Mechanisms of Interferon Action and Resistance in Hepatitis C Virus Infection

为了了解干扰素在体内的耐药性，我们先前在黑猩猩模型中检测了人干扰素(h干扰素)-α、-γ和共识干扰素的动态反应。我们发现，在感染丙型肝炎病毒的黑猩猩中，存在着对干扰素的缺陷反应，特别是在肝脏中，这种缺陷可能是通过激活SOCS3来调节的。进一步研究丙型肝炎病毒感染对黑猩猩体内干扰素效应途径的抑制机制，可能为解决干扰素治疗无应答的临床问题提供新的思路。为了进一步探索干扰素在丙型肝炎患者中的作用和耐药性的机制，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。在接受治疗的患者中，患者在活组织检查前24小时接受单独的聚乙二醇干扰素α-2a或利巴韦林72小时和聚乙二醇干扰素-α。将患者分为快速反应组(RR)和慢反应组(SR)。治疗前的活检标本取自匹配的对照组。根据随后的治疗反应，将治疗前患者分为RR组或SR组。使用Affymetrix微阵列技术进行基因表达谱分析。已知的ISG在接受治疗的患者中被诱导。在治疗前组中，未来慢反应组(SR)的ISG表达高于快速反应组(RR)。在治疗时，RR和SR具有相似的ISG绝对表达，但当使用治疗前组校正基线表达时，RR显著诱导ISG，而SR显示上调干扰素抑制途径。接受利巴韦林治疗的患者干扰素相关基因的诱导增强，与干扰素抑制和肝星状细胞(HSC)激活相关的基因下调。这些数据表明，ISG的诱导性对治疗反应很重要，利巴韦林可能通过增强肝脏对聚乙二醇干扰素的基因反应来改善预后。总的来说，这些机制可能为提高联合治疗的疗效提供了分子基础。 为了进一步探讨利巴韦林的作用机制，我们评估了聚乙二醇干扰素联合或不联合利巴韦林治疗期间的早期病毒动力学、血清细胞因子表达和病毒突变。50例1型感染患者随机分为两组，分别接受聚乙二醇α2a干扰素联合利巴韦林(A组)和不联合利巴韦林(B组)治疗，疗程均为1个月。然后，所有患者都接受了整整48周的联合治疗。对第一和第二阶段的病毒动力学进行了评估。分别于0、12h、3d和7d检测血清IP10、MIG1和MCP1水平，并在0d、7d和28d对跨越NS5A和NS5B部分的1772个核苷酸进行病毒测序，计算变异率。第一阶段下降在两组之间相似。接受利巴韦林治疗的患者有更快的第二时相动力学，但仅在第一时相下降良好的患者(&gt；0.5logIU/ml)(中位数-0.72vs-0.38log/ml/周，p=0.039)。在第一时相下降良好的患者中，A组有14/15(93%)的患者第二时相斜率继续良好(&lt；0.3log10IU/ml/周)，而B组有11/18(61%)(p=0.046)。接受利巴韦林治疗的患者在12小时时血清IP10的诱导率高于单独接受聚乙二醇干扰素的患者(7.4vs3.8倍，p=0.01)，但与第二时相斜率相似，第一时相下降良好的患者IP10诱导率的差异更为明显。在接受利巴韦林治疗的患者中，12小时的IP10诱导与第一时相(p=0.0004，r2=0.40)和第二时相(p=0.001，r2=0.4)动力学相关，而在接受聚乙二醇干扰素的患者中则不相关(第一时相p=0.27r2=0.05p0.6，r2=0.013)。接受利巴韦林治疗的患者在第3天和第7天的IP10诱导率也较高，但仅在第一时相动力学良好的患者中。使用MIG1和MCP1数据的结果与使用IP10的结果相似。对19名患者(11名A组，8名B组)的测序分析显示，接受利巴韦林治疗的患者与仅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%对B 83%，SVR A 52%对B 48%，p=NS)。在这项研究中，利巴韦林改善了早期病毒动力学，但仅在对聚乙二醇干扰素有初步反应的患者中使用。在接受利巴韦林治疗的患者中，干扰素刺激的细胞因子的诱导作用更强，与病毒动力学的相关性更强，这表明利巴韦林的作用可能是通过增强干扰素信号来介导的。 我们还在传染性丙型肝炎病毒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的方式有效地抑制JFH-1感染Huh7.5.1细胞，这种抑制作用跨越了体内利巴韦林的生理浓度。基因芯片分析和随后的定量PCR分析表明，利巴韦林治疗导致了一组特定的干扰素刺激基因(ISGs)的诱导，包括IRF7、IRF9和ISG15，这些基因在抗丙型肝炎病毒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，不同于已知的与干扰素作用和细胞内双链RNA传感途径(如RIG-I和MDA5)相关的机制。RNA干扰研究排除了利巴韦林作用下Toll样受体和NF-Kappa B通路的激活。其他实验证明，参与了一个短暂的转录抑制因子，其活性被利巴韦林抑制，导致这些抗病毒基因上调。我们的研究表明，利巴韦林通过一种新的先天机制发挥作用，增强干扰素-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识别可与干扰素联合使用的新的抗病毒分子具有价值。

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.