Functional Stability of HIV-1 Envelope Trimer Affects Accessibility to Broadly Neutralizing Antibodies at Its Apex

Abstract

The trimeric envelope glycoprotein spike (Env) of HIV-1 is the target of vaccine development to elicit broadly neutralizing antibodies (bnAbs). Env trimer instability and heterogeneity in principle make subunit interfaces inconsistent targets for the immune response. Here, we investigate how functional stability of Env relates to neutralization sensitivity to V2 bnAbs and V3 crown antibodies that engage subunit interfaces upon binding to unliganded Env. Env heterogeneity was inferred when antibodies neutralized a mutant Env with a plateau of less than 100% neutralization. A statistically significant correlation was found between the stability of mutant Envs and the MPN of V2 bnAb, PG9, as well as an inverse correlation between stability of Env and neutralization by V3 crown antibody, 447-52D. A number of Env-stabilizing mutations and V2 bnAb-enhancing mutations were identified in Env, but they did not always overlap, indicating distinct requirements of functional stabilization versus antibody recognition. Blocking complex glycosylation of Env affected V2 bnAb recognition, as previously described, but also notably increased functional stability of Env. This study shows how instability and heterogeneity affect antibody sensitivity of HIV-1 Env, which is relevant to vaccine design involving its dynamic apex.IMPORTANCE The Env trimer is the only viral protein on the surface of HIV-1 and is the target of neutralizing antibodies that reduce viral infectivity. Quaternary epitopes at the apex of the spike are recognized by some of the most potent and broadly neutralizing antibodies to date. Being that their glycan-protein hybrid epitopes are at subunit interfaces, the resulting heterogeneity can lead to partial neutralization. Here, we screened for mutations in Env that allowed for complete neutralization by the bnAbs. We found that when mutations outside V2 increased V2 bnAb recognition, they often also increased Env stability-of-function and decreased binding by narrowly neutralizing antibodies to the V3 crown. Three mutations together increased neutralization by V2 bnAb and eliminated binding by V3 crown antibodies. These results may aid the design of immunogens that elicit antibodies to the trimer apex.

Keywords: envelope; gp120; gp41; human immunodeficiency virus; neutralizing antibodies; protein stability; vaccines.

FIG 1

Neutralization of ADA and Comb-mut by V2 bnAbs, V3 crown antibodies, and CD4bs non-bnAbs. HIV-1 ADA and Comb-mut virions were tested in neutralization assays against the V2 bnAbs PG9 and PG16 (A) and the V3 crown antibodies 447-52D and F425-B4e8 and the CD4 binding site (CD4bs) non-bnAbs b6 and F105 (B). In panel A, the maximum percent neutralization (MPN) for each virus-antibody pair is noted.

FIG 2

Alignment of V1V2 and V3 sequences. The V1V2 (A) and V3 (B) amino acid sequences of the group M consensus (ConS), ADA, Comb-mut, BG505, and 16055 were aligned using DNASTAR Lasergene ClustalW with an HxB2 numbering scheme. Highlighted in gray are residues that differ from ConS. Residues in red indicate N-linked glycosylation sites at N156 and N160. Highlighted in yellow are positions 165, 169, and 315; the former two were mutated I165L/V169K to generate ADA-LK and CM-LK, and the latter was mutated R315Q to eliminate neutralization by V3 crown antibodies. Amino acids represented in green are >50% conserved among all isolates, and those boxed in red are >50% conserved among subtype B (hiv.lanl.org). The beta-sheet structure and labeling are indicated by black arrows, based on data published by McLellan et al. for V2 (21) and Huang et al. for V3 (87).

FIG 3

Neutralization (MPN) of mutant Envs of HIV-1 ADA and Comb-mut by PG9. Substitutions were introduced into ADA pseudovirus and mutants were tested for sensitivity to PG9 neutralization at a constant concentration of 5 μg/ml. A subset of substitutions that showed a significant increase in MPN was then similarly tested in a Comb-mut Env background. Certain mutations that were predicted by structural analysis to potentially affect V2 and V3 antibody neutralization were also tested in Comb-mut but not ADA. Mutations that were not tested in a given background are labeled “nd” (not determined) or “ni” (not infectious). “N186-188” represents the triple mutant N186G/D187S/N188S. The red and green dashed lines indicate MPNs for wild-type ADA and Comb-mut, respectively. Red dots indicate mutants that show enhanced PG9 neutralization relative to wild-type ADA, green dots indicate enhanced PG9 neutralization of both ADA and Comb-mut. Each bar represents an average of biological triplicate values.

FIG 4

Comb-mut containing V2 substitutions I165L and V169K is neutralized completely and efficiently by PG9 and PG16. ADA (A) and Comb-mut (B) parental pseudoviruses (black circles), as well as I165L V169K double mutants (red squares), were assayed for neutralization against PG9 (top panels) and PG16 (bottom panels). Experiments were performed in triplicate.

FIG 5

Stability of Env correlates with enhanced MPN of PG9 and decreased neutralization by 447-52D. (A) Mutants of ADA were tested for thermostability and neutralization by PG9. T90 values are plotted against the percent neutralization at 5 μg/ml of PG9. A statistically significant relationship was observed between increased Env stability and an increase in PG9 neutralization. (B) Infectivity decay at 37°C of ADA, Comb-mut, and CM-LK. The results of a representative experiment performed in triplicate are shown, and the error bars represent the standard deviations. (C) Physical thermostability of Comb-mut and CM-LK Env trimers. BN-PAGE Western blot analysis of trimeric Envs solubilized from heat-treated virus shows that I165L/V169K do not change the trimer stability of Comb-mut.

FIG 6

Neutralization of Comb-mut and CM-LK by V2 antibodies. (A) Comb-mut and CM-LK pseudotyped viruses were tested against various V2 bnAbs, as well as control antibodies PGT128 and VRC01. (B) The MPN of V2 bnAbs is shown against Comb-mut, CM-LK, and the constituent mutations of CM-LK, I165L and V169K. (C) ADA, ADA-LK, Comb-mut, and CM-LK pseudotyped viruses were tested in TZM-bl neutralization assays against gp120-gp41 interface bnAbs and the soluble form of the HIV-1 receptor, CD4. Data shown are from a representative experiment performed in triplicate, and error bars represent the standard deviations from the mean.

FIG 7

Neutralization properties of glycosylation-modified HIV-1 ADA, ADA-LK, Comb-mut, and CM-LK Env by V2 bnAbs. ADA (A), ADA-LK (B), Comb-mut (C), and CM-LK (D) pseudotyped viruses were generated in 293T cells, in 293S cells, or in the presence of swainsonine. Virions of Comb-mut and CM-LK that had the N156S mutation, which eliminates the glycosylation site at this position, were also generated in 293T cells. Viruses were tested in neutralization assays against V2 bnAbs PG9, PGT145, CH01, and VRC26.08. The MPN findings are shown in panels A to D. (E) The stability of ADA wild-type and ADA-LK virion Envs produced in 293T or 293S cells or in the presence of kifunensine or swainsonine was tested in a T90 assay. A statistically significant increase in the functional stability of Env was found with each treatment relative to producing virus in 293T cells. The statistical significance was determined using a paired two-tailed t test. (F) Six different viruses from different clades were produced with or without the addition of kifunensine, and the functional stability was tested in the T90 assay.

FIG 8

BN-PAGE gel mobility shift of Comb-mut and CM-LK trimeric Env in the presence of various bnAbs. (A) Comb-mut and CM-LK virions, displaying >95% cleaved Env, were incubated in the presence of the IgGs specified (25 μg/ml) and analyzed using BN-PAGE Western blot. Binding of an antibody causes the Env trimer to run more slowly on the gel, thus shifting the band upward on the blot. 2G12 and DEN3 were used as positive- and negative-control antibodies, respectively. (B) Virion-antibody complexes were fixed using the chemical cross-linker BS³—after antibody binding but before adding detergent—to prevent antibodies from falling off during the gel run. (C) Comb-mut and CM-LK virions were incubated with 447-52D or F425-B4e8 IgGs that bind to V3, or control IgG 2G12, and then Env trimers were solubilized using detergent and analyzed using a BN-PAGE Western blot.

FIG 9

V3 crown neutralization correlates inversely with trimer thermostability and PG9 neutralization (MPN). (A) T90 values of mutants of ADA are plotted against the IC₅₀ of V3 antibody 447-52D. A statistically significant relationship is seen between increased Env stability and a decrease in 447-52D neutralization. (B) The IC₅₀ of 447-52D is plotted against the MPN at 5 μg/ml of PG9. Mutants in the V2 domain (between amino acids 160 and 190) that might directly affect PG9 binding were removed from the analysis. (C) Neutralization of Comb-mut, CM-LK, and CM-LK R315Q pseudovirions by a panel of antibodies shows that R315Q abrogates neutralization by V3 crown antibody 447-52D (middle panel) without altering the neutralization properties of PG9 (top panel) and VRC01 (bottom panel). Data shown are from a representative experiment performed in triplicate, and error bars represent the standard deviations from the mean.

FIG 10

Location on Env of mutations that increase trimer stability or V2 neutralization. (A) Mutations that were found to increase PG9 neutralization or increase trimer stability are indicated as spheres on the crystal structure of JRFL envelope (PDB 5FYK). In the inset images, key residues at positions 165 and 169 (B) and position 315 (C) of gp120 are shown with residues nearby that may interact with them. Hydrogen bonds between neighboring amino acid residues, predicted using PyMOL, are shown using red dashed lines. I165 and R315 are shown in both inset images for orientation.