ELISA-based quantification of neuraminidase in commercial influenza vaccines using virus-derived reference antigens

Most commercial influenza vaccines are produced using inactivated viruses decorated with hemagglutinin (HA) and neuraminidase (NA) antigens from the recommended strains. However, only the HA antigen content is monitored, leaving the NA antigen content, stability, and potential contributions to efficacy unclear. We quantified functional NA amounts in commercial vaccines from two seasons by coupling NA enzyme-linked immunosorbent assays (ELISAs) with NA reference antigens that were isolated from egg-propagated vaccine strains (H1N1, H3N2, and type B) by Immobilized NA Active site Affinity Chromatography (INAAC). Our results show that nonexpired egg-based vaccines for the 2024-25 season contained the three expected NAs and that cell-based vaccines may require cell propagated NA reference antigens. NA amounts in egg-based vaccines varied by strain and manufacturer, ranging from 0.7 to 1.7 µg/dose or NA:HA ratios between 1:10 and 1:20. We confirmed the ELISA results with an NA activity analysis, indicating functional NA amounts were measured. In expired egg-based vaccines (2022-23 season), some NAs were at low or undetectable levels, indicative of structural integrity decay over time. These findings provide a foundation for quantifying NA content and stability in vaccines to evaluate NA's potential efficacy contributions and optimize the design of future influenza vaccines that contain both NA and HA.IMPORTANCEThe influenza virus surface antigens hemagglutinin (HA) and neuraminidase (NA) are both targeted by the immune system during infection. Although most licensed influenza vaccines contain both the HA and NA antigens from the recommended strains, only the HA content is monitored. Here, we measured functional NA amounts in commercial vaccines using a stability-indicating ELISA with NA reference antigens isolated from the vaccine viruses. Our results show that current egg-based vaccines contain the expected NAs at amounts that vary by strain and manufacturer. Expired vaccines had reduced or undetectable NA levels, suggesting NA may degrade over time. This methodology can be applied to measure NA content and stability in current vaccines and help to optimize the design of future vaccines that contain both NA and HA antigens.