Abstract

Simple SummaryThe hemagglutinin (HA) protein of influenza A virus is cleaved into HA1 and HA2 subunits during maturation. However, whether the subsequent conformational change alters HA immunogenicity remains unclear. In this study, we identified a circular loop structure flanking the cleavage site that is critical for HA expression (P7~P1, P′1~P′12). We generated a panel of mutants that either increased or decreased the cleavage efficiency of the HA0 precursor. To investigate the impact of conformational change on immunogenicity, we assessed these HA variants in a mouse model. Specifically, the Dlt5 (P6~P1, P′1~P′13) mutant reduced cleavage efficiency, resulting in elicited immunogenicity and significantly higher antibody titers in mice. In contrast, all other cleavable mutants induced similar antibody levels, suggesting the stabilized, conformationally locked HA protein possesses greater immunogenicity.Avian influenza (AI) significantly threatens poultry health and causes major economic losses in the poultry industry. Vaccination remains crucial for AI prevention and control. The major protective epitopes of influenza viruses are located on hemagglutinin (HA), a surface glycoprotein essential for viral infection. Most influenza vaccines induce neutralizing antibodies against HA to block viral entry. HA maturation requires the HA0 precursor to be proteolytically cleaved at a conserved site by host proteases to yield HA1 and HA2 subunits. A subsequent acidic condition triggers HA conformational changes, enabling viral–host membrane fusion. However, whether HA conformational variations affect immunogenicity remains unclear. In this study, the cleavage site of the HA gene from an H9N2 avian influenza virus was modified to block the proteolytic cleavage of the HA protein. Our results revealed distinct proteolytic patterns of certain mutants, which exhibited either increa