The emergence of new SARS-CoV-2 variants presents challenges for existing therapeutics. The spike glycoprotein plays a crucial role not only in initial viral entry but also in the transmission of SARS-CoV-2 components through syncytia formation. Spike-mediated cell-to-cell transmission exhibits strong resistance to extracellular therapeutic and convalescent antibodies via a mechanism that remains elusive.

In this study, we investigated two clinical SARS-CoV-2 isolates, XBB.1 and XBB.1.5, which differ by a single amino acid substitution in the S protein. Through biochemical and cell-based assays, we assessed entry kinetics, syncytia formation, and the neutralizing efficacy of convalescent sera, correlating these features with S-driven cell-cell fusion. Our findings reveal that this single mutation significantly alters viral entry dynamics and enhances syncytia formation, without compromising serum neutralization efficacy. Importantly, the mutation increases the efficiency of spike-mediated cell–cell fusion, suggesting a mechanism by which viral transmissibility and partial resistance to antibody-based interventions may be enhanced. These results underscore how even subtle changes in the S protein can profoundly affect SARS-CoV-2 transmissibility and immune evasion. A deeper understanding of spike-mediated fusion is critical to anticipating the impact of future variants and guiding the development of next-generation antiviral strategies.