A bivalent inactivated influenza vaccine incorporating epitope-optimized surface proteins confers cross-protective immunity against H9N2 influenza virus

The H9N2 avian influenza virus (AIV) has caused substantial economic losses to the global poultry industry and poses a zoonotic threat to humans. Vaccination constitutes a pivotal strategy for the prevention and control of H9N2 AIVs. However, the ongoing antigenic evolution of the viruses pose a persistent challenge to the protective efficacy of existing vaccines. Therefore, the development of a broadly protective H9N2 influenza vaccine capable of eliciting cross-reactive immune responses is crucial for mitigating both the disease burden and the risk of pandemics. Here, we developed a bivalent chimeric inactivated vaccine, designated cHANA, by combining two individually rescued chimeric inactivated viruses, cHANA1 and cHANA2. Each recombinant virus carries one set of Epigraph-designed HA and NA immunogens, and the two sets were computationally optimized from global H9N2 HA and NA sequence datasets to complement each other in epitope coverage across the H9N2 viral population. Compared to the WHO-recommended candidate vaccine virus (CVV), AL/39, cHANA elicited more potent cross-reactive antibody responses and T cell immunity in mice. Furthermore, it elicited effective cross-protection against lethal challenge with heterologous H9N2 virus and significantly reduced pulmonary viral loads of mice. By conferring broad protective immunity, this vaccine represents a promising universal vaccine candidate for controlling H9N2 outbreaks.