Construction of nontoxic derivatives of cholera toxin and characterization of the immunological response against the A subunit

Abstract

Using computer modelling, we have identified some of the residues of the A subunit of cholera toxin (CT) and heat-labile toxin that are involved in NAD binding, catalysis, and toxicity. Here we describe the site-directed mutagenesis of the CT gene and the construction of CT mutants. Nine mutations of the A subunit gene were generated. Six of them encoded proteins that were fully assembled in the AB 5 structure and were nontoxic; these proteins were CT-D53 (Val-533Asp), CT-K63 (Ser-633Lys), CT-K97 (Val-973Lys), CT-K104 (Tyr-1043Lys), CT-S106 (Pro-1063Ser), and the double mutant CT-D53/K63 (Val-533Asp, Ser-633Lys). Two of the mutations encoded proteins that were assembled into the AB 5 structure but were still toxic; these proteins were CT-H54 (Arg-543His) and CT-N107 (His-1073Asn). Finally, one of the mutant proteins, CT-E114 (Ser-1143Glu), was unable to assemble the A and the B subunits and produced only the B oligomer. The six nontoxic mutants were purified from the culture supernatants of recombinant Vibrio cholerae strains and further characterized. The CT-K63 mutant, which was the most efficient in assembly of the AB 5 structure, was used to immunize rabbits and was shown to be able to induce neutralizing antibodies against both the A and B subunits. This molecule may be useful for the construction of improved vaccines against cholera.

Intranasal Immunization of Mice with CpG DNA Induces Strong Systemic and Mucosal Responses That Are Influenced by Other Mucosal Adjuvants and Antigen Distribution

“…Here we found CTB to have weak adjuvant activity, but this might be due to contamination with residual holotoxin, since CTB purified from native CT was used (30,31). Likewise, with genetically detoxified mutants of CT and LT (7)(8)(9)(10)(11)(12)(13)(14)(15)(16), the importance of residual enzymatic activity is not clear. In some studies, mutants lacking enzymatic activity fail to generate antibody responses against coadministered antigen (3); whereas, in other studies they appeared to retain the adjuvant properties of the native toxin (10,13,32,33).…”

“…Two strategies have been proposed to circumvent the toxicity of the native toxins: (i) the use of the non-toxic B subunits that lack enzymatic activity (5,6); or (ii) genetically detoxified mutants of CT or LT that have little or no remaining enzymatic activity. Such compounds, which have greatly reduced toxicity, retain some adjuvanticity in animal models (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). The efficacy of these new adjuvants has yet to be proven in humans.…”

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 .…”

Protein engineering studies of A‐chain loop 47‐56 of Escherichia coli heat‐labile enterotoxin point to a prominent role of this loop for cytotoxicity

“…5. Substitutions of residues Arg-7 (Burnette et a/., 1991;Lobet eta/., 1991), Asp-9 (Pizza eta/., 1994), Ser-61 (Harford eta/., 1989), Ser-63 (Fontana et a/., 1995), and Glu-112 (Tsuji eta/., 1991) have resulted in lossof catalytic activity. The postulated binding mode of NAD in LT demonstrates the need for loop 47-56 to be displaced in order to accommodate the substrate in the active site.…”