Characterization of the precise epitopes for broadly neutralizing antibodies (bNAbs) is an important step for the design of an effective HIV-1 vaccine. Recent X-ray crystal structural studies indicate that the epitope of PG9 constitutes two N-glycans (at N160 and N156 positions) and a strand of peptide in the V1V2 domain of gp120. However, the precise nature of the neutralizing epitopes, particularly the fine structures of the N-glycans at N156 remains to be characterized. Further mapping of the epitopes is complicated by the complexity and heterogeneity of glycosylation of HIV-1 gp120. To address this problem, we launched a project aiming to identify the precise epitopes by synthesis and evaluation of a series of homogeneous cyclic glycopeptides corresponding to the V1/V2 domain. A chemoenzymatic method was developed to make such complex HIV-1 glycopeptides in which defined N-glycans were installed at the predetermined glycosylation sites. SPR binding studies with PG9 revealed that the fine structures of N-glycans at both the N156 and N160 sites were critical for the recognition of the synthetic antigens by PG9. In particular, a complex type sugar at the N156, rather than a Man5GlcNAc2 glycan, seems critical for the high affinity binding of PG9 to the antigen. The synthetic HIV-1 glycopeptides will be valuable to serve as coating antigens for detecting glycan-dependent neutralizing antibodies from sera of HIV-infected patients or those involved in HIV vaccine trials. These studies provide important insights for HIV-1 vaccine design.
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