ABSTRACTWe have been exploring the use of the live attenuated Salmonella entericaserovar Typhi Ty21a vaccine strain as a versatile oral vaccine vector for the expression and delivery of multiple foreign antigens, including ShigellaO-antigens. In this study, we separately cloned genes necessary for the biosynthesis of the Shigella flexneriserotype 2a and 3a O-antigens, which have been shown to provide broad cross-protection to multiple disease-predominant S. flexneriserotypes. The cloned S. flexneri2a rfboperon, along with bgtand gtrII, contained on the SfII bacteriophage, was sufficient in Ty21a to express the heterologous S. flexneri2a O-antigen containing the 3,4 antigenic determinants. Further, this rfboperon, along with gtrA, gtrB, and gtrXcontained on the Sfx bacteriophage and oaccontained on the Sf6 bacteriophage, was sufficient to express S. flexneri3a O-antigen containing the 6, 7, and 8 antigenic determinants. Ty21a, with these plasmid-carried or chromosomally inserted genes, demonstrated simultaneous and stable expression of homologous S. Typhi O-antigen plus the heterologous S. flexneriO-antigen. Candidate Ty21a vaccine strains expressing heterologous S. flexneri2a or 3a lipopolysaccharide (LPS) elicited significant serum antibody responses against both homologous S. Typhi and heterologous ShigellaLPS and protected mice against virulent S. flexneri2a or 3a challenges. These new S. flexneri2a and 3a O-antigen-expressing Ty21a vaccine strains, together with our previously constructed Ty21a strains expressing Shigella sonneior Shigella dysenteriae1 O-antigens, have the potential to be used together for simultaneous protection against the predominant causes of shigellosis worldwide as well as against typhoid fever.