A novel trivalent inactivated Salmonella vaccine formulated with CpG ODNs to enhance the cellular immunity in chickens.

This study aimed to develop and evaluate a CpG oligodeoxynucleotide (CpG ODN)-adjuvanted trivalent inactivated Salmonella vaccine including S. enterica subsp. Enterica serovar Typhimurium, Salmonella enterica subsp. Enterica serovar Enteritidis, and Salmonella enterica serotype Infantis, for its immunogenic efficacy in chickens. The immunomodulatory effects of various CpG ODNs were assessed based on proinflammatory cytokine secretion and the expression levels of CD80, CD86, and MHC-II in the chicken cell lines HD11 and DT40. According to the results, CpG ODNs D35 3CG PO, D35 3CG MB, 1466 Acore PO, 1466 Acore MB, and K3 which exhibited non-cytotoxicity in both HD11 and DT40 cell lines, were selected for vaccine formulation. To evaluate their effects under in vivo conditions, chicks (n = 25) were randomly assigned to fourteen groups (G1: only sterile pyrogen-free saline solution, G2: only inactivated vaccine, G3: inactivated vaccine with 150 mg/dose of ALUM, G4: commercial Salenvac T vaccine, G5-G14: various experimental vaccine formulations which included different CpG ODNs combined with inactivated bacterial strains, with or without ALUM). Immune responses were analyzed through serological assays for antigen-specific antibody titers and ex vivo splenocyte cultures for cytokine secretion. Flow cytometry was performed to assess T-cell activation and IFN-γ production. The results demonstrated that the CpG ODNs-adjuvanted vaccine formulations significantly enhanced both humoral and cellular immunity compared to the commercial vaccine. Specifically, the Vac#5+ ALUM formulation, which included the K3 CpG ODN, induced robust antibody responses against Salmonella antigens and significantly increased IFN-γ secretion, nearly two-fold higher than the commercial vaccine. This effect was primarily mediated by CD4+ helper and CD8+ cytotoxic T cells. These findings highlight the potential of CpG ODNs as effective vaccine adjuvants in poultry. To the best of our knowledge, this is the first study to investigate the use of CpG ODNs as adjuvants in inactivated Salmonella vaccine formulations. Future studies should focus on evaluating the long-term protective efficacy of this vaccine formulation and its ability to provide cross-protection against a broader spectrum of Salmonella serovars.