DISSOCIATION OF ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN ADJUVANTICITY FROM ADP-RIBOSYLTRANSFERASE ACTIVITY

The heat-labile enterotoxin (LT) of Escherichia coli is immunologicall y and physiochemically related to cholera enterotoxin. A number of stu dies have been performed to determine the relationship of the ADP-ribo sylating enzymatic activity of these enterotoxins to toxicity and adju vanticity, These studies have generally examined the effect of abolish ing the ADP-ribosyltransferase activity of A(1) by a variety of chemic al or genetic manipulations. In every case, loss of enzymatic activity was associated with loss of biological activity and also with the abi lity of the molecules to function as oral adjuvants. Consequently, we explored an alternate approach to detoxification of LT without alterin g its adjuvanticity. Specifically, we generated a novel mutant form of LT by genetic modification of the proteolytically sensitive residues that join the A(1) and A(2) components of the A subunit. This mutant c ontains a single amino acid substitution within the disulfide subtende d region joining A(1) and A(2), This mutant toxin, designated LT((R192 G)), is not sensitive to proteolytic activation, has negligible activi ty on mouse Y-1 adrenal tumor cells, and is devoid of ADP-ribosyltrans ferase activity. Nonetheless, LT((R192G)) retains the ability to funct ion as a mucosal adjuvant, increasing the serum immunoglobulin G (IgG) and mucosal IgA responses to coadministered antigen (OVA) beyond that achieved with administration of that antigen alone, Further, LT((R192 G)) prevented the induction of tolerance to coadministered antigen and did not induce tolerance against itself, as demonstrated by the prese nce of significant serum anti-LT IgG and mucosal anti-LT IgA antibodie s in immunized mice,