Reporter cell lines for detection of pertussis toxin in acellular pertussis vaccines as a functional animal-free alternative to the in vivo histamine sensitization test

Abstract

Detoxified pertussis toxin (pertussis toxoid) is a major antigen in acellular pertussis vaccines. Testing these vaccines on the presence of residual pertussis toxin (PTx) and reversion to toxicity is performed by the regulatory required in vivo Histamine Sensitization test (HIST). Lack of mechanistic understanding of the HIST, technical handicaps and animal welfare concerns, have promoted the development of alternative methods. As the majority of the cellular effects of PTx depend on its ability to activate intracellular pathways involving cAMP, the in vitro cAMP-PTx assay was developed. Although this assay could be used to detect PTx activity, it lacked sensitivity and robustness for use in a quality control setting. In the present study, novel reporter cell lines (CHO-CRE and A10-CRE) were generated that stably express a reporter construct responsive to changes in intracellular cAMP levels. These reporter cell lines were able to detect PTx in a concentration-dependent manner when combined with fixed amounts of forskolin. The CHO-CRE cell line enabled detection of PTx in the context of a multivalent vaccine containing aP, with a sensitivity equal to the HIST. However, the sensitivity of the A10-CRE cells was insufficient for this purpose. The experiments also suggest that the CHO-CRE reporter cell line might be suitable for assessment of cellular effects of PTd reverted to PTx. The CHO-CRE reporter cell line provides a platform that meets the criteria for specificity and sensitivity and is a promising in vitro model with potential to replace the HIST.

iGIST - a kinetic bioassay for pertussis toxin based on its effect on inhibitory GPCR signaling

“…The copyright holder for this this version posted September 30, 2020. ; https://doi.org/10.1101/2020.09.29.318451 doi: bioRxiv preprint 13 iGIST bioassay detects PTX spiked into Boostrix pertussis acellular vaccine -The commonly acknowledged limitation of CFA 19 and other proposed animal-free bioassays to detect PTX 27,29 is their poor compatibility with the final vaccine product due to cytotoxicity of the aluminum-based adjuvants 17 . This problem is pending, despite that several approaches to mitigate adjuvant toxicity,…”

“…Isoproterenol binds to β-adrenergic receptors 28 , which leads to activation of Gαs and thereby to subsequent stimulation of the cAMP-producing ACs. In an extension of their work, Hoonakker et al detected PTX effects in A10 and CHO cells with a cAMP response element (CRE)driven luciferase reporter 29 . In agreement with their earlier cAMP ELISA study 27 , PTX did not increase the CRE-reporter activity by itself, but it did enhance cAMP responses to isoproterenol or forskolin (FSK) 29 .…”

“…In an extension of their work, Hoonakker et al detected PTX effects in A10 and CHO cells with a cAMP response element (CRE)driven luciferase reporter 29 . In agreement with their earlier cAMP ELISA study 27 , PTX did not increase the CRE-reporter activity by itself, but it did enhance cAMP responses to isoproterenol or forskolin (FSK) 29 . FSK activates ACs by intercalating the C1 and C2 subunits of ACs into the catalytically active cAMP-producing form 30 .…”

“…The CRE-reporter assay has a low ng/ml-range sensitivity for PTX 29 , comparable to CFA 19 , yet the question of its practical use in vaccine industry awaits further studies.…”

Assays for Determining Pertussis Toxin Activity in Acellular Pertussis Vaccines

“…They improved the system by using novel reporter cell lines (CHO-CRE and A10-CRE) that stably express a reporter construct that is responsive to cAMP levels. Both cell lines can detect PTx in a concentration-dependent manner but only the CHO-CRE cell line can detect PTx in a multivalent vaccine matrix at levels similar to HIST [147]. The sensitivity of this method has been reported as 0.2 IU/mL for pure PTx [112].…”