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Abstract Despite the importance of immunity against neuraminidase (NA), NA content and immunogenicity are neglected in current influenza vaccines. To address this, a recombinant N1/N2 NA vaccine (NAV) was developed. Stability assays were used to determine optimal temperature and buffer conditions for vaccine storage. The effect of divalent cation-related enhancement of NA stability and activity on N1 and N2 immunogenicity and efficacy against viral challenge was assessed. Differences in activity between N1 and N2 and cation-related activity enhancement did not translate into differences in immunogenicity or efficacy. NAV-vaccinated mice showed robust antibody titers against N1 and N2, and after challenge with influenza A (H1N1) virus, decreased viral titers and decreased antiviral and inflammatory responses by transcriptomic analysis. These findings provide guidance for optimal storage and assessment of NA-based vaccines and confirm the importance of NA in influenza vaccination strategies in attenuating viral replication and limiting inflammatory responses necessary to clear infection.