Synthetic glycopeptides reveal the glycan specificity of HIV-neutralizing antibodies

Abstract

A new class of glycan-reactive HIV-neutralizing antibodies, including PG9 and PG16, has been recently discovered that seem to recognize previously uncharacterized glycopeptide epitopes on HIV-1 gp120. However, further characterization and reconstitution of the precise neutralizing epitopes are complicated by the heterogeneity of glycosylation. We report here the design, synthesis and antigenic evaluation of new cyclic V1V2 glycopeptides carrying defined N-linked glycans at the conserved glycosylation sites (Asn160 and Asn156 or Asn173) derived from gp120 of two HIV-1 isolates. Antibody binding studies confirmed the necessity of a Man₅GlcNAc₂ glycan at Asn160 for recognition by PG9 and PG16 and further revealed a critical role of a sialylated N-glycan at the secondary site (Asn156 or Asn173) in the context of glycopeptides for antibody binding. In addition to defining the glycan specificities of PG9 and PG16, the identified synthetic glycopeptides provide a valuable template for HIV-1 vaccine design.

Figure 1. Structures of the designed V1V2 glycopeptides derived from HIV-1 ZM109 and CAP45 strains

Figure 2. Chemoenzymatic synthesis of ZM109 V1V2 glycopeptides carrying defined N-glycans at the N160 site

Figure 3. Controlled glycosylation and HPLC separation of mono- and doubly glycosylated ZM-glycopeptides

a) synthetic scheme; b) the HPLC separation of the mono- and doubly glycosylated ZM-glycopeptides. The reverse phase HPLC was performed on a C18 column with a linear gradient of 15–30% MeCN in 30 min.

Figure 4. Chemoenzymatic synthesis and ESI-MS characterization of the doubly glycosylated ZM-glycopeptides

a) the synthetic scheme; b) ESI-MS of ZM-GP6; c) ESI-MS of ZM-GP7; d) ESI-MS of ZM-GP6d; and e) ESI-MS of ZM-GP8.

Figure 5. SPR analysis of the binding of synthetic V1V2 glycopeptides to PG9/PG16 Fabs

Biotinylated glycopeptides were immobilized on streptavidin chips and antibody PG9 Fab or PG16 Fab flowed through as analytes. The surface-plasmon resonance sensorgrams were recorded with 2-fold serial dilutions starting at the highest concentration of 100 µM. a) PG9 Fab and ZM-GP6; b) PG9 Fab and CAP-GP6; c) PG16 Fab and ZM-GP6; d) PG16 Fab and CAP-GP6ZM-GP6. The fitted curves were shown in orange color. The binding of ZM-GP2, ZM-GP5, ZM-GP8, CAP-GP2, CAP-GP5 and CAP-GP8 was shown in Supplementary Fig. 11. The following ZM- and CAP-glycopeptides did not show apparent binding responses at up to 100 µM: ZM-GP1, ZM-GP3, ZM-GP4, ZM-GP7, ZM-GP9; CAP-GP1; CAP-GP7, CAP-GP9; and those non-glycosylated peptides.