COEXPRESSION OF THE B-SUBUNIT OF SHIGA TOXIN-1 AND EAEA FROM ENTEROHEMORRHAGIC ESCHERICHIA-COLI IN VIBRIO-CHOLERAE VACCINE STRAINS

A promoterless gene for the Shiga toxin 1 B subunit (stxB(1)) has been placed under transcriptional control of the Vibrio cholerae heat shoc k gene htpG. A chromosomal enterohemorrhagic Escherichia coli fragment containing eaeA and 400 bp of upstream DNA was added to the construct , downstream of stxB(1); no transcription terminators were located bet ween the two genes, The plasmid construct was confirmed by DNA sequenc ing; in vitro transcription-translation studies demonstrated expressio n of EaeA from the plasmid, The htpGp-->stxB(1), eaeA construct was in serted into lacZ on the chromosome of Peru2, an El Tor V. cholerae str ain with both attRSI sequences and the entire cholera toxin genetic el ement deleted, and into lacZ in JRB10, a PeruZ derivative that has a s econd copy of htpGp-->stxB(1) also inserted in the V. cholerae virulen ce gene irgA. Two plasmid constructs, one containing stxB, under the c ontrol of the tac promoter and another containing htpGp-->stxB(1),eaeA , were transformed into Peru2, Expression of StxB1 by these constructs ,vas quantified by enzyme-linked immunosorbent assay and was highest i n the plasmid construct with stxB, under the control of the tac promot er, Localization of EaeA to the outer membrane of the vector strains w as demonstrated both by Western blotting and by immunofluorescence wit h an anti-EaeA antibody, A rabbit model for colonization by V. cholera e was used to compare the immune responses to the two heterologous ant igens, StxB1 and EaeA, expressed by these strains, Rabbits immunized w ith Peru2 transformed with a plasmid carrying tac-->stxB(1) developed neutralizing serum anti-StxB1 immunoglobulin G antibody responses, One of two rabbits immunized with a strain carrying a chromosomal copy of eaeA developed a marked immune response against EaeA, The plasmid con struct containing htpGp-->stxB(1),eaeA was unstable, producing low lev els of StxB1 in vitro and not evoking anti-EaeA antibody responses in vivo following oral immunization, Chromosomal insertion of eaeA may be preferred for future expression of this antigen in V. cholerae vaccin e constructs.