Animal Models for the Study of Hepatitis C Virus Infection and Related Liver Disease

Transcription

1 GASTROENTEROLOGY 2012;142: Animal Models for the Study of Hepatitis C Virus Infection and Related Liver Disease Jens Bukh Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre; and Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Hepatitis C virus (HCV) causes liver-related death in more than 300,000 people annually. Treatments for patients with chronic HCV are suboptimal, despite the introduction of directly acting antiviral agents. There is no vaccine that prevents HCV infection. Relevant animal models are important for HCV research and development of drugs and vaccines. Chimpanzees are the best model for studies of HCV infection and related innate and adaptive host immune responses. They can be used in immunogenicity and efficacy studies of HCV vaccines. The only small animal models of robust HCV infection are T- and B- cell deficient mice with human chimeric livers. Although these mice cannot be used in studies of adaptive immunity, they have provided new insights into HCV neutralization, interactions between virus and receptors, innate host responses, and therapeutic approaches. Recent progress in developing genetically humanized mice is exciting, but these models only permit studies of specific steps in the HCV life cycle and have limited or no viral replication. Keywords: Pathogenesis; Liver Disease; Safety; Drug Development. Advances in basic and clinical studies of hepatitis C virus (HCV) infection and associated acute and chronic liver diseases require representative in vitro and in vivo models. 1 Current in vitro systems are limited in the types of cells and virus strains that can be studied. 1,2 There is no established, small animal model that can be used to study the entire viral life cycle of HCV infection and associated immunity and pathogenesis. Chimpanzees are the only animals that can be used to completely study HCV infection; they can be infected with isolates of the 6 epidemiologically important genotypes and have innate and adaptive immune responses similar to those observed in infected humans. 3,4 Studies in chimpanzees led to the discovery of HCV and have improved our understanding of its pathogenic mechanisms. 5 These studies provided insight into the nature of immune response, particularly the intrahepatic response, to primary and secondary infections. 3,6,7 It is important to understand the components of an effective immune response to design successful vaccines. The immunogenicity and efficacy of potential vaccines against HCV can be tested experimentally in only chimpanzees; such studies have shown that vaccines designed to induce neutralizing antibodies or T-cell responses can control HCV titers and disease following challenge and potentially reduce the rate of chronicity Most human vaccine trials performed or initiated for HCV were based on results from studies of chimpanzees. Finally, it would not have been possible to demonstrate the infectivity of HCV clones without chimpanzees ; this research led to the development of HCV culture systems and small, genetically humanized animal models that can be challenged with modified forms of HCV. 22 A number of small animal models of HCV infection, developed during the last 10 years, are the only alternatives to research in chimpanzees: other primates do not appear to be susceptible to HCV infection. These models, and their utility and shortcomings in HCV research, are reviewed, and the future role of chimpanzees in HCV studies is addressed. An overview of HCV animal models and their application in HCV research, and in drug and vaccine development, is shown in Figure 1. Abbreviations used in this paper: DAA, direct-acting antiviral; HCV, hepatitis C virus; HVR1, hypervariable region 1; Ig, immunoglobulin; mir, microrna; NIH, National Institutes of Health; NS, nonstructural; SCID, severe combined immunodeficiency; SR-BI, scavenger receptor class B type I; upa, urokinase-type plasminogen activator by the AGA Institute /$36.00 doi: /j.gastro

2 1280 JENS BUKH GASTROENTEROLOGY Vol. 142, No. 6 Figure 1. Proposed animal models in HCV research. For each indicated model, specific characteristics on utility and applications are indicated. For all small animal models there are areas of research that are not currently feasible, indicated with not relevant. a Tested by intrahepatic transfection with RNA transcripts from full-length HCV clones. b Activates a cellular reporter permitting bioluminescence imaging. c Chimpanzees are not necessarily good models of chronic liver disease and HCV-associated hepatocellular carcinoma. Mouse Models T- and B-cell deficient mice (severe combined immunodeficiency [SCID] mice), grafted with human hepatocytes, are the only small animals that can be robustly infected with HCV. 23,24 Because these mice are immunedeficient, they have impaired utility for studies of adaptive immunity. However, in recent years, researchers have focused on developing mouse models of HCV infection and pathogenesis Although there have been some ingenious and sophisticated approaches, the developed models have restricted replication of HCV or support only part of the HCV life cycle, limiting the utility of these models. The first HCV infection studies in a mouse model were performed by Mercer et al in This study demonstrated that SCID mice with a genetic alteration that led to degeneration of their hepatocytes (they over expressed a urokinase-type plasminogen activator [upa]) transgene) could be engrafted with primary human hepatocytes and subsequently infected with HCV. However, mortality is high in this upa-scid model because fresh or cryopre-

3 May 2012 ANIMAL MODELS FOR HCV INFECTION DISEASE 1281 served primary hepatocytes have to be transplanted intrasplenically within the first 2 weeks of life. 23,30,31 Susceptibility to HCV infection requires repopulation of most of the mouse liver (70% 80%) with functional human hepatocytes that secrete a variety of human hepatic proteins. The transplanted human liver cells appear normal, with the exception of glycogen deposition. 30 Typically, these mice have a successful graft if their level of human albumin is 1 mg/ml. 31 The HCV infection rate of these mice correlates with a human-like lipoprotein profile. 32 Another model, developed from immunodeficient mice with genetic alterations, is also efficiently repopulated with human hepatocytes if a large number are transplanted and is therefore susceptible to HCV infection. 33 Up to 95% of the liver in these mice (the Fah -/- Rag2 -/- IL2rg -/- [FRG] model) can be engrafted with human hepatocytes. The advantage of this model is that the effect of genetic alteration that causes hepatocyte degeneration (absence of the gene that encodes fumeral acetoacetate hydrolase) can be blocked by oral administration of a drug, so the mice remain healthy until engraftment. To generate mice with human chimeric livers, the drug is withdrawn, and the mice are injected with human hepatocytes. In experiments, the human liver chimeric mice are infected with virus inocula from in vivo 23,30,33 or in vitro HCV infections. 19,33,34 They can also be infected via intrahepatic injection of RNA from HCV clones, 34,35 permitting reverse genetic studies. 36,37 Following infection, the HCV titer increases rapidly; genome titers of 10 7 IU/mL have been detected as early as 2 weeks after infection, with efficient passage to naïve mice. 31,33,34 The composition of the HCV particles produced in the chimeric livers appears to resemble that from infected humans or experimentally infected chimpanzees. 19 Finally, long-term infection (up to 10 months) is possible. 23,33 Mice with human chimeric livers therefore permit robust HCV infection and can be used to study infection with prototype isolates representing the 6 major HCV genotypes and important subtypes. 4 The obvious shortcoming is that these mice cannot be used in studies of the adaptive immune response to HCV. They therefore cannot be used to study many aspects of immunity and pathogenesis or, more importantly, as challenge models for vaccine studies. However, studies during the last 10 years in human liver chimeric upa-scid mice have contributed to our understanding of virus neutralization, virus-receptor interactions, novel therapeutics, and innate host responses. Finally, this model has been used to evaluate the in vivo relevance of functional findings from studies of recombinant HCV in Huh7 cell-based culture systems. Studies of Antibody-Based Strategies to Prevent Infection The generation of neutralizing antibodies during chronic HCV infection was originally demonstrated in chimpanzees. 3,38 More recently, studies in in vitro systems, using HCV pseudoparticles and HCV cell culture-grown virus, have confirmed the development of HCV antibodies with neutralizing capacity in most, if not all, HCV-infected individuals. 39 It will most likely be important for a HCV vaccine to induce high titers of neutralizing antibodies, as recently reported from tests of recombinant vaccine candidates in chimpanzees. 11,40 In addition, HCVspecific human monoclonal antibodies with in vitro neutralizing activity have been developed. 39 Finally, several HCV coreceptors have been identified, including CD81 and scavenger receptor class B type I (SR-BI), and receptor-blocking antibodies that inhibit HCV infection in vitro have been identified. 41,42 The human liver chimeric mouse model offers a unique opportunity to evaluate the abilities of these antibodies to prevent HCV infection in vivo. Chimpanzees were previously used to test the neutralizing activity of HCV-directed antibodies in research that defined the first recognized HCV neutralization epitope. 3,43 In a series of proof-of-concept studies in human liver chimeric mice, immunoglobulin (Ig) G purified from blood of a patient with chronic HCV genotype 1a infection completely neutralized the homologous HCV but only partially neutralized heterologous strains of HCV. 44,45 This chronicphase sample had previously been found to contain a high titer of neutralizing antibodies against HCV genotypes 1a, 4a, 5a, and 6a pseudoparticles and cell culture-grown viruses. 46,47 In fact, the in vitro titers of neutralizing antibodies against the heterologous strains tended to be higher than those against the homologous strain. However, 7 of 7 mice injected with the patient s IgG (at 1 mg/g) were protected against subsequent challenge with homologous HCV, measured 2 weeks after the challenge; all 4 mice given an unrelated IgG (controls) had high-titer HCV infections at this time point ( 10 7 IU/mL). When mice injected with the patient s IgG were challenged with HCV genotype 4a or 6a, only 2 of 6 and 2 of 4, respectively, were protected from infection 2 weeks after the challenge. However, the HCV titers in these mice were significantly lower than in the corresponding control mice. These studies demonstrated that neutralization titers observed in vitro do not always correlate with response to in vivo challenge. The effective neutralization of the homologous strain encourages further attempts at passive immunotherapy and HCV vaccine development. However, the relatively low level of cross-genotype neutralization indicates the challenges to developing antibodies that are effective against heterogeneous HCV. Interestingly, Law et al 48 reported that human liver chimeric mice infused with HCV E2-specific human monoclonal antibodies, directed against a conserved epitope, were protected against heterologous HCV. Anti-HCV might therefore be used in immunoprophylaxis or immunotherapy, although the effects of such antibodies after exposure are unclear. The high variability of HCV strains challenge the development of broadly protective, HCV-specific antibodies, so there has been interest in developing antibodies against host-cell HCV receptors, which might prevent HCV infection or progression of an established infection. In human

4 1282 JENS BUKH GASTROENTEROLOGY Vol. 142, No. 6 liver chimeric mice, monoclonal antibodies that block CD81 or SR-BI protected against subsequent HCV challenge with different genotypes; only anti-sr-bi controlled an already established infection. 41,42 Studies are needed to determine whether these antibodies can prevent infection of transplanted livers in patients. Studies of Drugs Approximately 50% of patients with HCV infection are cured by treatment with a combination of interferon- and ribavirin; response depends on HCV genotype and host genetic factors. Many patients are not treated because of contraindications and adverse effects of these drugs. Direct acting antiviral (DAA) agents have therefore been developed against specific HCV proteins, 49 including the nonstructural (NS)3/NS4A protease, which processes the viral polyprotein; the NS5B polymerase, which replicates the HCV RNA genome; and NS5A, which is involved in viral replication and assembly. DAAs that target the NS3/NS4A protease have been licensed for treatment of chronic HCV genotype 1 infection, initially in combination with interferon- and ribavirin. However, HCV can become resistant to DAA agents, given alone or in combination with interferon- and ribavirin, via single site mutations in the targeted protein. Nevertheless, HCV infection can be cured with these drugs, so more DAAs, against multiple viral targets, are being developed. Interferon-free treatment regimens that combine 2 or more DAAs are also being developed; these therapeutic strategies could be studied in human liver chimeric mice Chimpanzees and human liver chimeric mice each have limitations for studies of interferon treatment. 50,53 The mice can only be used to study the first phase of the interferon effect because the second phase involves the cellular immune response. Nonetheless, interferon has a clear antiviral effect in human liver chimeric mice infected with HCV genotype 1a, 1b, or 2a; mice with HCV genotype 2a infection responded better than those with genotype 1a, in line with observations in humans. 34,54 The mice were used to show that a naturally occurring HCV genotype 1b/2k recombinant could respond to interferon treatment 55 and also to support a model of evolving resistance mutations in the core protein during interferon treatment. 56 In a recent study, however, the interferon response did not appear to be affected in mice infected with recombinant HCV with engineered core or NS5A interferon resistance mutations. 37 Interestingly, mice repopulated with primary hepatocytes from donors with different interleukin-28b genotypes had different responses to interferon, similar to patients. 37 Human liver chimeric mice have also been used to study hepatocyte genes expressed during interferon treatment. 57 However, this mouse model is likely to have limitations for testing the safety and efficacy of therapeutic strategies that target host factors, such as locked nucleic acid directed against the microrna (mir)-122; this reagent is effective against HCV infection in chimpanzees. 58 Human liver chimeric mice might, however, be used in preclinical analyses of DAA agents. Studies are needed to determine how results from this model, which has relatively short-term infection that is limited to human hepatocytes, correlate with results from humans, who have chronic HCV infections and possible second sites of virus replication. Chimpanzees with chronic HCV infections have been used to test DAAs, but a smaller, more affordable animal model is needed for large-scale preclinical analyses of the many possible combination therapies. Human liver chimeric mice were used in studies of the first protease inhibitor shown to have direct antiviral effect in humans, BILN ,63 Short-term treatment reduced levels of viremia, and a study showed how rapidly resistance mutants were selected in vivo. 54 Another study demonstrated signs of cardiotoxicity, an adverse effect originally observed in primates that led to discontinuation of development of this drug. 63 Later studies in these mice showed the effects of other individual inhibitors, including the protease inhibitor telaprevir, now licensed for treatment of patients with HCV genotype 1 infection. 36,64 66 Virus isolated from patients who developed telaprevir resistance during monotherapy resulted in an attenuated infection of the mice, indicating loss of fitness. 36 Resistance to telaprevir developed within 2 weeks in mice infected with wild-type virus. 36 Finally, mice infected with recombinant HCV with engineered putative resistance mutations were in fact resistant to telaprevir. 36 Many results from studies of DAA agents in human liver chimeric mice are therefore similar to those from studies of chronic HCV patients. 49 Ohara et al 67 showed that the combination of telaprevir and an NS5B inhibitor, with or without interferon, led to rapid eradication of HCV infection and sustained clearance after the therapy was stopped. Five mice given high doses of protease or NS5B inhibitors had a 4 5 log decrease in viral titers and loss of viremia 1 week later (detection limit, 1000 IU/mL). Based on these findings, future treatments for HCV infection look promising, in line with preliminary results from clinical trials of DAA combinations. 68,69 Human liver chimeric mice are also being used in studies of drugs that increase the host innate response or affect viral entry and in adoptive immunotherapy Given their lack of adaptive immune response, these mice cannot not be used to evaluate therapeutic vaccines. Studies of HCV Pathogenesis and the Innate Immune Response Studies of the adaptive immune response to HCV infection cannot be performed in human liver chimeric mice because of their immunodeficient background. HCV infection of mice with chimeric human livers can persist for several months without liver injury because HCV apparently is not cytopathic in vivo. However, the innate immune response can be studied in these mice. Walters et al 79 analyzed the gene expression profiles in mice that received human hepatocytes from different donors, but were infected with the same strain of HCV (genotype 1a), and observed different responses, including an innate

5 May 2012 ANIMAL MODELS FOR HCV INFECTION DISEASE 1283 immune response. Therefore, there are specific immune responses to HCV in the absence of the adaptive immune system. However, the different responses did not affect HCV titers; studies in chimpanzees reported that viral titers decreased only when the HCV-specific adaptive immune response was detected weeks after the activation of the innate immune system. 80,81 This study also confirmed the influence of HCV on the lipid metabolism, as originally reported in studies of infected chimpanzees. 82 A study from the same authors showed that HCV infection affects pathways that sensitize the infected hepatocyte to apoptosis or cell death. 83 There have also been gene expression studies of human liver chimeric mice infected with HCV genotype 1b. 57 Finally, this model is being used to determine how HCV infection affects activities of specific host enzymes, such as those involved in cholesterol biosynthesis. 78,84 Studies of Cell Culture-Derived HCV Robust, true cell culture systems for HCV were reported in 2005; all required the strain JFH1 (genotype 2a), which has unique replication capacity in human hepatoma-derived cell lines. 21 This system included an intragenotypic 2a/2a recombinant, named J6/JFH1, which included the structural genes (Core, E1, and E2), p7, and the nonstructural (NS) gene 2 from the strain J6. 85 The recombinant J6/JFH1 cell culture-derived viruses robustly infected human liver chimeric mice, and recovered viruses had high specific culture infectivity. 19,86 Subsequent studies showed that this HCV recombinant did not require adaptive mutations in culture, but mutations were consistently identified in mice with robust J6/JFH1 infections, particularly in the NS2 gene. 87 Because human liver chimeric mice can be infected with the J6/JFH1 recombinant, they can be used to study culture-derived mutants. In recent studies, cultured J6/JFH1 viruses that lack hypervariable region 1 (HVR1) or 40 amino acids within NS5A domain 2 were found to be fully viable 87,88 ; a virus with enhanced green fluorescent protein inserted in NS5A domain 3 was viable, but the heterologous, enhanced green fluorescent protein-expressing sequence was eventually deleted. 88 The original JFH1 recombinant requires adaptive mutations for efficient growth in culture A recombinant that had 6 adaptive mutations was found to be fully viable in human liver chimeric mice, producing high viral titers by 2 weeks after inoculation. 89 However, recovered viruses had reversion of specific adaptive mutations, indicating impaired fitness of the culture-derived JFH1. 89 Furthermore, JFH1 genomes with mutations that lead to cytopathic effects in culture robustly infected human liver chimeric mice, but these mutations were eventually lost in vivo. 92 The JFH1 strain has also been reported to accumulate adaptive mutations in infected chimpanzees. 93,94 Most other cultured viruses are intergenotypic recombinants, depending on the replication capacity of JFH1. These recombinants require adaptive mutations, which could also affect in vivo infectivity. 89,95 98 It will be important to determine to what extent these mutations affect the utility of the human liver chimeric mice for studies of culture-derived viruses. The development of HCV replicons was an important contribution to studies of HCV RNA replication and to the development of DAAs. Their efficient replication in human hepatoma-derived cell lines requires adaptive mutations (also called replication-enhancing mutations) in the nonstructural proteins of HCV. A study in chimpanzees that used an infectious clone of the HCV strain (Con1; genotype 1b), from which the replicon was developed, demonstrated that genomes with replicon adaptive mutations in NS3 and NS5A were nonviable or highly attenuated in vivo. This observation could explain why fulllength genomes with these mutations did not yield virus production in culture. 99 More recent cell culture studies showed that the adaptive mutations interfere with HCV assembly. 100 Studies in human liver chimeric mice confirmed that the wild-type Con1 genome was viable, whereas Con1 genomes with combinations of replicationenhancing mutations were nonviable. 100 However, a Con1 genome with a replication-adaptive mutation in NS4B was fully viable in vivo, indicating that it might be possible to identify replication-enhancing mutations that lead to development of new HCV culture systems: this was demonstrated for the strain H77C. 101,102 Other Humanized Mouse Models for HCV Infection In 1995, Galun et al described immunodeficient mice with heterotopic human liver grafts. 103 To create these mice, liver fragments were transplanted at extrahepatic sites. However, the risk of graft loss is a major limitation to this model, which also yielded only low levels of viremia. The model has been used to evaluate the anti-hcv potential of a human monoclonal antibody and a DAA; dose-dependent inhibition was observed. 104 Two additional humanized mouse models were described in ,105 These models were designed to overcome the lack of adaptive immunity in the SCID-based models. Their use is restricted, however, by limited viral replication: they have no detectable level of viremia. One genetically modified model (AFC8-huHSC/hep mice) supports engraftment of human hepatocyte progenitor cells and hematopoietic stem cells, resulting in liver repopulation with human hepatocytes and immune reconstitution with human leukocytes. 105 When these mice were inoculated with sera from patients with high titers of HCV genotype 1a, HCV RNA was detected in the livers of about 50% of the mice but not in blood. Livers of mice with detectable HCV RNA were infiltrated with human immune cells and had evidence of hepatitis and increased levels of liver enzymes; HCV-specific T-cell responses were detected, and about half of these mice developed liver fibrosis. This model might have been the first small animal model of HCV infection that could be used to study adaptive immunity and pathogenesis. However, it is not clear whether infectious HCV particles are produced and

6 1284 JENS BUKH GASTROENTEROLOGY Vol. 142, No. 6 how the effects observed in the liver compare with those of humans with productive HCV infection. Also, these mice lack fully functional human B cells, so development of HCV specific antibodies is impaired. This is an important limitation, given the likely importance of HCV-neutralizing antibodies in controlling progression of HCV infection and for vaccine development. In the second genetically humanized model (Rosa26- Fluc mice), a subpopulation of the liver cells from immunocompetent mice expressed human cell-surface receptors required for HCV entry. 22 Entry of HCV into mouse liver cells could be tracked because the virus was genetically modified to induce expression of luciferase. However, most likely due to host restrictions, there was no evidence for HCV replication in the liver, and HCV could not be detected in blood of entry-positive mice. Several lines of evidence indicated that entry of the humanized liver cell occurred via pathways that resemble those established for HCV entry in humans. This model was used to study passive immunoprophylaxis against HCV challenge and protection against entry by a recombinant vaccine that induced HCV-neutralizing antibodies. Other Small Animal Models of HCV Infection There have been reports of HCV infection in New and Old World monkeys, but most evidence indicates that these primates are not susceptible to HCV infection. 106,107 Tree shrews (Tupaia belangeri, non-rodent small mammals that are easy to maintain and reproduce) have been proposed as a model for HCV infection. Even though these animals can apparently be infected with HCV, they have not found widespread use. The low and variable infection rates and HCV titers are problematic. The reported development of persistent infection and evidence for chronic liver disease are, however, attractive features of this model. In 2005, Wu et al 111 reported the ability to generate tolerized, immunocompetent rats with transplanted human hepatoma cells. Following inoculation with HCV sera (genotype 1), HCV antigen and replicating HCV RNA could be detected in human liver cells. Furthermore, the rats had increasing levels of viremia, with peak titers at 8 and 12 weeks after inoculation. In addition, the infected rats had signs of hepatitis. The utility of this animal model, however, remains in question; it has not been widely used and could not be used to study the cellular immune responses against HCV-infected cells. Surrogate Animal Models GB virus B (GBV-B) is a virus with an unknown natural host that produces high viral titers and acute hepatitis in experimentally infected tamarins, owl monkeys, and common marmosets Within the Flaviviridae family, the GBV-B genome clusters with HCV variants and has therefore been proposed as a surrogate for in vivo studies of HCV. 112,116 However, experimental GBV-B infections only rarely develop into chronic infections Clones of GBV-B that infect tamarins have been developed, 112 permitting functional in vivo studies, 118,120 and researchers have created chimeric viruses that contain genetic elements of HCV, such as a domain within the 5= untranslated region, HVR1, and p These might be used in functional HCV studies. However, it is not clear whether this model could have a role in HCV vaccine studies. A virus more closely related with HCV, named Canine Hepacivirus, was identified in dogs with respiratory illness. 124 It however remains to be determined whether an experimental animal model can be established for this virus and whether it could serve as a surrogate model for HCV studies. 125 Animal Models That Are Not Susceptible to HCV Infection Mice that express transgenes that encode HCV protein elements are mainly used in studies of HCV pathogenesis. Although a discussion of models that do not permit natural steps in the viral life cycle is outside the scope of this review, it should be mentioned that these models have contributed to understanding of HCV pathogenesis. Detailed information can be found in comprehensive reviews on this topic. 25, Associated research has indicated the direct role of HCV proteins, of the HCV core protein in particular, in HCV-associated carcinogenesis. Furthermore, studies in these mouse models have contributed to our understanding of how HCV affects lipid metabolism, which was originally observed in gene expression studies in infected chimpanzees. 82,129 Several animals that are not susceptible to HCV infection and therefore cannot be used to study viral challenge following vaccination (common rodents, baboons, and rhesus monkeys) have been used to test the immunogenicity of putative HCV vaccine antigens. 9 Those were useful preliminary studies, but the conclusions drawn from such studies were limited, because it is not possible to completely determine how these findings pertain to humans. Immunogenicity studies in such models are, however, a prerequisite for immunogenicity studies in humans; they are frequently also performed before candidates are tested in chimpanzees. Chimpanzees in HCV Research Chimpanzees are primarily available for HCV research in the United States, where several animal facilities can perform studies in a suitable environment following established guidelines for the care and use of laboratory animals. 3,130 The chimpanzees currently used have all been bred in captivity and represent a genetically unselected population. The National Institutes of Health (NIH) has provided funding for HCV studies in this expensive model in recognition of its unique importance for advancement of this research field. Other public and private funding agencies and companies have supported HCV research of vaccines and drug candidates in chimpanzees. It remains unclear, however, how a recent report from the Institute of Medicine (released December 15, 2011), assessing the necessity of us-

7 May 2012 ANIMAL MODELS FOR HCV INFECTION DISEASE 1285 ing this model in biomedical research, will influence future HCV research in chimpanzees, particularly for NIH-funded research. 131 The Institute of Medicine report recommends limiting the use of chimpanzees in biomedical research to studies that meet the following 3 criteria: (1) there is no other suitable model available, such as in vitro, nonhuman in vivo, or other models, for the research in question; (2) the research in question cannot be performed ethically on human subjects; and (3) foregoing the use of chimpanzees for the research in question will significantly slow or prevent important advancements to prevent, control, and/or treat life-threatening or debilitating conditions. Furthermore, the committee reported a number of findings 131 that would, if implemented by the NIH, limit this in vivo research. The NIH has established a new oversight group of independent experts to judge chimpanzee research studies in progress and new proposals and evaluate whether they meet criteria spelled out in the report. Whether this in the end will restrict research on HCV in chimpanzees remains to be determined. There are several areas of HCV research in which studies in currently available small animal models cannot provide data (Figure 1). These are studies of the protective immunity and vaccine and drug development. An evaluation of whether the information obtained from chimpanzee studies could potentially be obtained from naturally infected humans is outside the scope of this review, which aims to describe available animal models. However, it is apparent that controlled challenge studies can only be performed in chimpanzees. Controlled Challenge Studies Given the lack of small animal models for studies of the adaptive immune response, chimpanzees are the only model that can be used to study immunity associated with acute resolving infection and protective immunity against HCV reinfection following clearance. Learning more about the components of immune response against HCV would improve rational vaccine design. There are several examples of things we have learned from chimpanzees that we could not have learned from human studies. Early events in the immune response can be analyzed in a manner that is not possible in humans with acute infections: liver tissue can be collected at frequent intervals, and HCV-specific T-cell responses or gene expression profiles can be analyzed. 3 The acute resolving infection was associated with strong, intrahepatic, HCVspecific CD4 and CD8 T-cell responses. 81,132 HCV persistence was associated with weaker responses and/or with development of viral escape mechanisms, including mutations in viral epitopes recognized by CD8 T cells. 133,134 Detailed features of the host responses and their importance for the outcome of acute HCV infection are being mapped in a systematic fashion. 80,132, Changes in gene expression patterns have been characterized in liver samples of acutely infected chimpanzees. These studies have shown that acute HCV infection influences the expression of a large number of genes, including those involved with the innate and acquired immune responses, as well as those involved with metabolism (including lipid metabolism), apoptosis, and cell cycle regulation. 82,129,138 Studies in chimpanzees have been of unique importance for defining protective immunity against HCV following re-exposure. In general, the correlates of viral clearance following rechallenge were similar to those associated with clearance following a primary infection Importantly, the role of CD4 and CD8 T-cell responses was defined in studies in which chimpanzees with resolved HCV infections were depleted of CD4 and CD8 T cells using specific antibodies. After depletion of CD8 T cells, viremia was prolonged after challenge, and virus clearance coincided with the reappearance of these T cells in the liver. 142 After depletion of CD4 T cells, HCV persisted after challenge, indicating that an inadequate CD4 T-cell response affects the outcome of HCV infection. 140 Such controlled intervention studies provide essential information about protective immunity and can only be performed in chimpanzees. The chimpanzee remains the only animal model that permits HCV challenge after vaccination, and defined HCV challenge pools of genotypes 1 6 have been titrated in chimpanzees. 4 Vaccination and challenge can be performed with matched antigen and virus to demonstrate a protective effect. Of the protective correlates of an HCV vaccine (production of cellular immune responses and neutralizing antibodies), only the effect of antibodies can be tested in other systems; robust T-cell responses most likely will be required for development of an effective HCV vaccine. There are at least 2 examples of vaccine studies that have led to clinical phase I vaccine trials. The first promising HCV vaccine candidate was developed by Houghton and others 9 and aimed to prevent infection by inducing neutralizing antibodies. Chimpanzees were immunized with recombinant genotype 1a E1/E2 heterodimers produced in mammalian cells. Homologous and heterologous genotype 1a HCV challenge studies were performed. 9,10,146 Following homologous challenge, only 2 of 12 vaccinated chimpanzees were persistently infected, in contrast to 7 of 10 control chimpanzees. Several vaccinated animals were apparently not infected. Induced immune responses have been analyzed in detail and included the development of broadly reactive neutralizing antibodies. 9,40 Following heterologous genotype 1a challenge, all animals became infected. However, most immunized animals resolved the acute infection, whereas most control animals progressed to chronic infections. The recombinant E1/E2 vaccine therefore protected against challenge with a low dose of homologous HCV and reduced the carrier rate after homologous and heterologous challenges. This recombinant E1/E2 vaccine was subsequently tested for safety and immunogenicity in phase I clinical trial. 147 The studies of this vaccine in chimpanzees demonstrated that it might be possible to significantly reduce the rate of chronic infection through HCV vaccination, which could reduce HCV-associated disease. In 2006, Folgori et al 148 described a prime boost regimen in chimpanzees designed to induce a T-cell response.

8 1286 JENS BUKH GASTROENTEROLOGY Vol. 142, No. 6 The animals were initially vaccinated with adenovirus expressing the HCV NS3-NS5B protein of an HCV genotype 1b strain then given boosts of plasmids that encoded the same HCV antigen and then challenged with a heterologous 1a virus. 10,148 The final outcome of the vaccinated animals did not differ significantly from the control group (4 of 5 vaccinated animals and 3 of 5 controls cleared the infection). However, the virologic and clinical courses of infection differed between groups. The vaccinated animals controlled the virus earlier, indicated by significantly lower viremia and levels of liver enzymes. The vaccinated animals also developed potent, cross-reactive T-cell responses, indicated by the presence of HCV-specific interferon- producing CD8 T cells, including intrahepatic memory CD8 T cells, in the 4 animals with viral clearance. The promising results from this study led the investigators to develop an adenovirus-based HCV vaccine, which has recently been tested for safety and immunogenicity in a phase I clinical trial. 149 The chimpanzee study showed that it was possible to effectively control HCV viremia with vaccine-induced T-cell responses. It is not clear whether these responses will be sufficient to reduce the rate of chronic HCV infection in vaccinated populations because, in HCV rechallenge studies in chimpanzees, HCV was able to evade pre-existing cellular immunity, 139,150 even when the animals were challenged with autologous virus. 139 Vaccines should therefore be developed to induce production of neutralizing antibodies. Overall chimpanzees have permitted groundbreaking studies of HCV vaccines, permitting controlled challenge studies and providing detailed information on immune responses associated with viral control. These studies can serve as benchmarks for immunogenicity studies in humans. It will be important to use chimpanzees to define components of effective innate and adaptive immune responses against HCV 150 and for proof of principle studies of new HCV vaccine approaches. 146,148,151 Testing Therapeutics That Target Host Factors Various host factors affect HCV replication, such as mir-122, which binds to highly conserved sites in the 5= untranslated region of the HCV genome to regulate its replication. 152 In vitro replication of HCV genotypes 1 6 is inhibited by antagonists of mir The mir-122 antisense locked nucleic acid SPC3649 efficiently suppressed chronic infection of chimpanzees with HCV genotypes 1a and 1b, with no evidence of viral resistance or adverse effects. 58 This chimpanzee study led to phase 1 and 2 trials of this host-directed therapeutic. Small animal models are not necessarily useful for studies of reagents that target human host factors involved in HCV infection, and human studies cannot be performed without preclinical analyses of efficacy and safety. Future Directions Many complex approaches have been used to humanize mice and permit their infection with HCV; these have created new possibilities for HCV research. Human liver chimeric mice have contributed to studies of infection and passive imunoprophylaxis and are important for testing the in vivo relevance of in vitro findings. The model seems useful for preclinical analyses of DAAs, but such studies are not required to bring these drugs into the clinic. 29 Human liver chimeric mice are not useful for studies of reagents that target host factors to inhibit HCV replication; their effects on other human organ systems could be missed. Chimpanzees have important roles in these types of studies, exemplified by results from studies of the antagonist of mir The complex approaches taken to genetically humanize mice and permit studies of specific steps in the HCV life cycle could lead to development of more advanced humanized models. The creation of mice with human hematopoietic and liver cells has allowed researchers to reproduce HCVinduced liver fibrosis in mice. 105 This model is of particular importance because one of the shortcomings of chimpanzees for studies of HCV infection is that they are not necessarily good models of chronic liver disease and HCV-associated hepatocellular carcinoma. Mouse models are restricted in their utility for studies of HCV infection and progression, and for prophylactic and therapeutic vaccines. Chimpanzees currently remain the only model available for these studies. (Figure 1). 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Antiviral Res 2008;80: Received January 24, Accepted February 15, Reprint requests Address requests for reprints to: Jens Bukh, MD, Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Kettegaard Alle 30, DK-2650 Hvidovre, Denmark. jbukh@sund.ku.dk; fax: (45) Conflicts of interest The author discloses no conflicts.