Development of the cAMP reporter assay to replace the in vivo safety test for acellular pertussis vaccines

Abstract: Pertussis toxin (PTx) is one of the major virulence proteins of Bordetella pertussis and can intoxicate immune cells. Since pertussis toxoid (PTd) - the detoxified form of PTx - contributes to protection, this toxoid is the key component of all acellular pertussis (aP) vaccines. Despite careful monitoring of the production and inactivation procedure, there is fear for the toxic effects of residual toxin and reversion to toxicity. To examine possible toxin content, each batch of vaccine is subjected to the in vivo Histamine Sensitization test (HIST). In the last decades its intrinsic limitations - including a lack of mechanistic understanding, technical handicaps and animal welfare concerns - have pushed the search for alternative methods.

A promising alternative method is based on PTx-induced clustered growth of CHO cells, though the manual reading and the limited capacity to discriminate between levels of clustering, hamper quantitative detection of PTx levels. On a cellular level, PTx primarily interferes with intracellular pathways that involve cAMP. Based on this phenomenon, we generated a CHO cell-based reporter line that stably expresses a reporter construct responsive to changes in intracellular cAMP levels. This cell line enables the detection of PTx in a concentration-dependent manner with a sensitivity well below the levels detected with the in vivo HIST. More importantly, the cell line can detect PTx in the context of an aluminium phosphate adjuvated aP multivalent vaccine, with a sensitivity equal to the HIST. In addition, preliminary results show that the cell line might allow assessment of the cellular effects caused by reversion of PTd to PTx.

These results demonstrate that the CHO reporter cell line enables simple, quantitative and concentration-dependent detection of PTx. The cell line-line based assay therefore offers a promising in vitro alternative to replace the suboptimal in vivo HIST and in vitro CHO clustering test.

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