Investigation of pertussis toxin A- and B-subunit activities in acellular vaccines by enzymatic and carbohydrate-binding assays

Pertussis toxin (PT) is a major virulence factor produced by Bordetella pertussis. In its detoxified form (PTd), it is an important component of acellular pertussis vaccines although some residual FT activity may be present because of the limitations of the detoxification processes used. The in vivo histamine sensitisation test (HIST) in mice is currently used for the safety testing of these vaccines to determine the level of their residual FT activity. However, an alternative test is needed because of large assay variability and ethical concerns with regard to animal usage. The main objective of this study was to search for an alternative test to the HIST. The ADP-ribosylation enzyme activity of FT is thought to be the major factor responsible for the histamine-sensitising activity detected in vivo. In the present study, the enzymatic activities in different acellular pertussis-based combination vaccine formulations were measured by a recently-developed ADF-ribosylation assay and compared with their reactivities in the HIST. The results indicated that different products showed differences in ADP-ribosylation activity and, these did not correlate with their reactivity in the HIST. FT has two functionally-distinct domains: the enzymatic A-protomer and the B- oligomer that facilitates host-cell binding and entry of FT into the cell. This dual biological function could explain why the residual enzyme activity of FT in vaccines did not fully reflect the in vivo reactivity observed by the HIST. Thus, refinement of the in vitro test to include a step which monitored the B-subunit activity of FT was attempted. A quantitative FT carbohydrate-binding assay using glycoproteins or defined oligosaccharides was developed. PT was found to bind preferentially to multiantennary N-glycans, with the highest binding towards the fully sialylated structures. In contrast, PTd lost the ability to bind to sialylated multiantennary structures but retained some capacity to bind to neutral multiantennary structures. Different vaccine preparations had different levels of PT binding activity as well as enzymatic activity. It was concluded that, although the enzymatic activity of PT plays a more important role in the death of mice in the HIST, a high binding activity of the B- subunit could increase the in vivo toxic effect by aiding the accessibility of the A- subunit to its cellular targets. A mathematical equation was devised to establish a preliminary relationship between the enzymatic, carbohydrate-binding and HIST assays in a product-dependent manner. Further studies with a larger number of vaccines are required for a more meaningful statistical analysis. However the methods form a sound basis for the future development of an alternative assay to the histamine challenge test. The in vitro assays could also be useful for investigating the mechanisms of PT detoxification. Comparisons of A- and B-subunit activities of purified PT and vaccine preparations of PTd indicated that both subunits are modified after chemical detoxification. Different vaccine products had different levels of enzymatic and binding activities and it was concluded that different detoxification procedures, as well as formulation factors, could contribute to this variation. A CHO cell clustering assay is used as an alternative in vitro test to the HIST for assessing residual PT activity at the bulk stage of vaccine production. In a parallel study to the above, comparative proteomics was used to gain insights into the mechanism of PT-induced CHO cell clustering with a view to developing a mechanistic-based alternative assay for the safety testing of pertussis-based combination vaccines. A proteomic map of CHO cells was established and PT-induced CHO cell clustering appeared to be a complex process involving subtle changes in various cellular functions, mainly related to intracellular transport, cell stress and the cell cycle. The information obtained will be useful for future studies into the possible mechanisms of the effect of PT on CHO cells.