Cholera toxin (CT) and E. coli heat-labile enterotoxin (LT) have demonstrated potent adjuvant properties in a variety of vaccination strategies. Although similar in structure and function, these molecules exhibit different properties with respect to the nature of the immune enhancement they elicit. Our goal in this study has been to determine the basis for these differences by constructing and characterizing chimeric CT/LT molecules. Plasmids were constructed by subcloning gene fragments, facilitated by site-directed mutagenesis, in order to create two constructs, each expressing the enzymatically active A subunit of one toxin and the B subunit (responsible for binding) of the other toxin as confirmed by sequence analysis. These hybrid toxins were then purified from E. coli and the composition and assembly of CT-A/LT-B and LT-A/CT-B were demonstrated by ELISA, SDS-PAGE and immunodiffusion. These hybrid molecules exhibited differential toxicity in the Y-1 adrenal tumor cell assay, and varied in their ability to elicit accumulation of cyclic AMP in Caco-2 cells as a measure of enzymatic activity. LT-A/CT-B exhibited reduced enzymatic activity, and reduced enterotoxicity in the Patent Mouse model when compared to the parent toxins or CT-A/LT-B. When used as adjuvants intranasally, the hybrid toxins enhance both the humoral and cell-mediated immune response to tetanus toxoid to the same extent as the parent toxins. The use of LT and LT-A/CT-B enhances the production of IFN-gamma by antigen restimulated splenocytes, while CT and CT-A/LT-B enhance production of IL-5
