Intranasal delivery of a COBRA-based protein vaccine adjuvanted with c-di-AMP enhances breadth of protective immune responses against seasonal influenza viruses.

Seasonal influenza vaccines provide variable protection against antigenically drifted influenza A (H1N1 and H3N2) and influenza B viruses. Broadly protective approaches using computationally optimized broadly reactive antigens (COBRAs) can enhance immunity against diverse strains. Bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) is a mucosal adjuvant that boosts vaccine-induced immune responses. Here, we hypothesized that intranasal multivalent COBRA hemagglutinin (HA) and neuraminidase (NA) vaccination with c-di-AMP would induce broad humoral and cellular immunity in mice. Immunologically naïve and pre-immune DBA/2J mice were intranasally immunized with pentavalent vaccine formulations containing COBRA recombinant HA and NA or corresponding wild-type antigens. Formulations were administered at low or high antigen doses, with or without the mucosal adjuvant c-di-AMP. Humoral and cellular immune responses were evaluated, along with protection following influenza A (H1N1 and H3N2) and influenza B viral challenge. Pentavalent COBRA HA/NA intranasal vaccination adjuvanted with c-di-AMP induced strong humoral and cellular immune responses in murine models. Vaccination induced robust, dose-dependent total IgG and hemagglutination inhibition (HAI) titers, along with high frequencies of antigen-specific antibody-secreting cells and interferon-γ (IFN-γ)-producing cells in pre-immune mice. Naïve mice were also seroprotected mainly against H1N1 at higher vaccine doses. Following lethal challenge with H1N1, H3N2, and influenza B viruses, vaccinated mice exhibited reduced weight loss, decreased lung viral titers, and enhanced survival, with the strongest protection observed in adjuvanted groups across both naïve and pre-immune models. These findings highlight the value of combining broadly protective COBRA vaccine candidates with advanced mucosal adjuvants to enhance efficacy against antigenically drifted influenza virus strains.