title: 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.
authors.affiliation: Institute for Translational Vaccinology (Intravacc), P.O. Box 457, 3720 AL Bilthoven, The Netherlands. Electronic address: Marieke.Hoonakker@intravacc.nl.
authors.forename: Marieke E
authors.initials: ME
authors.lastname: Hoonakker
authors.affiliation: Institute for Translational Vaccinology (Intravacc), P.O. Box 457, 3720 AL Bilthoven, The Netherlands.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Verhagen
authors.affiliation: Institute for Translational Vaccinology (Intravacc), P.O. Box 457, 3720 AL Bilthoven, The Netherlands.
authors.forename: Larissa
authors.initials: L
authors.lastname: van der Maas
authors.affiliation: Institute for Translational Vaccinology (Intravacc), P.O. Box 457, 3720 AL Bilthoven, The Netherlands.
authors.forename: Arjen
authors.initials: A
authors.lastname: Sloots
authors.affiliation: Institute for Translational Vaccinology (Intravacc), P.O. Box 457, 3720 AL Bilthoven, The Netherlands; Department of Animals in Science and Society, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3508 TD Utrecht, The Netherlands.
authors.forename: Coenraad F M
authors.initials: CFM
authors.lastname: Hendriksen
mesh_terms: D000818:Animals; D001681:Biological Assay; D016466:CHO Cells; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D018703:Founder Effect; D017930:Genes, Reporter; D006632:Histamine / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D037342:Pertussis Toxin / Q000032:analysis*; D010567:Pertussis Vaccine / Q000032:analysis / Q000737:chemistry*; D051381:Rats; D020218:Response Elements; D012680:Sensitivity and Specificity; D022282:Vaccines, Acellular
chemical_list: D010567:Pertussis Vaccine; D022282:Vaccines, Acellular; D005576:Colforsin; D006632:Histamine; D000242:Cyclic AMP; D008156:Luciferases; D037342:Pertussis Toxin
keywords: Acellular pertussis vaccine; Histamine sensitization test; In vitro alternative; Pertussis toxin