Mechanisms of Interferon Action and Resistance in Hepatitis C Virus Infection

为了了解干扰素抗性在体内，我们以前研究了动态响应人干扰素（hIFN）-α，-γ和共识干扰素在黑猩猩模型。我们发现，HCV感染的黑猩猩对IFN的反应有缺陷，特别是在肝脏中，这种缺陷可能是通过SOCS 3的激活介导的。进一步研究HCV感染对IFN效应通路的抑制机制，可能为IFN治疗无效的临床问题提供新的见解。为了进一步探讨干扰素的作用机制和HCV患者的耐药，我们比较了患者在聚乙二醇干扰素和利巴韦林治疗前和治疗期间的肝脏基因表达。在治疗组中，患者在活检前接受聚乙二醇干扰素α-2a或利巴韦林72小时和聚乙二醇干扰素α-24小时。将患者分为快速反应者（RR）和缓慢反应者（SR）。从匹配的对照组获得治疗前活检标本。根据后续治疗应答，将治疗前患者分组为RR或SR。基因表达谱分析使用Affyssin微阵列技术进行。在接受治疗的患者中诱导了已知的ISG。在预治疗组中，未来慢反应者（SR）的预治疗ISG表达高于快速反应者（RR）。在治疗期间，RR和SR具有相似的绝对ISG表达，但当使用预治疗组校正基线表达时，RR显着诱导ISG，而SR显示IFN抑制途径上调。利巴韦林预处理的患者IFN相关基因的诱导增强，以及IFN抑制和肝星状细胞（HSC）激活相关基因的下调。这些数据表明，ISG诱导对于治疗反应是重要的，利巴韦林可能通过增强肝脏基因对聚乙二醇干扰素的反应来改善结果。总的来说，这些机制可以为联合治疗的疗效改善提供分子基础。 为了进一步探讨利巴韦林的作用机制，我们评估了在聚乙二醇干扰素治疗期间（有或无利巴韦林）的早期病毒动力学、血清细胞因子表达和病毒诱变。50例基因型1感染的患者被随机分为两组，A组接受聚乙二醇干扰素α 2a联合利巴韦林治疗，B组不接受利巴韦林治疗。所有患者随后接受了整整48周的联合治疗。评价了第一和第二阶段病毒动力学。在时间0、12小时、第3天和第7天测量血清IP 10、MIG 1和MCP 1水平。在第0天、第7天和第28天对跨越NS 5A和NS 5 B部分的1772个核苷酸区域进行病毒测序，并计算突变率。第一阶段下降在组间相似。接受利巴韦林治疗的患者具有更快的第二阶段动力学，但仅限于第一阶段下降良好的患者（>0.5 log IU/ml）（中位数-0.72 vs -0.38 log/ml/周，p=0.039）。在第一阶段下降良好的患者中，A组14/15（93%）继续具有良好的第二阶段斜率（<0.3 log 10 IU/ml/周），而B组11/18（61%）（p=0.046）。在12小时时，接受利巴韦林的患者的血清IP 10诱导高于单独接受聚乙二醇干扰素的患者（7.4对3.8倍，p=0.01），然而，与第二阶段斜率相似，IP 10诱导的差异在具有良好的第一阶段下降的患者中更明显。在接受利巴韦林治疗的患者中，12小时的IP 10诱导与第一阶段（p=0.0004，r2= 0.40）和第二阶段（p=0.001，r2=0.40）动力学相关，但在单独接受聚乙二醇干扰素治疗的患者中不相关（第一阶段p=0.27，r2=0.05，第二阶段p=0.6，r2=0.013）。接受利巴韦林治疗的患者在第3天和第7天的IP 10诱导也较高，但仅在第一阶段动力学良好的患者中。MIG 1和MCP 1数据的结果与IP 10的结果相似。19例患者（A组11例，B组8例）的测序分析显示，接受利巴韦林治疗的患者与接受聚乙二醇干扰素治疗的患者的同义和非同义突变率相似。两组之间的早期和持续病毒应答相似（EVR A 73% vs B 83%，SVR A 52% vs B 48%，p=NS）。在这项研究中，利巴韦林改善了早期病毒动力学，但仅限于对聚乙二醇干扰素有初始应答的患者。干扰素刺激的细胞因子的更大的诱导和利巴韦林治疗的患者中与病毒动力学的相关性表明，利巴韦林的作用可能是通过增强干扰素信号传导介导的。 我们还在感染性HCV细胞培养系统中测试了利巴韦林。与干扰素-α类似，利巴韦林以剂量依赖性方式有效抑制Huh 7.5.1细胞的JFH-1感染，其跨越利巴韦林在体内的生理浓度。微阵列分析和随后的定量PCR测定表明，利巴韦林治疗导致诱导一组特定的干扰素刺激基因（ISG），包括IRF 7，IRF 9和ISG 15，已知在抗HCV应答中发挥重要作用。 利巴韦林上调这些抗病毒基因是由一种新的机制介导的，该机制不同于已知与干扰素作用和细胞内双链RNA传感途径（如RIG-I和MDA 5）相关的机制。RNA干扰研究排除了利巴韦林作用中Toll样受体和NF-κ B通路的激活。另外的实验证明了一种短寿命的转录抑制因子的参与，其活性被利巴韦林抑制，导致这些抗病毒基因的上调。我们的研究表明，利巴韦林，通过一种新的先天机制，加强干扰素α治疗丙型肝炎的抗病毒作用。了解利巴韦林的作用机制对于鉴定可与干扰素联合使用的新型抗病毒分子是有价值的。

To understand IFN resistance in vivo, we previously examined the dynamic responses to human IFN (hIFN)-alfa, -gamma and consensus IFN in the chimpanzee model. We showed that there is a defective response, particularly in the liver, to IFNs in HCV-infected chimpanzees, and this defect is possibly mediated through the activation of SOCS3. Further study on the inhibitory mechanism of IFN effector pathway by HCV infection in chimpanzee may provide novel insights into the clinical issue of nonresponse to IFN therapy. To further explore the mechanisms of IFN action and resistance in HCV patients, we compared hepatic gene expression in patients prior to and during peginterferon and ribavirin therapy. In the on-treatment group patients received either peginterferon alfa-2a alone or ribavirin for 72 hours and peginterferon-alfa 24 hours prior to biopsy. Patients were grouped into rapid responders (RR) and slow responders (SR). Pre-treatment biopsy specimens were obtained from a matched control group. Pre-treatment patients were grouped as RR or SR based on subsequent treatment response. Gene expression profiling was performed using Affymetrix microarray technology. Known ISGs were induced in treated patients. In the pre-treatment group, future slow responders (SR) had higher pretreatment ISG expression than rapid responders (RR). On treatment, RR and SR had similar absolute ISG expression but when corrected for baseline expression using the pre-treatment group, RR had marked induction of ISGs while SR showed up-regulation of IFN-inhibitory pathways. Patients pretreated with ribavirin had heightened induction of IFN-related genes as well as down-regulation of genes involved in IFN-inhibition and hepatic stellate cell (HSC) activation. These data suggest that ISG inducibility is important for treatment response and ribavirin may improve outcomes by enhancing hepatic gene responses to peginterferon. Collectively these mechanisms may provide a molecular basis for the improved efficacy of combination therapy. To further explore the mechanism of ribavirin action, we evaluated early viral kinetics, serum cytokine expression and viral mutagenesis during peginterferon treatment with or without ribavirin. 50 patients with genotype 1 infection were randomized to receive peginterferon alfa 2a with (Group A) or without (Group B) ribavirin for the first month of treatment. All patients then received a full 48 weeks of combination therapy. First and second phase viral kinetics were evaluated. Serum IP10, MIG1 and MCP1 levels were measured at time 0, 12 hours, day 3 and day 7. Viral sequencing of a 1772 nucleotide region spanning part of NS5A and NS5B was performed at day 0, 7 and 28 and mutation rates were calculated. First phase decline was similar between groups. Patients receiving ribavirin had more rapid second phase kinetics, but only in those patients with a good first phase decline (>0.5 log IU/ml) (median -0.72 vs -0.38 log/ml/week, p=0.039). Of the patients with a good first phase decline, 14/15 (93%) in Group A went on to have a good second phase slope (<0.3 log10IU/ml/week), compared to11/18 (61%) in Group B (p=0.046). The induction of serum IP10 at 12 hours was higher in patients receiving ribavirin than those receiving peginterferon alone (7.4 vs 3.8 fold, p=0.01), however similar to second phase slope, the difference in IP10 induction was more apparent in those with a good first phase decline. IP10 induction at 12 hrs correlated with first (p=0.0004, r2=040) and second phase (p=0.001, r2=0.40) kinetics in patients who received ribavirin, but not in those receiving peginterferon alone (phase 1 p=0.27, r2=0.05, phase 2 p=0.6, r2=0.013). IP10 induction at day 3 and 7 was also higher in patients receiving ribavirin, but only in those with good first phase kinetics. Results with MIG1 and MCP1 data were similar to those with IP10. Sequencing analysis in 19 patients (11 Group A, 8 Group B) revealed similar synonymous and non-synonymous mutation rates in patients receiving ribavirin as those receiving peginterferon alone. Early and sustained viral responses were similar between groups (EVR A 73% vs B 83%, SVR A 52% vs B 48%, p=NS). In this study, ribavirin improves early viral kinetics, but only in those patients with an initial response to peginterferon. The greater induction of interferon-stimulated cytokines and the correlation with viral kinetics in ribavirin-treated patients, suggest that the effect of ribavirin may be mediated through augmentation of interferon signaling. We also tested ribavirin in the infectious HCV cell culture systems. Similar to interferon-alfa, ribavirin potently inhibits JFH-1 infection of Huh7.5.1 cells in a dose-dependent manner, which spans the physiological concentration of ribavirin in vivo. Microarray analysis and subsequent quantitative PCR assays demonstrated that ribavirin treatment results in the induction of a specific set of interferon stimulated genes (ISGs) including IRF7, IRF9, and ISG15 which are known to play an important role in anti-HCV response. Upregulation of these antiviral genes by ribavirin is mediated by a novel mechanism different from those known to be associated with interferon action and intracellular double stranded RNA sensing pathways such as RIG-I and MDA5. RNA interference studies excluded the activation of the Toll-like receptor and NF-Kappa B pathways in the action of ribavirin. Additional experiments demonstrated the involvement of a short-lived transcriptional repressor whose activity is inhibited by ribavirin resulting in the upregulation of these antiviral genes. Our study suggests that ribavirin, acting via a novel innate mechanism, potentiates the antiviral effect of interferon-alfa in the treatment of hepatitis C. Understanding the mechanism of action of ribavirin is valuable in identifying novel antiviral molecules that could be used in combination with interferon.