Differential Biological and Adjuvant Activities of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin Hybrids

Infectious diseases remain one of the leading causes of death in adults and children worldwide. In 1998, for example, infectious diseases killed more than 13 million people and were responsible for 63% of deaths in children less than 4 years old (3). In addition to suffering and death, infectious diseases impose an enormous financial burden on society. Recently, a great deal of attention has been focused on mucosal vaccination as an alternative to parenteral immunization and on its associated costs and risks. Recent studies have shown that mucosal immunization via intranasal (IN), oral, or rectal routes can elicit both humoral and cell-mediated immune responses in both the mucosal and the systemic compartments. Despite the attractiveness of mucosal vaccination, mucosally administered antigens are frequently not immunogenic. A number of strategies have been developed to facilitate and enhance the immune response obtained after mucosal immunization. Among these strategies are the use of attenuated mutants of bacteria (i.e., Salmonella spp.) as carriers of heterologous antigens, encapsulation of antigens into microspheres, gelatin capsules, different formulations of liposomes, adsorption onto nanoparticles, lipophilic immune stimulating complexes, and addition of bacterial products with known adjuvant properties. While a number of substances of bacterial origin have been tested as mucosal adjuvants (17, 21, 25), it is clear that the two bacterial proteins with the greatest potential to function as mucosal adjuvants are cholera toxin (CT), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli (7, 10, 18, 26, 27). Although LT and CT have many features in common, they are clearly distinct molecules with biochemical and immunologic differences which make them unique (9). In addition, LT has an unusual affinity for carbohydrate-containing matrices (6, 8). LT binds not only to agarose in columns used for purification but also, more importantly, to other biological molecules containing galactose, including glycoproteins and lipopolysaccharides. This lectin-like binding property of LT results in a broader receptor population on mammalian cells for LT than for CT, which binds only to ganglioside GM1 (1, 8, 12). Moreover, LT and CT generally activate different subsets of T-helper cells. CT promotes CD4+ type 2 cytokine responses and help for immunoglobulin G1 (IgG1), IgE, and mucosal IgA, while LT induces CD4+ type 1 and type 2 cytokine responses and help for IgG1, IgG2a, IgG2b, and mucosal IgA (19, 26). This distinction between LT and CT may be important in terms of selecting a mucosal adjuvant for use with specific categories of pathogens, assuming that the type 2 bias reported for CT is also seen in humans. Possible sources for this bias include the availability of different receptors for LT and CT, differences in intracellular localizations based upon differences in endoplasmic reticulum signal sequences between CT and LT (5, 14, 15), and differences in the activation of intracellular signaling pathways. The purpose of the present study was to construct and evaluate hybrid toxins, consisting of the A subunit of one toxin in conjunction with the B subunit of the other toxin, in order to provide information about the potential roles of the A and B subunits in making CT and LT unique. The hybrid toxins 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 demonstrated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunodiffusion with specific anti-A-subunit and anti-B-subunit antibodies. Hybrid toxins were evaluated for enzymatic activity, as measured by the accumulation of cyclic AMP (cAMP) in Caco-2 cells, and for enterotoxicity in a patent-mouse assay (4). Finally, hybrid toxins were evaluated for the ability to function as mucosal adjuvants for tetanus toxoid (TT).