INAAC: An affinity chromatography strategy enabling characterization and quantification of influenza neuraminidase...

Seasonal influenza vaccines are commonly produced using viruses containing hemagglutinin (HA) and neuraminidase (NA) antigens from recommended strains. However, NA amounts are not monitored due to lack of strategies for isolating NA reference antigens from viruses, limiting the ability to evaluate any contributions from NA for more than 50 years. Here, we developed an influenza neuraminidase active-site affinity chromatography (INAAC) strategy that uses an active-site binding antibody to isolate functional NAs from influenza A and B vaccine strains. INAAC recovered 20 to 60% of detergent-solubilized NA activity from vaccine viruses and recombinant sources, with CaCl₂ elution proving most effective. ELISA results with the isolated NA from the H1N1 strain and recombinant full-length N1 showed that a commercial vaccine contains functional N1 and H1 at a ratio of ∼1:10. Structure determination of the isolated N1 by cryo-electron microscopy confirmed the native tetrameric conformation and provided insight into the stalk conformation of a virus-derived NA. Finally, comparative analyses of NAs isolated from recent egg-propagated vaccine strains (H1N1, H3N2, and type B) revealed a unique proteolytic susceptibility of type B NA and strain-specific differences in sialic acid affinities and catalytic rates. These results demonstrate that INAAC supports multiple applications from NA structural analysis to producing NA vaccine antigens and reference standards, addressing longstanding challenges for incorporating NA into influenza vaccine development and quality control.