BECC-engineered live-attenuated vaccine candidates display reduced endotoxicity with robust immunogenicity in mice.

. infection contributes significantly to the global disease burden, primarily affecting young children in developing countries. Currently, there are no FDA-approved vaccines against and the prevalence of antibiotic resistance is increasing, making therapeutic options limited. Live-attenuated vaccine strains WRSs2 () and WRSf2G12 ( 2a) are highly immunogenic, making them promising vaccine candidates, but possess an inflammatory lipid A structure on their lipopolysaccharide (LPS; also known as endotoxin). Here, we utilized bacterial enzymatic combinatorial chemistry (BECC) to ectopically express lipid A modifying enzymes in WRSs2 and WRSf2G12, as well as their respective wild-type strains, generating targeted lipid A modifications across the backgrounds. Dephosphorylation of lipid A, rather than deacylation, reduced LPS-induced TLR4 signaling and dampened endotoxic effects . These BECC-modified vaccine strains retained the phenotypic traits of their parental strains, such as invasion of epithelial cells and immunogenicity in mice without adverse endotoxicity. Overall, our observations suggest that BECC-engineered live attenuated vaccines are a promising approach to safe and effective vaccines.