A low-endotoxic Salmonella vector with dual bacterial-host promoter expression of Lawsonia intracellularis antigens elicits protective immunity in a murine model

Lawsonia intracellularis, the etiological agent for proliferative enteropathy, remains a major cause of productivity loss and economic burden in the global swine industry. Currently available vaccines provide suboptimal protection and raise safety concerns, highlighting the need for improved immunization strategies. In this study, we developed Salmonella Typhimurium-based vaccine candidates against L. intracellularis in a murine model, incorporating immunoprotective epitopes from the surface autotransporter protein LatA, the flagellar protein FliC, and the heat shock protein 60 (HSP60). These antigens were expressed using dual bacterial-host plasmids, pJHL270 and pJHL305, containing prokaryotic (Ptrc) and eukaryotic (CMV) promoters to ensure both extracellular and intracellular antigen presentation. The attenuated Salmonella delivery strain JOL3086 (Δlon ΔpagL) exhibited a high safety profile with markedly reduced endotoxicity. Western blot analysis confirmed antigen expression in both bacterial and mammalian systems in vitro. Immunization with the recombinant Salmonella strains elicited significant humoral (systemic IgG and mucosal IgA) and cell-mediated (CD4⁺, CD8⁺ T cells) immune responses, accompanied by significantly increased levels of Ifng, Il6, Il4, and Il10 cytokines. Among the three vaccine strains, JOL3149 (pJHL305 expressing FliC-HSP60) induced the most pronounced immunological responses and conferred superior protection in C57BL/6 mice challenged with L. intracellularis, as evidenced by decreased fecal bacterial shedding, reduced intestinal bacterial load, and alleviated intestinal lesions postchallenge. Collectively, these findings demonstrate the potential of the Salmonella-mediated dual expression platform as a safe and effective delivery system for L. intracellularis antigens, offering a promising foundation for next-generation vaccines against L. intracellularis.