Optimising a linear ‘Doggybone’ DNA vaccine for influenza virus through the incorporation of DNA targeting sequences and neuraminidase antigen

Influenza virus represents a challenge for traditional vaccine approaches due to its seasonal changes and potential for zoonotic transmission. Nucleic acid vaccines can overcome some of these challenges, especially through the inclusion of multiple antigens to increase breadth of response. RNA vaccines were an important part of the response to the COVID-19 pandemic, but for future outbreaks DNA vaccines may have some advantages in terms of stability and manufacturing cost that warrant continuing investigation to fully realise their potential. Here we investigate influenza virus vaccines made using a closed loop linear DNA platform, Doggybone™ DNA (dbDNA), produced by a rapid and scalable cell-free method. Influenza vaccines have mostly focussed on Haemagglutinin (HA), but the inclusion of Neuraminidase (NA) may provide additional protection. Here we explored the potential of including NA in a dbDNA vaccine, looking at DNA optimisation, mechanism and breadth of protection. We showed that DNA targeting sequences (DTS) improved immune responses against HA but not NA. We explored whether NA vaccine induced protection against influenza virus infection was cell mediated but depletion of CD8 and NK cells made no impact, suggesting it was antibody mediated. This is reflected in restriction of protection only homologous strains of influenza virus. Importantly, we saw that including both HA and NA in a single combined vaccine did not dampen the immune response to either one. Overall, we show that linear dbDNA can induce an immune response against NA which may offer increased protection in instances of HA mismatch where NA remains more conserved.