Synthesis of hybrid molecules between heat-labile enterotoxin and cholera toxin B subunits: potential for use in a broad-spectrum vaccine (original) (raw)
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Infection and immunity, 1999
Vibrio cholerae secretes cholera toxin (CT) and the closely related heat-labile enterotoxin (LT) of Escherichia coli, the latter when expressed in V. cholerae. Both toxins are also potent immunoadjuvants. Mutant LT molecules that retain immunoadjuvant properties while possessing markedly diminished enterotoxic activities when expressed by E. coli have been developed. One such mutant LT molecule has the substitution of a glycine residue for arginine-192 [LT(R192G)]. Live attenuated strains of V. cholerae that have been used both as V. cholerae vaccines and as vectors for inducing mucosal and systemic immune responses directed against expressed heterologous antigens have been developed. In order to ascertain whether LT(R192G) can act as an immunoadjuvant when expressed in vivo by V. cholerae, we introduced a plasmid (pCS95) expressing this molecule into three vaccine strains of V. cholerae, Peru2, ETR3, and JRB14; the latter two strains contain genes encoding different heterologous an...
Gene, 1991
A simple and effective electroporation method for the transformation of Vibrio cholerae with nonmobilizable plasmids is described. Expression plasmids directing the synthesis of fusion proteins with the subunit B of Escherichiu coli heat-labile enterotoxin B (LT-B) were transformed into nontoxinogenic V. cholerue vaccine strains. A protein consisting of two overlapping immunodominant antibody-binding sites of the hepatitis B virus (HBV) middle surface antigen fused to the C terminus of full-length LT-B was secreted into the supematant of I/. cholerae cultures, whereas two other LT-B/HBV fusion proteins were mostly retained within the cells or rapidly degraded in the culture supernatant.
Protein Expression and Purification, 2002
Cholera toxin B subunit (CTB) has been extensively studied as immunogen, adjuvant, and oral tolerance inductor depending on the antigen conjugated or coadministered. It has been already expressed in several bacterial and yeast systems. In this study, we synthesized a versatile gene coding a 6XHis-tagged CTB (359 bp). The sequence was designed according to codon usage of Escherichia coli, Lactobacillus casei, and Salmonella typhimurium. The gene assembly was based on a polymerase chain reaction, in which the polymerase extends DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was amplified, cloned, and expressed in E. coli in an insoluble form, reaching levels about 13 mg of purified active pentameric rCTB per liter of induced culture. Western blot and ELISA analyses showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodies against the cholera toxin. The ability to form the functional pentamers was observed in cell culture by the inhibition of cholera toxin activity on Y1 adrenal cells in the presence of recombinant CTB. The 6XHis-tagged CTB provides a simple way to obtain functional CTB through Ni 2þ -charged resin after refolding and also free of possible CTA contaminants as in the case of CTB obtained from Vibrio cholerae cultures. Ó
Intranasal Immunogenicity and Adjuvanticity of Site-Directed Mutant Derivatives of Cholera Toxin
Infection and Immunity, 1997
Genetically modified derivatives of cholera toxin (CT), harboring a single amino acid substitution in and around the NAD binding cleft of the A subunit, were isolated following site-directed mutagenesis of the ctxA gene. Two mutants of CT, designated CTS106 (with a proline-to-serine change at position 106) and CTK63 (with a serine-to-lysine change at position 63), were found to have substantially reduced ADP-ribosyltransferase activity and toxicity; CTK63 was completely nontoxic in all assays, whereas CTS106 was 10 4 times less toxic than wild-type CT. The mucosal adjuvanticity and immunogenicity of derivatives of CT were assessed by intranasal immunization of mice, with either ovalbumin or fragment C of tetanus toxin as a bystander antigen.
Production of Pentameric Cholera Toxin B Subunit in Escherichia coli
Avicenna Journal of Medical Biotechnology, 2012
Cholera toxin B subunit (CTB) has been extensively studied as an immunogen, adjuvant, and inducer of oral tolerance in many investigations. Production of CTB has been carried out in the bacterial, plant, insect and yeast expression systems. In this study the expression of the CTB containing a 6XHis-tagged was performed by Escherichia coli (E.coli) M15. The yield of purified pentameric recombinant CTB was about 1 mg/l. Western blot analysis demonstrated that the recombinant CTB was antigenically active. In addition, GM1-ganglioside ELISA showed that recombinant CTB binds to GM1-gangelioside receptor, confirming disulfide bond formation and proper folding of the recombinant protein in E.coli. Overall, in regard to the vast applications of CTB in medicine, this bacterial expression system will be a fast, cost-effective and simple system for production of pentameric CTB and CTB conjugated proteins.
Infection and Immunity, 2001
Although cholera toxin (Ctx) and Escherichia coli heat-labile enterotoxin (Etx) are known to be potent mucosal adjuvants, it remains controversial whether the adjuvanticity of the holotoxins extends to their nontoxic, receptor-binding B subunits. Here, we have systematically evaluated the comparative adjuvant properties of highly purified recombinant EtxB and CtxB. EtxB was found to be a more potent adjuvant than CtxB, stimulating responses to hen egg lysozyme when the two were coadministered to mice intranasally, as assessed by enhanced serum and secretory antibody titers as well as by stimulation of lymphocyte proliferation in spleen and draining lymph nodes. These results indicate that, although structurally very similar, EtxB and CtxB have strikingly different immunostimulatory properties and should not be considered equivalent as prospective vaccine adjuvants.
Production of cholera toxin subunit B by a mutant strain of Vibrio cholerae
Applied Microbiology and Biotechnology, 1990
The B subunit (CTB) of cholera toxin (CT) can be used as a carrier protein for conjugate vaccines designed to elicit antipolysaccharide antibodies. A defined medium, AGM4, was designed to grow a highproducing mutant of Vibrio cholerae expressing only the B subunit of CT: V. cholerae 0395-NI. AGM4 contains four amino acids, asparagine, glutamic acid, arginine and serine, salts and a trace element solution. The carbon source is glucose. The fermentations performed in AGM4 indicated that CTB production paralleled the growth of the organism but that there was a maximal release of CTB during the stationary phase. There was a clear optimum of productivity at pH 8.0 and 30 ° C. The pH had an influence on CTB production and not only on its release. Analysis of the amino acids present in the medium showed a correlation between their consumption rates and CTB productivity.
Infection and immunity, 1995
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 AB5 structure and were nontoxic; these proteins were CT-D53 (Val-53-->Asp), CT-K63 (Ser-63-->Lys), CT-K97 (Val-97-->Lys), CT-K104 (Tyr-104-->Lys), CT-S106 (Pro-106-->Ser), and the double mutant CT-D53/K63 (Val-53-->Asp, Ser-63-->Lys). Two of the mutations encoded proteins that were assembled into the AB5 structure but were still toxic; these proteins were CT-H54 (Arg-54-->His) and CT-N107 (His-107-->Asn). Finally, one of the mutant proteins, CT-E114 (Ser-114-->Glu), was unable to assemble the A and the B subunits and produced only the B oligomer. The six nontoxi...
Fine Epitope Mapping of the Vibrio cholera Toxins A, B, and P and an ELISA Assay
Oral immunization with the choleric toxin (CT) elicits a high level of protection against its enterotoxin activities and can control cholera in endemic settings. However, the complete B-cell epitope map of the CT responsible for protection remains to be clarified. Here, we have mapped the B-cell linear epitopes of the three chains of the CT protein (CTP) prepared by Spot synthesis. The immunoreactivity of sera from mice immunized with an oral, inactivated vaccine (Schankol†™) was measured against membrane-bound peptides for mapping. Results: Eighteen IgG epitopes were identified; eight in the CTA, three in the CTB, and seven in the protein P. Three epitopes, TQTGFVRHDDGYVST (aa 66-77, Vc/TxA-3), KNGAIFQVE VPGSQN (aa 64-78, Vc/TxB-11), and LNDEHK (aa 90-95, Vc/TxP-16), were chosen to synthesize a multiple antigen peptide that was used to coat ELISA plates to screen immunized mouse sera as a test for an in vitro diagnostics for cholera. Conclusion: Vaccination with inactive CT-generat...