A practical influenza neutralization assay to simultaneously quantify hemagglutinin and neuraminidase-inhibiting...

Influenza vaccine immunogenicity is commonly assessed by determining hemagglutination inhibition (HAI) titers in serum samples. HAI titers have been used to predict vaccine efficacy, but this often fails when live attenuated vaccines are evaluated, because it does not encompass all immune mediators of protection. Although antibodies that inhibit viral neuraminidase (NA) also contribute to protection against disease, there is currently no routine assessment of NA inhibition titers. A serological method with the capacity to measure functional inhibition of both HA and NA would be valuable. We developed a high-throughput virus neutralization assay that uses viral NA activity to quantify influenza replication (the AVINA assay), and showed its capacity to identify antivirals with a broad range of target specificities. In this report we demonstrate that antibodies with specificity for either HA or NA are detected in this assay, whereas a commonly used virus neutralization assay only detects those with HA-specificity. We also compared human responses to seasonal influenza vaccines measured by HAI, micro-neutralization, NA inhibition and AVINA assays. The response rates to both trivalent inactivated and live attenuated vaccines were greater when measured by the AVINA than the other assays, reflecting the dual antigen reactivity and increased sensitivity of the assay. The potential of this single assay to predict protection against influenza-induced tachypnea was demonstrated in vaccinated cotton rats. The AVINA assay is therefore a practical, comprehensive method to determine influenza vaccine immunogenicity and potential efficacy.