HETEROLOGOUS ANTIGEN EXPRESSION IN VIBRIO-CHOLERAE VECTOR STRAINS

Live attenuated vector strains of Vibrio cholerae were derived from Pe ru-2, a Peruvian El Tor Inaba strain deleted for the cholera toxin gen etic element and attRS1 sequences, which was developed as a live, oral vaccine strain, A promoterless gene encoding the Shiga-like toxin I B subunit (slt-IB) was inserted in the V. cholerae virulence gene irgA by in vivo marker exchange, such that slt-IB was under transcriptional control of the iron-regulated irgA promoter, slt-IB was also placed u nder transcriptional control of the V. cholerae heat shock promoter, h tpGp, and introduced into either the irgA or lacZ locus, or both loci, on the chromosome of Peru-2, generating JRB10, JRB11, or JRB12, respe ctively, A new technique was used to perform allelic exchange with lac Z, This method uses plasmid p6891MCS, a pBR327 derivative containing c loned V. cholerae lacZ, to insert markers of interest into the V. chol erae chromosome, Recombinants can be detected by simple color screenin g and antibiotic selection, In vitro measurements of Slt-IB produced b y the vector strains suggested that expression of Slt-IB from the irgA and htpG promoters was synergistic and that two copies of the gene fo r Slt-IB increased expression over a single copy, The V. cholerae vect ors colonized the gastrointestinal mucosa of rabbits after oral immuni zation, as demonstrated by very high serum antibody responses to V. ch olerae antigens. Comparison of the serologic responses to the B subuni t of cholera toxin (CtxB) following orogastric inoculation either with the wild-type C6709 or with Peru-10, a strain containing ctxB regulat ed by htpGp, suggested that both the cholera toxin and heat shock prom oters were active in vivo, provoking comparable immunologic responses. Orogastric inoculation of rabbits with vector strains evoked serum im munoglobulin G (IgG) responses to Slt-IB in two of the four strains te sted; all four strains produced biliary IgA responses, No correlation was observed between the type of promoter expressing slt-IB and the le vel of serum IgG or biliary IgA response, but the vector strain contai ning two copies of the gene for slt-IB evoked greater serum IgG respon ses than strains containing a single copy, consistent with the increas ed expression of Slt-IB from this strain observed in vitro. A comparis on of the serum and biliary antibody responses to Slt-IB expressed fro m htpGp versus CtxB expressed from the same promoter suggested that Ct xB is a more effective orally delivered immunogen.