title: Quantitative detection of RT activity by PERT assay: feasibility and limits to a standardized screening assay for human vaccines.
abstract: The detection of adventitious retroviruses has always been critical for assessing the safety concerns associated with viral vaccines. Assays for the enzymatic activity of reverse transcriptase (RT) are used as general methods for the detection of both known and unknown retroviruses. Several studies using newly-developed ultrasensitive PCR-based RT assays reported RT activity in viral vaccines grown in chicken cells. Here, we have assessed the performances of such a PCR-based RT assay--PERT assay--for the quantitative detection of RT activity in vaccines. Sensitivity, linearity and reproducibility of the method were studied on purified RT and viral vaccines treated to release RT from potentially contaminant retroviruses. The level of RT activity detected in chicken cell-derived vaccines was higher for live attenuated vaccines compared to inactivated ones. Contrary to other studies, RT activity was found in some mammalian cell-derived vaccines. AZT-TP sensitivity of RT activities detected in these vaccines and discrimination between retroviral and RT-like activities was further investigated. Feasibility and limits of PERT assay as a broad-spectrum retroviruses detection method in vaccines are discussed.
authors.affiliation: Agence Française de Sécurité Sanitaire des Produits de Santé, Direction des Laboratoires et des Contrôles, Unité de Contrôle des Médicaments Immunologiques, Lyon, France.
authors.forename: M
authors.initials: M
authors.lastname: André
authors.forename: S
authors.initials: S
authors.lastname: Morgeaux
authors.forename: F
authors.initials: F
authors.lastname: Fuchs
mesh_terms: D000818:Animals; D002460:Cell Line / Q000821:virology; D002642:Chick Embryo; D002645:Chickens; D002522:Chlorocebus aethiops; D004340:Drug Contamination; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D005240:Feasibility Studies; D006801:Humans; D008322:Mammals; D016133:Polymerase Chain Reaction; D011786:Quality Control; D012194:RNA-Directed DNA Polymerase / Q000032:analysis*; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012449:Safety; D012680:Sensitivity and Specificity; D014613:Vaccines, Attenuated / Q000737:chemistry / Q000592:standards; D014709:Vero Cells / Q000821:virology; D014764:Viral Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors; D014765:Viral Vaccines / Q000737:chemistry* / Q000592:standards; D014776:Virus Cultivation; D015215:Zidovudine / Q000494:pharmacology
chemical_list: D018894:Reverse Transcriptase Inhibitors; D014613:Vaccines, Attenuated; D014764:Viral Proteins; D014765:Viral Vaccines; D015215:Zidovudine; D012194:RNA-Directed DNA Polymerase