Viral DNA contamination is responsible for Epstein–Barr virus detection in cytotoxic T lymphocytes stimulated in vitro with Epstein–Barr virus B-lymphoblastoid cell line |
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Authors: | Mathilde Berthomé Géraldine Gallot Régine Vivien Béatrice Clémenceau Jean-Michel Nguyen Marianne Coste-Burel Henri Vié |
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Institution: | 1. Institut de Recherche Thérapeutique de l’Université de Nantes, UMR INSERM, U892, 8 quai Moncousu, BP 70721, 44007, Nantes Cedex 1, France 2. Univ Nantes, 44000, Nantes, France 3. Centre Hospitalo Universitaire de Nantes, 44093, Nantes, France
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Abstract: | Epstein–Barr virus (EBV)-transformed B-lymphoblastoid cell lines (LCLs) are used to prepare human EBV-specific T lymphocytes
(EBV-CTL) in vitro. Within an LCL, up to 5–7% the cells release infectious EBV, and this has fostered safety concerns for
therapeutic applications because of the exposure of T cells to EBV. The release of infectious viruses can be prevented by
ganciclovir, but this drug may seriously affect LCL growth. In the wake of these concerns, the present work was designed to
compile safety data on EBV-CTL preparation for the purpose of submission to a regulatory agency. We showed that further to
supernatant exclusion, the number of EBV genome copies (EBVc) associated with the EBV-CTL always made up a constant proportion
of the EBVc number detected in the culture supernatant. In addition, such was the case whether infectious virus could be produced
by the LCL or not, suggesting that the EBV signal detected was due to a DNA contamination rather than an infection. Furthermore,
we demonstrated that the number of EBVc associated with the EBV-CTL was highly sensitive to DNAse treatment, and finally that
EBVc could no longer be detected after the EBV-CTL had been amplified in the absence of LCL. Consequently, during in vitro
EBV-CTL preparation, either T cells cannot be infected or they die rapidly after EBV infection. |
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