CHEANG, N. Y. Z.; YAP, W. C.; TULLETT, K. M.; Qian, X.; TAN, P. S.; Purushotorman, K.; TAN, W. Y.; MAH, S. Y. Y.; MacAry, P. A.; TAN, C. W.; Lahoud, M. H.; ALONSO, S.

Short-lived, clade-specific immune responses with limited mucosal priming are limitations faced by current COVID-19 mRNA vaccines against sarbecoviruses. We have developed a nasal booster vaccine candidate that induced robust and sustained, cross-clade, systemic and mucosal protective immunity. Two recombinant Clec9A-specific monoclonal antibodies fused to the Receptor Binding Domain (RBD) from Omicron XBB.1.5 and SARS-CoV-1, respectively were generated. In Comirnaty mRNA-vaccinated mice, boosting with each individual Clec9A-RBD construct induced immune responses that either were limited in breadth or waned over time; while boosting with both constructs combined (Clec9AOMNI) elicited robust cross-clade neutralizing antibodies (nAb) and T cell responses that were significantly more sustained compared to Bivalent Comirnaty (BC) mRNA vaccine booster. The persistence of RBD-specific follicular helper CD4+ T cells, germinal centre B cells, and long-lived plasma cells that facilitated affinity maturation in Clec9AOMNI-boosted mice, correlated with the detection of triple cross-reactive B cells that bind to ancestral SARS-CoV-2 ancestral, SARS-CoV-2 XBB.1.5 and SARS-CoV-1 RBD. Remarkably, intranasal boosting with Clec9AOMNI generated robust and sustained mucosal immune responses in the upper and lower respiratory compartments, including RBD-specific IgA, cross-clade nAb and cellular immunity together with functional tissue-resident memory T cells, without compromising the systemic immune responses. Correspondingly, Clec9AOMNI booster conferred superior protection against Omicron BA.1 compared to BC booster when challenge was performed at six months post-boost. Hence, Clec9AOMNI is a promising nasal booster vaccine candidate that has the potential to mitigate pandemic threats from emerging sarbecoviruses.