Dual role of glycosylation in resistance to CD4-binding site broadly neutralizing antibodies

Broadly neutralizing antibodies (bNAbs) provide a useful tool for HIV cure strategies because of their ability to target conserved regions on the envelope (Env) protein in the context of both virions and infected cells. One of the best-studied bNAbs is the CD4 binding site (CD4bs) antibody VRC01 and others in its class. A major obstacle to effective cure strategies with bNAbs is viral immune escape. A deeper understanding of escape pathways from VRC01-class antibodies in genetically diverse samples is needed. Using an in vitro viral escape assay where infected CD4⁺ T cells were cultured in the presence of increasing VRC01 concentrations, complete resistance to VRC01 was detected in Env 246.F3 by day 42. We determined that resistance was due to a mutation at position N276 that resulted in elimination of the glycan-attachment site. As the loss of a glycan at this site is known to conversely increase virus sensitivity to CD4bs antibodies, we explored this finding further. Specifically, we introduced the N276D mutation into the 12-virus global Env panel and measured neutralization susceptibility to a panel of CD4bs bNAbs. This N276D mutation increased resistance or sensitivity depending on the Env and bNAb in question, emphasizing a dual role of glycan N276 in VRC01-class bNAb neutralization. The role of this glycan in escape was demonstrated to be dependent on the context of the Env, the bNAb, and the glycosylation complexity of the virus as determined by producer cells. These findings underscore the complexity of glycosylation in antibody escape in genetically diverse HIV.IMPORTANCEDespite decades of research, an HIV cure remains elusive, largely due to the virus's immense genetic variability and ability to evade immune clearance. Broadly neutralizing antibodies (bNAbs), particularly those targeting the conserved CD4 binding site such as VRC01 and others in its class, offer promise for cure by targeting both circulating virus and infected cells. However, viral escape from bNAbs remains a critical hurdle. In this study, we demonstrate that glycan-mediated escape from VRC01-class bNAbs is highly context-dependent-shaped by Env, the bNAb, and the glycosylation patterns introduced by the producer cell. These findings emphasize the dual role of glycans in affecting antibody sensitivity (shielding the virus to increase antibody resistance or sometimes providing an epitope that increases antibody sensitivity) and underscore the importance of viral and host factors in shaping effective bNAb-based cure strategies across diverse HIV-1 strains.