The discovery of glycan-dependent broadly neutralizing antibodies (bnAbs) targeting regions of the envelope glycoprotein gp120 has driven the need to elucidate their epitope structures for vaccine development. Several bnAbs, including PGT128, PGT121, and 10-1074, have been reported to target glycan-dependent epitopes on the V3 region of gp120, but their glycan specificity remains to be fully characterized. We sought to clarify this by using the chemoenzymatic approach to synthesize homogeneous glycopeptides modeled on the V3 region of gp120 and with glycosylation at the conserved Asn332 residue, which was shown essential for antibody recognition. We utilized solid phase peptide synthesis (SPPS) to synthesize the GlcNAc-peptide precursor in which a GlcNAc-Asn was inserted at the Asn-332 site. Then various high-mannose and complex type N-glycans were attached at the pre-determined site via chemoenzymatic glycosylation using glycosynthase as the enzyme and glycan oxazoline as the donor substrate. Our SPR binding studies indicated that the PGT121 antibody recognized the V3 peptide epitope with a complex-type glycan at the Asn332 site while the PGT128 and antibody 10-1074 could efficient bind to the synthetic V3 glycopeptides having a high mannose Man9 glycan at the Asn332 site. Interestingly, free N-glycans or non-glycosylated V3 peptides showed no or only marginal binding to those antibodies under the same conditions, showing the requirement of both the appropriate glycan and the peptide portion for high-affinity interactions. Further studies are ongoing to probe the effect of other glycosylation sites in the V3 domain on the recognition by these HIV-neutralizing antibodies.

Synthesis of homogeneous HIV-1 V3 glycopeptides for characterizing the glycan specificity of glycan-dependent HIV-neutralizing antibodies

247th National Meeting (2014)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.