In Vivo Iterative Adjuvant Screening Identifies an Intranasal Vaccine Formulation for Elicitation of Protective Mucosal Immune Responses Against SARS-CoV-2

<b>Background:</b> Intranasal (I.N.) vaccination holds promise to elicit mucosal immunity that counters respiratory pathogens at the site of infection. For subunit protein vaccines, immunostimulatory adjuvants are typically required. <b>Methods:</b> We screened a panel of 22 lipid-phase adjuvants to identify which ones elicited antigen-specific IgA with I.N. immunization of liposome-displayed SARS-CoV-2 receptor-binding domain (RBD). <b>Results:</b> Initial screening showed the TLR-4 agonist Kdo2-Lipid A (KLA) effectively elicited RBD-specific IgA. A second round of screening identified further inclusion of the invariant NKT cell ligands α-Galactosylceramide (α-GalCer) and its synthetic analog 7DW8-5 as complementary adjuvants for I.N. immunization, resulting in orders-of-magnitude-greater mucosal IgA response relative to intramuscular (I.M.) immunization. The inclusion of cationic lipids conferred capacity for mucosal adhesion and maintained immune responses. In K18 hACE2 transgenic mice, vaccination significantly reduced viral replication and prevented mortality from SARS-CoV-2 challenge. <b>Conclusions:</b> These results point towards the potential for the use of KLA and α-GalCer for I.N. subunit vaccines.