title: Stable expression of Shigella sonnei form I O-polysaccharide genes recombineered into the chromosome of live Salmonella oral vaccine vector Ty21a.
abstract: Live, attenuated Salmonella enterica serovar Typhi strain Ty21a, a licensed oral typhoid fever vaccine, has also been employed for use as a vector to deliver protective antigens of Shigella and other pathogens. Importantly, lipopolysaccharide (LPS) alone has been shown to be a potent antigen for specific protection against shigellosis. We reported previously the plasmid cloning of heterologous LPS biosynthetic genes and the expression in Ty21a of either S. sonnei or of S. dysenteriae 1 LPS's. The resulting plasmids encoding Shigella LPS's were reasonably stable for >50 generations of growth in nonselective media, but still contained an antibiotic resistance marker that is objectionable to vaccine regulatory authorities. Deletion of this antibiotic-resistance marker inexplicably resulted in significant plasmid instability. Thus, we sought a method to insert the large ∼12kb S. sonnei LPS gene region into the chromosome, that would allow for subsequent removal of a selectable marker and would result in 100% genetic stability. Toward this objective, we optimized an existing recombination method to mediate the insertion of a ∼12kb region encoding the S. sonnei LPS genes into the Ty21a genome in a region that is nonfunctional due to mutation. The resulting strain Ty21a-Ss simultaneously expresses both homologous Ty21a and heterologous S. sonnei O-antigens. This chromosomal insert was shown to be 100% genetically stable in vitro and in vivo. Moreover, Ty21a-Ss elicited strong dual anti-LPS serum immune responses and 100% protection in mice against a virulent S. sonnei challenge. This new vaccine candidate, absolutely stable for vaccine manufacture, should provide combined protection against enteric fevers due to Salmonella serovar Typhi as shown previously (and some Paratyphi infections) and against shigellosis due to S. sonnei.
authors.affiliation: Laboratory of Enteric and Sexually Transmitted Diseases, U.S. FDA-Center for Biologics Evaluation and Research, NIH campus Bldg. 29, 8800 Rockville Pike, Mail Code HFM440, Bethesda, MD 20892, USA.
authors.forename: Madushini N
authors.initials: MN
authors.lastname: Dharmasena
authors.forename: Brock W
authors.initials: BW
authors.lastname: Hanisch
authors.forename: Tint T
authors.initials: TT
authors.lastname: Wai
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Kopecko
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000276:immunology*; D053898:Biosynthetic Pathways; D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004195:Disease Models, Animal; D004337:Drug Carriers / Q000008:administration & dosage*; D016503:Drug Delivery Systems; D004405:Dysentery, Bacillary / Q000517:prevention & control; D005260:Female; D042822:Genomic Instability; D051379:Mice; D008807:Mice, Inbred BALB C; D008967:Molecular Biology / Q000379:methods; D008969:Molecular Sequence Data; D019081:O Antigens / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D012485:Salmonella typhi / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012764:Shigella sonnei / Q000235:genetics / Q000276:immunology*; D014613:Vaccines, Attenuated / Q000008:administration & dosage / Q000276:immunology; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D004269:DNA, Bacterial; D004337:Drug Carriers; D019081:O Antigens; D014613:Vaccines, Attenuated; D014614:Vaccines, Synthetic