Unprecedented Role of Hybrid <i>N-</i>Glycans as Ligands for HIV‑1 Broadly Neutralizing Antibodies

The development of an HIV vaccine has been hampered by the extraordinary mutability and genetic diversity of the virus, particularly the substantial sequence diversity of gp120 and gp 41 envelope glycoproteins existing in more than 2000 HIV variants. The highly diverse glycans on HIV spikes are commonly considered as immunologically silent self-antigens; however, the discovery of highly potent broadly neutralizing antibodies (bNAbs) from HIV patients targeting the viral surface glycans has raised a major question about the origin of their antigens. Recent epitope mapping studies of the bNAb PG9 indicated a requirement of a properly spaced high mannose and a complex type glycan connected by a short peptide spacer. We have recently discovered that a 1:1 mixture of Man₅ and sialyl biantennary glycan with well-defined distance and without the peptide spacer is well recognized by PG9 with high avidity and, thus, proposed that a hybrid glycan with oligomannose and complex-type arm could be the proper ligand of PG9. To verify this proposition, we first designed and chemo-enzymatically synthesized a series of unusual hybrid-type <i>N</i>-glycan structures, which may exist on HIV surface glycoproteins through the host-guided <i>N</i>-glycosylation pathway. The synthetic hybrid glycans were then used to prepare glycan arrays for the binding studies of PG9 and several other highly potent bNAbs, including PG16, PGT121, PGT128-3C, 2G12, VRC13, VRC-PG05, VRC26.25, VRC26.09, PGDM1400, 35O22, and 10-1074. Our results demonstrated that PG9 and some other bNAbs bind with strong avidity (subnanomolar <i>K</i>_d) to certain hybrid structures, suggesting that these unusual glycans may serve as epitopes for the design of vaccines against HIV