title: Development of an approach for the laboratory toxicological evaluation of Bordetella pertussis adenylate cyclase genetic toxoid constructs as multipurpose vaccines.
abstract: Detoxified recombinant CyaA constructs have been developed as potential viral and tumoral epitope carriers for immunoprophylactic and therapeutic vaccination and as antigen candidates for inclusion in acellular pertussis vaccines. In this work, we attempt to explore a test system for the laboratory safety evaluation of CyaA preparations. Endotoxin was determined in vitro by the Limulus amoebocyte lysate assay. Cytotoxicity was measured by a tetrazolium salt test and a lactate dehydrogenase release assay using murine and human phagocytic cell lines. Cell viability was < 50% at concentrations > 1 microg/mL of the wild type toxin and concentrations in the nanogram range inhibited the zymosan-induced oxidative burst as measured by chemiluminescence. However, no effects were observed for detoxified and non-acylated forms of CyaA at comparable and higher toxin concentrations. Effects found in the in vitro assays could not be related to the in vivo mouse weight gain test used as a general toxicity test. In vitro cytotoxicity and oxidative burst studies on phagocytes, and the evaluation of endotoxin levels are useful for safety screening of CyaA constructs. However, in vivo specific toxicity tests and potential toxic implications of enzymatic activity-independent effects of CyaA on cell function should be investigated.
authors.affiliation: Division of Bacteriology, National Institute for Biological Standards and Control (NIBSC), South Mimms, Potters Bar, Hertfordshire, UK. sprior@nibsc.ac.uk
authors.forename: Sandra
authors.initials: S
authors.lastname: Prior
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Corbel
authors.forename: Dorothy K L
authors.initials: DK
authors.lastname: Xing
mesh_terms: D000166:Acridines; D000262:Adenylyl Cyclases / Q000235:genetics* / Q000276:immunology*; D000818:Animals; D001886:Bordetella pertussis / Q000201:enzymology / Q000235:genetics* / Q000276:immunology*; D002478:Cells, Cultured; D004731:Endotoxins / Q000737:chemistry / Q000276:immunology; D005260:Female; D005434:Flow Cytometry; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008033:Limulus Test; D049449:Luminescence; D051379:Mice; D008809:Mice, Inbred C3H; D010567:Pertussis Vaccine / Q000276:immunology*; D010586:Phagocytes / Q000276:immunology / Q000502:physiology; D016897:Respiratory Burst / Q000187:drug effects / Q000502:physiology; D013329:Structure-Activity Relationship; D013778:Tetrazolium Salts; D013844:Thiazoles; D014121:Toxoids / Q000235:genetics* / Q000276:immunology*; D015430:Weight Gain / Q000187:drug effects
chemical_list: D000166:Acridines; D004731:Endotoxins; D010567:Pertussis Vaccine; D013778:Tetrazolium Salts; D013844:Thiazoles; D014121:Toxoids; C033472:10,10'-dimethyl-9,9'-biacridinium; D007770:L-Lactate Dehydrogenase; D000262:Adenylyl Cyclases; C022616:thiazolyl blue