title: A replicon-based bioassay for the measurement of interferons in patients with chronic hepatitis C.
abstract: Overall treatment results of chronic hepatitis C have improved markedly with the introduction of pegylated interferon-alpha (PEG-IFN-alpha) and ribavirin combination therapy. However, cure rates in the most common genotype 1 infection are still unsatisfactory. IFN-alpha dose-response studies on viral kinetics suggest that inadequate dosing might be a key factor but drug levels have hardly been tested, which is in part due to difficulties in measuring this cytokine in patient samples. We have shown recently that hepatitis C virus (HCV) replicons are highly sensitive to IFN-alpha. In this report we tested whether the replicon system could be used as a sensitive bioassay to determine the amount of biologically active IFN-alpha in serum or heparinized plasma of patients under therapy. To facilitate the measurements, a stably replicating subgenomic HCV RNA was developed that carries the gene encoding the firefly luciferase. Dose response studies with IFN-alpha demonstrate that the amount of expressed luciferase directly correlates with the level of HCV replication. By using this cell-based assay, serum samples of HCV patients treated with different types and doses of IFN-alpha were analyzed in parallel to IFN-alpha standards made by serial dilutions of the same type of IFN-alpha the patient was treated with. Based on nonlinear logistic models serum concentrations corresponding to 1.3-19 U/ml were determined in patients under standard or high dose IFN-alpha therapy, and from 3.8 to 4.1 ng/ml in patients treated with PEG IFN-alpha. In conclusion, the HCV-replicon based bioassay allows determining the levels of biologically active IFN-alpha in serum and heparinized plasma of patients under treatment.
authors.affiliation: Department of Hepatology and Gastroenterology, Erasmus University Medical Center, Rotterdam, The Netherlands.
authors.forename: Jan M
authors.initials: JM
authors.lastname: Vrolijk
authors.forename: Artur
authors.initials: A
authors.lastname: Kaul
authors.forename: Bettina E
authors.initials: BE
authors.lastname: Hansen
authors.forename: Volker
authors.initials: V
authors.lastname: Lohmann
authors.forename: Bart L
authors.initials: BL
authors.lastname: Haagmans
authors.forename: Solko W
authors.initials: SW
authors.lastname: Schalm
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
mesh_terms: D000998:Antiviral Agents / Q000097:blood / Q000627:therapeutic use; D001681:Biological Assay; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics; D019698:Hepatitis C, Chronic / Q000188:drug therapy / Q000821:virology; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000097:blood* / Q000627:therapeutic use; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D011092:Polyethylene Glycols / Q000627:therapeutic use; D012367:RNA, Viral / Q000096:biosynthesis; D011994:Recombinant Proteins; D012093:Replicon; D012254:Ribavirin / Q000627:therapeutic use; D014407:Tumor Cells, Cultured; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D012367:RNA, Viral; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D012254:Ribavirin; D008156:Luciferases; C100416:peginterferon alfa-2a