Novel Intranasal Replication-Deficient NS1ΔC Flu Vaccine Confers Protection from Divergent Influenza A and B Viruses in Mice

<b>Background/Objectives</b>: The current strategy for seasonal influenza prophylaxis relies on updating the vaccine components annually to account for the rapid antigenic drift of viruses and the low cross-protective efficacy of available vaccines. Mutant influenza viruses with truncated or deleted NS1 protein are known to stimulate cross-specific T-cell immune response and provide protection against heterosubtypic influenza A and B viruses. <b>Methods</b>: We generated NS1ΔC influenza A and B viruses with C-terminal NS1 deletions by reverse genetics. In a mouse model, we assessed the safety and immunogenicity of the B/Lee/NS1ΔC strain upon intranasal administration, as well as the mechanism of its cross-protective efficacy against sublethal B/Victoria and B/Yamagata challenges. We then investigated the potential of the intranasal Flu/NS1ΔC vaccine-a trivalent formulation of NS1ΔC A/H1N1, A/H3N2, and B influenza viruses-to protect mice from lethal influenza infection with homologous, heterologous, and antigenically drifted influenza A and B viruses. <b>Results</b>: Intranasal immunization with the B/Lee/NS1ΔC strain was safe in mice. It activated cross-specific T-cell responses in the lungs and protected animals against heterologous challenge by reducing viral load, inflammation, and lung pathology. Immunization with the trivalent Flu/NS1ΔC vaccine formulation improved survival and reduced weight loss and viral load upon challenge with A/H1N1pdm, A/H2N2, A/H5N1, and B/Victoria viruses. <b>Conclusions</b>: The trivalent intranasal Flu/NS1ΔC influenza vaccine is a promising tool to improve seasonal influenza protection and preparedness for an influenza pandemic.