InFlux: a highly multiplexed neutralization assay to measure anti-influenza humoral immunity 4817

Abstract Description Influenza A (IAV) poses a significant global health threat, with annual vaccine effectiveness remaining low (20–60%) due to rapid escape mutations in the hemagglutinin (HA) protein. Understanding population-level immunity to IAV is critical for designing better vaccines and therapeutics. Comprehensive anti-influenza antibody profiles can reveal lifetime infection and vaccination histories, but existing serological assays like ELISA cannot measure neutralizing activity, which is crucial for assessing protection. To address this, we have developed a multiplexed neutralization assay to profile HA-neutralizing antibodies in human sera against a panel of single-replication viruses distinguished by short RNA barcodes integrated into their HA segments. This panel encompasses ∼65 strains from 1918 to 2021 representing the entire antigenic diversity of H1N1 and H3N2 hemagglutinin. Our results show that these HA-specific RNA barcodes reliably measure viral infectivity and neutralization titers. This innovative assay will enable rapid generation of population-level profiles of HA-neutralizing antibodies, providing critical insights into protective immunity and guiding the development of more effective IAV vaccines and therapeutics. Funding Sources NIH/NIAID UO1 Topic Categories Technological Innovations in Immunology (TECH)