PI-6 Construction and analysis of V3 glycopeptide antigens for HIV-1 vaccine design

Abstract

The heavy glycosylation on HIV-1 envelope protein (Env) plays an important role in viral evasion and is critical for the viral plasticity and diversity in the circulating strains. The discovery of a new class of broadly neutralizing antibodies (bnAbs), including PG9, PGT128, PGT121, and 10-1074, that recognize conserved glycopeptide epitopes from the V1V2 and V3 domains provide a valuable template for vaccine design. Structural and mutational studies have provided basic information on the nature and structure of the glycopeptide epitopes of these antibodies. Nevertheless, due to the tremendous heterogeneity of envelope glycosylation, the fine glycan specificity of these antibodies remains to be fully characterized. Moreover, it remains a challenging task to reconstruct glycosylation-well defined antigenic structures even when the glycan specificity is defined. To address these problems, we have launched a chemical biology program aiming to reconstitute a library of structurally well-defined glycopeptides and glycoproteins to probe the glycan specificity of these glycan-reactive broadly neutralizing antibodies. Through the chemoenzymatic synthesis and antibody-binding studies of a library of V3 glycopeptides, we found that the PGT128 requires a high-mannose N-glycan on V3 domain with promiscuous site of glycosylation at either N332, N301, orN295); the PGT121 shows binding specificity towards a sialylated N-glycan at N301 site; and the 10-1074 prefers a high-mannose type glycan at N332 site. From the mapping of the epitopes, we designed glycopeptide-based immunogens derived from the V3 domain through chemical and biological approaches. The synthetic glycopeptide immunogens obtained by chemoenzymatic synthesis and the glycosylation-defined V3 glycopeptide-Fc fusion proteins obtained through mammalian cell expression both showed potent binding towards multiple V3-targeting bnAbs. Rabbit immunization experiments revealed that the synthetic V3 glycopeptide could elicit glycan-dependent antibody responses. These results together suggested that our glycosylation-defined V3 glycopeptide immunogens could serve as a valuable vaccine component in the combination with other vaccine candidates for further immunization studies.