Relationship of HIV-1 and SIV envelope glycoprotein trimer occupation and neutralization

Abstract

Insights into the process of HIV-1 neutralization may assist rational vaccine design. Here, we compared antibody neutralization against the JR-FL primary isolate and trimer binding affinities judged by native PAGE. Monovalent Fab-trimer binding and neutralization showed a direct quantitative relationship, implying that neutralization begins as each trimer is occupied by one antibody. At saturation, three Fab or soluble CD4 molecules engaged each trimer. In contrast, a maximum of one soluble CD4 molecule bound to functional SIV trimers with a truncated a gp41 tail. Remarkably, soluble CD4 was found to trigger dramatic enhancement of this virus. Unlike Fabs, a quantitative correlation between JR-FL trimer binding and neutralization was unclear for some, but not all IgGs, as neutralization was markedly increased, but trimer affinity was largely unchanged. In addition, only one molecule of certain gp41-specific IgGs appeared to be able to bind each trimer. We discuss the implications of these findings in weighing the relative contributions of size, multivalent binding and other possible effects of IgGs to explain their increased potency.

Profiling the Specificity of Neutralizing Antibodies in a Large Panel of Plasmas from Patients Chronically Infected with Human Immunodeficiency Virus Type 1 Subtypes B and C

“…Recently, a native polyacrylamide gel electrophoresis (PAGE) method was described that visualizes Ab binding to Env trimers exposed on pseudovirions (12)(13)(14)42). Only NAbs were found to bind to these trimers, confirming the role of trimer binding as a key requirement for neutralization, as suggested previously by others (19,56).…”

“…Blue native PAGE (BN-PAGE) band shifts were used to analyze Ab binding to native, virion-derived Env trimers as described previously (12)(13)(14)42 were then washed with PBS and gently solubilized in 0.12% Triton X-100 in 1 mM EDTA-1.5 M aminocaproic acid and one microliter of a protease inhibitor cocktail (Sigma). An equal volume of 2ϫ sample buffer containing 100 mM morpholinepropanesulfonic acid (MOPS), 100 mM Tris-HCl, pH 7.7, 40% glycerol, and 0.1% Coomassie blue was then added.…”

Polyclonal B Cell Responses to Conserved Neutralization Epitopes in a Subset of HIV-1-Infected Individuals

“…Blue native polyacrylamide gel electrophoresis (BN-PAGE) band shifts were used to analyze Ab binding to native, virion-derived Env trimers as described previously (19)(20)(21)70). Trimer binding was measured via depletion of the unliganded trimer using UN-SCAN-IT densitometry software (Silk Scientific) (19).…”

“…Blue native polyacrylamide gel electrophoresis (BN-PAGE) band shifts were used to analyze Ab binding to native, virion-derived Env trimers as described previously (19)(20)(21)70). Trimer binding was measured via depletion of the unliganded trimer using UN-SCAN-IT densitometry software (Silk Scientific) (19). Briefly, ID 50 s were calculated with reference to control lanes in each gel, in which the density of a uncomplexed control trimer band is taken as 100% and the density of a band that is fully liganded by a nAb (either b12, 2G12, or a high concentration of plasma) is taken as 0%.…”

Intraprotomer masking of third variable loop (V3) epitopes by the first and second variable loops (V1V2) within the native HIV-1 envelope glycoprotein trimer

“…1B) Env constructs. In considering interpretation of possible experimental outcomes, it is critical to note previous conclusions that antibody binding to a single subunit within an HIV-1 trimer is sufficient to neutralize its function, based on experimental data coupled with computational modeling (25)(26)(27)(28). This conclusion has been reached in studies by multiple research groups with a variety of mAbs against diverse determinants on both gp120 (CD4 binding site, CD4-induced epitopes, oligomannose determinants) and gp41 (membrane proximal external region) and against multiple HIV-1 isolates.…”