Glycan masking of NTD loops with a chimeric RBD of the spike protein as a vaccine design strategy against emerging SARS‐CoV‐2 Omicron variants

Abstract The N‐terminal domain (NTD) of the SARS‐CoV‐2 S protein comprises five exposed protruding loops. Deletions, insertions, and substitutions within these NTD loops play a significant role in viral evolution and contribute to immune evasion. We reported previously that introducing the glycan masking mutation R158N/Y160T in the NTD loop led to increased titers of neutralizing antibodies against the SARS‐CoV‐2 Wuhan‐Hu‐01 strain, as well as the Alpha, Beta, and Delta variants. In this study, we conducted further investigations on 10 additional glycan‐masking sites in the NTD loops. Our findings indicate that the introduction of glycan masking mutations, specifically N87/G89T, H146N/N148T, N185/K187T, and V213N/D215T significantly enhanced neutralizing antibody titers against the Delta variant. The combination of dual glycan‐masking mutations R158N/Y160T+V213N/D215T and R158N/Y160T+G219N results in a shift toward the Omicron BA.1. Furthermore, the introduction of the Omicron receptor binding domain (RBD) alongside these two dual glycan masking mutations of Wuhan‐Hu‐1 and XBB.1 NTD sequences resulted in a noticeable shift in antigenic distances, aligning with the Omicron BA.4/5, BA.2.75.2, BQ.1.1, and XBB.1 subvariants on the antigenic map. This strategic combination, which involves the dual glycan masking mutations R158N/Y160T+V213N/D215T and R158N/Y160T+G219N in the NTD loops, along with the domain swap incorporating the Omicron RBD, emerges as a promising vaccine design strategy for the continuous development of next‐generation SARS‐CoV‐2 vaccines.