Development of novel model systems to assess replication of patient derived hepatitis C virus in tissue culture

1. Introduction: The development of novel therapies for chronic hepatitis C virus (HCV) infection could potentially be streamlined by using patient-derived model systems in which drugs can be tested on patient derived viral strains prior to embarking upon clinical trials. Such models have been hampered by difficulties in maintaining primary human hepatocytes (PHHs) from infected individuals in culture and by the lack of a replication model system for patient derived virus. Most in vitro models to date do not comprise individual patient virus from all strains of HCV and hence novel antivirals cannot be tested on them. The aim of this research was to develop novel models of replication of HCV that will allow new drugs to be tested against all viral genotypes. We have examined two models - an ex vivo liver biopsy model and an HCV monocyte-hepatocyte fusion model. 2. Methods: Liver biopsy fragments from patients with chronic HCV infection were studied to determine whether or not they could be used as a replication model. Biopsy fragments were incubated with DMEM alone or with antiviral drugs for 24h and 48h and viral replication assayed by RT-PCR for HCV RNA. Similar experiments were done with isolated primary human hepatocytes (PHHs) from patients with chronic HCV infection which were then compared with liver biopsy fragments. Various methods were used to extend the longevity of 6 PHHs. In later experiments we examined a novel model in which CD14 positive monocytes derived from patients with active HCV infection were fused with different liver cell lines in order to observe HCV replication. In this later model viral replication was observed and drug validation experiments were performed examining the effect of telaprevir on infected hepatocytes from genotype 1 and genotype 3 patients. 3. Results: Viral replication was observed in both models. The mean viral RNA concentration in biopsies from patients with genotype 1 HCV was significantly lower after 24 hours' incubation with telaprevir compared to unsupplemented medium (P=0.0015). Telaprevir did not have an effect on HCV RNA levels in biopsies from patients with genotypes 2 or 3 HCV. Viability of PHHs was reduced compared to intact biopsy fragments. In the HCV-replication fusion model, HCV expression was higher in fused cells compared to unfused monocytes (p=0.0007). Optimal fusion conditions were achieved by using monocytes and Huh 7.5s in a 1:1 ratio and using 3 minutes incubation in PEG with pH of 7. HCV expression was highest during the first 7 days in fused cells and there was a significant decline in HCV RNA during subsequent days (p=0.002). Viral protein production was observed in fused cells by indirect immunofluorescence, which confirmed viral replication. HCV was successfully transferred to Huh7.5 cells using the monocyte 'capture-fusion' approach. Telaprevir showed greater antiviral efficacy in fused/infected cells with genotype 1 compared to those with genotype 3 strain of HCV. Interferon's activity was compromised by transfecting Huh 7.5 7 cells with PIV5 plasmid. This however did not increase HCV expression in these cells after fusion with CD14 positive monocytes. 4. Conclusion: Fragments of tissue from human liver biopsies can be maintained in tissue culture for at least 24 hours. Our data demonstrates the principle that antiviral agents can be tested ex vivo using surplus liver biopsy material and that the results reflect the findings of clinical studies in that telaprevir has efficacy in genotype 1 but not genotype 2 or 3 HCV. Moreover, monocytes may act as sanctuary sites for HCV virions and could be used to transfer virus to hepatocytes in vitro. The HCV fusion model can be used to test drug sensitivities for different genotypes as well as individual patient sensitivity to antiviral agents. Further analysis of the clinical utility of these model systems is indicated.