Deletion of steD gene and yejAE operons in LPS-deficient Salmonella elicits strong t cell immunity and complete protection against homologous serotypes.

Non-typhoidal Salmonella (NTS) infections are a major cause of foodborne illness worldwide, and rising antimicrobial resistance underscores the need for effective vaccines. Live attenuated Salmonella strains have potential, but developing an effective vaccine remains a challenge. In this study, we built on the previously constructed attenuated Salmonella Typhimurium strain RAST002, which carries deletions of manA and steD, by introducing a yejAE operon deletion to generate RAST003, aiming to further enhance immune responses. Oral immunization with this single strain induced robust systemic IgG and mucosal sIgA responses, significantly increased CD4+ and CD8+ T cell frequencies, and markedly elevated IFN-γ production, indicating a dominant Th1-type cellular immune response, alongside increased IL-2 and TNF-α levels supporting overall T cell activation. Immunized mice were fully protected against homologous S. Typhimurium challenge and exhibited significantly improved survival following heterologous S. Enteritidis exposure. These results demonstrate that combining LPS deficiency with targeted deletions of immune evasion genes generates a single, highly immunogenic Salmonella strain capable of inducing broad, protective immunity, providing a promising strategy for NTS vaccine development.