HIV-1 prehairpin intermediate inhibitors show efficacy independent of neutralization tier

Abstract

HIV-1 strains are categorized into one of three neutralization tiers based on the relative ease by which they are neutralized by plasma from HIV-1-infected donors not on antiretroviral therapy; tier-1 strains are particularly sensitive to neutralization while tier-2 and tier-3 strains are increasingly difficult to neutralize. Most broadly neutralizing antibodies (bnAbs) previously described target the native prefusion conformation of HIV-1 Envelope (Env), but the relevance of the tiered categories for inhibitors targeting another Env conformation, the prehairpin intermediate, is not well understood. Here, we show that two inhibitors targeting distinct highly conserved regions of the prehairpin intermediate have strikingly consistent neutralization potencies (within ~100-fold for a given inhibitor) against strains in all three neutralization tiers of HIV-1; in contrast, best-in-class bnAbs targeting diverse Env epitopes vary by more than 10,000-fold in potency against these strains. Our results indicate that antisera-based HIV-1 neutralization tiers are not relevant for inhibitors targeting the prehairpin intermediate and highlight the potential for therapies and vaccine efforts targeting this conformation.

Keywords: 5-Helix; HIV-1; prehairpin intermediate; tier-2 virus; tier-3 virus.

Fig. 1.

The HIV-1 PHI represents an additional neutralization target. (A) HIV-1 strains are organized into neutralization tiers based on their sensitivity to patient sera: tier-1 strains are particularly sensitive to neutralization, while tier-2 and tier-3 strains are more resistant. (B) HIV-1 Env (linear schematic) is a transmembrane viral protein composed of a trimer of gp120/gp41 heterodimers. The native, prefusion conformation of Env (Left) engages cell-surface receptors CD4 and CCR5/CXCR4. The gp41 NHR and CHR are critical regions in membrane fusion exposed in the PHI (Center) before collapsing into a stable trimer-of-hairpins structure (Right). Env-directed inhibitors target either the native prefusion conformation (Left) or the PHI (Center). Most bnAbs target the native prefusion state, while fusion inhibitors like 5-Helix, D5_AR IgG, and enfuvirtide prevent the transition between the PHI and the trimer-of-hairpins structure. Cartoons are based on high-resolution crystal and cryogenic electron microscopy structures [Protein Data Bank codes: 5FUU (9), 6MEO (10), 1AIK (11), 2X7R (12) and a common model for the PHI (–18). (C) The PHI inhibitors D5_AR IgG and 5-Helix target the NHR and CHR, respectively. D5_AR IgG binds a highly conserved hydrophobic pocket in the NHR trimeric coiled-coil (orange), and recent work has shown that the neutralization potency of D5_AR IgG is greatly enhanced >1,000-fold when target cells express Fc gamma receptor I (FcγRI; yellow) (19, 20). 5-Helix is an engineered protein designed to mimic the post-fusion trimer-of-hairpins structure and binds the highly conserved α-helical face of the CHR (blue) (21). Highly conserved residues are highlighted in red.

Fig. 2.

Inhibitors targeting the PHI have broad activity across neutralization tiers. (A) CHR-targeting inhibitor 5-Helix shows activity across a roughly 10-fold range against viral strains in neutralization tiers 1/2/3. In A–E, IC₅₀ values from TZM-bl neutralization assays performed in biological triplicate are plotted as mean ± SD and colored by neutralization tier: tier 1 (blue), tier 2 (red), and tier 3 (green). (B) NHR-targeting D5_AR IgG shows activity against viral strains in neutralization tiers 1/2/3 that is potentiated roughly 1,000-fold for cells expressing FcγRI. Mean IC₅₀ values ± SD of repeated neutralization assays done in at least biological triplicate using either TZM-bl (x-axis) or TZM-bl/FcγRI (y-axis) cells are shown. (C) HIV-1 strains with identical CHR sequences (HXB2 and HXB2 L565Q) show differences in 5-Helix sensitivity. Two-tailed unpaired t tests were performed with the indicated P value (**P < 0.01). (D) Increased binding of 5-Helix to CHR sequences measured by biolayer interferometry does not correspond to increased sensitivity to inhibition by 5-Helix. Reported K_D values (mean ± SD) were determined from repeated experiments (n = 2) for each peptide across a range of concentrations of 5-Helix. (E) Sensitivity to 5-Helix does not predict sensitivity to D5_AR IgG. IC₅₀ values of 5-Helix and D5_AR IgG fall over notably different ranges and show only a modest association. A linear regression (black) with 95% confidence bands (dashed) has a correlation coefficient of R² = 0.06.

Fig. 3.

PHI inhibitors show highly consistent and broad HIV-1 neutralization independent of tier, unlike best-in-class bnAbs. (A) 5-Helix and D5_AR IgG show highly consistent neutralization activity across tiers. Neutralization IC₅₀ values were determined against a panel of tier-1/2/3 strains for immunoglobin purified from pooled HIV-1 sera (HIVIG) and five bnAbs employed in clinical trials 10E8v4, VRC01, 3BNC117, PGDM1400, and 10-1074. Using HIVIG as an indicator of tier, only 5-Helix and D5_AR IgG (in TZM-bl cells ± FcγRI expression) show highly consistent activity among strains across tiers. The limit-of-quantitation (LOQ) is indicated by dashed lines. Values are plotted as mean ± SD and colored by neutralization tier: tier 1 (blue), tier 2 (red), and tier 3 (green). Epitopes of each inhibitor are indicated: MPER, CD4 binding site (CD4bs), gp120 V1/V2 apex, and gp120 V3 loop. (B) Box-and-whisker plots of IC₅₀ values in A show that 5-Helix and D5_AR IgG have narrower ranges than HIVIG or best-in-class bnAbs. The logarithms of the IQR and the range are reported. The LOQ for the antibody neutralization datasets is indicated by the dashed line. (C) Proposed model for incorporating inhibitors targeting the PHI into current HIV-1 prevention approaches. Viruses from different neutralization tiers show different susceptibility to current best-in-class bnAbs but similar vulnerability to inhibitors targeting the PHI. High neutralization potency is represented as thicker layers of protection with no potency represented as a gap in the layer. Eliciting antibodies that neutralize via the PHI may provide an additional layer of protection that would be useful even if moderate but very broad.