The heat-labile enterotoxin (LT) of Escherichia coli is immunologically and physiochemically related to the cholera enterotoxin (CT) produced by Vibrio cholerae. A number of studies have been performed to determine the relationship of the ADP-ribosylating enzymatic activity of these enterotoxins to toxicity and adjuvanticity. These studies have generally examined the effect of abolishing the ADP-ribosyltransferase activity of A$\sb1$ by a variety of chemical or genetic manipulations. In every case, loss of enzymatic activity was associated with loss of biological activity and also with the ability of the molecules to function as oral adjuvants. Consequently, we explored an alternate approach to detoxification of LT without altering its adjuvanticity. Specifically, we generated a novel mutant form of LT by genetic modification of the proteolytically sensitive residues that join the A$\sb1$ and A$\sb2$ components of the A subunit. This mutant contains a single amino acid substitution within the disulfide subtended region joining A$\sb1$ and A$\sb2$. This mutant toxin, designated LT$\rm\sb{(R192G)}$, is not sensitive to proteolytic activation by trypsin, chymotrypsin or pepsin, is devoid of in vitro ADP-ribosyltransferase activity, retains a basal level of activity on mouse Y-1 adrenal tumor cells and is able to induce production of cyclic adenosine monophosphate (cAMP) by those cells. Importantly, this mutant retains the ability to function as a mucosal adjuvant, increasing the serum immunoglobulin G (IgG) and mucosal IgA responses to coadministered antigen above that achieved by administration of antigen alone. Moreover, LT$\rm\sb{(R192G)}$ prevented the induction of oral tolerance both to itself and to coadministered antigen as evidenced by the presence of significant serum and mucosal anti-LT antibodies in inoculated mice. In addition, LT$\rm\sb{(R192G)}$ is non-toxic in the patent mouse model and is capable of inducing anti-toxin immunity which protects mice against challenge with native toxin. Additionally, this mutant may be useful as a non-toxic oral adjuvant and as one component of a subunit vaccine to protect against enterotoxin mediated diarrheal diseases. The mutant has the ability to elicit the production of serum IgG and mucosal IgA against both itself and against antigens with which it is delivered, which raises the possibility of an effective immunization program for a variety of pathogens involving the simultaneous oral administration of LT$\rm\sb{(R192G)}$ with killed or attenuated organisms or with relevant antigens of a specific pathogen (Walker & Clements, 1993). The potential use of LT$\rm\sb{(R192G)}$ to enhance the immunogenicity of mucosally administered vaccines presents us with one promising means by which to facilitate mucosal immunization. This raises the possibility of an effective immunization program for a variety of pathogens involving the simultaneous oral administration of non-toxic LT$\rm\sb{(R192G)}$ with killed or attenuated organisms or with relevant antigens of a specific pathogen (Walker & Clements, 1993)
