Yeast-elicited cross-reactive antibodies to HIV Env glycans efficiently neutralize virions expressing exclusively high-mannose N-linked glycans

Abstract

The HIV envelope (Env) protein uses a dense coat of glycans to mask conserved domains and evade host humoral immune responses. The broadly neutralizing antibody 2G12, which binds a specific cluster of high-mannose glycans on HIV Env, shows that the glycan shield can also serve as a target for neutralizing antibodies. We have described a triple mutant Saccharomyces cerevisiae strain that expresses high-mannose glycoproteins that bind to 2G12. When used to immunize rabbits, this yeast elicits antibodies that bind to gp120-associated glycans but fail to neutralize virus. Here we sought to determine the reason for these discordant results. Affinity purification of sera over columns conjugated with three 2G12-reactive yeast glycoproteins showed that these proteins could adsorb 80% of the antibodies that bind to gp120 glycans. Despite binding to monomeric gp120, these mannose-specific antibodies failed to bind cell surface-expressed trimeric Env. However, when Env was expressed in the presence of the mannosidase inhibitor kifunensine to force retention of high-mannose glycans at all sites, the purified antibodies gained the abilities to bind trimeric Env and to strongly and broadly neutralize viruses produced under these conditions. Combined, these data show that the triple mutant yeast strain elicits antibodies that bind to high-mannose glycans presented on the HIV envelope, but only when they are displayed in a manner not found on native Env trimers. This implies that the underlying structure of the protein scaffold used to present the high-mannose glycans may be critical to allow elicitation of antibodies that recognize trimeric Env and neutralize virus.

FIG. 1.

2G12-reactive TM yeast glycoproteins capture the majority of gp120-reactive serum antibodies. (A) Sera (weeks 9 to 14) from each animal were passed over a column conjugated with the 2G12-reactive yeast glycoproteins Ecm33, Gp38, and PstI. Raw sera and the YGP column flowthrough were tested at a dilution of 1:100 for binding to LAI-BH8 gp120 in an ELISA to determine the fraction of gp120-reactive antibodies captured by the column. Data represent the averages for 3 independent experiments ± standard errors of the means. (B) The gp120-binding capacity of the purified antibodies was confirmed using immunoprecipitation of gp120 in solution. Lane 1, 100 ng JRFL gp120; lanes 2 and 4, gp120s precipitated with 5 μg/ml protein A-purified prebleed sera from rabbits 1226 and 1238, respectively; lanes 3 and 5, gp120s precipitated with 5 μg/ml YGP column-purified postimmunization sera from rabbits 1226 and 1238, respectively; lane 6, gp120 precipitated with 2 μg/ml 2G12. One-eighth of the final precipitated immunocomplex sample, equivalent to 125 ng gp120 starting material, was loaded per lane. The upper panel was probed with 1 μg/ml of rabbit anti-gp120 antibody 3824, which was raised with a synthetic peptide, and the lower panel was probed with 0.5 μg/ml 2G12. Binding to JRFL gp120 is shown, while similar results were obtained for binding to R2 gp120.

FIG. 2.

Elicited antibodies display different gp120 strain preferences and bind clade B gp120s with lower affinity than 2G12. (A) YGP-purified serum IgG at 10 μg/ml (green bars) and 2G12 at 2 μg/ml (blue bars) were tested for binding to gp120s from various strains in ELISA. YGP-purified serum from each rabbit was tested for binding to each strain. For each individual serum, the strain that was recognized most efficiently was set as 100%, and the relative percent binding to all other strains was calculated. Green bars represent the average percent gp120 binding across the 6 animals for each strain. Error bars represent the variability between animals for each gp120, indicating the maximum and minimum relative percent binding across all 6 rabbit sera as a measure of strain preference. Data represent 3 independent experiments. (B) The ability of YGP-purified serum IgG to prevent binding of MAb 2G12 or b12 to LAI-BH8 gp120 was measured using ELISA, with percent inhibition of MAb binding indicated. Data represent the averages for 3 independent experiments ± standard errors of the means. (C) Relative affinities of YGP-purified sera and 2G12 for 4 gp120s were measured using 2-fold dilutions of antibody in ELISA. EC₅₀s are indicated in the table. Data represent the averages for 3 independent experiments ± standard errors of the means.

FIG. 3.

TM yeast-elicited antibodies preferentially bind terminal Manα1,2-Manα1,2 trisaccharides. (A) Schematic representation of the synthetic mannose-containing carbohydrate structures present on the CFG-printed glycan array, version 4.1. (B) YGP-purified serum IgG from each animal was tested at 10 μg/ml for binding to the various mannose-containing synthetic carbohydrates. Fluorescent antibodies were used to detect serum antibody binding, which was measured in relative light units.

FIG. 4.

Serum antibodies bind cell surface oligomeric Env poorly. (A) MAbs b12 and 2G12 were tested at 10 μg/ml for binding to LAI-BH8 Env expressed on the surfaces of quail fibroblast cells (red) to determine envelope expression levels. Binding to cells not expressing Env (blue) was measured in parallel. (B) Protein A-purified prebleed sera (PB) or YGP-purified postimmune sera were tested at 10 μg/ml for binding to Env-negative and Env-positive cells. Data are from a single representative experiment.

FIG. 5.

Retention of Man₉ N-linked glycans on Env increases serum antibody recognition of Env trimers. (A) Binding of MAbs b12 and 2G12 to Env-negative and Env-positive cells grown in the absence or presence of 25 μg/ml kifunensine (Kif). (B) Binding of protein A-purified prebleed sera (PB) and YGP-purified antibodies to Env-negative and Env-positive cells produced in the presence of 25 μg/ml kifunensine. Data are from one representative experiment.

FIG. 6.

Serum antibodies are able to neutralize virions produced in the presence of kifunensine. (A) Pseudovirions expressing the indicated Envs on a luciferase viral core were tested for neutralization by protein A-purified prebleed sera, yeast glycoprotein-purified postimmune sera, MAb 2G12, pooled HIV-positive human serum (HIV Ig), and MAb b12 at 50 μg/ml. Percent inhibition by YGP-purified sera relative to prebleed sera is indicated in the top panel, while percent inhibition by the control antibodies and human serum is indicated in the bottom panel. (B) Pseudovirions produced in the presence of 25 μg/ml kifunensine were tested for neutralization as described above. (C to F) To measure the neutralization potency of each rabbit serum relative to that of 2G12, serial antibody dilutions were tested for neutralization of clade B pseudovirions bearing the envelopes of HIV strains JRFL (C), LAI (D), PVO (E), and R2 (F). Data represent the averages for 3 independent experiments ± standard errors of the means.