Vrolijk Jan M, Kaul Artur, Hansen Bettina E, Lohmann Volker, Haagmans Bart L, Schalm Solko W, Bartenschlager Ralf
Department of Hepatology and Gastroenterology, Erasmus University Medical Center, Rotterdam, The Netherlands.
J Virol Methods. 2003 Jun 30;110(2):201-9. doi: 10.1016/s0166-0934(03)00134-4.
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.
随着聚乙二醇化干扰素-α（PEG-IFN-α）和利巴韦林联合疗法的引入，慢性丙型肝炎的总体治疗效果有了显著改善。然而，最常见的1型感染的治愈率仍然不尽人意。关于病毒动力学的干扰素-α剂量反应研究表明，剂量不足可能是一个关键因素，但药物水平几乎未得到检测，部分原因是在患者样本中测量这种细胞因子存在困难。我们最近发现丙型肝炎病毒（HCV）复制子对干扰素-α高度敏感。在本报告中，我们测试了复制子系统是否可用作一种灵敏的生物测定法，以确定接受治疗患者血清或肝素化血浆中生物活性干扰素-α的含量。为便于测量，构建了一种稳定复制的亚基因组HCV RNA，其携带编码萤火虫荧光素酶的基因。干扰素-α的剂量反应研究表明，表达的荧光素酶量与HCV复制水平直接相关。通过使用这种基于细胞的测定法，对接受不同类型和剂量干扰素-α治疗的HCV患者的血清样本与通过对患者接受治疗的同类型干扰素-α进行系列稀释制备的干扰素-α标准品进行了平行分析。基于非线性逻辑模型，确定了接受标准或高剂量干扰素-α治疗患者的血清浓度对应于1.3 - 19 U/ml，接受聚乙二醇干扰素-α治疗的患者血清浓度为3.8至4.1 ng/ml。总之，基于HCV复制子的生物测定法能够确定接受治疗患者血清和肝素化血浆中生物活性干扰素-α的水平。
