at mediating antibody-dependent cell-mediated cytotoxicity (ADCC) are poorly exposed on the unliganded envelope trimer. Here we investigated the mechanism of exposure of these epitopes and found that envelope interaction with the HIV-1 CD4 receptor is required to expose some of these epitopes. Moreover, our results suggest that HIV-1 CD4 downregulation might help avoid the killing of HIV-1-infected cells by this immune mechanism.

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Figures

FIG 1
FIG 1
Interaction of HIV-1 Env with coexpressed CD4 induces conformational changes that expose inner domain as well as complex CD4-induced epitopes. (A to D) Interaction of coexpressed CD4 with HIV-1YU2ΔCT Env enhances recognition by A32 (A), C11 (B), ADCC-mediating antibodies isolated from RV144 trial recipients (C) (33), and cluster A-recognizing antibodies (D) (35). Epitope exposure requires CD4-gp120 interaction as shown by decreased recognition of these epitopes with either a mutant of CD4 (F43H) with decreased capacities to interact with gp120 or a CD4-binding site Env variant (D368A). (E and F) Coexpression of Env and CD4 also induces conformational changes in Env that enhance CD4i epitopes (17b and 412D) (E) and decrease recognition by quaternary-dependent Abs such as PG9 and PG16 (F). Data shown are representative of those obtained in at least 3 independent experiments performed in triplicate (mean ± standard deviation [SD]). Env signals were normalized to that obtained with the gp120 outer domain-recognizing antibody 2G12 (A, B, C, and D), and this ratio was normalized to the absence of coexpressed CD4 for panels E and F. −, absence of CD4. The increasing blue bar indicates a stepwise increase in the amount of CD4 expressor being transfected. wt, wild type.
FIG 2
FIG 2
Coexpressed CD4 competes for ligands that recognize the Env CD4-binding site. Cells expressing HIV-1YU2ΔCT Env together with increasing concentrations of human CD4 were stained by the anti-CD4 OKT4 antibody (A) or the CD4-binding site ligands CD4-Ig, b12, VRC03, and VRC01 (B), using the cell-based ELISA described in Materials and Methods. Data are representative of those obtained in at least three independent experiments performed in triplicate (mean ± SD). Signals were normalized to that obtained with the gp120 outer domain-recognizing antibody 2G12 in the absence of coexpressed CD4.
FIG 3
FIG 3
Conformational changes induced by coexpressed CD4 require Env to transit to the CD4-bound conformation. The HIV-1YU2ΔCT layer 1 (H66A) Env variant with a decreased propensity to sample the CD4-bound conformation (5, 54) exhibits decreased exposure of A32 (A) and C11 (B) epitopes upon coexpression of CD4. However, a gp120 change that fills the Phe43 cavity and favors a conformation closer to the CD4-bound conformation (58) enhances the CD4-induced exposure of these epitopes and is sufficient to revert the phenotype of the layer 1 variant. Data shown are representative of those obtained in at least 3 independent experiments performed in triplicate (mean ± SD). Signals were normalized to that obtained with the gp120 outer domain-recognizing antibody 2G12.
FIG 4
FIG 4
Envelope conformational changes induced by coexpressed CD4 are conserved among HIV-1 and HIV-2/SIVmac Env. Laboratory-adapted HIV-1HxBc2 (A), primary HIV-1JRFL (B), HIV-1ADA (C), transmitted/founder HIV-1 clades C (C1086) (D) and D (190049) (E), and HIV-27312 (F) and SIVmac239 (G) envelope glycoprotein expressors were transfected into HOS cells together with increasing concentrations of a human CD4 expressor. At 48 hours posttransfection, Env conformation was assessed by cell-based ELISA with antibodies A32 and C11 recognizing the HIV-1 gp120 inner domain, as described in Materials and Methods. For HIV-2 and SIVmac239 envelopes, the recently described CD4i (1.4H) antibody was used (52). Data are representative of those obtained in at least three independent experiments performed in triplicate (mean ± SD). Signals were normalized to that obtained with the gp120 outer domain-recognizing antibody 2G12 or PGT121 (for C1086) for HIV-1 Env. For HIV-2 and SIVmac239 Envs, signals were normalized to that obtained with serum from SIV-infected macaques.
FIG 5
FIG 5
Env conformational changes induced by coexpressed CD4 are conserved when Env is expressed within replication-competent proviruses and are indirectly modulated by Nef and Vpu proteins. pNL4.3 GFP ADA and pNL4.3 GFP ADA Vpu− Nef− were transfected into permissive, BST2-free HOS cells (66) together with increasing concentrations of a human CD4 expressor. At 48 hours posttransfection, Env conformation was assessed by cell-based ELISA with A32 (A) and C11 (B) Abs, as described in Materials and Methods. Importantly, Env conformational changes require CD4-gp120 interaction, as shown by decreased recognition by these Abs of an Env variant with a change (D368A) in the CD4-binding site. (C) Anti-CD4 OKT4 antibody was used to monitor levels of CD4. Data are representative of those obtained in at least three independent experiments performed in triplicate ± SD. Signals were normalized to that obtained with the gp120 outer domain-recognizing antibody 2G12 (A and B).
FIG 6
FIG 6
Env conformational changes induced by surface CD4 increase susceptibility of HIV-infected cells to antibody-dependent cellular cytotoxicity. (A to D) CEM.NKR cells infected with VSV-G pseudotyped NL4.3 GFP ADA either wild-type (wt), Nef− (N−), Vpu− (U−), or Nef− Vpu− (N− U−) encoding wt or D368A Env were stained against surface CD4 (OKT4 Ab) (A) or Env 2G12 (B), A32 (C), and CH54 (D) at 48 h postinfection and analyzed by flow cytometry as described in Materials and Methods. Signals were normalized to the mean signal of the wt virus (fold change) for Env epitopes and to the mock control for surface CD4. (E) Alternatively, the susceptibility to A32-mediated lysis by PBMC effector cells of those cells was analyzed by flow cytometry as described in Materials and Methods. (F) Preincubating with the Fab fragment of A32 prevented A32-mediated lysis. (G) Susceptibility to A32-, CH54-, CH94-, L9-i1-, L9-i2-, N5-i5-, N12-i3-, and N26-i1-mediated lysis by PBMC effector cells of wt versus N-U-infected CEM.NKR cells was analyzed as described for panel E. Data shown are the results of at least three independent infections ± standard error of the mean (SEM). ADCC was measured using PBMCs from three different healthy donors using the gating and formula presented in Fig. S3 in the supplemental material. Statistical significance was tested using an unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
FIG 7
FIG 7
Nef and Vpu prevent the exposure of A32-like epitopes at the surface of infected primary CD4+ T cells. PHA-activated CD4+ T cells isolated from PBMCs of three healthy donors were infected with VSV-G-pseudotyped NL4.3 GFP ADA, either wild type (wt), Nef− (N−), Vpu− (U−), or Nef− Vpu− (N− U−), encoding wt or D368A Env and were stained with A32 (A), CH54 (B), and anti-cluster A (C) Abs at 48 h postinfection and analyzed by flow cytometry as described in Materials and Methods. Signals were normalized to the mean signal of the wt virus (fold change). Data shown are the results of at least three independent infections ± SEM. Statistical significance was tested using an unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
FIG 8
FIG 8
The A32 epitope is potentially accessible in the CD4-bound Env trimer. The cryoelectron tomographic map of the CD4-bound and 17b Fab-bound HIV-1 Env trimer (EMDB 5020) (67) is viewed from the perspective of the target cell. The density associated with the 17b antibody Fab has been removed for clarity. The Env trimer axis is designated with a black triangle. Three CD4-bound gp120 cores (PDB 3DNO) were fit to the density map, and the CD4-bound gp120 with complete N and C termini (PDB 3JWO) (61) was aligned with one subunit. On this subunit, CD4 domains 1 and 2 are shown (blue ribbon). The gp120 residues implicated by mutagenesis in binding the A32 antibody in this study and a previous study (5) are depicted in CPK mode (red, significant effects on A32 binding; orange, moderate effects on binding). The hypothesized angle of approach of A32-like antibodies that mediate ADCC is represented by the green arrow.
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