Identification and characterization of broadly neutralizing epitopes on the HIV-1 envelope are important steps in the design of an effective HIV-1 vaccine. Recently, a new class of broadly neutralizing antibodies (bNAbs), including PG9, PG16, and the PGT class antibodies, has been isolated from HIV-infected elite controllers. These bNAbs neutralize primary HIV-1 strains with remarkable breadth and potency. A common feature of antigen recognition by these bNAbs is that they all target glycan-dependent quaternary epitopes at the V1/V2 and/or V3 regions of gp120. Recent X-ray crystal structural studies indicate that PG9 binds to N-glycans at N160 and N156 in the context of V1/V2 domain, and PGT128 recognizes conserved N-glycans at N322 and N301 sites in the context of V3 domain. However, the precise nature of the neutralizing epitopes, particularly the fine structures of the N-glycans at N156 and N301 remains to be characterized. Further mapping of the epitopes is complicated by the complexity and heterogeneity of glycosylation of HIV-1 gp120. We hypothesize that unique V1/V2 and V3 glycopeptides constitute the neutralizing epitopes for these bNAbs. To test this hypothesis, we will perform experiments described in two specific aims. Aim 1 is to design and synthesize cyclic V1/V2 and V3 HIV-1 glycopeptides with defined N-glycans being attached at the conserved N- glycosylation sites, by a novel chemoenzymatic method. Aim 2 is to characterize antigen recognition by the neutralizing antibodies through binding and structural studies with the synthetic glycopeptides. In addition, the synthetic glycopeptides will be used to detect glycan- dependent, V1/V2 and V3-specific neutralizing antibodies in sera from HIV-infected non- progressors. These studies are likely to provide important insights for HIV-1 vaccine design. PUBLIC HEALTH RELEVANCE: HIV/AIDS is a serious global epidemic that threatens human health and social stability. The proposed research aims to characterize the neutralizing epitopes of broadly neutralizing antibodies, which will provide important insights in HIV-1 vaccine design.