Role of glycosylation mutations at the N-terminal domain of SARS-CoV-2 XEC variant in immune evasion, cell-cell fusion, and spike stability.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, producing new variants that drive global coronavirus disease 2019 surges. XEC, a recombinant of KS.1.1 and KP.3.3, contains T22N and F59S mutations in the spike protein's N-terminal domain (NTD). The T22N mutation, similar to the DelS31 mutation in KP.3.1.1, introduces a potential N-linked glycosylation site in XEC. In this study, we examined the neutralizing antibody (nAb) response and mutation effects in sera from bivalent-vaccinated healthcare workers, BA.2.86/JN.1 wave-infected patients, and XBB.1.5 monovalent-vaccinated hamsters, assessing responses to XEC alongside D614G, JN.1, KP.3, and KP.3.1.1. XEC demonstrated significantly reduced neutralization titers across all cohorts, largely due to the F59S mutation. Notably, removal of glycosylation sites in XEC and KP.3.1.1 substantially restored nAb titers. Antigenic cartography analysis revealed XEC to be more antigenically distinct from its common ancestral BA.2.86/JN.1 compared to KP.3.1.1, with the F59S mutation as a determining factor. Similar to KP.3.1.1, XEC showed reduced cell-cell fusion relative to its parental KP.3, a change attributed to the T22N glycosylation. We also observed reduced S1 shedding for XEC and KP.3.1.1, which was reversed by ablation of T22N and DelS31 glycosylation mutations, respectively. Molecular modeling suggests that T22N and F59S mutations of XEC alter hydrophobic interactions with adjacent spike protein residues, impacting both conformational stability and neutralization. Overall, our findings underscore the pivotal role of NTD mutations in shaping SARS-CoV-2 spike biology and immune escape mechanisms.IMPORTANCEThe continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the emergence of novel variants with enhanced immune evasion properties, posing challenges for current vaccination strategies. This study identifies key N-terminal domain (NTD) mutations, particularly T22N and F59S in the recent XEC variant, which significantly impacts antigenicity, neutralization, and spike protein stability. The introduction of an N-linked glycosylation site through T22N, along with the antigenic shift driven by F59S, highlights how subtle mutations can drastically alter viral immune recognition. By demonstrating that glycosylation site removal restores neutralization sensitivity, this work provides crucial insights into the molecular mechanisms governing antibody escape. Additionally, the observed effects on spike protein shedding and cell-cell fusion contribute to a broader understanding of variant fitness and transmissibility. These findings emphasize the importance of monitoring NTD mutations in emerging SARS-CoV-2 lineages and support the need for adaptive vaccine designs to counteract ongoing viral evolution.