Recombinant outer-membrane vesicles (OMVs) from Salmonella Enteritidis as multivalent candidate vaccines for enteric fevers and invasive non-typhoidal Salmonella infections

Invasive non-typhoidal Salmonella (iNTS) infections, typhoid, and paratyphoid fever are major global health burdens, particularly considering increasing antimicrobial resistance. These diseases often colocalize geographically, making broadly protective multivalent vaccines highly desirable. Multicomponent vaccine candidates are currently being developed to achieve protection against multiple systemic Salmonella diseases. The aim of this study was to create a vaccine candidate against iNTS and typhoid fever consisting of bacterial outer-membrane vesicles (OMVs) expressing simultaneously multiple key immunogenic antigens on their membrane surface. The abequose synthase ( abe ) gene was inserted into Salmonella Enteritidis to achieve expression of the O:4 surface polysaccharide antigen, alongside the naturally expressed O:9 antigen. The Vi polysaccharide was expressed using a plasmid containing the viaB operon. Reactogenicity of lipopolysaccharide molecules was reduced by deleting the msbB and pagP genes to alter Lipid A acylation. The tolR gene was deleted to increase the yield of OMVs by the mutant bacteria. Immunization of mice with the O:4, O:9, and Vi-expressing OMVs resulted in antibody responses against all three target antigens and reduced bacterial burdens in the livers and spleens of the animals after challenging with virulent Salmonella Typhimurium and Salmonella Enteritidis.