A monocytic detour to replicate patient‐derived hepatitis C virus in hepatoma cells and its use for phenotypic analyses (2014)

Potential conflict of interest: Nothing to report. See Article on Page 1192 With approximately 170 million infected individuals worldwide, hepatitis C virus (HCV) represents a major public health concern. In spite of a mild, often unapparent course of acute infection, the virus persists in most cases and these individuals are at high risk to develop liver fibrosis, liver cirrhosis, and eventually hepatocellular carcinoma. Although a prophylactic vaccine to prevent HCV infection is not in sight, tremendous progress has been made with respect to treatment of chronic hepatitis C (CHC). Until 2011, the standard of care was based on pegylated‐interferon (IFN)‐α and ribavirin, which were poorly tolerated and of limited efficacy. The introduction of the first direct‐acting antiviral (DAA) drugs, the HCV protease inhibitors, telaprevir (Incivek) and boceprevir (Victrelis), profoundly improved the therapeutic benefit, especially for patients infected with genotype 1 viruses. With the recent approval of new classes of DAAs targeting the viral polymerase (sofosbuvir) or the phosphoprotein nonstructural protein (NS)5A (daclatasvir and ledipasvir) as well as second‐generation protease inhibitors (simeprevir), IFN‐free therapy with high virus‐elimination rates has become a reality. In addition, new generations of highly efficient DAAs as well as drugs targeting the host cell factor, cyclophilin A (e.g., alisporivir), and suppressing HCV replication, are in late‐stage clinical development.1 Although these agents and drugs that are expected to be approved in the near future will offer to physicians novel treatment modalities, efficacies of existing drugs depend on viral genotype, virus diversity (i.e., preexisting resistance mutations), treatment history of patients, and disease status. In addition, currently available DAAs used for IFN‐free therapy are very costly, and therefore, in most countries, only patients with advanced liver disease, if any, benefit from these new treatments. In order to wisely select the most efficacious and economic therapy to control HCV infection, one should ideally predict the therapeutic benefit of a given combination therapy. For instance, the presence of preexisting resistance mutations in HCV populations circulating in patients can be predictive for treatment failure, especially in case of DAAs with a low resistance barrier, such as first‐generation protease or NS5A inhibitors. Genotyping of HCV by deep‐sequencing analyses can detect resistant variants.2 However, resistance profiles can be unpredictably complex, thus requiring phenotypic analysis of virus populations. Although phenotypic assays have been established, they require time‐consuming construction of replicon RNA libraries and reflect sequence variability only in single selected genome regions (e.g., NS3 in case of patients treated with protease inhibitors).3 However, assays based on propagating HCV that circulates in serum of infected patients are not available. For reasons that are still poorly defined, most HCV genomes isolated from patients or experimentally infected animals are unable to replicate in cultured cell lines.5 In this issue of Hepatology, Cunningham et al. report on a new method to propagate patient‐derived HCV in cell culture and use this approach for phenotypic analysis and prediction of treatment response to telaprevir and alisporivir.6 The assay relies on the fusion of highly permissive Huh7.5 human hepatoma cells with monocytes containing HCV RNA (Fig. 1). Surprisingly, although HCV does not replicate in monocytic cells, replication occurs in fused heterokarya and can be quantified using highly sensitive polymerase chain reaction (PCR)‐based assays. Two sources of monocytes were used: first, CD14(+) monocytes isolated from chronically infected patients and, second, cells of the monocytic cell line, THP‐1, that had been activated with phorbol 12‐myristate 13‐acetate (PMA) and IFN‐γ before inoculation with HCV‐containing patient serum. The latter “capture‐fusion” approach is fast, works for all six HCV genotypes, and allows propagation of patient‐serum HCV. In this way, the investigators could recapitulate genotype‐dependent inhibition of patient‐derived HCV by telaprevir and alisporivir. Moreover, the assay phenocopied IFN‐α/ribavirin/telaprevir treatment response of patients, who experienced virological breakthrough, including 1 case where lack of virological response to telaprevir could not be linked to known resistance mutations. Importantly, the assay predicted sustained virological response to telaprevir for genotype 3 viruses that respond to this DAA in only ∼30% of cases. It will be interesting to determine whether this assay allows accurate prediction of treatment response for other DAAs alone or in combination.Figure 1: Principle of the capture‐fusion model. CD14(+) primary monocytes isolated from chronically infected patients (upper left) or cells of the monocytic cell line, THP‐1 (lower left), treated with PMA and IFN‐γ before inoculation with HCV‐containing patient serum are fused with cells of the highly permissive hepatoma cell line, Huh7.5. In fused cells (heterokarya), HCV undergoes a full replication cycle, which can be detected by RT‐qPCR and passage of virus to naïve Huh7.5 cells. Cultures of fused cells can be treated with antiviral drugs and thus be used for phenotypic analysis of patient‐derived HCV to determine replication fitness and drug sensitivity. It is unclear how HCV enters stimulated THP‐1 cells and how viral RNA persists in these cells (indicated with the question mark). Positive and negative HCV‐RNA strands are depicted as red and dark‐blue waved lines, respectively. Abbreviation: RT‐qPCR, quantitative reverse‐transcriptase PCR.Although this novel capture‐fusion assay has great promise for clinical application, several fundamental questions remain to be determined. Most notably, it is unclear why patient‐derived HCV needs to be introduced into Huh7.5 cells by monocytic cells, rather than by direct inoculation. One possibility is to avoid induction of an innate immune response that might occur in the latter case, although Huh7.5 cells have defects in Toll‐like receptor 3– and retinoic acid‐inducible gene I–signaling pathways. Equally unclear is the HCV entry pathway into monocytic cells. Uptake of HCV by THP‐1 cells did not require the known receptors, CD81, claudin‐1, and occludin. It was proposed that PMA and IFN‐γ pretreatment increases Fc‐receptor abundance on the cell surface, arguing that HCV‐antibody complexes might have been taken up by this receptor.7 Alternatively, exosomes present in serum of HCV‐infected patients and containing infectious viral RNA8 might have been transmitted into THP‐1 cells. The assay developed by Cunningham et al. undoubtedly constitutes a breakthrough for characterizing clinical HCV isolates of any genotype. However, replication efficiency is rather low: on average, ∼20 HCV‐RNA copies per fused cell, which is ∼100‐fold lower than Huh7 cells infected with Japanese fulminant hepatitis type 1–derived virus.9 For this reason, detection of HCV replication by less‐sensitive methods, such as western blotting or immunofluorescence, was not successful. Nevertheless, the investigators provide evidence that a full replication cycle occurs in fused cells, including virus production. Surprisingly, this virus was infectious for naïve Huh7.5 cells, although efficiency was very low. Though these results are at variance to earlier attempts to propagate patient‐derived HCV in this cell line, the capture‐fusion assay might constitute a new cell system to study replication properties of nonadapted clinically relevant HCV isolates and identify host factors that might be of relevance for such variants and for improvement of this assay. Finally, HCV‐containing primary monocytes and serum‐derived HCV from the same patient could be compared in this novel system to reveal possible determinants of tissue tropism and immunological features of relevance for pathogenesis. Although all‐oral, IFN‐free, DAA‐based therapy of CHC has just been approved for genotype 1, IFN‐based therapy, in combination with first‐generation protease inhibitors, will still be used in many countries where new DAAs are not affordable. Thus, phenotypic assays suitable to predict treatment responses will be of great value to select the most efficacious, least costly drug cocktail for a given patient. Author names in bold designate shared co‐first authorship.