CONSTRUCTION AND CHARACTERIZATION OF A POTENTIAL LIVE ORAL CARRIER-BASED VACCINE AGAINST VIBRIO-CHOLERAE O139

The rfb region from Vibrio cholerae O139 strain MO45 was cloned from c osmid gene banks established in Escherichia coli HB101, using an immun oblot assay for screening of the correct clones, Immunoblot analysis o f lipopolysaccharide (LPS) preparations revealed the presence of two t ypes of positive clones: (i) those expressing only a short core-linked O polysaccharide (SOPS) and (ii) those also expressing a highly polym erized capsular polysaccharide (CPS) not bound to the E. coli K-12 LPS core, In addition, the latter clones appear to contain a locus which may encode a putative regulator of SOPS acid CPS chain length, Further characterization in E. coli showed that CPS constitutes a barrier aga inst large particles such as the bacteriophage Ffm but not against bac teriophage lambda or P1, In addition, a portion of the K-12 LPS core m ay not be substituted with SOPS, Loci associated with the two clonal t ypes were transferred into V. cholerae CH19, an rfbAB deletion mutant of CVD103-HgR deficient in the production of the homologous Inaba O po lysaccharide, This resulted in the stable expression of SOPS, alone or together with CPS, that was indistinguishable from that of wild-type V. cholerae O139. Strains CH25 and CH26, which correspond to CH19 bear ing the V. cholerae O139 rfb region integrated into the chromosome, we re found to be genetically stable and essentially identical to the par ent CVD103-HgR with respect to physiological properties such as cell m otility, mercury resistance, toxicity, and production of the cholera t oxin B subunit. Rabbits immunized with CH25 elicited high titers of an ti-O139 SOPS- and CPS-specific serum antibodies, These strains possess characteristics desirable in candidate Live oral vaccines against V. cholerae O139.