Cell culture systems for the hepatitis C virus and their application for development of specifically targeted antiviral therapy for hepatitis C (Stat-C)

A hallmark of the hepatitis C virus (HCV) is its high propensity to establish a persistent infection. It is estimated that up to 80% of infected individuals are unable to eliminate the virus, and persistently infected persons have a high risk to develop chronic liver disease including steatosis, fibrosis, liver cirrhosis and eventually hepatocellular carcinoma.

Current therapy of chronic hepatitis C is based on the combination of pegylated interferon-alpha (IFN-α) and ribavirin. Although sustained viral response rates could be improved profoundly, this therapy has several disadvantages, including the limited efficacy against infections with genotype 1 viruses and multiple side-effects affecting compliance and continuation of therapy. For these reasons, enormous efforts are undertaken in academia and industry to develop drugs that directly target the virus. Such developments have been greatly facilitated by the establishment of cell culture systems allowing the propagation of infectious HCV in the laboratory under standardized conditions.

The first cell culture system for HCV was based on the use of genetically modified viral “mini-genomes” (designated replicons) that replicated efficiently in human hepatoma cells [5]. This system recapitulates all intracellular steps of the viral replication cycle and has been used to develop and validate drugs that target the viral serinetype protease residing in nonstructural protein 3 (NS3) and the RNA-dependent RNA polymerase residing in NS5B. In addition, the replicon system has been used for high throughput screening of compound libraries leading to the discovery of highly potent compounds interfering with the viral replication factor NS5A [2]. In addition, the replicon system is instrumental in understanding the mode-of-action of these drugs and to identify mutations in the HCV genome conferring antiviral drug resistance. Notably, resistance mutations identified in the replicon system have also been found in patients in the course of clinical trials demonstrating the usefulness of this HCV cell culture model [6].

Although the replicon system has been of enormous help for drug discovery, its use was limited because it did not support production of infectious virus particles and therefore neither the early nor the late steps of the viral replication cycle could be studied (entry and assembly, respectively). This limitation has been overcome by the identification of a particular HCV isolate that was molecularly cloned from a Japanese patient suffering from fulminant hepatitis (hence this genome was called JFH-1). This genome replicates extremely well in cultured human liver cells and supports the production of virus particles that are infectious both in cell culture and in vivo [8] [9]. Initially limited by low virus yields, this culture system has been improved steadily during the last few years up to a level allowing “classical” virological assays such as determination of infectivity titers or monitoring of viral spread in a cell culture [4] [7] [9] [10].

Apart from targeting viral factors and enzymes, more recent studies have identified host cell factors that are essential for efficient replication of HCV. These host factors include cyclophilin A (CypA), phosphatidlyinositole 4-kinase III alpha and microRNA-122 (miR-122). Importantly, efficient CypA antagonists have been developed and clinical trials suggest that their use in combination with standard of care is superior over current therapy [1]. Likewise, a proof-of-concept study conducted with HCV-infected chimpanzees has demonstrated that sequestration of miR-122 greatly reduces viral loads in these animals [3]. Thus, drugs targeting host cell factors might be an additional, complementary approach for treatment of chronic hepatitis C.

In conclusion, progress in development of Stat-C has been closely linked to the establishment of adequate HCV cell culture systems. Multiple directly acting antiviral drugs are currently in different phases of preclinical and clinical development. It is estimated that during 2011/2012 the first generation of protease inhibitors to be used in combination with peg-IFN-α and ribavirin will be approved for treatment of chronic hepatitis C increasing the sustained viral response rate even further. It remains to be determined whether IFN-free treatment will be possible and if so, how many directly acting antiviral drugs will be required for treatment of chronic hepatitis C.

• References

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