Development of alternatives to histamine sensitisation test for pertussis vaccines by in vitro biochemical and biological assays

Pertussis vaccines are commonly used worldwide for the prevention of whooping cough disease. Currently a significant number of animals are routinely used in the histamine sensitisation test (HIST) for control of pertussis toxin (PTx) presented in pertussis vaccines. This test is a lethal challenge test and large variations in test performance have been observed. This normally leads to repeating tests and large numbers of animals being used by manufacturers and control laboratories. Unfortunately the introduction of acellular pertussis vaccines has led to an increased use of HIST. Thus, there is an urgent need to develop an alternative to the HIST. We have developed an in vitro assay system based on a combination of E-HPLC for measurement of the enzymatic activity of the A-subunit and carbohydrate-binding assay which measures the B-subunits binding function of the toxin. Although preliminary study using the developed assay system for examination of limited number of vaccine batches showed promising results, it is far from reaching statistical power to be able to establish the correlation between the new in vitro assay system and the in vivo challenge test. Furthermore, most marketed products are now pertussis based combination vaccines and more antigen components in the combination vaccine formulations add further complications to the validation of the in vitro assay system against the HIST. It is unknown if the interaction between PTx and vaccine components/or formulation factors in the combination vaccine could also affect the outcome of HIST. In this application, we propose to investigate also the biological and physiological effects of PTx on a HUVEC cell system in particular in combination with other vaccine components/final formulation to establish the link between the biochemical activities of PTx found by E-HPLC/binding assays with the biological and physiological effects of PTx on cells. Further validation using a large number batches of different types of vaccine with the newly developed assaying system and establishment of the correlation between in vitro systems and the in vivo system using existed HIST data will be done. With the combined results obtained from the E-HPLC/binding assay, biological functional investigation on cell based assay and the in vivo HIST, we will set up product dependent specifications for the in vitro alternative assay system. If time permits, we would also like to organise a small scale collaborative study to further validate the new assay system. If successful, recommendations will be made to change European Pharmacopoeia and WHO guidelines.