Multifaceted immune responses and protective efficacy elicited by a recombinant autolyzed Salmonella expressing FliC flagellar antigen of F18⁺ Escherichia coli

Porcine edema disease (ED) caused by F18⁺ Shiga toxin 2e-producing Escherichia coli (STEC) has imposed significant economic losses in the swine industry worldwide, resulting in sudden deaths in post-weaned piglets. The flagellin protein of F18⁺ STEC, a structural component of the flagellar filament, is a known virulence factor that mediates adhesion and invasion to porcine epithelial cells. In this study, Salmonella inactivated by the E lysis gene and expressing the flagellin (fliC) antigen was genetically engineered utilizing a plasmid (pMMP184) carrying an efficient heterologous antigen delivery system. The resulting strain JOL1485 producing FliC was successfully inactivated by the E lysis gene cassette. Following the lysis procedure, FliC secretion and production of JOL1485 was validated by immunoblot analysis. To evaluate protective immunogenicity elicited by the constructed strain, BALB/c mice were injected with 1 × 10⁸ lysed cells via the intramuscular route. The markedly elevated titers of FliC-specific IgG, IgG1 and sIgA antibodies were observed, indicating a robust Th2-associated humoral immune response was raised in the immunized mice. The proportion of CD3⁺ CD4⁺ splenic T cells and proliferative activity were also elevated in in vivo and in vitro stimulated mice splenocytes. Further, JOL1485 successfully elicited upregulated gene expression of cytokines IL-6, IL-8, IL17, IL-21, IFN-γ and TNF-α in naïve porcine peripheral blood mononuclear cells (PBMCs). The overall immune response elicited by JOL1485 conferred a significant rise of protection against a lethal virulent F18⁺ STEC challenge whereas all non-immunized mice died following the challenge. Our results demonstrate that fliC efficiently expressed in the genetically inactivated Salmonella strain has immunostimulatory and protective effects against a F18⁺ STEC lethal challenge, and may be promising as a potential vaccine candidate against ED infection.