Structural rationality and enhanced immunoprotective efficacy of endogenously expressed OmpX-IL-9 fusion protein...

Salmonella infection severely threatens the broiler industry. OmpX, a promising vaccine candidate, exhibits limited immunogenicity as a single agent, while exogenous IL-9 adjuvants have inherent application deficiencies. This study is the first to demonstrate that endogenously fused IL-9 acts as an effective intramolecular adjuvant for OmpX, thereby providing a novel strategy for developing exogenous adjuvant-free subunit vaccines against Salmonellosis. Recombinant pET-28a-ompx-il-9 plasmids were constructed, and the fusion proteins were expressed, purified, and identified by Western blot. The structural integrity of OmpX-IL-9 was confirmed via SOPMA and SWISS-MODEL. Broilers were immunized with OmpX-IL-9 alone, or OmpX adjuvanted with Mn²⁺ or aluminum hydroxide (AH). After the second immunization, splenic lymphocyte proliferation, pro-inflammatory cytokine (IL-1β, IL-6, TNF-α) mRNA expression, and OmpX-specific IgG titers were evaluated. Following Salmonella challenge, bacterial loads in visceral organs and survival rates were determined. Our results confirm that OmpX-IL-9 maintains a stable structure with dual functional domains intact. The OmpX-IL-9 group significantly promoted lymphocyte proliferation, elevated cytokine mRNA levels and specific antibody titers, reduced post-challenge bacterial loads, and improved survival rates, with efficacy markedly superior to the OmpX-only group. This study validates IL-9 as a potent intramolecular adjuvant that effectively enhances OmpX immunogenicity, providing an important foundation for the development of broiler Salmonella vaccines.