An LPS-Deficient vaccine candidate with steD deletion confers enhanced protection against homologous and heterologous Salmonella.

Nontyphoidal Salmonella infection is a leading cause of foodborne illness globally, contributing to significant public health concerns and exacerbating the growing problem of antimicrobial resistance. Effective strategies for controlling Salmonella infections are urgently needed, particularly within the livestock and poultry industries. In this study, we developed a novel attenuated Salmonella Typhimurium (S. Typhimurium) strain, RAST002, by introducing a deletion of the steD gene into an LPS-deficient background. The steD gene plays a key role in modulating host immune responses by interfering with T cell activation and suppressing antigen presentation. We hypothesized that deleting this gene would enhance CD4⁺ T cell-mediated immunity, thereby improving the immunogenicity of the vaccine candidate. Phenotypic characterization confirmed that the gene deletions (ΔmanA ΔsteD) did not impair bacterial growth or motility, suggesting that essential physiological functions were preserved. Notably, these genetic modifications also improved the safety profile of the vaccine candidate. Immunization with RAST002 induced strong cross-reactive antibody responses and significantly enhanced cellular immunity, as evidenced by an increased proportion of CD4⁺ T cells in the spleen compared to controls. Protective efficacy was assessed through challenge studies, demonstrating up to 80% protection against the homologous strain. Furthermore, RAST002 provided protection ranging from 60% to 80% against various clinically isolated heterologous Salmonella Enteritidis (S. Enteritidis) strains. These findings underscore the potential of combining immune evasion gene deletions with LPS-deficient Salmonella strains as an effective strategy for broad-spectrum protection against Salmonella infections.