Differential biological and adjuvant activities of cholera toxin and Escherichia coli heat-labile enterotoxin hybrids

Two bacterial products that have been demonstrated to function as mucosal a djuvants are cholera toxin (CT), produced by various strains of Vibrio chol erae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli, Although LT and CT have many features in comm on, they are clearly distinct molecules with biochemical and immunologic di fferences which make them unique. The goal of this study was to determine t he basis for these biological differences by constructing and characterizin g chimeric CT-LT molecules, Toxin gene fragments were subcloned to create t wo constructs, each expressing the enzymatically active A subunit of one to xin and the receptor binding B subunit of the other toxin. These hybrid tox ins were purified, and the composition and assembly of CT A subunit (CT-A)- LT B subunit (LT-B) and LT A subunit (LT-A)-CT B subunit (CT-B) were confir med. Hybrids were evaluated for enzymatic activity, as measured by the accu mulation of cyclic AMP in Caco-2 cells, and the enterotoxicity of each toxi n was assessed in a patent-mouse assay, The results demonstrated that LT-A- CT-B induces the accumulation of lower levels of cyclic AMP and has less en terotoxicity than either wild-type toxin or the other hybrid. Nonetheless, this hybrid retains adjuvant activity equivalent to or greater than that of either wild-type toxin or the other hybrid when used in conjunction with t etanus toroid for intranasal immunization of BALB/c mice. Importantly, the ability of LT to induce a type 1 cytokine response was found to be a functi on of LT-A. Specifically, LT-A-CT-B was able to augment the levels of antig en-specific gamma interferon (IFN-gamma) and interleukin 5 to levels compar able to those achieved with native LT, while CT-A-LT-B and native CT both p roduced lower levels of antigen-specific IFN-gamma, Thus, these toxin hybri ds possess unique biological characteristics and provide information about the basis for differences in the biological activities observed for CT and LT.