Toll like receptor 7 and hepatitis C virus infection☆
Article Outline
Chronic hepatitis C virus (HCV) infection affects more than 170 million people, 3% of the world’s population, and is one of the most frequent causes of cirrhosis, hepatocellular carcinoma and end-stage liver disease [1]. Treatment for chronic HCV has improved substantially during the last decade and is now based on the combination of a pegylated interferon-α (pegIFN) alfa and the nucleoside analog ribavirin [1]. Apart from multiple side effects, their therapeutic regimen results in a sustained virological response (SVR) in about 40–90% of patients, only depending on the patient’s age, gender, weight, race and the genotype of HCV. Therefore, novel antiviral strategies are needed. In addition to HCV-specific antiviral concepts that directly inhibit HCV replication, such as protease and polymerase inhibitors [2], agents, such as toll like receptor (TLR) agonists, that selectively stimulate the production of IFN may be of potential use.
TLRs are a family of at least 12 membrane proteins that trigger innate immune responses [3]. TLR-family members are pattern-recognition receptors (PRR) that collectively recognize lipid, carbohydrate, peptide and nucleic-acid structures that are broadly expressed by different groups of microorganisms. Pathogen recognition through TLR and other PRR serves distinct but related functions such as the sensing of the presence and type of pathogen, the provoking of an innate immune response (such as the production of IFN) and the priming of a long-lasting adaptive immune response [3]. These central functions of TLR have sparked great interest to explore their possible role during the natural course of infection in viral infections and their therapeutic potential for triggering the innate immunity with TLR agonists [4]. Two articles in this issue address these two aspects in HCV infection.
HCV is recognized by innate virus-sensing mechanisms and induces a rapid IFN response [5], [6]. Indeed, HCV infection is followed by the rapid induction of IFN-regulated genes, such as MxA or 2‘5‘Oligoadenylate synthetase, within the first two weeks of infection [7], [8], [9]. The source of IFN is not completely clear but it is likely that next to infected hepatocytes, plasmacytoid dendritic cells (pDC), known to secrete high amounts of IFN, contribute to the rapid secretion of this cytokine. Of note, TLR 7 is highly expressed on pDC making it a promising candidate for an immune mediator in HCV infection. Although the direct involvement of TLR 7 in natural HCV infection has not been shown, indirect evidence suggests a possible role. For example, a proof of concept study by Horsmans et al. has shown that a once daily 7-day treatment with intravenous isatoribine, an agonist of TLR 7, reduced plasma virus concentrations in chronically HCV infected patients [10]. Furthermore, a recent in vitro study demonstrated that TLR 7 can induce HCV immunity not only by IFN induction but also through an IFN-independent mechanism [11]. Indeed, a synthetic TLR 7 activator reduced HCV mRNA and protein levels in isolated Huh-7 hepatocytes, whereas other TLRs were ineffective. The antiviral activity of the synthetic TLR 7 activator was associated with stimulation of antiviral genes such as the IFN response factor 7, but not with the activation of the IFN-responsive STAT-1 transcription factor. More importantly, the antiviral effect was not neutralized by the addition of anti-IFNα receptor antibodies, clearly suggesting that potent synthetic TLR 7 ligands may inhibit HCV replication not only by stimulation of IFN production but also by direct activation of antiviral mechanisms in hepatocytes [11]. In this context, immunohistochemical analyses revealed that TLR 7 is expressed in normal as well as HCV infected hepatocytes [11]. Thus, these combined results have supported the conclusion that TLR 7 may indeed play a role during natural infection.
This hypothesis is now further supported by the report of Schott et al. in this issue of the Journal who analyzed the prevalence of TLR 7 single nucleotide polymorphisms (SNPs) in a large cohort of patients chronically infected with HCV and their effect on liver fibrosis [12]. TLR 7 was sequenced in 52 women and subsequently SNPs with an allele frequency >5% were further analyzed in a cohort of 807 patients. Five different single SNPs, three with a frequency of >5%, were detected. Of note, one variant, c.1-120T
>G, was associated with less inflammation and fibrosis in male patients. Since TLR 7 is located on the X-chromosome it is not entirely surprising that this effect was solely observed in males. While the biological mechanisms responsible for the effect of the c.1-120T>G variation remain to be determined, Schott et al. observed an increased secretion of interleukin 6 (IL-6) in peripheral blood mononuclear cells derived from these patients. IL-6 is an interesting cytokine in this context because it is released after TLR 7 ligation and since it has potential antifibrotic effects. Taken together, these data clearly support a biological role of TLR 7 in chronic HCV infection, i.e. specific TLR 7 SNPs may have a significant effect on fibrosis progression in patients with chronic HCV infection.
Another study in this issue addresses the potential therapeutic effects of TLR 7 agonists. Pockros et al. analyzed the clinical efficiency, safety, pharmacokinetics and pharmacodynamics of resiquimod, a TLR 7 and 8 agonist [13]. Two randomized, double-blind phase IIa studies were performed in a heterogeneous group of chronically HCV infected patients, including IFN non-responders or relapsers. Resiquimod was administered orally twice a week for four consecutive weeks in both studies. In a U.S. multicenter study 12 subjects received 0.01mg/kg resiquimod and four placebo. In a French single center study six subjects received 0.01mg/kg, 11 0.02mg/kg resiquimod and six received placebo. Overall, oral administration of 0.01mg/kg resiquimod was well tolerated with no serious adverse events. However, severe side-effects were reported for 22% of patients receiving 0.01mg/kg and for 73% of subjects receiving 0.02mg/kg resiquimod. Two patients receiving 0.02mg/kg discontinued treatment because of IFN-like side-effects. Cytokine induction was consistently observed at a dose of 0.02mg/kg and 5/11 subjects treated with this dose have at least a 1-log reduction of HCV RNA levels after the first dose. However, an immediate viral rebound was observed. Only one patient was considered a responder per protocol (>2 log reduction) at the end of the four week treatment and this was not sustained on follow-up. Overall, these results support the proof-of concept study by Horsmans et al. [10] demonstrating an effect of TLR 7 agonists in HCV infection. The reported response rates are rather disappointing however and the high rate of side effects is troublesome. Thus, for the time being, TLR 7 agonists are no promising option for the treatment of chronic HCV infection with no substantial advantage over standard treatment. How can the poor antiviral effect of TRL 7 agonists be explained? The simplest explanation is underdosing. Indeed, oral administration of 0.01mg/kg resiquimod had only a minimal effect on cytokine induction and HCV RNA level reduction with variable pharmacokinetics of resiquimod. At the same time, resiquimod at a concentration of 0.02mg/kg had already substantial side effects. Biological reasons may in part explain the low response rate to TLR 7 agonists, i.e. pDC from chronically HCV infected patients showed an impaired response to TLR ligation [14]. Since pDC, one of the major sources of IFN, express TLR 7 on their surface, this impairment could significantly alter the response to TLR agonists. The hyporesponsiveness may also be determined by host genetics, especially in this cohort of previous non-responders and relapsers [15]. Clearly, additional studies are required to address these aspects and to determine the long-term clinical efficiency and safety of TLR 7 agonists in the treatment of chronic HCV infection.
Taken together, the studies by Schott et al. and Pockros et al. provide new insights into the possible role of TLR 7 in the natural course of HCV infection and its antiviral actions. A better understanding of the innate immune responses operative during acute and chronic HCV infection, its interplay with the adaptive immune response and its effects on the natural course of HCV infection are areas of major biological and clinical relevance. A better understanding of these complex interactions should allow to translate this knowledge into new antiviral strategies.
References
- . Viral hepatitis C. Lancet. 2003;362:2095–2100
- . The hepatitis C virus life cycle as a target for new antiviral therapies. Gastroenterology. 2007;132:1979–1998
- . Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol. 2007;7:179–190
- . Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists. Nat Med. 2007;13:552–559
- . Flying under the radar: the immunobiology of hepatitis C. Annu Rev Immunol. 2007;25:71–99
- . A target on the move: innate and adaptive immune escape strategies of hepatitis C virus. Antiviral Res. 2006;69:129–141
- . DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection. J Virol. 2001;75:7059–7066
- Genomic analysis of the host response to hepatitis C virus infection. Proc Natl Acad Sci USA. 2002;99:15669–15674
- Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease. Proc Natl Acad Sci USA. 2002;99:15661–15668
- Isatoribine, an agonist of TLR7, reduces plasma virus concentration in chronic hepatitis C infection. Hepatology. 2005;42:724–731
- Activation of anti-hepatitis C virus responses via Toll-like receptor 7. Proc Natl Acad Sci USA. 2006;103:1828–1833
- A Toll-like receptor 7 single nucleotide polymorphism protects from advanced inflammation and fibrosis in male patients with chronic HCV-infection. J Hepatol. 2007;47:203–211
- Oral resiquimod in chronic HCV infection: Safety and efficacy in 2 placebo-controlled, double-blind phase IIa studies. J Hepatol. 2007;47:174–182
- TLR ligand-dependent activation of naive CD4 T cells by plasmacytoid dendritic cells is impaired in hepatitis C virus infection. J Immunol. 2007;178:4436–4444
- Changes in gene expression during pegylated interferon and ribavirin therapy of chronic hepatitis C virus distinguish responders from nonresponders to antiviral therapy. J Virol. 2007;81:3391–3401
☆ The authors declare that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
PII: S0168-8278(07)00300-5
doi:10.1016/j.jhep.2007.05.009
© 2007 European Association for the Study of the Liver. Published by Elsevier Inc. All rights reserved.
