title: Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.
abstract: Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the importance of new approaches for IFN potency determination.
authors.affiliation: Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil. Electronic address: edsonrao@ioc.fiocruz.br.
authors.forename: Edson R A
authors.initials: ER
authors.lastname: de Oliveira
authors.forename: Bruna M M P
authors.initials: BM
authors.lastname: Lima
authors.forename: Wlamir C
authors.initials: WC
authors.lastname: de Moura
authors.forename: Ana Cristina M de A
authors.initials: AC
authors.lastname: Nogueira
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001681:Biological Assay / Q000592:standards*; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects*; D003588:Cytopathogenic Effect, Viral / Q000187:drug effects*; D016174:Hepacivirus / Q000187:drug effects; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology*; D011786:Quality Control; D012015:Reference Standards; D050794:STAT1 Transcription Factor / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D016898:Interferon-alpha; D050794:STAT1 Transcription Factor
keywords: Antiviral assay; Cell viability; Hep-2C; IFN-alpha