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Novel Robust Hepatitis C Virus Mouse Efficacy Model
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Antimicrobial Agents and Chemotherapy, October 2006, p. 3260-3268, Vol. 50, No. 10
Qing Zhu,1* Yoko Oei,1 Dirk B. Mendel,1 Evelyn N. Garrett,2 Montesa B. Patawaran,1 Paul W. Hollenbach,3 Sharon L. Aukerman,1, and Amy J. Weiner4,
Departments of Pharmacology,1 Experimental Pathology,2 Translational Medicine,3 Vaccines, Chiron Corporation, 4560 Horton Street, Emeryville, California 946084
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-) 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- 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.
INTRODUCTION
Human liver disease caused by hepatitis C virus (HCV) has emerged as a major challenge to public health, affecting an estimated 175 million people worldwide (2). Greater than 50% of infections lead to chronic liver disease with a risk of developing liver cirrhosis and hepatocellular carcinoma (1). Infection with HCV has also been identified as the most common indication for liver transplantation in the United States and Europe (9). Treatment options for chronic HCV infection are limited to a combination of pegylated alpha interferon (IFN-) and ribavirin (RB) (11), which is only partially effective and is often associated with troublesome side effects. Patients with genotype 1 HCV, the predominant genotype worldwide, are the most resistant to IFN- and RB treatment (14). Thus, development of novel therapies for HCV is greatly needed, yet has progressed slowly.
HCV is an enveloped, positive-strand RNA virus and a member of the family Flaviviridae (10). Efficient replication of an HCV subgenomic replicon in in vitro cell culture has provided a valuable tool for molecular characterization of HCV, investigating virus host interactions, screening antiviral compounds, and developing new drug targets (4, 26, 29). Recently, an in vitro cell culture system capable of producing an infectious genotype 2a HCV has also been described (25, 39, 41). While the cell-based assays have provided a useful tool for screening compounds, they have not proved sufficient to predict the activity of compounds in vivo (21, 35, 38). The limited pipeline of new HCV antivirals may, in part, be attributed to the absence of robust small-animal models, which are typically used for simultaneously assessing drug action, efficacy, and toxicity. Difficulty in developing animal models is largely a result of the narrow host range of HCV, which infects only humans and chimpanzees. Over the past few years, several small-animal models such as the HCV-Trimera and chimeric scid-Alb/uPA Hepatech mouse models have been developed and demonstrated to be useful for studying HCV infection and drug evaluation (8, 17, 19, 21, 30, 40). However, low throughput, technical difficulty, and high cost significantly limit the utility of these models for drug discovery. Recently, a mouse model that permits the evaluation of the HCV NS3/4A serine protease target only has been described (33). The disadvantages of this model include the following (i) only antiviral compounds targeting HCV protease can be evaluated;(ii) the target is not in the context of the native replisome and thus may not accurately reflect the conformation of the target in infected cells; and (iii) the viral vectors used to deliver the protease construct may affect the cell biology of the host, which may, in turn, influence the dynamics of the assay.
In this report we describe the development, validation, and application of a simple, reproducible noninfectious HCV mouse efficacy model for evaluating antiviral compounds against multiple viral targets. The model utilizes a mouse-adapted replicon-containing Huh-7 human hepatoma cell line expressing a luciferase reporter linked to the HCV subgenome. These cells can be implanted subcutaneously (SC) or directly into the liver of gamma ()-irradiated SCID mice. The replicon used in this model expresses the HCV nonstructural proteins that comprised the replisome and is transfected into human hepatoma Huh-7 cells (32). The replication level of HCV RNA replicon in individual mice was monitored by measuring luciferase activity using a noninvasive whole-body, real-time Xenogen IVIS imaging system (Fig. 1A). Both the SC and liver models were validated by demonstrating a statistically significant reduction in the viral RNA replication levels after treatment with IFN- 2b (7) or a small-molecule HCV NS3/4A protease inhibitor (BILN 2061) (23). These results indicate that the model recapitulates the clinical activity of these compounds and has potential for contributing to the rapid evaluation of novel treatments and combination therapies for HCV.
DISCUSSION
Anti-HCV compounds are often advanced into clinical trials based on demonstration of in vitro activity in cell-based assays and an acceptable pharmacokinetic (PK) and safety profile in the absence of in vivo efficacy. One drawback of this approach is that it is difficult to accurately predict the exposure necessary to achieve in vivo activity based on in vitro systems. In addition, determination that a compound does have sufficient theoretical exposure in the target organ of a normal animal based on PKs does not guarantee the efficacy of that compound in an animal containing the drug target. Major limitations of current small-animal efficacy models include low throughput, the requirement for highly specialized surgical skills, high cost, and access to human liver and HCV-infected sera. Therefore, only a very limited number of compounds can be evaluated. Additionally, because these systems use live infectious virus, specialized containment is required, and researchers are unnecessarily exposed to HCV, for which there is only a partially effective therapy available at the present time. The mouse HCV RNA replication model described here provides a noninfectious, reproducible, time- and cost-effective tool for evaluating the efficacy, PK, and pharmacodynamic (PD) relationship of potential HCV therapies simultaneously in vivo. Since our initial efforts failed to produce a replicon cell line that could persist in vivo sufficiently to generate a usable efficacy model, we reconsidered previous studies in which it was shown that RNA adaptation and/or cell adaptation were advantageous in the selection of conditions most supportive for viral replication in cell culture systems (3, 5, 15, 20, 22, 27, 28, 31, 36). By taking advantage of the host's natural ability to apply selective pressure on the replicon-containing cells passaged in vivo, we were able to select T7-11 cells, which supported high levels of HCV RNA replication in vivo for several weeks.
We found that the immune status of the host mouse strain significantly influenced the magnitude and duration of the HCV RNA replication in vivo. Whole-body irradiation causes marked depletion of hematopoietic cells, which in turn impairs functional immune response. A partial reconstitution of the host immune system typically occurs after 2 weeks (18). -Irradiated SCID and SCID/bg mice best supported HCV replicon-containing cells but for only approximately 1 week postimplantation. Since a rapid decrease in bioluminescence signal was immediately observed in nonirradiated SCID and SCID/bg mice that had T and B lymphocyte deficiencies (24), the results suggested that viral replication was either directly or indirectly inhibited by the residual host immunity associated with the innate immune responses. Whole-body irradiation of genetically immunodeficient mice resulted in the optimal conditions for maintaining viral and tumor cell replication.
Interestingly, the bioluminescence signal in mice bearing T7-11 cells was able to recover after approximately 2 weeks, while the signal in mice bearing S6.1-6 cells continued to decline, coincident with the expected reconstitution of innate immune response. Since the tumor growth rates of S6.1-6 and T7-11 cells in -irradiated mice were similar, the loss of bioluminescence signal in S6.1-6 cells can best be explained by the reduction in HCV RNA replication. Preliminary data showed that RNA replication in T7-11 cells was five- to eightfold more resistant to human IFN- in vitro than that in parental S6.1-6 cells, suggesting that the antiviral responses in the T7-11 cells appeared to be attenuated, a condition that would account for the observed increased stability of replicon RNA in vivo. This decreased sensitivity to IFN- was associated with adaptive mutations acquired at the cellular level, not at the viral RNA level. This was demonstrated by transient transfection of an identical replicon RNA (rep114/ET) into either S6.1 or cured T7-11 cells cleared of replicon RNA by IFN- treatment in vitro. Only cured T7-11 cells maintained the IFN- insensitivity phenotype as determined by the 50% effective concentration of IFN- in vitro (unpublished data). However, both T7-11 and S6.1 cells possessed functional Jak/STAT antiviral signaling pathways, as demonstrated by immunoblot analysis of expression of phosphorylated Stat1 proteins. This result suggested that downstream effectors of Jak/STAT might potentially be impaired in T7-11 cells (unpublished data).
Validation of the animal efficacy model was performed using human IFN- and protease inhibitor BILN 2061, two agents that have demonstrated anti-HCV activity in patients. Dose-dependent reduction of bioluminescence and rebound of signal after withdrawal of treatments clearly demonstrated the expected antiviral activity of these two agents. The careful examination of tumors from these studies indicated that the decline of bioluminescence resulted from a specific antiviral effect rather than a cytotoxic effect of the treatment. A good correlation was observed between the mouse model and what is seen in the clinic with respect to the in vivo therapeutic effects of these two agents (7, 16, 23). Importantly, we showed that the combination IFN-/BILN 2061 treatment significantly improved the antiviral effect on HCV RNA replication compared with either single agent, strongly suggesting that the combination therapy of small-molecule anti-HCV inhibitor and IFN- could be a promising therapeutic approach to the treatment of HCV. The results suggest that combination therapies (i.e., protease and polymerase inhibitors), which have not yet been tested in humans, can be assessed in this model.
Major advantages of the models described in this report include the following: (i) the simultaneous evaluation of multiple parameters such as efficacy and PKs/PDs can be performed since HCV replication in individual animals can be frequently monitored in vivo using the IVIS imaging system; (ii) the models can achieve statistical significance since large numbers of animals in multiarm studies can be implanted within a short period of time; (iii) using a noninfectious system supports a high-throughput mode for compound evaluation; and (iv) combination therapies can be rapidly assessed for one or more antiviral targets. Importantly, our findings showed that IFN- and BILN 2061 efficacy studies were comparable in both subcutaneous and liver models. Thus, the subcutaneous model provides a powerful tool for initial rapid in vivo compound screening and evaluation of tolerability and efficacy of compounds, while the intrahepatic model, which is a closer approximation of the human disease, can be used to more effectively evaluate the PKs/PDs and safety of drugs prior to evaluation in clinical trials.
Our data are the first to show that Huh-7 cells containing an HCV replicon can be stably adapted to grow in immunodeficient mice for more than 1 month. The workable time for drug evaluation is approximately 2 to 3 weeks. Preliminary data suggest that the stability of the viral RNA in the T7-11 cells in vivo is dependent upon the cell adaptations rather than the genetic constitution of the replicon contained within the mouse-adapted cell line (unpublished data). Therefore, while the data presented in this report are based on a mouse-adapted Huh-7 cell line harboring a subgenomic genotype 1b replicon, it is worth noting that this approach may be applied to other HCV genotypes using the appropriate replicons (infectious or noninfectious) and may potentially be applied to other viruses.
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