Structural and immunological studies of pertussis toxin: effect of toxoiding

Fowler, Sarah (2002). Structural and immunological studies of pertussis toxin: effect of toxoiding. PhD thesis The Open University.



A detoxified form of pertussis toxin (PT) is used as a component of acellular pertussis vaccines, to prevent whooping cough in children. In this study five commercial PT toxoids, and a series of formaldehyde-treated (0 - 1.00 %, w/v) native and non-toxic mutant PT (PT-9K/129G) preparations were analysed, to investigate the effects of detoxification on the structure and immunogenicity of PT.

Native PT and a non-toxic mutant PT, compared using physico-chemical methods and molecular modelling, were found to be structurally comparable. Analysis of the PT toxoids demonstrated the variation in folding, conformation and subunit association induced by the different chemical methods of detoxification. A good murine IgG response to the toxoids was dependent on the maintenance of local structure such as the integrity of the protective SI epitope, but molecular aggregation did not appear to be detrimental. Sizing analysis of formaldehyde-treated mutant and native PT preparations, illustrated that both mutant and native PT molecules increased in size and heterogeneity, as a function of formaldehyde concentration. Immunoblotting of the formaldehydetreated preparations confirmed the presence of covalent inter-subunit cross-linking. Immunological analysis of formaldehyde-treated mutant PT preparations demonstrated that a small degree of aggregation may be beneficial for the immune response in mice, but that increasing amino acid modification, cross-linking and aggregation alters the type of immune response and causes a decrease in the neutralising antibodies. Toxicity assays demonstrated that formaldehyde detoxification of PT is due to modification of both the A- and the B-domain, and is subject to reversion. Evidence for antibody suppression and enhancement by different amounts of residual active PT was also obtained. The observations made here will help us to understand the complex relationships between antigen structure and immune response in vaccines containing PT.

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