Recombinant HA1-ΔfliC enhances adherence to respiratory epithelial cells and promotes the superiorly protective immune responses against H9N2 influenza virus in chickens

H9N2 subtype avian influenza virus (AIV) is an ongoing threat causing substantial loss to the poultry industry and thus necessitating the development of safe and effective vaccines against AIV. Given that inactivated vaccines are less effective in activating the mucosal immune system, we aimed to generate a vaccine that can actively engage the mucosal immunity which is the front line of the immune system. We generated a group of flagellin-based hemagglutinin globular head (HA1) fusion proteins and characterized their immunogenicity and efficacy. We found that Salmonella typhimurium flagellin (fliC) lacking the hypervariable domain (called herein as HA1-Delta fliC) was recognized by TLR5 and induced a moderate innate immune response compared to N-terminus of fliC (HA1-fliC) and C-terminus of fliC (fliC-HA1). The HA1-Delta fliC protein had increased adherence to the nasal cavity and trachea than HA1-fliC and fliC-HA1 and significantly increased the HA-specific sIgA titers. Our in vivo results revealed that chickens treated with HA1-Delta fliC had a significantly reduced level of viral loads in the cloaca and throat compared with chickens treated with inactivated vaccine. Overall, these results revealed that HA1-Delta fliC can protect chickens against H9N2 AIV by eliciting the efficient mucosal immune responses.