Development and evaluation of a multi-epitope subunit vaccine against Salmonella Enteritidis infection.

Salmonella Enteritidis (S. Enteritidis) is a major pathogen causing foodborne diseases worldwide, posing a serious threat to public health security and the sustainable development of the poultry industry. To overcome the limitations of conventional vaccines in terms of efficacy, safety, and cross-protection, a novel multi-epitope subunit vaccine (SEMV) was designed and constructed using an immunoinformatics-based strategy. Through systematic screening of epitopes from key immunogenic proteins of S. Enteritidis (SseB, SipC, OmpD and TolC), a total of 8 B-cell epitopes, 4 MHC-I-restricted epitopes, and 8 MHC-II-restricted epitopes were identified. These epitopes were linked together with optimized linkers and an adjuvant to produce the recombinant SEMV protein. Bioinformatic evaluation confirmed that the vaccine candidate exhibited favorable antigenicity, non-allergenicity, and structural stability. Molecular docking analysis showed that SEMV formed a stable complex with TLR15, with a docking score of -479.08, a confidence score of 0.9986, and an MM/GBSA binding free energy of ‑36.34 kcal/mol. Molecular dynamics simulation further demonstrated that the complex reached equilibrium after 95 ns (RMSD ≈ 0.8 nm), maintained an average of approximately six hydrogen bonds, and exhibited an MM/PBSA binding free energy of ‑25.94 kcal/mol; key residues such as B:ILE258, A:PRO706, and A:GLU740 played critical roles in binding. In chicken immunization and challenge experiments, SEMV induced high levels of specific antibodies and cellular immune responses, significantly reduced bacterial loads in visceral organs, and alleviated histopathological damage. The vaccine demonstrated reliable immune protective efficacy against both homologous and heterologous Salmonella challenges. Collectively, these findings indicate that SEMV is a highly promising multi-epitope subunit vaccine candidate against Salmonella, laying an important foundation for subsequent preclinical and clinical studies.