Epitope-Optimized Influenza Hemagglutinin Nanoparticle Vaccine Provides Broad Cross- Reactive Immunity against H9N2 Influenza Virus

The H9N2 avian influenza virus (AIV) represents a growing threat to the global poultry industry and poses an ongoing risk of human infections. Vaccination is a crucial strategy for the prevention and control of H9N2 AIVs. However, the continuous evolution of the virus consistently challenges the immune protection efficiency. Therefore, developing a universal H9N2 influenza vaccine capable of eliciting a broad-spectrum immune response is essential for epidemic prevention and control. In this study, we report an epitope-optimized nanoparticle (NPs) vaccine that elicits broad cross-reactive immunity against the H9N2 influenza virus. Utilizing Epigraph, a computational algorithm, we first designed three globular heads of H9 hemagglutinin (HA1) with optimized epitopes. Each antigen was subsequently conjugated to mi3 NPs, and the three constructs were mixed at equimolar ratios to generate the Epigraph vaccine. We compared the Epigraph vaccine to the currently recommended candidate vaccine virus (CVV), AL/39. The Epigraph vaccine effectively induced cross-reactive antibodies in mice and activated both CD4+ and CD8+ T cell immune responses. Moreover, it provided effective protection against lethal challenges of diverse H9N2 strains and significantly reduced the viral load in the lungs of the mice. This study provides a promising universal vaccine candidate for combating future H9N2 epidemics.