EP1436410A1 - Use of hcv core antigen assay for screening of anti-viral compounds - Google Patents

Abstract

The present invention is directed to a simple and efficient methof for in vitro screening of antiviral drugs. In one embodiment, an immunoassay for measuring the amount of HCV core protein in an in vitro HCV replicating system is used. This method can be used for high throughput screening of drugs that can potentially be used to treat HCV infection in patients.

Description

USE OF HCV CORE ANTIGEN ASSAY

FOR

SCREENING OF ANTI-VIRAL COMPOUNDS

Background

Following its discovery in 1989 as the causative agent implicated in the majority of non-A, non-B hepatitis (1), hepatitis C virus (HCV) has become a focus of considerable diagnostic and anti-viral research (16). HCV is transmitted primarily through exposure to infected blood or blood products. Following the initial acute infection, around 85% of infected individuals progress to chronic hepatitis. Currently there are an estimated 170 million individuals worldwide infected with HCV and at risk of developing liver disease (28). The introduction of diagnostic tests used in routine blood screening in the 1990s has led to a downward trend in HCV incidence, at least in the U.S.A. However, given the slow progression to the end-stage liver disease associated with HCV infection, even the existing infections will continue to present a serious medical and economic burden for decades (17).

A number of diagnostic tests for HCV are used to help prevent infection by the routine screening of blood and blood products, and to aid in the clinical management of HCV-infected patients. Following infection, anti-HCV antibodies (anti-HCV) are generated by seroconversion, and detection of these antibodies by enzyme-linked immunosorbent assays (ELISAs) during blood screening has become the frontline diagnostic technique for HCV infection. Second and third generation assays displayed improved sensitivity over the first generation ELISAs. Although the ELISA formats allow for automation and deployment in clinical hospital settings, they have several limitations; these include sub-optimal sensitivity, particularly in immunosuppressed liver transplant recipients, which obviously have only limited efficacy in diagnosing primary HCV infection due to the variable time required for antibody generation. In addition, since patients that have resolved HCV infection may remain anti-HCV positive, serological data does not allow an accurate assessment of viremia and can not distinguish between active and resolved infections. Therefore, a number of nucleic acid based assays have been developed for both detection and quantitation of viral load, as well as line- probe assays for HCV genotyping.

The quantitative RNA detection assays rely on reverse transcription- polymerase chain reaction (RT-PCR) methods of nucleic acid amplification (HCV Amplicor, Roche) or signal amplification using branched-DNA technology (Quantiplex HCV bDNA assay, Bayer Diagnostics). Later generation RNA assays display improved dynamic range, sensitivity, and genotype coverage over the earlier assays, with the HCV Amplicor 2.0 (Roche) and the bDNA HCV 3.0 (Bayer Diagnostics) assays exhibiting lower limits of detection of 600 and 500 lU/ml HCV RNA respectively (23). Pre-treatment viral loads have been proposed as decision thresholds in modifying the treatment durations in patients infected with certain HCV genotypes (21). Together with biochemical assessment of serum alanine transferase levels as a marker for liver damage, these virological assays have found application in the monitoring of patient response to therapeutic agents in the clinic. In addition, qualitative RNA methods such as transcription-mediated amplification (TMA) have been used to classify end-of-treatment responders. However, despite their utility and sensitivity, HCV RNA detection methods remain relatively costly, and can require specialized equipment and expertise. As an alternative approach, an ELISA based on detection of HCV antigen in serum or plasma was developed using monoclonal antibodies specific for the HCV core protein. This assay has been adapted for HCV monitoring in patient serum with the addition of a pretreatment step to dissociate any HCV core antigen-antibody complexes, which may decrease the sensitivity of detection. The assay has recently been commercialized (Ortho^® trak-C™; Ortho-Clinical Diagnostics, Inc., Raritan, NJ) and HCV core detection shown to be an accurate indirect marker for HCV replication in the clinic for viral load determination before and during anti-viral therapy.

In contrast to the rapid pace of development of HCV diagnostics, the development of HCV anti-virals has been hampered by the lack of robust, reliable and efficient cell culture and small animal models that support hepatitis C virus replication and pathogenesis. In the absence of cellular models, much effort has focused on biochemical screening of virally-encoded targets (29). The recent development of selectable subgenomic HCV replicons functional in the Huh-7 hepatoma cell line should aid detailed molecular studies of HCV and the development of anti-viral drugs (3, 19). Following the initial study discussed in Lohmann et al., cell lines harboring various HCV replicons have been characterized in detail with regard to adaptive mutations, protein and RNA expression, and the genetic requirements for efficient HCV replication (4, 12, 15, 18, 22). Several reports

have demonstrated the activity of both interferons α and γ in blocking

replication in HCV replicon cells (4, 10, 11 , 13), although the exact mechanisms remain unknown. In addition, the ability of transgenic homozygous Alb-uPa/SCID mice to support growth of human hepatocytes and their subsequent infection with HCV patient sera may further provide an effective small animal model for the testing of HCV anti-virals (20). Even with the efficient cell culture systems, the monitoring of virus by molecular methods is still a rate-limiting step. The aforementioned methods can be time consuming, expensive and require highly trained personnel. The result is that the drug discovery effort against HCV has been a slow process. For the foregoing reasons, there is a need for a method for screening of anti-viral compounds that is amenable to the high throughput automation currently employed in drug discovery research. Summary

The present invention is directed to for a method for screening of antiviral compounds that satisfies this need. A method for determining whether a substance is capable of modulating, inhibiting or interfering with HCV comprises treating an HCV replication system with a test substance, and performing an immunoassay for detection of HCV core antigen to measure the ability of said substance to modulate, inhibit or interfere the HCV replication system as compared to a control. The immunoassay can be used for measuring the amount of HCV core protein in an in vitro HCV replicating system or model of HCV infection. Similarly the immunoassay could be used to detect HCV core protein in a human or human sera. The use of HCV core antigen assay for screening for anti-viral compounds will provide a new tool in the drug discovery effort against HCV. Brief Description of the Drawings

These and other features, aspects, and advantages of the present invention will become better understood with the following description, appended claims, and accompanying figures where: Figure 1. Shows the correlation of HCV core antigen to HCV RNA in an HCV infected patient population;

Figure 2. Shows a standard curve of HCV core protein in a total HCV core antigen ELISA;

Figure 3. Shows a response curve of HCV core expression in Huh-21-5 replicon cells to doses of interferon;

Figure 4. Shows a response curve of HCV core expression in Huh-21-5 replicon cells to interferon over time;

Figure 5A. Shows a Western blot analysis with monoclonal antibodies;

Figure 5B. Shows HCV core detection from genomic replicon cells and supernatant;

Figure 6. Shows a range HCV core detection in extractions;

Figure 7. Huh 21-5 cells were plated in 96 well plates and treated with human

IFN-alpha for 24 hrs. prior to cell lysis and processing through the Ortho^® trak-

C™ ELISA; and Figure 8. Shows dose dependent inhibition of HCV core expression upon Cpd. Detailed Description The term "substance" or "test substance" as used herein refers to any compound that would be suitable for testing for use to treat HCV by inhibiting

HCV replication, or modulates, inhibits or interferes with the expression of HCV core antigen (hereinafter collectively referred to as "modulating, inhibiting, or interfering with the HCV replication system"). Such substances or test substances may be a wide variety of molecules including, but not limited to, DNA, RNA, ribonucleosides, ribonucleoside mono-, di-, or tri-phosphates, protein, peptides, or other small organic or inorganic molecules. The term "HCV replication system" as used herein refers to any cell based system such as replicons and non-human animal models, as well as patients whose serum can be tested.

Until recently, the search for anti-viral drugs that can be used to treat HCV has been hampered by the lack of reliable and efficient cell culture systems that can be used to demonstrate hepatitis C virus replication and pathogenesis. Dr. Ralph Bartenschlager and colleagues have developed HCV replicons functional in a cell culture and provided a basis for long-sought cellular system that should allow detailed molecular studies of HCV and the development of anti-viral drugs (15, 18, 19). The details of the construction of the original HCV subgenomic replicons are given in the European Patent

Application 1043399. In brief, a full-length consensus genome was constructed from viral RNA from an infected human liver, and a subgenomic fragment containing a neomycin phosphotransferase gene (neoR) in place of the HCV structural genes was transfected into human Huh-7 hepatoma cells; upon G418 selection, stable cells were found to contain autonomously replicating HCV replicons which displayed faithful HCV gene expression and polyprotein processing.

Further improvement to the HCV replicon model came from Dr. Bartenschlager's laboratory and its subsequent inclusion of the HCV structural genes, including core, to the genome sequence used to generate the HCV replicons. This modified full-length HCV replicon now expresses HCV core antigen. Another means for achieving this expression could come from other genetically manipulated systems, like replicon models, which actually harbor the part of the HCV genome encoding the core antigen. Moreover, a murine model for HCV infection based on successful implantation of human hepatocytes and subsequent viral infection has been reported (20). As this model also exhibits HCV gene expression, it is an example of a non-human animal model with potential use in HCV anti-viral testing.

The clinical utility of the Ortho-Clinical Diagnostics Ortho® trak-C™ ELISA assay in monitoring HCV viral load in patients undergoing anti-viral therapy has recently been demonstrated (5). To further evaluate if this assay had application in the area of anti-viral testing, we selected the recently described cellular HCV replicon model and used the assay to monitor viral replication and the anti-viral effects of IFN. In the clinical Ortho® trak-C™ assay procedure, sample is first incubated with a dissociation buffer in a pretreatment step prior to dilution and addition to the ELISA plate microwells coated with monoclonal antibodies directed against HCV core. Detection of bound core is effected through addition of an HRP conjugate and subsequent enzymatic detection of HRP activity using an absorbance readout. Using recombinant purified HCV core antigen as a standard, the assay displays a lower limit of detection of 1.5pg core protein, and linearity of detection up to over 100pg (Ortho-Clinical Diagnostics, Inc.; trak-C Assay product sheet). As the recombinant core protein supplied as the standard is a fusion protein containing non-core amino acids, the actual HCV portion of the antigen represents 37.9% of the mass; therefore 1 pg of the kit standard is equivalent to 0.379pg of HCV core protein. Clinical studies using patient sera have previously demonstrated that 1 pg of HCV core (as detected by the Ortho® trak- C™ kit, without mass correction) is equivalent to approximately 8000 international units (IU) of HCV RNA (5). Given the sensitivity of the assay, we then evaluated it for monitoring HCV core levels in extracts of HCV replicon containing cells.

Parental Huh-7 cells, and Huh 21-5 cells containing the bicistronic, full- length genomic HCV l389neo/Core-3' replicon sequence, were seeded and grown for 24 hours, prior to freezing at -800 C. The replicon sequence carried in the 21-5 cells contains the HCV 5' non translated region plus nucleotides 342-389 of the core sequence, the neomycin resistance gene, the EMCV- IRES, and the complete HCV polyprotein coding sequence concluding with the HCV 3' non-translated region (11 ). Using Western blot analysis with monoclonal antibodies (Fig. 5A), expression of HCV core and NS5B at the expected sizes (-21 kd and 68kD respectively) are readily detectable in total extracts of 105 21-5 cells, but not from control Huh-7 cell extracts, confirming previous analysis of HCV polyprotein processing in these cells (11 ).

In parallel, cell pellets and culture medium supernatants from both Huh- 7 and 21-5 cells were processed using the Ortho® trak-C™ assay. In pilot experiments it was determined that the pretreatment step included in the Ortho® trak-C™ protocol for clinical samples, used to dissociate any potential antigen-antibody complexes in serum, was dispensable when assaying cell extracts (data not shown). Therefore, cell extracts and supernatants were processed in the absence of that pretreatment step. When testing aliquots equivalent to 3x104 cells (Fig. 5B), or higher (data not shown), the assay was consistently saturated for HCV core detection from Huh 21-5 genomic HCV replicon cells, with no detectable core present in Huh-7 cell extracts, or from the clarified growth media supernatants from Huh 21-5 or Huh-7 cells, or control growth medium (Fig. 5B). This failure to detect HCV core in the supernatant of the 21-5 cells argues against any productive particle formation and release from these full-length replicon cells, consistent with the reported inability to transfer G418 resistance from the supernatants of other full-length HCV replicon containing cells to fresh cultures (14). To more accurately determine the sensitivity of HCV core detection from Huh 21-5 cultures, cells were seeded in 96-well plates at different densities and grown for 24 hours prior to freezing. Following cell disruption with the addition of water, an aliquot of each extract was transferred to a new microplate. Upon processing through the ELISA, HCV core could be detected in extracts from as few as 63 Huh 21-5 cells, equivalent to ~3pg core, well above the limit of detection (Fig. 6). Thus the assay was sensitive enough to monitor HCV replication at low cell numbers, an advantage in cell-based compound screening. After adjusting by a factor of 0.379 for the mass of the recombinant HCV protein used as a standard, we calculate that, in this particular experiment (Fig. 6), a single Huh 21-5 cell contains approximately 21 (+/- 0.7) fg of HCV core antigen, equivalent to approximately 6x105 core molecules per cell.

To validate the assay for monitoring anti-viral efficacy, Huh 21-5 cells

were plated in 96-well plates and treated with human IFN-αfor 24 hours prior to

cell lysis and processing through the Ortho® trak-C™ ELISA. Figure 7 (black bars, left axis) shows the dose response of HCV core expression (relative to non-treated cells) following 10 or 500IU/ml IFN-D treatment. Core levels decreased to 42% and 18% of control following treatment with 10 or 500IU/ml

respectively. Similar decreases were observed using recombinant IFN-γ (data

not shown). To compare the anti-viral data obtained with Ortho® trak-C™ HCV core detection to existing methods of RNA detection, total RNA was prepared in parallel from identically treated 21-5 cells and quantified for HCV genomic RNA using the bDNA 3.0 signal amplification system. As seen in Figure 7

(shaded bars, right axis), the relative inhibitory effect of human IFN-α on HCV

RNA levels was highly similar (dropping to 34% and 13% of control) to that observed following core detection. These data are consistent with published

data for cells harboring subgenomic replicons treated with IFN-α. (4, 10, 13)

and 21-5 cells treated with IFN-γ (11). Moreover, they support the use of the

HCV core ELISA in generating anti-viral data comparable to that obtained with RNA-based methods.

Having demonstrated the utility of the core assay using IFN-α as a

reference anti-viral compound, we further used Ortho® trak-C™ detection of core as a read-out to confirm HCV anti-viral activity of several small molecules identified through systematic compound screening efforts (data not shown). Using one of these compounds (Cpd. A), we addressed the inter-assay variability of core detection by testing, on two separate days, replicate aliquots from cell extracts prepared from Huh-21-5 cells incubated with varying concentrations of Cpd. A. Figure 8 shows the dose dependent inhibition of HCV core expression upon Cpd. A exposure, with the mean and standard deviation from the different day replicates shown. Using the data from all three doses, with detection values spanning the majority of the range of core detection in the assay, an average coefficient of variation (C.V.) of 4.8% was derived. This level of inter-assay variability when testing identical samples is consistent with other reported diagnostic assays, and is acceptable for cell- based compound screening (data not shown).

Following primary HCV infection which leads to viremia, most immunocompetent hosts mount an antibody seroconversion directed at multiple HCV antigens. The exact role of that antibody response in controlling HCV pathogenesis still remains unclear, given the inability of such responses to prevent reinfection in chimpanzee studies (9). Nevertheless, the presence of circulating antibodies has provided a basis for both the initial identification of the HCV genome and the development of diagnostic assays to HCV (7, 25). More recently, diagnostic assays based on direct detection of HCV antigens have been developed, for example to the HCV core protein. Commercialized by Ortho-Clinical Diagnostics, the Ortho® trak-C™ assay is an ELISA-based method already demonstrated to have clinical utility in the monitoring of patient responses to therapy (5). In this study, the utility of the same assay in monitoring viral load in two preclinical models of HCV replication was addressed.

Previous studies using subgenomic HCV replicon-containing Huh-7 cells have demonstrated their sensitivity to the anti-viral effects of interferons, as monitored by either RT-PCR or Northern blot analysis of viral RNA (4, 10, 11 , 13). More recently, selectable full-length genomic HCV replicons have also

been shown to be sensitive to inhibition by IFN-γ by RNA detection, and

Western blotting of HCV proteins (11). Which exact mediators of the Type I IFN response conduct the HCV anti-viral effect remains unknown, however evidence from replicon studies already suggests that the Mx proteins are not responsible (10). Taking advantage of HCV core expression in the full-length HCV 21-5 replicon cells, we demonstrated the ability of the Ortho® trak-C™ assay to detect core protein from as few as 63 cell equivalents. This correlates to approximately 3pg antigen, above the lower limit of detection of 1.5pg. In contrast, detection of core in the same cell extracts using Western blotting and chemiluminescent detection has a lower markedly sensitivity, requiring an input of >104 cell equivalents in our laboratory (data not shown).

Interferon inhibition studies performed in 96-well format with 21-5 replicon cells yielded inhibition data consistent with those previously reported, confirming the utility of the method for cellular anti-viral screening of chemical compound collections. Indeed, we have used the assay to verify anti-viral activity of a number of chemical compounds, and using one of these, also demonstrated that the level of inter-assay variability was below 5%. The HCV core assay would presumably also find application in other cellular models of HCV replication, for example the T7 polymerase-dependent binary HCV expression system (8), or in infection models using primary human hepatocytes and human HCV positive serum (6).

Homozygous Alb-uPa/SCID mice have recently been shown to develop chimeric mouse/human livers upon transplantation of primary human hepatocytes, and are able to support successful HCV infection following injection of patient sera. The mouse system can be used for screening antiviral compounds. Although we envisage that the Ortho® trak-C™ assay would similarly detect HCV core antigen in other animal models of infection, for example the murine HCV-Trimera model or the chimpanzee (reviewed in (26), we have not addressed those models in this study. Advantages of the Ortho® trak-C™ assay over RNA-based methods for HCV monitoring include the lack of any requirement for RNA extraction procedures, which may be prone to nuclease contamination. Moreover, the ELISA format requires only standard lab equipment, and exhibits high inter- experimental reproducibility and sensitivity of detection. Compared to other protein detection methods like Western blotting or immunofluorescence, the Ortho® trak-C™ format allows for a 96-well based format and more rapid quantitation of data using automated plate readers.

The present invention measures the amount of HCV core protein in an in vitro HCV replicating system, any non-human animal model for HCV, from patient serum, or from any other source of HCV core protein. For example, another source could be a recombinantly made protein. Example 1

HCV core antigen ELISA was used to determine the effect of the anti- viral drug interferon on HCV core expression in a Huh-7 hepatoma cell line designated as Huh-21-5 carrying a persistent HCV replicon (l389neo/Core- 375.1) harboring a full length RNA replicon with the cell culture adapted HCV sequence NK5.1 (cell line and replicon were obtained from Reblikon GmBh, Mainz, Germany). Growth of the 21-5 cell line in the presence of G418 (at 250ug/ml) in DMEM, 10% foetal calf serum, 2mM glutamine, 1x non-essential amino acids, and antibiotic/antimycotic, is strictly dependent on the successful replication of the full-length HCV replicon RNA initially introduced into the cell by transfection which harbors a neomycin resistance gene cassette located 5' to the HCV genome sequence RNA. This cell line therefore harbors an autonomously replicating HCV genome and expresses all of the HCV viral proteins, including the core antigen. Detailed conditions for establishment, growth and maintenance of the 21.5 cell line are essentially as described for the original subgenomic HCV replicons described in Lohmann et al. (19) and in the European Patent Application 1043399.

There was a significant reduction in HCV core detected when the cell was treated with interferon and the reduction in core antigen was dependent on the amount and the time of interferon treatment (see Figures 3 and 4). The ELISA method used was based on Ortho-Clinical Diagnostic, Inc.'s microplate

HCV core antigen for diagnostic application (the Ortho® trak-C™).

One embodiment of the invention is directed to a method for determining whether a substance is capable of modulating, inhibiting or interfering with HCV comprising the steps of treating an HCV replication system with said substance, and performing an immunoassay for the detection of HCV core antigen as a means of measuring the ability of said substance to modulate, inhibit or interfere the HCV replication system as compared to a control HCV replication system that was not treated with said substance. The test substance selected can be DNA, RNA, ribonucleosides, ribonucleoside mono-, di-, or tri-phosphates, protein, peptides, or other small organic and inorganic molecules or analogs thereof .The following examples are intended to demonstrate the advantages and utility of the invention by describing the treatment of the cells and measurement of HCV core antigen. One skilled in the art would recognize there are other ways of replicating HCV, for example in mouse or chimpanzee animal models. These examples are meant to illustrate, but not limit, the spirit and scope of the invention. Example 2 Huh-21-5 cells harboring the l389neo/Core-375.1 full length HCV replicon were split from sub-confluent monolayers (by trypsin addition) and seeded (1000 cells/well) the day prior to drug treatment into 96-well microplates in DMEM, 10% foetal calf serum, 2mM glutamine, 1x non-essential amino acids, and antibiotic/antimycotic. At 24 hours post-seeding, the existing medium was replaced with

medium supplemented with recombinant human Interferon alpha (IFNα)

(Biosource International. Inc., Camarillo, CA) at the concentrations indicated (0, 10 or 500 Units/ml medium). Cells were cultured for the times indicated in 370C / 5% C02 before the medium was removed from the cell monolayer and the plate frozen at -400C. Example 3

The core antigen was measured quantitatively using Ortho-Clinical Diagnostics Total HCV Core Antigen ELISA Assay. The cells to be tested (normally grown or cells grown in presence of the drug being tested) were suspended in 100 microliter of phosphate buffered saline (PBS). 50 microliters of Ortho Total HCV Core Antigen ELISA Pretreatment Buffer was added to the suspended cells and the contents were heated to 56 degrees C for 30 minutes.100 microliters of cooled pretreated cells were added to microwells coated with HCV anti-core monoclonal antibodies containing 100 microliters of Ortho HCV Total Core Antigen ELISA Reaction Buffer. The microwells were then incubated at 25 degrees C for 60 minutes and washed six times with Ortho Wash Buffer with phosphate buffered saline (PBS) and 0.05% TWEEN 20 (polyoxyethylenesorbitan mono laurate) urea. 200 microliters of a solution containing anti-core monoclonal antibodies conjugate to horse radish peroxidase were added to each well and the contents incubated for 30 minutes at 25 degrees C.

The wells were then washed and 200 microliters of substrate solution, a buffer containing HRP, was added to each well. After 30 minutes at room temperature in the dark 50 microliters of a Stop solution are added to each well. The Stop solution was 4N sulfuric acid. Addition of the acid stops the enzymatic reaction. The microwells are read at 492nm using 600-650 references.

To estimate the core antigen concentration a series of recombinant HCV core protein dilutions were run on each plate (Figure 2).

The results of HCV Core Antigen ELISA testing of Huh-21-5 cells harboring the full length HCV replicon treated with varying dosages of interferon and measurements taken over varying time intervals are shown in Tables 1 and 2. Figures 3-4 show the dose response curves. Table 1. Shows the results of HCV Core Antigen ELISA testing of 500 units per ml. of interferon-treated Huh-21-5 cells at varying time intervals. Table 1a

Mean STD Table 1 b

Mean STD

Table 1 c

Mean STD

Table 2. Shows the results of HCV Core Antigen ELISA testing of Huh-21-5 cells treated with varying dosages of interferon and measured over varying time intervals.

Time IFN Dose Mean Hours Units/ml OD STD

12 0 1.602 0.209 12 10 0.971 0.275 12 500 0.689 0.099

Time IFN Dose Mean Hours Units/ml OD STD

24 0 2.815 0.293 24 10 1.177 0.395 24 500 0.501 0.097

Example 4

The strategy and generation of stably transfected Huh-7 cells carrying autonomously replicating HCV replicons have been described elsewhere (3, 19). The Huh-21-5 cell line used here in harbors the bicistronic, full-length HCV l₃₈gneo/Core-3' replicon with the cell culture adapted HCV 1 b sequence NK5.1

(11 , 15). Growth of the 21-5 cell line in the presence of G418 (at 250 μg/ml) in

Dulbecco's minimum essential medium (MEM) with Earle's salts (Gibco BRL, Grand Island, NY), 10% foetaL calf serum (Gemini BioProducts, Woodland, CA), 2mM glutamine, 1x non-essential amino acids, and 100U/ml penicillin,

100 μg/ml streptomycin, is strictly dependent on the replication of the HCV

RNA (introduced into the cell by transfection) which harbors a neomycin resistance gene located 5' to the HCV genome sequence RNA. Huh-7 cells used as control did not harbor replicon RNA. Example 5

Recombinant human interferon alpha (IFNα) and gamma (IFNγ) were

obtained from Biosource International (Camarillo, CA). Compound "A" was obtained from Johnson and Johnson Pharmaceutical Research and Development (Raritan, NJ). Compound "A" and IFNs were dissolved in 30% dimethyl sulphoxide, 50mM Hepes pH7.9, and stored at -800C. Example 6

Huh-21-5 cells were split from sub-confluent monolayers (by trypsin addition) and seeded (1000 cells/well) the day prior to drug treatment into 96- well microplates in DMEM, 10% foetal calf serum, 2mM glutamine, 1x non- essential amino acids, and antibiotic/antimycotic. After 24 hours, the medium was replaced with medium supplemented with recombinant IFNα at the

concentrations indicated (0, 10 or 500 International Units/ml medium). Cells were cultured for the times indicated in 370C / 5% C02 before the medium was removed from the cell monolayer and the plate frozen at -400C, prior to thawing and processing using the ELISA. Example 7

The core antigen was measured quantitatively using Ortho-Clinical Diagnostics Ortho® trak-C™ assay. The cells were grown in the absence or presence of the drug being tested, for the incubation times stated, before the medium was removed and the cells frozen at -400C. Upon thawing, the cells were resuspended in 100ul of water and incubated at room temperature for 10 minutes. In the Ortho® trak-C™ assay instructions for testing clinical serum samples, 50ul of Ortho ELISA Pretreatment Buffer is then added to the 100ul samples, and the contents heated to 560C for 30 minutes; when testing extracts from HCV replicon cells we found this step to be unnecessary, and therefore it was omitted. Therefore, 10 ul of cell extract (10% of the starting

material) was mixed with 75μl Ortho® trak-C™ Diluent Buffer and added to

microwells coated with multiple HCV anti-core monoclonal antibodies containing 100 ul of Ortho ELISA Reaction Buffer. The microwells were then incubated at 25 0C for 60 minutes and washed six times with 1x Ortho Wash Buffer with PBS and 0.05% TWEEN 20 (polyoxyethylenesorbitan mono laurate) urea to remove any unbound material. 200 ul of a solution containing anti-core monoclonal antibody Fab fragments conjugated to horseradish peroxidase were added to each well and the contents incubated for 30 minutes at 25 0C. The wells were then washed and aspirated using 1x Wash Buffer with urea to remove unbound conjugate. 200 ul of substrate solution, containing o-phenylenediamine (OPD) and hydrogen peroxide, were then added to each well. After 30 minutes at room temperature in the dark, 50 ul of a Stop solution (4N sulfuric acid) were added to each well. Absorbance was read at 492nm using 600-650 references with a standard plate reader. To estimate the core antigen concentration a series of recombinant HCV core protein dilutions (containing 0, 1.5, 5, 10, 50, 100 pg core) were run on each plate without the Pretreatment step, to generate a standard curve. The absorbance measured from blank wells (containing no cell extract) processed with all subsequent reagents was background subtracted from all other absorbance values prior to evaluation of the standard curve and sample core determinations. The concentration of HCV core in test samples was determined by interpolation to the standard curve of recombinant core protein obtained using linear regression analysis (GraphPad Prism; San Diego) using the formula y = (O.D. sample- y-axis intercept)/slope, where y is the sample concentration (in pg). Example 8

Total cell extracts were prepared from Huh-7 and Huh-21-5 cells by resuspending trypsinized cell pellets (105 cells) in SDS-PAGE loading buffer and passage through a QIAshredder (Qiagen, Valencia, CA). Samples were heated by boiling and separated by SDS-PAGE on 4-20% gradient gels, before transfer to membrane and standard immunoblotting procedures and chemiluminescent detection (Amersham Pharmacia Biotech, Piscataway, NJ). The expression of HCV core protein and HCV polymerase were detected by using anti-core and anti-NS5B monoclonal antibodies. The primary monoclonal antibodies 10G5H4 (core) and 1A11A1 (NS5B) were used at a dilution of 1/5000. For results see Figure 5A. And as a control for cellular protein

expression, human α-tubulin was detected with monoclonal antibody DM1A

(Sigma-Aldrich, St. Loius, MO, USA) at a dilution of 1/5000. Example 9

Total RNA was prepared from control or IFN-treated cells in 96-well plates using the RNeasy kit (Qiagen, Valencia, CA). Quantitation of the HCV genomic RNA in 10ng total RNA was performed using the HCV RNA bDNA 3.0 Assay at the Bayer Diagnostics Reference Testing Laboratory (Berkeley, California). All samples tested fell within the reporting range of the assay (2,500 to 40,000,000 HCV copies).

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Claims

We claim:

1. A method for determining whether a substance is capable of modulating, inhibiting or interfering with HCV comprising the steps of: a) treating an HCV replication system with a test substance, and b) performing an immunoassay for detection of HCV core antigen to measure the ability of said substance to modulate, inhibit or interfere the HCV replication system as compared to a control.

2. The method of claim one wherein said test substance is selected from the group consisting of DNA, RNA, ribonucleosides, ribonucleoside mono-, di-, or tri-phosphates, protein, peptides, or other small organic and inorganic molecules or an analog thereof.