Heat-labile enterotoxin (LT), produced by enterotoxigenic strains of E. coli, and cholera toxin (CT), produced by V. cholerae strains, are the causative agents of traveller's diarrhoea and cholera, respectively [Spangler (1992) Microbiol Rev 56:622]. LT and CT show 80% homology in the primary structure and an identical tertiary structure. They are composed of two functionally distinct domains: the enzymatically-active A subunit and the B pentamer, which contains the receptor-binding site. The A subunit ADP-ribosylates the target protein Gs, a GTP-binding protein which regulates the intracellular levels of cAMP [Rappuoli & Pizza (1991), in Sourcebook of bacterial protein toxins, Academic Press NY]. Enhancement in cAMP levels can alter ion transport, inducing secretion of water and chloride ions into the intestine.
CT and LT are both powerful immunogens and potent mucosal adjuvants when co-administered with antigens at the mucosal level [eg. Jackson et al. (1993) Infect Immun 61:4272; WO95/17211]. Immunogenicity and adjuvanticity of wild-type CT and LT have been extensively studied in animals [eg. Rollwagen et al. (1993) Vaccine 11:1316], but their toxicity has precluded their use in humans. In an attempt to overcome the problems generated by the use of active holotoxins, two different approaches have been followed, one based on the use of the B pentamer, the non-toxic domain of the holotoxin [eg. Holmgren et al. (1992) Vaccine10:911], and the other based on the generation of genetically detoxified derivatives of LT and CT [eg. Fontana et al. (1995) Infect Immun 63:2356]. Site-directed mutagenesis on both A and B subunits has provided a tool to explore the basis of the immunological and adjuvant responses induced by these molecules.
Examples of such experiments can be found in:                Harford et al. [Eur J Biochem (1989) 183:311] made LT-A carrying Ser-61-Phe and Gly-79-Lys substitutions.        Tsuji et al. [J Biol Chem (1990) 265:22520] produced LT-A carrying a Glu-112-Lys substitution.        Burnette et al. [Infect Immun (1991) 59:4266] produced CT-A carrying a Arg-7-Lys substitution. This work can also be seen in WO92/19265.        WO93/13202 described non-toxic CT and LT carrying mutations at Val-53, Ser-63, Val-97, Tyr-104, and Pro-106        
A mutant in the receptor binding site of the B subunit of LT, the G33D mutant, has been reported to lack the immunological properties of the native B subunit [Nashar et al. (1996) PNAS 93:226], suggesting that binding to the receptor is important for the immunogenicity. It has also been shown that non-toxic derivatives of LT carrying mutant A subunits retain the immunological properties of wild-type LT [eg. Magagnoli et al. (1996) 64:5434], suggesting that ADP-ribosylation activity is not essential for immunogenicity.
In relation to adjuvanticity, much data has been generated but many questions remain unanswered. Some studies have suggested that LT-B and CT-B have adjuvant activity, but the conclusions drawn have been compromised by contamination with active toxin [Wilson et al. (1993) Vaccine 11:113]. Studies with recombinant LT-B and CT-B have suggested that they behave as poor mucosal adjuvants [eg. Douce et al. (1997) Infect. Immun. 65:2821]
Attempts to define the role of ADP-ribosylation activity in the adjuvanticity of LT has generated conflicting results. Lycke et al. [Eur J Immunol (1992) 22:2277] have described a non-toxic LT derivative (LT-E112K) which, when administered with KLH by oral route in mice, lacked the adjuvant properties of wild-type LT, thus suggesting that adjuvant activity is linked to ADP-ribosylation activity. LT derivatives such as LT-K7 and LT-K63 [eg. Douce et al. (1997) supra; Douce et al. (1996) PNAS 92:1644; DiTommaso et al. (1996) Infect Immun 64:974], however, which are devoid of toxicity both in vitro and in vivo, have been shown to be able to elicit an antibody response against a co-administered antigen in intranasally immunised mice. LT-K63 has been shown to induce measle-specific CTL response after intranasal immunisation with a synthetic peptide [Partidos et al. (1996) Immunol 89:483], and strongly enhances protection against H. pylori following intragastric immunisation with H. pylori antigens. The antibody titres induced by these non-toxic LT mutants were lower than those obtained with wild-type toxin, however, and were only detected after two mucosal immunisations [Douce et al. (1997) supra].
It is an object of the invention to provide forms of LT which are detoxified, so that they might be suitable for use in humans, but which retain the adjuvant and immunogenic properties of LT as far as possible.