Adjuvanted mucosal vaccination enhances protection and prevents influenza virus transmission in the guinea pig model

Influenza virus infects the respiratory mucosa, highlighting the importance of mucosal immunity for early protection and transmission control. Here, we evaluated whether intranasal (IN) vaccination with recombinant trimeric hemagglutinin protein from A/Michigan/45/2015 (triHA) formulated with a combined mucosal adjuvant, nanoemulsion plus IVT, an RNA-based RIG-I agonist (NE/IVT), could protect guinea pigs against heterologous A/Netherlands/602/2009 challenge and reduce viral transmission. To compare mucosal and parenteral immunization, IN triHA/NE/IVT was benchmarked against IN triHA alone, IM triHA/AddaVax (IM triHA/Advx), and standard IM quadrivalent inactivated influenza vaccine (QIV). We also tested whether IN triHA/NE/IVT could boost IM QIV- primed immunity and included animals previously infected with A/Michigan/45/2015 to model pre-existing infection- induced immunity. Transmission was assessed by co-housing naïve sentinels with vaccinated, challenged donors. IN triHA/NE/IVT induced systemic humoral responses comparable to IM triHA/Advx while generating superior nasal mucosal IgA responses. Unexpectedly, IM triHA/AddaVax also induced detectable, albeit lower, mucosal IgG and IgA, contrasting with prior mouse data and highlighting species-specific differences. IN triHA/NE/IVT boosting after IM QIV enhanced serum IgG and mucosal IgA compared with QIV prime-boost alone and increased cross-neutralizing activity against antigenically distinct A/Victoria/4897/2022. Both IN triHA/NE/IVT and IN Michigan/15 prior- infection prevented detectable viral shedding after challenge, and naïve sentinels co-housed with IN triHA/NE/IVT- vaccinated donors remained seronegative. Together, these findings support NE/IVT as a potential mucosal platform capable of inducing robust systemic and mucosal immunity and boosting IM vaccine-primed responses.