Glycoprotein G enables HSV-2 neuroinvasion and provides protection as a glycosylated vaccine antigen.

The role of glycoprotein G (gG-2) of herpes simplex virus type 2 (HSV-2) in viral pathogenesis remains poorly understood. gG-2 is cleaved into a secreted form (sgG-2) and a membrane-associated form (mgG-2), but the in vivo function of mgG-2 and the contribution of its glycosylation to immune responses have not been defined. Here, we provide a comprehensive characterization of the N- and O-linked glycosylation profile of mgG-2 and investigate its functional relevance for viral spread and vaccine-induced immunity. Using a mouse genital infection model, we show that an mgG-2-deficient HSV-2 mutant replicates in vaginal epithelial cells but is severely impaired in dissemination to dorsal root ganglia and the central nervous system, identifying mgG-2 as a key determinant of neuronal spread in vivo. In parallel, immunization with recombinant mgG-2 elicited strong humoral and Th1-polarized CD4 + T-cell responses and conferred protection against genital HSV-2 challenge. Importantly, glycosylation of mgG-2 was required for optimal immunogenicity and protection, as deglycosylated variants induced reduced CD4 + T-cell responses and allowed increased viral spread to neuronal tissues. Mechanistically, our findings suggest that glycosylation of mgG-2 modulates antigen recognition and shapes adaptive immune responses that limit viral dissemination after vaccination. Together, these results demonstrate that mgG-2 plays a critical role in HSV-2 pathogenesis and provide a strong rationale for targeting glycosylated mgG-2 in the development of both prophylactic and therapeutic vaccines against HSV-2.