Viral Hepatitis
Authors: Harvey Alter
Journal: Hepatology (2006)

The movie Field of Dreams movingly resurrected some of baseball's greatest legends. This article, "Field of Themes," will resurrect some of the legendary hepatitis virus articles published in Hepatology over the past 4 years. Severe space constraints force this review to be restricted to hepatitis C virus (HCV), a virus that accounted for 71% of the 81 articles previously highlighted. I apologize to HBV aficionados that I cannot B all that I can B. A major advance in the study of HCV has been the development of subgenomic replicons capable of transiently infecting hepatoma cell lines. Diverse applications of this replicon system have been reported in Hepatology , and several are cited in the sections below. The replicon game has now moved to a bigger stage with the recent demonstration that a viral strain (JFH-1) from a patient with fulminant hepatitis could be grown in HuH7.5 cells and generate enveloped viral particles capable of passage in culture and transmission to the chimpanzee.1 Is this the long sought Holy Grail of in vitro HCV propagation? Perhaps, but the application is currently limited because only this one "fulminant strain" is capable of growth and passage in culture. Efforts are now underway in many laboratories to improve the efficiency of this liver cell line and to adapt other strains of HCV to grow in this model system. I foresee technological unemployment for chimpanzees. Another technological breakthrough is the development of HCV pseudotypes that represent the merging of genes coding for a retroviral core, HCV envelope, and green fluorescence protein.2 This pseudotype can readily infect HuH7 cells, and blockage of infection is a measure of neutralizing antibody (ntAb) response.3 NtAb to HCV do indeed exist, can achieve high levels, and can neutralize across genotypes, but they appear late in infection and probably do not play a key role in recovery from acute infection. Whether such ntAb serve to contain chronic viremia and whether they will be valuable in post-exposure or posttransplantation settings awaits further study. Lavillette et al.4 developed pseudotypes for each of the major genotypes and used these to study HCV receptors. Most liver cell lines, including HuH7 and Hep3B, were infected by each of the pseudotypes, but non-liver cells, including human T and B cells, could not be infected, suggesting that these lymphoid cells are not a major replication site or reservoir for HCV. Another interesting finding was that Hep G2 cells, which do not constitutively express CD81, could not be infected by any of the pseudotypes, but could be infected after transfection with CD81 and entry into cells could be substantially blocked by anti-CD81. Hence, the role of CD81 as an HCV receptor has been substantiated by pseudotype technology. However, although CD81 may be required, it is not sufficient, since other CD81-bearing cells do not allow HCV entry or replication. A critical co-receptor is still unidentified. Gene arrays represent still another emerging technology. Many genes can be shown to be up- or down-regulated after transfection of Huh7 cells with subgenomic HCV.5 However, interpretation of these gene perturbations is difficult, given their magnitude. Do these genetic fluctuations reflect important elements of pathogenesis, immune response, or treatment response, or are they merely epiphenomena? Clinical relevance is the challenge in these early days of gene array analysis, as it will also be for the emerging field of proteomics. HCV, hepatitis C virus; ntAb, neutralizing antibody; ALT, alanine aminotransferase; HCC hepatocellular carcinoma; IVDUs, intravenous drug users; TGF-α1, transforming growth factor alpha1; IL-10, interleukin 10; PBMCs, peripheral blood mononuclear cells; IFN-α, interferon alpha; PB, peripheral blood; HPCs, hepatic progenitor cells; LSM, liver stiffness measurement; SVR, sustained virological response: siRNA, sSmall interfering RNAs. As the natural history of HCV infection has unfolded in recent years and as referral bias have been minimized, it has become evident that spontaneous recovery rates may be 25% or even higher in adults and over 50% in children. Age at infection is perhaps the prime determinant of virological outcome, though age may serve only as a surrogate for the vigor of the immune response. In a common source outbreak among pregnancy-age women in Germany,6 51% had a spontaneous recovery and in those chronically infected, only 2% developed cirrhosis and 9% bridging fibrosis after 25 years. In Hepatology , Larghi et al.7 showed that of 14 patients (ages 21-45 years) involved in a common source outbreak, 57% spontaneously lost HCV RNA. Casiraghi et al.8 traced 31 recipients of neonatal mini-transfusions from a single HCV-infected donor; 35 years after their neonatal transfusion, 11 of 16 viremic patients with elevated alanine aminotransferase (ALT) underwent liver biopsy; none had more than mild inflammation, 82% had no or only 1+ fibrosis, and only 2 had bridging fibrosis. Thus, infection in the very young is characterized not only by high spontaneous recovery rates, but also by relatively benign outcomes over the first 2-3 decades after infection. Unknown is whether the severity of liver disease will escalate as these patients reach age 50 and beyond. Of more surprise was the relatively benign outcome of HCV infection in 207 intravenous drug users who underwent liver biopsy in a study by Rai et al.9 After an estimated mean duration of infection of 19.7 years, only 1% had cirrhosis and 9% bridging fibrosis. Also surprisingly, there was no relationship between outcome and HIV status, although persons with CD4 counts <200 were excluded from study. This exclusion may be the key because DiMartino et al.10 reached a different conclusion in studying 80 HCV-HIV–co-infected patients. HIV–co-infected patients had a higher viral load, more severe inflammation, and an accelerated progression to cirrhosis and liver-related mortality compared with those infected with HCV alone. To reconcile these studies, the effect of HIV-induced immunosuppression on fibrosis progression seems to only become manifest when CD4 counts fall below 200, the same point at which opportunistic infections begin to emerge. The most intriguing question in this setting and the liver transplant setting is why immunosuppression would lead to accelerated fibrosis when the mechanism of HCV-related liver disease is presumed to be immune-mediated. One of the perplexing issues of HCV natural history is the 8- to 10-fold higher incidence of hepatocellular carcinoma (HCC) in Japan compared with the United States, even though both countries have similar HCV prevalence. Part of the answer lies in age-adjusted prevalence, where it can be shown that peak prevalence in Japan is at age 70 compared with age 40 in the United States. Japanese investigators have long claimed that their high HCC rates simply reflect duration of infection in the respective populations. Molecular clock analysis11 now supports this supposition. These data predict that as our HCV-infected population ages, we will see increasing rates of HCC equivalent to those in Japan, unless infection is abrogated by successful treatments. In all such studies, it is difficult to distinguish the relative impact of duration of infection versus the immunomodulating effects of old age. In an interesting study of HCC pathogenesis, Kamegaya et al.12 studied the effect of HCV transgenes on tumor formation in the diethynitrosamine murine cancer model. HCV E1/E2 worsened outcome not by stimulating tumor proliferation but by inhibiting apoptosis. If these findings can extrapolated to humans, one could speculate that HCV may play a dual role (cirrhosis induction/apoptosis inhibition) in a multi-hit process of hepatocarcinogenesis. The relationship between viral persistence and CD4/CD8 T-cell dysfunction has been intensively studied (high MPI: manuscript proliferation index). Most studies have shown that recovered subjects have more vigorous and broadly reactive CD4 and CD8 T-cell responses than chronically infected patients and that these cells are not quantitatively diminished, but rather functionally anergic.13 The unresolved question is whether diminished HCV-specific T cell responses are the basis for persistent infection or the consequence of that infection. Cox et al.14 addressed this issue by studying CD8 responses during acute HCV infection in a population of at-risk intravenous drug users (IVDUs). Surprisingly, and in contrast to earlier studies, the magnitude of the CD8 response early in HCV infection did not differ significantly between those who cleared infection (21%) and the majority who became persistent carriers. In this prospective study, there was a progressive loss in the strength and breadth of reactivity to HCV antigens in those who developed chronic infection, suggesting that impaired CD8 responses are the consequence of functional T-cell exhaustion rather than the cause of persistent infection. Two other mechanisms of an impaired immune response that have received considerable recent attention are the induction of T-regulatory cells and the tolerizing effects of various HCV proteins. CD4+/CD25+/Fox P3+ T regulatory cells constitute 5% to 10% of CD4+ cells and have been shown to be more common in chronic than in resolved HCV infection and to have a dose-dependent suppressor effect on CD8 responses ex vivo; depletion of such cells enhances the CD8 response.15, 16 The suppressive effect of T-regs is primarily dependent on cell-to-cell contact, but they also secrete transportable suppressors, including transforming growth factor alpha1 (TGF-α1) and interleukin 10 (IL-10), the latter known to suppress Th1-cell function. Therapeutically, one would like to suppress the suppressor cells, but this runs the risk of causing severe CD-8–induced liver damage or inciting autoimmune phenomena. One concern is that measurements on peripheral blood mononuclear cells (PBMCs) might not reflect T-cell interactions in the liver. Spangenberg et al.17 and Penna et al.18 isolated intrahepatic T cells and showed that, as in peripheral blood, HCV-specific CD8 cells appear to be dysfunctional, particularly in their ability to secrete interferon alpha (IFN-α). Although some quantitative differences were seen between measurements in the peripheral blood (PB) and the liver, the general conclusion was that measurements in the PB are fairly reflective of the intrahepatic state, thus adding credence to many previous studies that focused solely on PBMCs. The rapid induction of type I IFNs (α/β) is a pivotal event in the innate anti-viral reponse. Using HCV replicon cells, Zhang et al.19 demonstrated that the primary mechanism of HCV-mediated suppression of intracellular IFN-α production was the ability of HCV non-structural proteins, particularly NS5A, to inhibit the actions of IRF-7. This and similar observations by Foy et al.20 and by Otsuka et al.21 on NS-3 inhibition of IRF-3 phosphorylation are very important additions to our understanding of how HCV uses its own proteins to escape the early intracellular innate immune response and thus gains a foothold that fosters viral persistence. In sum, viral persistence in HCV infection cannot be simply explained and must have multiple underlying components, including the suppressive effects of HCV antigens on the innate and adaptive immune response, early high-level viremia and a late effector T-cell response, diminished CD4 T-cell help, the suppressive effects of T-regulatory cells, a complex viral quasispecies with B and T-cell escape mutations, and a late-appearing and suboptimal neutralizing antibody response. Clouston et al.22 propose an interesting hypothesis for fibrosis formation wherein the fundamental defect is hepatocyte replicative arrest from any of a variety of insults, including viral infections. This arrest stimulates an alternative replicative pathway involving hepatic progenitor cells (HPCs) that can differentiate either into hepatocytes or bile ductular epithelium. HPCs are found primarily at the periphery of the portal tract in close proximity to bile ductular cells. Their hypothesis, supported by immunohistochemistry and image analysis, is that activation of the alternate pathway leads to a ductular reaction that expresses cytokines that attract and activate stellate cells, leading to collagen deposition at the portal tract interface. This is an appealing concept ("great deductule reasoning") because it provides a common pathway for fibrosis formation and because it explains why fibrosis appears to be initiated at the portal interface. The study does not, however, close the loop by measuring the signals the might bring stellate cells and myofibroblasts into the periportal area. Nonetheless, this is thinking outside the box and a potential paradigm shift in our concept of hepatic fibrosis. A study by Monto et al.23 shows a very strong association between steatosis and fibrosis (greater than age or alcohol) and between steatosis and obesity, generating the weighty thought that caloric restriction might be as important as alcohol reduction in the prevention of HCV-related fibrosis progression. Whether this is fat or fiction remains to be determined, but there is inherent logic in this strategy. More surprisingly, a meta analyisis24 of 19 studies involving 2,323 patients found a highly significant association between the in vitro demonstration of cryoprecipitate and the development of cirrhosis in patients with hepatitis C (χ2 = 142, P <.001). Although this relationship leaves me cold, there is no denying the strength of the association and the need to perform more basic studies to find a plausible explanation. Many investigators have developed algorithms to predict liver fibrosis in lieu of liver biopsy.25-29 These biochemical biopsies leave one between a ROC and hard place. Space does not permit a comparison of these various algorithms. Suffice it to say, all investigations established ROC curves to determine cut-off values and demonstrated reasonable positive and negative predictive values for including or excluding cirrhosis. Unfortunately, in most algorithms, approximately 50% of patients have values that fit between the cutoffs and thus cannot be interpreted. In addition, these predictive algorithms primarily distinguish cirrhosis from the absence of cirrhosis, but do not distinguish F0 or F1 fibrosis from stages F2, F3, or F4 (Ishak), stages at which critical treatment decisions have to be made. In making these decisions, the liver biopsy is most helpful. A noninvasive marker that could fine-tune the stage of fibrosis would have to be specific for stellate cell activation or collagen matrix formation and not be based simply on markers of inflammation, portal hypertension, or impaired liver synthetic function. The latter markers have their place in certain situations, but overall occur too late in the process to be sufficiently discriminatory. That is why direct measures of liver elasticity/stiffness (LSM) are more intrinsically appealing. Fibroscan30 is a device that emits a low-frequency vibration that induces an elastic shear wave through the liver; the velocity of the wave is directly related to liver stiffness such that the harder the tissue, the faster the wave. The test is non-invasive, highly reproducible, and can be repeated an unlimited number of times in the same examination or sequential examinations. The ability to perform frequent assessment over time is a major advantage over liver biopsy. In a study of 251 patients, LSM predicted a fibrosis stage of ≥F2 (Metavir) with 91% accuracy. In another study,31 Fibroscan was put into stiff competition with Fibrotest and won convincingly; 100% of 26 patients with minimal fibrosis could have been spared biopsy by Fibroscan compared with only 38% by Fibrotest and 100% of those with ≥F2 would have been identified by Fibroscan compared with 64% by Fibrotest. Liver stiffness measurement is a very promising young technique that, like good wine, should get better with age. Combination therapy with Peg-IFN and Ribavirin results in a sustained virological response in approximately 50% of genotype 1 infections and 80% in genotype 2 or 3 infections. This response rate is now tempered by the study of Radkowski et al.,32 which found residual HCV RNA in 88% of 17 patients followed for a mean of 64 months post-sustained virological response (SVR). HCV RNA was found predominantly in macrophages and lymphocytes, but also in serum and liver. However, residual viral loads were very low, and, despite persistent virus, liver biopsy showed significant improvement in both inflammation and fibrosis. Residual virus may maintain a persistent humoral and cellular immune response that can hold the virus in check once the viral load has been substantially lowered by therapy. Thus, clinical recurrence has not been a problem in patients who have been followed for up to 15 years. Despite these encouraging long-term clinical results, residual virus raises residual fear that clinical exacerbations could occur if such patients were subsequently immunosuppressed. Lindahl et al.33 tested the hypothesis that individualized ribavirin dosing to achieve a steady-state concentration of >15μmol/L, a level twice that achieved with current therapy, might enhance SVR in genotype 1 infection. They measured plasma levels every 2-4 weeks and adjusted the dose accordingly. By 24 weeks, the mean daily dose had increased to 2,540 mg/day and the highest dose given was 4,000 mg/day. Despite these high doses, no patient was removed from the study because of ribavirin side effects, although 2 of 10 patients required blood transfusion. The results were dramatic in that 9 of 10 previously untreated genotype1 patients attained an SVR. This is a small study that needs to be validated in a randomized controlled trail, but it is intriguing that such high response rates could be achieved with existing drugs by using a ribavirin dose-escalation strategy. Still other new approaches to treatment include morpholinos, an antisense molecule that has been modified to enhance hybridization, to provide nuclease resistance and to limit toxicity. McCaffrey et al.34 designed a morpholino directed to the HCV IRES and showed in the mouse model that this small molecule reduced IRES activity by 95% for at least 6 days. The duration of effect shows that the molecule was rendered nuclease resistant. Small interfering RNAs (siRNA) represent another novel approach to anti-viral therapy and have shown efficacy in HCV replicons as well as in Hep G2 cells transfected with HBV.35 However, a major obstacle to the therapeutic use of small inhibitory molecules is finding a way to deliver them to the liver in sufficient quantity to achieve therapeutic benefit. We now have proof of principle but not proof of clinical utility. It is a brave new world for HCV, particularly in terms of new technologies, insights into molecular virology and immunopathogenesis, and innovative treatment approaches. Vaccine development has been disappointingly slow and faces many of the same obstacles that confront vaccines for HIV. Nonetheless, a vaccine for HCV should be attainable because there is a natural mechanism for viral clearance; we just need to learn how to tap into it. Hopefully, a successful vaccine will be THE highlight of the next "Highlight of the Highlights."