Characterization of a Mutant Escherichia coli Heat-Labile Toxin, LT(R192G/L211A), as a Safe and Effective Oral Adjuvant

Despite the fact that the adjuvant properties of the heat-labile enterotoxins of Escherichia coli (LT) and Vibrio cholerae (CT) have been known for more than 20 years, there are no available oral vaccines containing these molecules as adjuvants, primarily because they are both very potent enterotoxins. A number of attempts with various degrees of success have been made to reduce or eliminate the enterotoxicity of LT and CT so they can safely be used as oral adjuvants or immunogens. In this report we characterize the structural, enzymatic, enterotoxic, and adjuvant properties of a novel mutant of LT, designated LT(R192G/L211A), or dmLT. dmLT was not sensitive to trypsin activation, had reduced enzymatic activity for induction of cyclic AMP in Caco-2 cells, and exhibited no enterotoxicity in the patent mouse assay. Importantly, dmLT retained the ability to function as an oral adjuvant for a coadministered antigen (tetanus toxoid) and to elicit anti-LT antibodies. In vitro and in vivo data suggest that the reduced enterotoxicity of this molecule compared to native LT or the single mutant, LT(R192G), is a consequence of increased sensitivity to proteolysis and rapid intracellular degradation in mammalian cells. In conclusion, dmLT is a safe and powerful detoxified enterotoxin with the potential to function as a mucosal adjuvant for coadministered antigens and to elicit anti-LT antibodies without undesirable side effects. Bacterially derived enterotoxins, such as the heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) and the closely related cholera enterotoxin (CT) produced by Vibrio cholerae, promote secretory diarrhea during microbial infection. Each enterotoxin is composed of an enzymatically active A-subunit noncovalently associated with a pentameric B-subunit. Induction of intestinal fluid secretion occurs after a series of events involving both changes to toxin structure and activation of intracellular signaling pathways (reviewed in reference 14). The B-subunit mediates binding and internalization of the toxin to cells; subsequent proteolytic cleavage and disulfide bond reduction separate the A-subunit into two domains, the enzymatically active A1 subunit and a smaller A2 peptide. Transport of A1 into the cytoplasm results in ADP-ribosylation of Gs, followed by irreversible activation of adenylate cyclase and increases in intracellular levels of cyclic AMP (cAMP). In intestinal epithelial cells, this induction of cAMP causes a disregulation of cAMP-sensitive ion transport mechanisms, inhibiting intracellular salt absorption, increasing electrolyte transport into the gut lumen, and creating an osmotic gradient favoring intestinal water secretion (12). An enzymatically active A-subunit is required for intestinal fluid secretion but also enables LT to act as an oral adjuvant, boosting the immune response to coadministered antigens and inducing both humoral and cellular immune responses in both the systemic and mucosal compartments. However, even low doses of LT (and CT) induce side effects, such as diarrhea,