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

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.