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Abstract

Glycosylation plays a pivotal role in modulating the structure and immunogenicity of viral antigens. Three glycosylation sites on the receptor-binding domain (RBD) of SARS-CoV-2, including N331, N343 (N-linked), and T323 (O-linked), are highly conserved and remain unchanged across multiple variant strains. To investigate their functional relevance, a series of site-directed glycosylation-deficient mutants were generated on the basis of the RBD-dimer antigen of the ZF2001 vaccine (developed by ZFSW Biologics), including N-N331-NA, N-N343-NA, O-T323-NA, and an RBD-NA control. Comparative analyses of glycosylation-deficient RBD variants revealed a site-specific hierarchy in immune modulation. In particular, disruption of the N343 site led to a pronounced reduction in antigen-specific antibody titers and T helper cell-related cytokine responses in immunized mice, whereas mutations at N331 and T323 had more limited effects. Furthermore, glycan profiling using liquid chromatography–tandem mass spectrometry (LC–MS/MS), secondary structure assessment using microfluidic modulation spectroscopy (MMS), and molecular dynamics simulations revealed that the N343 glycan contributes to local structural stability and preserves key antigenic features of the RBD. Together, these results identify N343 as a critical glycosylation hotspot that governs RBD immunogenicity and antigenicity, providing a mechanistic foundation for the structure-based optimization of SARS-CoV-2 vaccines targeting glycan-regulated epitopes.