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J. Biol. Chem., Vol. 277, Issue 36, 33369-33377, September 6, 2002

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Detoxification of Cholera Toxin without Removal of Its Immunoadjuvanticity by the Addition of (STa-related) Peptides to the Catalytic Subunit
A POTENTIAL NEW STRATEGY TO GENERATE IMMUNOSTIMULANTS FOR VACCINATION^*

Joaquín Sánchez^§¶, Gun Wallerström, Margareta Fredriksson, Jonas Ångström, and Jan Holmgren

From the  Department of Medical Microbiology and Immunology, Göteborg University and the Göteborg University Vaccine Research Institute, Guldhedsgatan 10A, Göteborg SE 413 46, Sweden, the ^§ Facultad de Medicina, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, Colonia Chamilpa, Cuernavaca, Morelos, CP62210, Mexico, and the  Institute of Medical Biochemistry, Göteborg University, P. O. Box 440, Göteborg SE 405 30, Sweden

Peptides related to the heat-stable enterotoxin STa were fused to the N terminus of the A-subunit of cholera toxin (CTA) to explore whether peptide additions could help generate detoxified cholera toxin (CT) derivatives. Proteins carrying APRPGP (6-CTA), ASRCAELCCNPACPAP (16-CTA), or ANSSNYCCELCCNPACTGCYPGP (23-CTA) were genetically constructed. Using a two-plasmid system these derivatives were co-expressed in Vibrio cholerae with cholera toxin B-subunit (CTB) to allow formation and secretion of holotoxin-like molecules (engineered CT, eCTs). Purified eCTs maintained all normal CT properties yet they were more than 10-fold (eCT-6), 100-fold (eCT-16), or 1000-fold (eCT-23) less enterotoxic than wild-type CT. The inverse correlation between enterotoxicity and peptide length indicated sterical interference with the ADP-ribosylating active site in CTA. This interpretation agreed with greater than 1000-fold reductions in cAMP induction, with reductions, albeit not proportional, in in vitro agmatine ADP-ribosylation, and was supported by molecular simulations. Intranasal immunization of mice demonstrated that eCTs retained their inherent immunogenicity and ability to potentiate immune responses to a co-administered heterologous protein antigen, although in variable degrees. Therefore, the addition of STa-related peptides to CTA reduced the toxicity of CT while partly preserving its natural immunoadjuvanticity. These results suggest peptide extensions to CTA are a useful alternative to site-directed mutagenesis to detoxify CT. The simplicity of the procedure, combined with efficient expression and assembly of derivatives, suggests this approach could allow for large scale production of detoxified, yet immunologically active CT molecules.

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