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Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD
Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 50, Room 6529, 50-South Dr. MSC 8009, Bethesda, MD 20892-8009
This is a US Government work. There are no restrictions on its use.
Chimpanzees remain the only recognized animal model for the study of hepatitis C virus (HCV). Studies performed in chimpanzees played a critical role in the discovery of HCV and are continuing to play an essential role in defining the natural history of this important human pathogen. In the absence of a reproducible cell culture system, the infectivity titer of HCV challenge pools can be determined only in chimpanzees. Recent studies in chimpanzees have provided new insight into the nature of host immune responses—particularly the intrahepatic responses—following primary and secondary experimental HCV infections. The immunogenicity and efficacy of vaccine candidates against HCV can be tested only in chimpanzees. Finally, it would not have been possible to demonstrate the infectivity of infectious clones of HCV without chimpanzees. Chimpanzees became infected when RNA transcripts from molecular clones were inoculated directly into the liver. The infection generated by such transfection did not differ significantly from that observed in animals infected intravenously with wild-type HCV. The RNA inoculated into chimpanzees originated from a single sequence, and the animals therefore had a monoclonal HCV infection. Monoclonal infection simplifies studies of HCV, because virus interaction with the host is not confounded by the quasispecies invariably present in a natural infection. It furthermore permits true homologous challenge in studies of protective immunity and in testing the efficacy of vaccine candidates. Finally, this in vivo transfection system has made it possible to test for the first time the importance of genetic elements for HCV infectivity. (HEPATOLOGY 2004;39:1469–1475.)
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HCV is a positive-strand RNA virus classified in a separate genus, Hepacivirus, of the Flaviviridae, a family of small enveloped viruses. This virus exhibits extensive genetic heterogeneity and has been classified into six major genotypes and numerous subtypes. In addition, each patient is infected with a quasispecies, which is a mixture of closely related but distinct genomes. Much has been learned about the natural history of HCV by careful studies of cohorts of patients who became exposed—for example, through blood transfusions. Only one out of every four patients acutely infected with HCV has clinical disease, and fulminant hepatitis caused by acute infection is extremely rare. However, as many as 80% of individuals with acute HCV become persistently infected, and HCV is a major cause of chronic liver disease (including liver cirrhosis and hepatocellular carcinoma) worldwide. In the United States, hepatitis C–related chronic liver disease is a leading cause of liver transplantation and causes thousands of deaths annually.
Although the clinical characteristics of HCV are well known to the readers of HEPATOLOGY, the critical role played by the chimpanzee model in the study of this elusive agent might be less well recognized. Years before the discovery of HCV, it was demonstrated that the etiological agent responsible for non-A, non-B hepatitis could be transmitted to chimpanzees, and the chimpanzee was subsequently used to determine the physicochemical properties of this agent.1 Furthermore, HCV was eventually cloned from plasma with a high non-A, non-B infectivity titer, as determined in the chimpanzee model; this plasma was collected from a persistently infected chimpanzee.2 Thus research performed in chimpanzees was the critical prelude for the current era of molecular and clinical studies of HCV.
Because of the limited availability and high cost of chimpanzees, an alternative animal model would be desirable; however, other primates do not appear to be susceptible to HCV, and there has been only limited success in developing a small animal model.3 The chimpanzee is primarily available for 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.4 The chimpanzees currently used are all bred in captivity and represent a genetically unselected population. The National Institutes of Health is providing funding for studies in this expensive model in recognition of its unique importance for the study of HCV.
The justification for using the chimpanzee model is to study important biological questions that cannot be studied by any other means or to generate reagents not otherwise attainable. For HCV, such requirements are easily met. A list of the various research topics addressed in the chimpanzee model is shown in Fig. 1. Because of the limited availability of chimpanzees, most studies involve only two to four animals. This limitation has implications for making conclusions that are based on statistical significance, and it must be acknowledged that observed correlations might represent biological variation among the animals. However, the chimpanzee has provided unique opportunities to study the mechanism of disease caused by HCV.
HCV, hepatitis C virus; E, envelope; HVR1, hypervariable region 1.
Natural History of HCV in Chimpanzees
Numerous studies have detailed the course of wild-type HCV infection in chimpanzees,1, 5 and the chimpanzee has proved to be a useful model of such infections. First, because of the close genetic relatedness of chimpanzees to humans, it is possible to use the same reagents and tests that have been developed for human studies. Second, challenge experiments can be performed in chimpanzees so that appropriate samples can be collected during the entire course of infection. Infections in humans are often not recognized until acute hepatitis develops 6–14 weeks after primary infection or infections are missed altogether because they are asymptomatic. Also, for ethical reasons, it is not possible to collect frequent liver tissue samples from infected patients. Finally, with the chimpanzee model it is possible to reproduce the infection and associated hepatitis observed in humans. In general, an acute experimental HCV infection is characterized by early appearance of viremia (rarely later than day 7) and peak viral titers of 105–107 genome copies per mL. Antibodies against HCV proteins are only rarely detected before week 8. Most acutely infected chimpanzees have evidence of hepatitis with elevated serum liver enzyme values and necroinflammatory changes in liver biopsies; typically the disease is somewhat milder than that observed in humans. Some acute experimental infections are resolved; however, most progress to chronicity, which is characterized by persistent viremia, seropositivity to numerous antigens, and mild hepatitis.
Monoclonal HCV Infection in Chimpanzees
The recent development of infectious clones of HCV is a good example of the unique importance of the chimpanzee model for the advance in HCV research.6, 7 Because there is no cell culture system that can be used to cultivate HCV reproducibly, it would not have been possible to demonstrate the infectivity of such clones without using chimpanzees. Chimpanzees became infected when genomic RNA transcripts synthesized in vitro from full-length HCV complementary DNA clones were inoculated directly into the liver (Fig. 2). The HCV infection generated by such transfection of chimpanzees did not differ significantly from that observed in animals infected intravenously with the original virus. Furthermore, chimpanzees transfected with molecular clones of HCV developed acute hepatitis, thus formally proving that HCV causes liver disease. Importantly, the RNA inoculated into chimpanzees was generated from a single HCV sequence; the animals, therefore, became infected with a monoclonal virus. Such monoclonal infection of chimpanzees simplifies studies of HCV, because virus interaction with the host is not confounded by the quasispecies invariably present in a natural infection. In this issue of HEPATOLOGY, Major et al.8 characterize acute hepatitis C in 10 chimpanzees experimentally infected with the same monoclonal virus, representing the consensus sequence of one of the most important prototype strains of HCV, strain H77. The authors provide a detailed account of virus kinetics in relation to disease, host responses, and outcome of infection. The main purpose of this concise review is to discuss the findings of this unique study in the context of previously published data obtained in chimpanzees and to provide an overview of the use of monoclonal HCV infection in performing more detailed and controlled studies of HCV.
Chronicity Rate of HCV in Chimpanzees
The high chronicity rate of HCV is a hallmark feature of this infection. The fact that six of the 10 animals infected with monoclonal virus became chronically infected indicates that the presence of a quasispecies virus population during the early acute phase of infection is not a requirement for persistence.8 It is notable that the 60% chronicity rate is higher than that reported in some other studies. Bassett et al.9 reported that, in a cross-sectional study of 46 chimpanzees experimentally infected with different strains of HCV, only 18 animals (39%) remained viremic and that, in a longitudinal study of six chimpanzees inoculated with the H77 strain, only two animals (33%) became persistently infected. However, in our experience, at least 60% of chimpanzees inoculated with the H77 strain (monoclonal or polyclonal) became persistently infected (unpublished data).10 In addition, a chronicity rate of greater than 60% has been reported for experimental infection of chimpanzees with other HCV strains, including the prototype HCV-1 strain.10–12 It is currently unclear which factors account for these discrepant results. The animals that became persistently infected in the study by Major et al.8 were followed for 82–216 weeks after infection and the virus titers remained at 104–105 genome copies per mL. However, it is possible that some of the animals might have viral clearance after the end of follow-up. We have observed late viral clearance in an animal that had HCV titers of 104–105 genome copies per mL during the first 2 years of follow-up (unpublished data). Whether gender, age, haplotype, or viral strain could influence the chronicity rate in chimpanzees remains to be determined. The use of animals that have not previously been exposed to HCV is essential, because prior exposure might lower the chronicity rate.
Most reported chimpanzees with acute resolving infection have clearance of viremia during weeks 12 to 24. However, among the four animals with resolving monoclonal infection, low-level intermittent viremia was observed even after week 24.8 In one case, HCV RNA was detected in sera collected at week 52, but not during subsequent weeks. Following homologous rechallenge of this animal, small amounts of HCV RNA also remained detectable.13. This raises the intriguing possibility that HCV might persist in most if not all cases, as has been reported for hepatitis B virus. It has been demonstrated that in patients with resolved infection, T cell responses can be detected many years after the acute infection, suggesting continuing antigen stimulation.14 However, it is also possible that extended low titer viremia can precede viral clearance. Finally, the possibility of false positive results caused by the highly sensitive polymerase chain reaction assays for HCV RNA should also be considered.
Viral Kinetics, Host Responses, and Outcome of Acute HCV Infection
The viral and host responses observed by Major et al.8 in 10 chimpanzees infected with monoclonal HCV are summarized in Table 1. It was impressive how similar HCV replication was during the early acute phase in chimpanzees with different outcomes of infection. Furthermore, the initial course of viremia was apparently not influenced by whether the animal became infected by intrahepatic injection of RNA transcripts or by intravenous inoculation of the monoclonal virus collected from a transfected chimpanzee. Sophisticated mathematical analysis demonstrated that all animals had a very rapid increase in virus titer during the first 2 weeks, followed by a significantly slower increase during subsequent weeks. What is preventing the virus from reaching higher titers? During the first 2 weeks of monoclonal infection of tamarins with GB virus B, which causes hepatitis and is the virus most closely related to HCV, the virus often reached titers that were three to four orders of magnitude higher than those of HCV in chimpanzees.15 If HCV, as believed, replicates primarily in hepatocytes, it is not likely that the number of target cells is the limitation. It is possible that HCV is somewhat attenuated in the chimpanzee model. The adaptive immune responses might play a role, but most virus-specific host responses occur weeks later, and selection of virus variants that might result from escape from such responses is usually not seen until even later. However, in previous studies,16, 17 as well as in the study by Major et al.,8 markers of the innate immune response (e.g., interferon α) were up-regulated in the liver at the earliest time points analyzed. It is reasonable to assume that the antiviral effect of such endogenous interferon α–related responses contributed to the slowing of virus production. However, a number of other genes not related to the interferon responses have previously been found to be up-regulated during the early acute phase of HCV and could potentially play an equally important role for the early control of viremia.16, 18 Importantly, these virus-induced early responses did not appear to determine whether an infection was eventually cleared.
Table 1. Viral and Immunological Events During Acute Monoclonal HCV Infection*
Acute Resolving infection (4 Animals)
Persistent infection (6 Animals)
Based on data published by Major et al.8 The weeks shown indicate the first detection of a given parameter in animals with acute resolving or persistent infection, respectively.
Induction of mRNA in liver tissue was determined by real-time PCR. Generally, a 2-fold increase was considered significant. Only 4 of the 6 animals that became persistently infected were studied.
Antibodies against the envelope proteins were detected only in animals that developed a persistent infection.8 The authors measured only antibodies against the envelope (E) 2 hypervariable region 1 (HVR1), but most likely these animals developed antibodies against other epitopes in E1 and E2 outside of the HVR1, as recently reported for two other persistently infected chimpanzees.19 Does this finding mean that neutralizing antibodies do not play a role in the control of acute HCV? Currently, applied HCV anti-envelope tests use recombinant proteins or peptides. Thus it cannot be determined whether or not animals with acute resolving infection actually develop antibodies against the envelope proteins that are incorporated into the infectious viral particles. However, a recent study using pseudotyped retrovirus bearing the E1 and E2 of HCV to detect neutralizing antibodies to HCV suggest that animals with acute resolving infection also do not develop neutralizing antibodies, whereas animals with persistent infection do develop such antibodies.19 The study by Major et al.8 provides a possible explanation for this phenomenon, because the duration of high-level viral replication prior to the initial decrease in titer observed in all animals was shorter in the animals with resolved infection. In fact, the decrease in viral titers in these animals occurred during weeks 6–9, whereas anti-HVR1 developed during weeks 11–15 in the persistently infected animals. Apparently, the development of anti-envelope antibodies required extended antigen stimulation, because all 10 animals developed antibodies to other proteins during weeks 6–14. It appears that the development of antibodies to the envelope proteins of HCV might be less frequent, less prompt, and less vigorous in chimpanzees compared with humans20, 21; perhaps this can be explained by a lower level of replication during the early acute phase in chimpanzees.
The chimpanzee model has recently contributed a great deal to our understanding of the cellular immune responses in acute HCV. The early immune events can be analyzed in a fashion that is not possible in acutely infected humans, because liver tissue can be collected frequently and can be used to detect HCV-specific T cell responses or messenger RNA profiles in the organ where the virus replicates. Cooper et al.22 found that animals with acute resolving infection had vigorous intrahepatic HCV-specific CD8+ T cell responses, whereas much weaker responses were detected in animals that developed a persistent infection. Subsequently, Shoukry et al.23 found that clearance was associated with intrahepatic CD4+ T cell responses that appeared early during the infection. Similarly, Thimme et al.17 demonstrated that animals with viral clearance had significant intrahepatic CD4+ and CD8+ T cell responses. However, some animals in which the virus persisted had equivalent intrahepatic CD4+ and CD8+ T cell responses; in these animals, a significant decrease in virus titer during the acute phase was followed by persistence at relatively low titers. Animals in which the virus persisted at the titers found during the acute infection did not have intrahepatic T cell responses. It should be noted that the peripheral T cell responses did not appear to differ among animals with different courses of infection.17, 24, 25 Thus, vigorous intrahepatic T cell responses correlated with a significant decrease in virus titer during the acute phase but did not always result in viral clearance. Major et al.8 did not perform a functional analysis of the intrahepatic T cell responses because they believed that the ex vivo expansion of liver-infiltrating T cells necessary for such assays might influence the results. Instead, they examined the messenger RNA expression of a few genes in the liver by using real-time polymerase chain reaction assays. This permits a quantitative analysis of mRNA expression, but it is a much less exhaustive analysis than those recently published in which microarray was used to characterize RNA extracted from liver samples of acutely infected chimpanzees.16, 18 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.
So have the studies in experimentally infected chimpanzees revealed the factors that determine the final outcome of primary HCV infection? It is evident that clearance does not occur in the absence of virus-specific host immune responses. All 10 animals inoculated with monoclonal HCV had similar interferon γ induction in the liver, coinciding with the initial decrease in virus titers and the development of hepatitis.8 Because interferon γ and interferon γ–induced genes in the liver are expressed as a result of the activation of immune cells and their homing to the liver, this result suggests equivalent intrahepatic cellular immune responses in all animals. However, only four animals actually cleared the infection. Is there then anything unique about these animals or their responses that might explain their ability to eradicate HCV? Even animals that developed a persistent infection had a significant decrease in virus titer from peak levels. The only association identified was that the peak titers were 0.5–1 log lower and that the initial decrease in virus titers occurred 1–2 weeks earlier in the animals with resolved infection. Only the resolved animals had significant induction of messenger RNA specific for the epsilon chain of CD3 and of messenger RNA specific for monocyte-induced protein 1 α, a cytokine involved with homing and activation of immune cells, which could indicate qualitative and/or quantitative differences in the intrahepatic cellular immune responses. Such differences may relate to which immune cells (e.g., T, natural killer, or natural killer T cells) are activated, or they may relate to the level of the cells' activation. The timing of these responses may also be crucial. Clearly, additional studies are required to examine these variables in the host immune response to HCV, including studies that address how the virus has developed strategies to circumvent such responses.26
Another possibility is that persistence resulted from the selection of virus mutants that escaped the cellular immune response. An association between persistent HCV infection in chimpanzees and the emergence of such cytotoxic T lymphocyte escape variants has been demonstrated previously.27, 28 Evolution of HCV in the infected chimpanzees was not reported by Major et al.8 It is evident, however, that mutants, including T cell escape mutants, are selected rapidly following the initial intrahepatic HCV-specific T cell responses in all animals in which HCV persists. The unanswered question is whether such mutations develop only in animals in which the virus persists or whether they are found also prior to clearance in animals with a resolved infection. It should be mentioned that the evolution of HCV in humans and chimpanzees might differ; in particular, it has been suggested that the envelope proteins of HCV, most notably the HVR1 region, undergo fewer mutations than in humans during the acute phase of infection in chimpanzees.29
Protective Immunity Against HCV
The chimpanzee model has been of unique importance for defining the nature of protective immunity to HCV following re-exposure. Experiments in chimpanzees demonstrated that infection with HCV does not provide complete protective immunity.30, 31 Chimpanzees that had resolved their acute experimental HCV infection developed viremia in practically all cases when challenged with the homologous virus, including monoclonal virus or heterologous viruses.13, 30–36 However, the duration of viremia in most cases was significantly shortened following reinfection and hepatitis was observed only in a few cases. Yet persistent infection was also observed in other cases following repeated heterologous rechallenge.30 Finally, in our laboratory we have recently observed two chimpanzees that became persistently infected following homologous monoclonal rechallenge (unpublished data). In general, the correlates of viral clearance following rechallenge were found to be similar to those associated with clearance following a primary infection. The critical role of CD4+ and CD8+ T cell responses was defined in two unique studies in which chimpanzees with resolved HCV infections were depleted of CD4+ and CD8+ T cells, respectively, by administration of specific antibodies.37, 38 Following CD8+ T cell depletion, viremia was prolonged after rechallenge and virus clearance coincided with the recurrence of this subset of T cells in the liver.38 Following CD4+ T cell depletion, HCV persisted after rechallenge, suggesting that an inadequate CD4+ T cell response plays a role in the outcome of HCV.37
Functional Analysis of HCV in Chimpanzees
The ability to test infectivity of molecular HCV clones in chimpanzees has permitted for the first time studies of the importance of viral genetic elements for virus infectivity. The critical importance of the active sites of various enzymatic functions, such as the protease, helicase, and polymerase, has been confirmed.39 A critical role of the p7 protein, which might represent an additional target for drug development, was recently demonstrated.40 Furthermore, the various regions of the 3′ untranslated region, except for a short region between the open reading frame and the polypyrimidine tract, are critical for infectivity.39, 41 Infectious chimeric viruses have been generated from different strains or genotypes and might permit further analysis of the function and interaction of different genetic components.24, 40, 42 Finally, it has been demonstrated that the E2 HVR1 is not critical for the viability of HCV43 and that outcome of infection with the virus lacking HVR1 was not determined by the deleted region, suggesting that factors other than the evolution of HVR1 or the immune response to this region determine the outcome of an HCV infection in chimpanzees.17, 18, 43
The development of HCV replicons was an important breakthrough for basic research on HCV RNA replication.44 However, efficient replication in Huh-7 cells depended on adaptive mutations in the nonstructural proteins of HCV.45 A study in chimpanzees using an infectious clone of the HCV strain from which the replicon was developed permitted analysis of the influence of such adaptive mutations for in vivo infectivity.46 It was found that genomes with adaptive mutations were highly attenuated in vivo. This finding has implications for understanding the biological relevance of data obtained in the in vitro replication systems, in particular studies of the virus–host cell interactions. It emphasizes the importance of confirming in a biologic system data generated in the test tube or in cell cultures. The chimpanzee represents the only such system for HCV.
Overall, the study of monoclonal HCV infection demonstrates the strength of studying HCV in the chimpanzee model. The detailed analysis of the early events of host responses that might correlate with a favorable outcome, in the absence of virus heterogeneity, can provide information of potential relevance for the design of vaccine candidates for HCV. It is expected that the chimpanzee will continue to play a critical role for testing the efficacy of such vaccine candidates.10, 47 Here the availability of monoclonal virus will permit true homologous challenge48 and a detailed knowledge of the natural history of primary monoclonal infection, as provided by Major et al.,8 is essential for evaluating the results. Chimpanzees are also expected to continue to be important in defining the role of immunoglobulins in preventing HCV infection47 and in testing the effect of passive immunoprophylaxis or therapy.49
The author thanks Dr. Robert H. Purcell for reviewing the manuscript.