Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells

• Published in: Journal of immunological methods 2013-12, Vol.400-401, p.97-105
• Main Authors: de Oliveira, Edson R.A., Lima, Bruna M.M.P., de Moura, Wlamir C., de A. Nogueira, Ana Cristina M.
• Format: Article
• Language: English
• Subjects: Antiviral Agents - pharmacology; Antiviral assay; apoptosis; Apoptosis - drug effects; bioassays; Biological Assay - standards; biopharmaceuticals; cell cycle; Cell Cycle Checkpoints - drug effects; cell growth; Cell Line, Tumor; Cell Survival - drug effects; Cell viability; chronic hepatitis C; Cytopathogenic Effect, Viral - drug effects; cytopathogenicity; flow cytometry; growth retardation; Hep-2C; Hepacivirus - drug effects; Humans; IFN-alpha; innate immunity; interferon-alpha; Interferon-alpha - pharmacology; logit analysis; Quality Control; Reference Standards; STAT1 Transcription Factor - metabolism; viruses
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2013.10.011

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 1000IU/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 1000IU/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.