A recombinant lipidated receptor-binding domain is a promising self-adjuvanting antigen for mucosal vaccine

The SARS-CoV-2 pandemic has resulted in millions of fatalities, and the continual emergence of variants highlights the vital need for effective mucosal vaccines. Novel antigen designs and delivery strategies can elicit robust, epitope-focused mucosal immune responses while minimizing side effects and avoiding immune imprinting are urgently needed. Here, we developed an innovative bacterial lipidation-based SARS-CoV-2 receptor-binding domain (RBD) mucosal nanovaccine using the SpyCatcher/SpyTag (SC/ST) orthogonal assembly system. The lipidated SpyCatcher (lipoSC), derived from Escherichia coli , was employed as a nano-scaffold to display the SARS-CoV-2 RBD glycoprotein on its surface. The lipoSC-RBDST exhibited favorable monodispersity and thermostability, effectively promoted T follicular helper (Tfh) cell and germinal center B cell proliferation in endobronchial lymph nodes following mucosal administration, and was well tolerated overall. A series of animal experiments showed the potent ability of lipoSC-RBDST to activate RBD-specific mucosal immunity in the respiratory tract, as evidenced by the presence of neutralizing antibodies (nAbs) and secretory IgA (sIgA) in bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NLF). Serum neutralizing antibody titers remained above 10 3 even eight months post-immunization. There was also a significant increase in lung-localized tissue-resident memory (T RM ) T cells. Furthermore, two doses of mucosal booster activated and retained the omicron-specific immune responses in the respiratory track in wild-type RBD-primed mice. In conclusion, these findings support the utilization of a bacterial lipidation-based antigen design as a promising strategy for mucosal delivery to elicit epitope-focused mucosal immunity against SARS-CoV-2.