IMMUNE-MECHANISMS AND PROTECTIVE ANTIGENS OF VIBRIO-CHOLERAE SEROGROUP O139 AS A BASIS FOR VACCINE DEVELOPMENT

We have characterized 11 isolates of Vibrio cholerae O139 Bengal with regard to properties deemed to be relevant for development of a vaccin e against O139 cholera. For most strains two colony variants, A and B, which are nonhemolytic and hemolytic, respectively, were detected on blood agar, The A and B variants were associated with high- and low-le vel production of soluble hemagglutinin-protease, respectively. Howeve r, on Luria-Bertani agar both types formed opaque colonies, which has been shown to be associated with capsule formation. Interestingly, und er the stationary tube-shaken flask culture conditions in yeast extrac t-peptone water medium which were used to stimulate the production of cholera toxin (CT) and toxin-coregulated pill, B variants constitutive ly produced CT and TcpA, two ToxR-regulated proteins, at 28 and 37 deg rees C, whereas the production of these proteins by A variants was dow nregulated at the higher temperature, One of the strains, 4260B, havin g a well-exposed O antigen and capsule and the capacity to produce lar ge amounts of TcpA, CT, and mannose-sensitive hemagglutinin pill but m inimal amounts of the proteolytic soluble hemagglutinin, was selected to produce antibacterial antisera and as a challenge strain in protect ion studies using the rabbit ileal loop model. Rabbit antisera to live , heat-killed, or formalin-killed O139 vibrios or to purified O139 lip opolysaccharide (LPS) as well as monoclonal antibodies (Mabs) to O139 LPS agglutinated all O139 isolates, However, when A and B variants of strain 4260 were tested for sensitivity to vibriocidal activity of the se antibody preparations, only the B variant was killed. All of the an tisera against live or killed O139 vibrios conferred passive protectio n against fluid accumulation induced by the challenge strain, The prot ective effects of the antisera were correlated to anti-LPS antibody ti ters rather than to titers against whole bacteria that had been grown for toxin-coregulated pilus expression. This protection was considerab ly higher than that conferred by antisera to classical, El Tor, or rec ombinantly produced (classical) CT or CTB. Furthermore, MAbs to O139 L PS and CTB-CT exhibited a strong synergistic protection against O139 c hallenge irrespective of the level of sensitivity of challenge strains to O139 LPS MAbs in vibriocidal assays in vitro.