title: Use of the Vibrio cholerae irgA gene as a locus for insertion and expression of heterologous antigens in cholera vaccine strains.
abstract: Vibrio cholerae may be a particularly effective organism for use in delivering heterologous antigens to stimulate a common mucosal immune response. A live attenuated vaccine strain of V. cholerae was constructed from the ctxA deletion mutant 0395-N1, containing the B subunit of Shiga-like toxin I under the transcriptional control of the iron-regulated irgA promoter. The B subunit of Shiga-like toxin I is identical to the B subunit of Shiga toxin (StxB). irgA encodes the major iron-regulated outer membrane protein of V. cholerae, which is a known virulence factor for this organism. Clones of the structural gene irgA from the classical V. cholerae strain 0395, with the gene for the Shiga-like toxin I B subunit inserted under the control of the irgA promoter, were used to introduce an internal deletion of irgA into the chromosome of 0395-N1 by in vivo marker exchange, using the suicide vector plasmid pCVD442. This plasmid contains the sacB gene from Bacillus subtilis, which allowed positive selection for loss of plasmid sequences on exposure to sucrose. The construction of vaccine strains was confirmed by Southern hybridization studies and outer membrane protein analysis. The expression of StxB in the vaccine strain VAC2 following growth in high- or low-iron conditions was shown to be tightly iron-regulated by Western blot analysis and by quantification of StxB using a sandwich enzyme-linked immunosorbent assay. The production of StxB by VAC2 under low-iron conditions was greater than that of the reference strain Shigella dysenteriae 60R.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Infectious Disease Unit, Massachusetts General Hospital, Boston 02114.
authors.forename: J R
authors.initials: JR
authors.lastname: Butterton
authors.forename: S A
authors.initials: SA
authors.lastname: Boyko
authors.forename: S B
authors.initials: SB
authors.lastname: Calderwood
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics; D015139:Blotting, Southern; D022121:Cholera Vaccines / Q000235:genetics* / Q000276:immunology* / Q000633:toxicity; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D007928:Lethal Dose 50; D046911:Macromolecular Substances; D016254:Mutagenesis, Insertional / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011956:Receptors, Cell Surface; D022622:Shiga Toxin 1; D014158:Transcription, Genetic / Q000235:genetics; D014613:Vaccines, Attenuated / Q000235:genetics / Q000276:immunology; D014734:Vibrio cholerae / Q000235:genetics* / Q000276:immunology*; D014774:Virulence
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D022121:Cholera Vaccines; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D022622:Shiga Toxin 1; D014613:Vaccines, Attenuated; C074789:irgA protein, Vibrio cholerae