USE OF THE VIBRIO-CHOLERAE IRGA GENE AS A LOCUS FOR INSERTION AND EXPRESSION OF HETEROLOGOUS ANTIGENS IN CHOLERA VACCINE STRAINS

Vibrio cholerae may be a particularly effective organism for use in de livering heterologous antigens to stimulate a common mucosal immune re sponse. A live attenuated vaccine strain of V. cholerae was constructe d from the ctxA deletion mutant 0395-N1, containing the B subunit of S higa-like toxin I under the transcriptional control of the iron-regula ted 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-regu lated outer membrane protein of V. cholerae, which is a known virulenc e factor for this organism. Clones of the structural gene irgA from th e classical V. cholerae strain 0395, with the gene for the Shiga-like toxin I B subunit inserted under the control of the irgA promoter, wer e used to introduce an internal deletion of irgA into the chromosome o f 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 expo sure to sucrose. The construction of vaccine strains was confirmed by Southern hybridization studies and outer membrane protein analysis. Th e expression of StxB in the vaccine strain VAC2 following growth in hi gh- or low-iron conditions was shown to be tightly iron-regulated by W estern blot analysis and by quantification of StxB using a sandwich en zyme-linked immunosorbent assay. The production of StxB by VAC2 under low-iron conditions was greater than that of the reference strain Shig ella dysenteriae 60R. This vaccine strain produced no detectable cytot oxicity in a HeLa cell assay, and showed no increased virulence over t he attenuated parent strain, 0395-N1, in a suckling mouse model. We su ggest that the V. cholerae irgA gene is a particularly useful locus fo r the insertion and expression of heterologous antigens in cholera vac cine strains for oral delivery.