A ∆ssaV deletion attenuates Salmonella Choleraesuis to generate a self-limiting, immunogenic vaccine candidate

Live attenuated Salmonella enterica serovar Choleraesuis (S. Choleraesuis) strains, a host-adapted swine pathogen with well-characterized virulence and strong immunogenicity, represents a rational choice for constructing live bacterial vaccines. The ssaV gene, a key component of the SPI-2 T3SS, was knocked out to construct a vaccine candidate designed to strike an optimal balance between attenuation and immunogenicity using CRISPR-Cas9. This mutant retained the ability to adhere and invade epithelial cells but showed markedly reduced survival in macrophages, indicating impaired SPI-2-dependent persistence. The ∆ssaV mutant exhibited a > 1000-fold increase in LD₅₀ and underwent rapid systemic clearance, collectively confirming its marked attenuation and efficient elimination in vivo. The ∆ssaV mutant vaccine elicited strong humoral and cellular immune responses in mice and achieved a 70% survival rate upon lethal challenge with the WT strain. These results demonstrate that ssaV deletion creates a rationally designed vaccine strain that is attenuated, immunogenic, and self-limiting, positioning it as a promising live vaccine candidate in the murine model.