Novel configurations of high molecular weight species of the pertussis toxin vaccine component.
Pertussis toxin (PT) is used in its formaldehyde-detoxified form in acellular pertussis vaccines for preventing whooping cough in children. The effects of formaldehyde treatment (up to 0.5% (w/v) formaldehyde) on the size, molecular association, folding and monoclonal antibody (mAb) binding of PT were studied to further define the structural nature of the high molecular weight species as related to their epitope integrity. Analytical ultracentrifugation (AUC) demonstrated that formaldehyde treatment of PT prevented the dissociation of the holotoxin. Together with results from size exclusion chromatography (SEC), SEC/multi-angle laser light scattering (MALLS) and immunoblotting it was demonstrated that PT increased in molecular weight and heterogeneity as a function of formaldehyde concentration, caused at least in part by covalent cross-linking. Five mAbs specific for PT subunits (S1-S5) bound to the cross-linked species, although there was some loss of epitopes in the larger aggregates. Intrinsic fluorescence spectroscopy gave evidence of progressive unfolding and re-association of PT. These findings demonstrate that a favourable balance between protein stabilisation and denaturation may be achieved by the treatment of pertussis toxin with formaldehyde, and provides a basis for determining the significance of high molecular weight cross-linked species of pertussis toxin in protection against whooping cough.
Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK.
Sarah
S
Fowler
Olwyn
O
Byron
Kornelia
K
Jumel
Dorothy
D
Xing
Michael J
MJ
Corbel
Barbara
B
Bolgiano
D019943:Amino Acid Substitution; D019540:Area Under Curve; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005557:Formaldehyde / Q000494:pharmacology; D008027:Light; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D037342:Pertussis Toxin / Q000235:genetics / Q000276:immunology*; D010567:Pertussis Vaccine / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence
D003432:Cross-Linking Reagents; D010567:Pertussis Vaccine; D005557:Formaldehyde; D037342:Pertussis Toxin