Search

During 2013–14 and 2015–16, A/H1N1pdm09 live attenuated influenza vaccine (LAIV) viruses replicated inefficiently in primary human nasal epithelial cells (hNEC). This led to reduced vaccine effectiveness (VE) in quadrivalent formulations, mediated by inter-strain competition. By mutating the haemagglutinin (HA) protein, we aimed to enhance hNEC replication of a novel A/H1N1pdm09 vaccine strain to overcome competition and improve VE. Combinations of N125D, D127E, D222G and R223Q substitutions were introduced to the HA protein of A/Slovenia/2903/2015 (A/SLOV15). A/SLOV15 S13, containing all four HA substitutions, produced approximately 1000-fold more virus than parental V1 during hNEC infection. Immunogenicity in ferrets was increased by approximately 10-fold, without compromising yield in eggs or antigenic match to wild-type (wt) reference strains. Despite S13 and V1 being antigenically similar, only S13 protected ferrets from wt virus shedding and fever post-challenge. Crucially, these data suggested that enhanced fitness allowed S13 to overcome inter-strain competition in quadrivalent LAIV (QLAIV). This improved efficacy was later validated by real-world VE data. S13 displayed increased binding avidity to a mammalian-like α-2,6 receptor analogue (6-SLN), relative to V1, while maintaining avian-like 3-SLN avidity. In silico modelling of the HA receptor binding site revealed additional interactions in the S13:6-SLN binding network and a mild increase in 6-SLN binding energy, indicating a possible mechanism for increased α-2,6 receptor-binding avidity. These data confirm that rational HA mutagenesis can be used to optimise hNEC replication and VE for A/H1N1pdm09 LAIV viruses. Author summary: Vaccine effectiveness for the pandemic H1N1 component of the FluMist/Fluenz live attenuated influenza vaccine was lower than expected during the 2013–14 and 2015–16 influenza seasons in the USA. Previous studies highlighted the importance of vaccine viruses replicating sufficiently well in human cells that they could compete in quadrivalent formulations and protect ferrets against influenza-like illness. Here, it was demonstrated that mutagenesis of the haemagglutinin surface protein can be used to improve vaccine effectiveness of a pandemic H1N1 vaccine strain. This was achieved by optimising replication in human cells while, crucially, leaving other key characteristics required for vaccine production unaffected. The mutated vaccine virus successfully overcame inter-strain competition, providing protection from influenza-like illness in ferrets, while its parent did not. Furthermore, it was demonstrated that enhanced binding to human-like receptors was the mechanism by which replicative fitness was being improved. These data suggested that improving replicative fitness, allowing the vaccine virus to drive a protective immune response in quadrivalent formulations, would result in increased effectiveness in patients. Subsequently, high vaccine effectiveness was demonstrated during the 2017–18 northern hemisphere influenza season, validating this approach. [ABSTRACT FROM AUTHOR]