Mutants of the Escherichia coli heat-labile enterotoxin with reduced ADP-ribosylation activity or no activity retain the immunogenic properties of the native holotoxin

Abstract

The Escherichia coli heat-labile enterotoxin (LT) is a potent inducer of mucosal immune responses. In a previous study (L. DeHaan, W. R. Verweij, M. Holtrop, E. Agsteribbe, and J. Wilschut, Vaccine 14:620-626, 1996), we have shown that efficient induction of an LTB-specific mucosal immune response by LT requires the presence of the LTA chain, suggesting a possible role of the enzymatic activity of LTA in the induction of these responses. In the present study, we generated LT mutants with altered ADP-ribosylation activities and evaluated their immunogenicity upon intranasal administration to mice. The results demonstrate that the mucosal immunogenicity of LT is not dependent on its ADP-ribosylation activity.

NALT- versus PEYER'S-patch-mediated mucosal immunity

“…Among the various candidates for mucosal antigen delivery, Sendai-virus-associated fusion protein seems particularly suited to function as a molecule that of cholera toxin (nCT) and heat-labile enterotoxin (nLT) cause severe diarrhoea and so are unsuitable for use in humans. To overcome these hurdles, researchers have substituted a single amino acid to generate non-toxic mutant forms of cholera toxin (mCT) and heat-labile enterotoxin (mLT) [85][86][87][88] ; these retain the adjuvanticity of the native forms but do not induce the ribosylation of ADP that is associated with toxic activity. Our efforts to devise a safe first generation toxin-based adjuvant have focused on mCT S61F (in which phenylalanine replaces serine at position 61) and mCT E112K (in which lysine replaces glutamic acid at position 112); these mutations were created by making a single amino-acid substitution in the active centre of the ADP-ribosyltransferase in the A subunit of cholera toxin 89 .…”

Mutational analysis of the role of ADP-ribosylation activity and GM1 -binding activity in the adjuvant properties of theEscherichia coli heat-labile enterotoxin towards intranasally administered keyhole limpet hemocyanin

“…LT mutants LT-T50G (LTA: Thr50 1 Gly) and LT-E112K (LTA: Glu112 1 Lys) were constructed, expressed and purified as previously described [22,23]. Both mutants retained affinity for the toxin receptor G M1 (Table 1, see also [22]).…”

“…In addition, these mutants failed to stimulate intracellular cAMP levels in a cAMP accumulation test. However, in contrast to LT-E112K, the T50G mutant retained the capacity of wild-type LT to change chinese hamster ovary (CHO) cell morphology [23,24], suggesting that LT-T50G had retained some enzymatic activity. Therefore, we now reexamined the enzymatic activity of these mutants in a refined DEABAG assay, as described by Soman et al [25].…”

“…LTB, LT, LTA mutants, LT-T50G (LTA: Thr50 1 Gly) and LT-E112K (LTA: Glu112 1 Lys), LTB mutants LTB-G33D (Gly33 1 Asp) and LT-G33D, double mutant LT-E112K/ G33D (LTA: Glu112 1 Lys; LTB: Gly33 1 Asp), a recombinant His-tagged form of LTA, LTA(His) 10 , and a His-tagged LTA mutant, LTA-E112K(His) 10 , were constructed, expressed and purified as described previously [23,24,40] (DeHaan et al, submitted). Protein pools were dialyzed against PBS and stored at 4°C.…”

Crystal structure of heat‐labile enterotoxin from Escherichia coli with increased thermostability introduced by an engineered disulfide bond in the A subunit

“…The current focus of engineering vaccines against LT-I or CT is to use the complete toxin including the A subunit, preferably inactivated to eliminate its toxic activity in the gut. Recently, several groups were able to remove the toxic activity of LT or CT by site-directed mutagenesis in the A subunit while retaining the adjuvant activity of the ABS hetero-hexamer (Dickinson & Clements, 1995;Douce et al, 1995;de Haan et al, 1996;Rappuoli et al, 1996). One potential disadvantage of vaccines containing these toxins is that, as the name heat-labile enterotoxin implies, the ABs holotoxin is not thermostable.…”