Mannosylated chitosan-PLGA delivers Ganoderma lucidum triterpenes for oral immunoprotection against H9N2 in broilers

The H9N2 subtype of avian influenza virus remains a major threat to poultry production and public health, creating a need for effective vaccines that induce mucosal immunity. Oral immunization is attractive for poultry because it is convenient and suitable for mass application, but its efficacy is often limited by antigen degradation in the gastrointestinal tract and poor intestinal uptake. In this study, we developed a mannosylated chitosan-poly(lactic-co-glycolic acid) nanoemulsion loaded with Ganoderma lucidum triterpenes (MPG) as an oral mucosal adjuvant and delivery system, and subsequently incorporated inactivated H9N2 virus to prepare an oral inactivated vaccine. The MPG nanoemulsion showed a mean particle size of 439.7 ± 2.7 nm, with a negative zeta potential, good stability, protection under simulated gastric conditions, rapid release under intestinal conditions, favorable mucoadhesive properties and a promising immunostimulatory activity in vitro. Broiler chickens were orally immunized twice, and immune responses and protective efficacy were evaluated. Compared with the other groups, the MPG-adjuvanted vaccine significantly increased serum hemagglutination inhibition antibody titers and intestinal H9N2-specific secretory immunoglobulin A levels. After homologous challenge, vaccinated chickens showed reduced oropharyngeal viral shedding and milder lung lesions. In jejunal tissue, the MPG-adjuvanted vaccine increased the RNA expression of immunoglobulin A, polymeric immunoglobulin receptor, J-chain, IL-17, TNF-α, and IL-10, suggesting enhanced intestinal antibody transport and local immune responses. These results indicate that MPG improves the oral adjuvant activity of Ganoderma lucidum triterpenes and enhances both systemic and intestinal immune responses to an inactivated H9N2 vaccine. This strategy may provide a practical approach for oral mucosal vaccination against H9N2 in poultry.