Novel robust hepatitis C virus mouse efficacy model

Abstract

The lack of a robust small-animal model for hepatitis C virus (HCV) has hindered the discovery and development of novel drug treatments for HCV infections. We developed a reproducible and easily accessible xenograft mouse efficacy model in which HCV RNA replication is accurately monitored in vivo by real-time, noninvasive whole-body imaging of gamma-irradiated SCID mice implanted with a mouse-adapted luciferase replicon-containing Huh-7 cell line (T7-11). The model was validated by demonstrating that both a small-molecule NS3/4A protease inhibitor (BILN 2061) and human alpha interferon (IFN-alpha) decreased HCV RNA replication and that treatment withdrawal resulted in a rebound in replication, which paralleled clinical outcomes in humans. We further showed that protease inhibitor and IFN-alpha combination therapy was more effective in reducing HCV RNA replication than treatment with each compound alone and supports testing in humans. This robust mouse efficacy model provides a powerful tool for rapid evaluation of potential anti-HCV compounds in vivo as part of aggressive drug discovery efforts.

FIG. 1.

(A) Schematic of the HCV replication animal model. Mouse-adapted replicon-containing T7-11 cells expressing a high level of luciferase (Luc) were implanted subcutaneously or directly into the liver parenchyma of γ-irradiated SCID mice. Drug evaluation was performed 2 to 3 weeks after cell implantation. The HCV replication level was monitored quantitatively by measuring the bioluminescence signal using the IVIS imaging system. (B) Structure of the subgenomic replicon rep114/ET derived from pFK I389 LucNS3-3′/5.1 (22) and pFK I341PI Luc NS3-3′/ET (27). 5′, HCV 5 ′ nontranslated region; 3′, 3′ HCV nontranslated region; PVI, poliovirus IRES; Luc, firefly luciferase; Ubi, ubiquitin cleavage site; Neo, neomycin phosphotransferase; EI, encephalomyocarditis virus IRES; *, adaptive mutations E1202G, T1280I, and K1846T.

FIG. 2.

Correlation between mean bioluminescence in vivo and replication of subgenomic RNA in T7-11 cell subcutaneous model. (A) Mean bioluminescence of mouse-adapted replicon-containing T7-11 cells and parental S6.1-6 cells in gamma-irradiated SCID mice. Mean bioluminescence was determined for each group (n = 10 per group) at different times after subcutaneous implantation of T7-11 and S6.1-6 cells. The images of representative mice from both groups at days 1, 10, 15, 25, and 31 are also shown. (B) Tumor growth rate of T7-11 and S6.1-6 cells. Mean tumor volumes (0.5 × width × width × length = mm³) from both groups measured at days 15, 22, 25, and 29 are plotted. The graph is representative of four independent experiments. Error bars indicate standard error. (C) Detection of HCV viral RNA in tumor tissue. Total RNA was isolated from subcutaneous tumors which were excised 26 days after the implantation of T7-11 cells and analyzed by Northern blot analysis (10 μg per lane). Blots were hybridized with ³²P-labeled RNA probes corresponding to the HCV NS5 region. A total of 1 × 10⁸ genomes of in vitro transcribed HCV RNA (vRNA) served as a marker and control for the hybridization reaction (lane 1), and 28S rRNA served as a control for the amount of RNA present in each sample analyzed. Lanes 2 and 3 were total RNA extracted from naïve Huh-7 and S6.1 cells as negative controls. Lane 4 is RNA from a T7-11 tumor. SE, standard error.

FIG. 3.

Antiviral activity of IFN-α 2b and NS3/4A protease inhibitor BILN 2061 (BI) in the subcutaneous T7-11 model. (A) Dose-dependent IFN anti-HCV effect. Subcutaneous IFN treatment was initiated on day 19 after cell implantation and was administered once daily for 7 days: control, 0.1 ml of 100 μg/ml human albumin in HBSS; 7,500 IU group, 7,500 IU of IFN per day per 18 g of mouse; 15,000 IU group, 15,000 IU of IFN per day per 18 g of mouse. n = 10 to 15 mice per group. (B) Tumor growth rate in subcutaneous T7-11 model. Mean tumor volumes (0.5 × width × width × length = mm³) are plotted from control, 7,5000 IU of IFN, and 15,000 IU of IFN groups measured at days 8, 12,15, 19, 22, 26, and 29. (C) IFN treatment was given as described in panel A with 15,000 IU/mouse/day in two groups: IFN QD 14 days, once daily administration for 2 weeks; IFN QD 3 days, once daily administration for 3 days. Individual mice were imaged on day 19 before treatment, followed by days 21, 22, 26, 29, and 33. (D) Immunohistochemical analysis of tumor tissues from untreated, IFN-treated (IFN QD 14 days), and IFN rebound (IFN QD 3 days) groups 33 days postimplantation as described in panel C. The expression of viral protein NS5B was detected with a polyclonal antibody against NS5B. Cell death was detected with cPARP antibody as described in Materials and Methods. (E) Antiviral effect of protease inhibitor BILN 2061. BILN 2061 treatment was given subcutaneously at 30 mg/kg once daily for 3 days starting on day 21 postimplantation. BILN 2061 treatment was then withdrawn on day 24. Individual mice were imaged prior to each day's treatment on days 21, 22, 23, 24, 30, and 32. Error bars indicate standard error (SE). QD, once daily. BI, BILN 2061.

FIG. 4.

Anti-HCV activity of IFN-α 2b and BILN 2061 in the intrahepatic mouse efficacy model. (A) Histology of a chimeric liver. Hematoxylin and eosin staining (A′ and B′) allows identification of the human hepatoma T7-11 cells (H) within mouse (M) parenchyma. Clusters of human T7-11 cells can also be visualized with antibody specific for human mitochondria (A and B) shown in brown. (B) Antiviral effect of IFN-α 2b (IFN). T7-11 cells were implanted directly into the parenchyma of liver in γ-irradiated SCID mice. IFN treatment was given SC once daily for 2 days starting on day 27 postimplantation. A dose of 15,000 IU/mouse/day of IFN was given to the treated group, and 10 μg/mouse/day of human albumin was given to the control group. (C) Antiviral effect of BILN 2061 (BI). A treatment of 30 mg/kg of BILN 2061 was initiated once daily subcutaneously for 3 days starting on day 25 postintrahepatic implantation. Individual mice were imaged daily prior to each day's treatment on days 25 through 28. Error bars indicate standard error (SE).

FIG. 5.

Antiviral effect of protease inhibitor BILN 2061 (BI) in combination with IFN-α 2b (IFN) in the T7-11 subcutaneous efficacy model. T7-11 cells were implanted subcutaneously into γ-irradiated SCID mice. BILN 2061 at 30 mg/kg and/or IFN at 15,000 IU per mouse was administrated once daily for 3 days starting on day 18 after cell implantation. Individual mice were imaged daily prior to each day's treatment on days 18 through 21.