The spike 486 site is a key immune evasion point and a determinant of the immunogenicity of the RBD-dimer mRNA vaccine against SARS-CoV-2 variants.

The COVID-19 pandemic, caused by SARS-CoV-2, has led to millions of hospitalizations and deaths worldwide. The rapid emergence of new SARS-CoV-2 variants significantly challenged the efficacy of licensed vaccines because of the immune evasion caused by key mutations in the receptor-binding domain (RBD). Whether incorporating these critical evolutionary site(s) into COVID-19 vaccines can enhance the immunogenicity needs to be elucidated. In this study, we developed an mRNA vaccine encoding tandem RBDs from the Delta and BA.4/5 variants (RBD-Dimer) to improve cross-variant coverage. Robust humoral and T-cell responses were induced by the vaccine in mice, leading to effective neutralization against the prototype, Delta, and BA.4/5 variants. However, neutralizing activity against BQ.1 and XBB variants was compromised. Pseudovirus-based mutation screening identified F486S as a crucial site for immune evasion. Incorporating this mutation into RBD-Dimer mRNA vaccine candidates significantly enhanced neutralizing antibody response against the XBB variant, while maintaining T-cell responses, indicating an essential role of F486S in broadening immunogenicity against XBB variants. These findings identified the Spike 486 site as a critical immune evasion site and a key determinant for the efficacy of COVID-19 vaccines against emerged variants, and underscored the importance of key sites in RBD in enhancing the breadth of immune protection of COVID-19 vaccines.