Modifications of the catalytic and binding subunits of pertussis toxin by formaldehyde: effects on toxicity and immunogenicity

A panel of pertussis toxin (PT) preparations with varying levels of residual toxicity was prepared by treatment of native PT with formaldehyde (0-1.00% (w/v)) with the purpose of investigating the effects of residual toxicity on immunogenicity. The catalytically inactive mutant PT (PT-9K/129G) was used for comparison. Results from in vitro ADP-ribosyl transferase and Chinese hamster ovary (CHO)-cell toxicity assays demonstrated a formaldehyde-dependent reduction in PT toxicity, and implied that both A and B domain functions of PT were modified. The in vivo histamine sensitisation and leukocyte proliferation tests suggested that the formaldehyde-treated native PT preparations were subject to reversion to toxicity. Reversion was confirmed by in vitro toxicity assays, which demonstrated recovery of A and B domain functions. The presence of high molecular weight aggregated and cross-linked species of PT in these preparations did not appear to be detrimental to the production of a neutralising antibody response. IgG responses to native and non-catalytic mutant PT suggested that low levels of residual activity in the native PT enhanced the antibody response, while higher levels of activity inhibited the response. Using the non-catalytic mutant PT showed that formaldehyde-induced changes were not detrimental to the magnitude of the PT-specific antibody response but did reduce the PT-specific neutralising activity. In conclusion, the residual toxicity of PT preparations following formaldehyde treatment may play an important role in the immune response to pertussis vaccine, potentially altering the quality, class and magnitude of the antibodies produced to PT. (C) 2003 Elsevier Science Ltd. All rights reserved.