Naringenin as a phytogenic adjuvant systematically enhances the protective efficacy of H9N2 inactivated vaccine through coordinated innate-adaptive immune priming in chickens

Although inactivated vaccines remain the primary strategy for preventing and controlling avian influenza virus, they fail to induce durable and systemic immune protection. Adjuvants are crucial for enhancing antigen immunogenicity and improving immune responses. In this study, we evaluated the adjuvant activity of naringenin (Nar) for H9N2 inactivated vaccine by detecting humoral immunity, cellular immunity, and viral challenge. The results demonstrated that Nar/H9N2 co-administration significantly increased IgG levels and hemagglutination inhibition (HI) titers. Nar/H9N2 promoted the formation of high-affinity antibodies by upregulating the expression of genes associated with B cell activation and germinal centers (GCs) formation, thus facilitating humoral immune responses. Concurrently, Nar/H9N2 vaccine enhanced T cell proliferation, CD4⁺and CD8⁺T cell differentiation, and the expression of Th1/Th2 cytokines, thereby promoting cellular immunity. Crucially, compared to the inactivated H9N2 vaccine alone, viral challenge experiments confirmed that Nar-adjuvanted immunization confers superior protection, markedly reducing viral shedding and minimizing damage to the trachea and lungs. These findings elucidate the capacity of naringenin to synchronize multifaceted immune activation through GCs optimization and T-cell modulation, establishing Nar as a viable candidate for poultry vaccine adjuvants.