Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants.

Abstract

A nontoxic mutant (LTK7) of the Escherichia coli heat-labile enterotoxin (LT) lacking ADP-ribosylating activity but retaining holotoxin formation was constructed. By using site-directed mutagenesis, the arginine at position 7 of the A subunit was replaced with lysine. This molecule, which was nontoxic in several assays, was able to bind to eukaryotic cells and acted as a mucosal adjuvant for co-administered proteins; BALB/c mice immunized intranasally with LTK7 and ovalbumin developed high levels of serum and local antibodies to ovalbumin and toxin. In addition, mice immunized intranasally with fragment C of tetanus toxin and LTK7 were protected against lethal challenge with tetanus toxin. Thus nontoxic mutants of heat-labile toxin can act as effective intranasal mucosal adjuvants.

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

“…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.…”

“…Due to its enhanced thermostability, the mutant toxin might have implications for optimal design of cholera vaccines that are based upon the presence of the LT-I or CT holotoxin. Furthermore, introduction of this stabilizing disulfide into the A subunit may be combined with the introduction of other mutations that reduce or abolish toxicity while maintaining potent activity as a mucosal adjuvant (Dickinson & Clements, 1995;Douce et al, 1995;Rappuoli et al, 1996).…”

The mucosal adjuvanticity of two nontoxic mutants of Escherichia coli heat-labile enterotoxin varies with immunization routes

“…Mutation of amino acids in the NAD binding sites (residues 58-76) or active sites (residues 7, 44, 61, 110 and 112) of the A subunit of cholera toxin resulted in inactivation of the ADP-ribosyltransferase activity and loss of toxicity in Y1 cells (Fontana et al, 1995). Similar point mutations in the A subunit of E. coli enterotoxin (LTK7, LTK63, LTR72, LTE110D, LTE112D, LTE112K and LTR192G) have produced inactive forms of LT (Tsuji et al, 1990;Lobet et al, 1991;Tsuji et al, 1991;Pizza et al, 1993;Grant et al, 1994;Dickinson et al, 1995;Douce et al, 1995;Giuliani et al, 1998). However, substitution of Ala to His or Glu at position 72 resulted in a protein with a toxicity indistinguishable from that of wild-type LT, whereas Ala to Arg substitution at the same position greatly reduced toxicity .…”

“…However, the highly-toxic nature of these proteins has precluded their clinical use in humans. Attempts have been made to overcome this problem with LT (Lobet et al, 1991;Douce et al, 1995;Di Tommaso et al, 1996;Marchetti et al, 1998) and with CT (Douce et al, 1997;Yamamoto et al, 1997a;Yamamoto et al, 1997b), using mutagenesis aimed at neutralization of toxicity without compromising immunogenicity. Mutation of amino acids in the NAD binding sites (residues 58-76) or active sites (residues 7, 44, 61, 110 and 112) of the A subunit of cholera toxin resulted in inactivation of the ADP-ribosyltransferase activity and loss of toxicity in Y1 cells (Fontana et al, 1995).…”

Development of two novel nontoxic mutants of Escherichia coli heat-labile enterotoxin

“…However, their toxi-cities have precluded usages in human (Douce et al, 1995). One approach to overcome the problem of toxicity is the generation of genetically detoxified derivatives of LT (Lobet et al ., 1991;Dickinson and Clements, 1995) and CT (Fontana et al, 1995;Yamamoto et al ., 1997b) by a site-directed mutagenesis of amino acids which are located on the β-strand that constitutes the 'floor' of NAD-binding cavity.…”