An Asymmetric Opening of HIV-1 Envelope Mediates Antibody-Dependent Cellular Cytotoxicity

Abstract

The HIV-1 envelope glycoprotein (Env) (gp120-gp41)₃ is the target for neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC). HIV-1 Env is flexible, sampling different conformational states. Before engaging CD4, Env adopts a closed conformation (State 1) that is largely antibody resistant. CD4 binding induces an intermediate state (State 2), followed by an open conformation (State 3) that is susceptible to engagement by antibodies that recognize otherwise occluded epitopes. We investigate conformational changes in Env that induce ADCC in the presence of a small-molecule CD4-mimetic compound (CD4mc). We uncover an asymmetric Env conformation (State 2A) recognized by antibodies targeting the conserved gp120 inner domain and mediating ADCC. Sera from HIV+ individuals contain these antibodies, which can stabilize Env State 2A in combination with CD4mc. Additionally, triggering State 2A on HIV-infected primary CD4⁺ T cells exposes epitopes that induce ADCC. Strategies that induce this Env conformation may represent approaches to fight HIV-1 infection.

Keywords: 17b; A32; ADCC; CD4i Abs; HIV-1; State 2A; cryo-EM; envelope glycoproteins; smFRET.

Figure 1.. CD4mc in combination with anti-cluster A and CoRBS Abs stabilize Env State 2A.

Histograms of FRET values (left) and TDPs resulting from HMM analysis (right) observed for HIV-1_JR-FL Env ΔCT (A) in the absence of bound ligands; and in the presence of (B) CD4mc BNM-lll-170; (C) both BNM-lll-170 and the CD4i Ab 17b; (D) BNM-lll-170, 17b Fab, and the anti-cluster A Ab A32; (E) BNM-lll-170, 17b, and A32; or (F) BNM-lll-170, 17b, and C11. In all cases the CD4mc was used at 100 pM, and Abs or Fab were used at 5 μg/ml. The FRET histograms were formed by compiling the indicated number (N) of smFRET traces. Overlaid on the histograms is the sum (red) of four Gaussian distributions with means and standard deviations determined by HMM analysis (0.18 ± 0.09, 0.38 ± 0.09, 0.65 ± 0.09, 0.90 ± 0.08; grey). Error bars reflect the standard deviation in the number of data points per histogram bin determined from three independent groups of smFRET traces.

Figure 2.. Cryo-EM imaging of AT-2-inactivated HIV-1 particles with CD4mc BNM-NM70 in the presence of 17b and A32 antibodies.

(A) AT-2-inactivated HlV-1 viral particles embedded in vitrious ice with spikes highlighted by green circles. (B) Magnified images of single membrane-bound spikes as indicated by red arrows. The images were collected with a K2 camera mounted on a Talos Arctica cryo TEM. For improved visualization purposes, the images shown here were filtered using Gaussian blur with 2.0 sigma and contrast enhanced with 4% saturated pixels. (C) Reconstructed ~13Å cryoEM map of the asymmetric HlV-1 Env in complex with the CD4 mimetic (BNM-lll-170) in the presence of the 17b and A32 anti-HIV-1 antibodies.

Figure 3.. Modeling the HIV-1 Env spike in State 2A.

The left column shows the side views of segmented masked cryo-EM density (at ~13 Å resolution) of Env in complex with BNM-III-170 and the 17b and A32 antibodies. The Env-associated density is colored cyan, blue and green with the membrane colored gray. (A) The upper panel represents the different side views of the complex projected at right angles. Orange, purple and yellow colored regions exhibit the additional densities corresponding to parts of the bound antibody Fab chains. (B) Side views of the membrane-bound asymmetric HIV-1 spike projected at right angles, same as displayed in panel A. (C) Top views of the densities corresponding to panels A and B, respectively.

Figure 4.. Membrane-bound CD4 exposes cluster-A epitopes and stabilizes Env State 2A.

(A) FRET histograms observed for HIV-1_JR-FL Env in the absence of Nef, and in the absence of any bound ligand, and (B) in the presence of membrane-bound CD4. Histograms are displayed as in Figure 1.

Figure 5.. Env cleavage decreases the spontaneous sampling of State 2A.

The impact of Env cleavage on cluster A epitope exposure was evaluated in the presence or absence of the CD4mc BNM-III-170 and CoRBS 17b (full antibody or Fab or Fab’2 fragments) by cell-surface staining of 293T cells transfected with HIV-1_JRFL ΔCT Env (Cl+) or its cleavage defective (Cl−) counterpart, using the A32 (A) or C11 (B) anti-cluster A Abs. Data are presented as means and SEM of the mean fluorescence intensity (MFI) from at least three independent experiments. Statistical significance was evaluated using multiple-comparison-one-way ANOVAs or Kruskal-Wallis test, * P < 0.05, ** P <0.01, *** P < 0.001, ****P <0.0001; ns, not significant. FRET histograms and TDPs for (C) unbound HIV-1_JR-FL CL- Env; and in the presence of (D) BNM-III-170, 17b Ab, and A32 Ab; or (E) BNM-III-170, 17b Ab, and C11 Ab. All ligands were used at the same concentrations as in Figure 1. FRET data are displayed as in Figure 1.

Figure 6.. HIV+ sera stabilize State 2A in the presence of CD4mc.

FRET histograms obtained for HIV-1_JR-FL Env ΔCT in the absence or presence of CD4mc BNM-III-170 (100 mM), as indicated, and in the presence of (A) sera from 9 chronically HIV-1-infected individuals, or (B) 3 healthy uninfected individuals. FRET histograms are displayed as in Figure 1. (C) The impact of CD4mc BNM-III-170 addition on FRET state occupancy for each of the 9 HIV+ sera. Statistical significance was evaluated using an unpaired t-test or Mann-Whiney test, ** P <0.01; ns, not significant.

Figure 7.. CD4mc-mediated exposure of cluster A epitopes sensitizes HIV-1-infected primary CD4+ T cells to ADCC and correlates with State 2A stabilization.

Primary CD4+ T cells infected with the HIV-1_JRFL infectious molecular clone were used to evaluate (A) the susceptibility of infected cells to ADCC mediated by sera from nine chronically HIV-1-infected individuals in the presence of CD4mc BNM-III-170, alone or in combination with Fab fragments of 17b, A32, or both. (B) State 2A occupancy (Figure 6) exhibited a positive correlation with the binding of sera from nine chronically HIV-1-infected individuals +/− CD4mc BNM-III-170 to primary CD4 T cells infected with the HIV-1_JRFL infectious molecular clone and (C) to the stabilized gp120 inner domain (ID2) (Tolbert et al., 2016) by ELISA. Data are presented as means and SEM from at least three independent experiments. Statistical significance was evaluated utilizing a multiple-comparison-one-way ANOVAs or a Pearson rank correlation, * P < 0.05, *** P < 0.001, ****P <0.0001; ns, not significant.