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We have been exploring the use of the live attenuatedSalmonella enterica serovar Typhi Ty21a vaccine strain as a versatile oral vaccine vector for the expression and delivery of multiple foreign antigens, includingShigella O-antigens. In this study, we separately cloned genes necessary for the biosynthesis of theShigella flexneri serotype 2a and 3a O-antigens, which have been shown to provide broad cross-protection to multiple disease-predominantS. flexneri serotypes. The clonedS. flexneri 2arfb operon, along withbgt andgtrII , contained on the SfII bacteriophage, was sufficient in Ty21a to express the heterologousS. flexneri 2a O-antigen containing the 3,4 antigenic determinants. Further, thisrfb operon, along withgtrA ,gtrB , andgtrX contained on the Sfx bacteriophage andoac contained on the Sf6 bacteriophage, was sufficient to expressS. flexneri 3a 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 homologousS . Typhi O-antigen plus the heterologousS. flexneri O-antigen. Candidate Ty21a vaccine strains expressing heterologousS. flexneri 2a or 3a lipopolysaccharide (LPS) elicited significant serum antibody responses against both homologousS . Typhi and heterologousShigella LPS and protected mice against virulentS. flexneri 2a or 3a challenges. These newS. flexneri 2a and 3a O-antigen-expressing Ty21a vaccine strains, together with our previously constructed Ty21a strains expressingShigella sonnei orShigella dysenteriae 1 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.

Stable Chromosomal Expression of Shigella flexneri 2a and 3a O-Antigens in the Live Salmonella Oral Vaccine Vector Ty21a