Journul of Hepatohg.v 2000; 32 (suppl.

1): 98-l 12 Printed ,n Drnnzark All vrghts reserved

Munkrgaard

Cupenhqen

Journal of Hepatology ISSN 0169-S/85

Pathogenesis, diagnosis and management of hepatitis C

Nathalie
Service d H6putologie,
Centre

Boyer

and Patrick

Marcellin
Cl-481. H6pital Bemjon, Clichy, France

de Recherche Claude Bernard SW les H&pat&s Virales and INSERM

The hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. It is estimated that about 170 million people are chronically infected with HCY Chronic hepatitis C is a major cause of cirrhosis and hepatocellular carcinoma and HCV-related endstage liver disease is, in many countries, the first cause of liver transplantation. HCV infection is characterized by its propensity to chronicity. Because of its high genetic variability, HCV has the capability to escape the immune response of the host. HCV is not directly cytopatbic and liver lesions are mainly related to immune-mediated mechanisms, which are characterized by a predominant type 1 helper cell response. Co-factors influencing the outcome of the disease including age, gender and alcohol consumption are poorly understood and other factors such as immunologic and genetic factors may play an important role. Recent studies have shown that the combination therapy with alpha interferon and ribavirin induces a sustained virological response in about 40% of patients with chronic hepatitis C. The sustained response rates are mainly dependent on the viral genotype (roughly 60% in genotype non-l and 30% in genotype 1). Reliable diagnostic tools are now available and useful for detecting HCV infection, to quantify viral load and to determine the viral type. The assessment of the viral quasispecies and the characterization of viral

sequences might be clinically relevant but standardized and simple techniques are needed. The lack of animal models and of in vitro culture systems hampers the understanding of the pathogenesis of chronic hepatitis C and the development of new antivirals. New therapeutic schedules with higher and/ or daily doses of alpha interferon do not seem to improve the efficacy greatly. The conjugation with polyethylene glycol (PEG) improved the pharmacodynamics and the efficacy of alpha interferon. Emerging new therapies include inhibitors of viral enzymes (protease, helicase and polymerase), cytokines (IL-12 and IL-lo), antisense oligonucleotides and ribozymes. The first candidate compounds should be available in the next few years. The development of an effective vaccine remains the most difficult and pressing challenge. Because of the high protein variability of HCY protective vaccines could be extremely difficult to produce and therapeutic vaccines seem more realistic. Considerable progress has been made in the field of HCV since its discovery 10 years ago but a major effort needs to be made in the next decade to control HCV-related liver disease.

Key words: Genotype; terferon; Quasispecies;

Hepatitis C; HCV RNA; InRibavirin.

C virus (HCV), the agent responsible for most cases of blood-borne hepatitis, was discovered by Choo et al. just 10 years ago (1). HCV is the leading cause of chronic liver disease worldwide. An estimated 3% of the world population is chronically infected with HCV, and HCV accounts for approxiEPATITIS Correspona ence: Patrick Marcellin, Service d Hkpatologie, HKpital Beaujon, 100 Bd. du G&n&al Leclerc, 92110 Clichy, France. Tel: +33 1 40 87 53 38. Fax: +33 1 47 30 94 40. e-mail: marcellin@bichat.inserm.fr

mately 20% of cases of acute hepatitis and 70% of cases of chronic hepatitis (2). Chronic hepatitis C is a major cause of cirrhosis and hepatocellular carcinoma. Moreover, HCV-related end-stage liver disease is, in many countries, the first cause of liver transplantation. The mechanisms responsible for the persistence of viral infection and for the liver lesions are not well understood. Despite an active immune response of the host, HCV has the capability to escape. It is believed that the quasispecies nature of HCV is one of the major mechanisms allowing the virus to cause chronic infection. HCV is not directly cytopathic and liver

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Management of hepatitis C

lesions are mainly related to immune mediated mechanisms. Co-factors influencing the outcome of the disease, including age, gender and alcohol consumption, are poorly understood and other factors, e.g. immunologic and genetic, may play an important role. Since the first study by Hoofnagle et al. in 1986 (3), many controlled trials have shown the efficacy of alpha interferon therapy in patients with chronic hepatitis C with a sustained response in a minority of patients. Recent studies have shown that the efficacy of treatment was significantly improved by the combination of interferon with ribavirin, which is nowadays the preferred treatment. Predictors of sustained response to therapy, mainly viral load and genotype, have been identified. Indeed, considerable progress has been made in the field of hepatitis C in the last 10 years but many challenges still remain. The purpose of this article is briefly to review our current understanding of the pathogenic mechanisms, the diagnostic tools available and the results obtained with current therapy. Future perspectives for the development of new treatment strategies are also considered.

Pathogenesis
HCV infection is characterized by its propensity to evolve into chronicity and by a wide clinical spectrum. About 85% of patients infected by HCV will develop chronic infection and resolution of acute hepatitis C is observed in only 15% (2). The severity of the liver disease varies widely from asymptomatic chronic infection, with normal liver tests and nearly normal liver, to severe chronic hepatitis, leading rapidly to cirrhosis and hepatocellular carcinoma. The mechanisms responsible for the persistence of HCV infection and for the liver lesions are not well understood. The lack of an efficient in vitro replication system or an animal model (the chimpanzee model is limited) has greatly hampered the study of these mechanisms.
Mechanisms involved in the persistence of HCV infection

The quality of the cellular immune response is crucial for the elimination or the persistence of HCV infection. CD4+ T cells and their cytokines with inflammatory and regulatory activities seem to play an important role in the immunopathogenesis of chronic HCV infection. CD4+ T cell responses are polarized into type 1 and type 2 helper T cell (Thl and Th2) responses. The Thl cells secrete interleukin 2 (IL-2) and interferon gamma, which are important stimuli for the development of the host antiviral immune responses, including cytotoxic T-lymphocyte (CTL) generation and NK-cell activation. The Th2 cells produce

IL-4 and IL-lo, which enhance antibody production and downregulate the Thl response. It is hypothesized that the imbalance between the Thl and the Th2 responses is implicated in disease progression and the inability to clear infection. Patients with acute HCV infection who clear the virus and have a self-limited acute hepatitis develop a strong Thl response, but a weak or absent Th2 response. Conversely, patients who develop a chronic infection show a predominant Th2 response, but a weak Th 1 response (4). These observations suggest that the effect of Thl cytokines is crucial for protection against HCV infection, whereas a preferential production of Th2 cytokines may have an inhibitory effect on the patient s immune system and therefore favors persistent HCV infection. The primary causes of these different early immune responses in acute HCV infection are not known. However, strategies to alter the Thl/Th2 balance through the use of cytokine therapy may have clinical implications in the treatment of chronic HCV infection. The presence of HCV infection has been shown in peripheral blood mononuclear cells, monocytes and lymphocytes. Furthermore, the detection of minus strand RNA in hematopoietic cells suggests that this is a possible extrahepatic site of replication for HCV (5). This extrahepatic site of HCV infection might also play a role in the persistence of HCV infection, possibly by altering the immune response or by favoring infection of liver cells. Interestingly, a cellular protein that binds E2 has recently been identified, called CD81 (6), which is expressed on the surface of several cell types, including lymphocytes and hepatocytes, and is currently believed to be an HCV receptor or co-receptor. Antibodies that neutralize infection by HCV appear to do so by preventing E2 binding to CD81. This finding opens an important avenue for future research. Antibody production is critical for neutralization of free viral particles and for preventing virus entry into the host. Studies showed that neutralizing antibodies are produced during natural HCV infection despite the high rate of chronic evolution (7). The most likely explanation for the ineffectiveness of the antibody response against HCV is that rapid occurrence of viral mutations within the epitopes recognized by neutralizing antibodies can abrogate antibody recognition of the new variant virus (8).
HCV heterogeneity

Indeed, escape from antibody and CTL responses as a result of viral mutations probably plays a major role in view of the extremely high variability of HCV The RNA-dependent polymerase of RNA viruses is highly error prone and lacks proofreading capabilities. The es99

N. Boyer & P. Marcellin

timated frequency of spontaneous nucleotide substitution is very high: lop2 to 10e3 substitutions per nucleotide per year. Consequently, within any given individual infected with HCV, the viral population consists of a heterogeneous mixture of closely related virions, which vary from each other by only l-9% of bases, termed quasispecies (9). The biological consequences of quasispecies include the development of escape mutants to humoral and cellular immunity, variable cell tropism (lymphotropic versus hepatotropic) vaccine failure and rapid development of drug resistance. Indeed, HCV has a high genetic variability, especially in the E2 and the NSl regions of the genome, with two hypervariable regions within E2 called HVRl and HVR2 (10,ll). The high mutation rate in these regions is believed to be the result of selective pressure by the host immune system. The development of HCV neutralizing antibodies is thus possible, but the extremely high degree of variability in the HVRl and the HVR2 regions enables the selection of escape mutants. This view is consistent with the extremely rapid viral production rate in patients with chronic hepatitis C, in the order of 10 -1012 virions per day and a short in vivo half-life of the virus in the order of 47 h (12,13). The high turnover rate of HCV explains the rapid generation of viral diversity and the opportunity for viral escape from the host immune response.
Mechanisms involved in liver lesions

gression of the liver disease have been suggested, including virus-related factors and host-related factors.
Virus-related factors

HCV is not directly cytopathic. The hepatitic process appears to result from the immune recognition and destruction of infected hepatocytes (4). Persistent HCV infection in the liver is continuously triggering an active T cell response, which is probably the main mechanism responsible for the liver lesions. Studies of the intrahepatic immune processes implicated in the pathogenesis of chronic hepatitis C are limited. However, HCV specific helper and cytotoxic T cells able to recognize structural and non structural HCV proteins (especially core and NS4 proteins) have been detected within the liver infiltrates (14). The predominant production of Thl cytokines is believed to play a role in enhancing necro-inflammatory lesions (4,15). This continuous necroinflammatory process, inefficient for clearing viral infection, is probably the main cause of the fibrogenesis mechanisms responsible for the progression of the liver disease. However, necro-inflammatory lesions and progression of fibrosis are not always well correlated, which suggests the role of cofactors.

The role of viral factors such as the viral load and the genotype are still being debated. Most studies argue for the absence of correlation between serum HCV RNA levels and the severity of the liver lesions (16,17). Interestingly, patients with chronic infection and normal serum aminotransferase levels and nearly normal liver histology may have high serum HCV RNA levels (2,lS). However, serum HCV RNA levels are an indirect reflection of intrahepatic HCV replication. We found no correlation between serum and liver HCV RNA levels (19) and, interestingly, the amount of liver HCV RNA was higher in patients infected with genotype 1 b than in those infected with genotype 2 or 3. More studies assessing the role of the intrahepatic HCV RNA levels and on minus strand RNA better reflecting HCV replication (5) are needed. The influence of viral genotype in the pathogenesis of the liver disease is still controversial. In many studies, HCV genotype 1b was found to be associated with a more severe liver disease including a higher frequency of cirrhosis and hepatocellular carcinoma (20,21). However, many of these studies were flawed by several possible biases, including confounding factors such as age, source of infection and duration of infection. Indeed, genotype lb is mostly found in old patients infected by transfusion and with a long duration of infection. In more careful studies with adjustment on these variables, the association between genotype lb and a more severe liver disease was not found (17,22). Interestingly, the distribution of genotypes is not different in patients with chronic hepatitis C and normal serum alanine aminotransferase (ALT) levels, as compared with those with increased serum ALT (2). Finally, genotype does not seem to be an important factor influencing the severity of liver disease. However, the association between genotype 1b and a more severe outcome in the transplant setting was found in some studies (23,24) but was not confirmed by other studies (25). Further studies are needed to better determine the possible role of genotype in the outcome of HCVrelated liver disease.
Host-relatedjbctors

Factors influencing the evolution of chronic hepatitis C

The evolution of liver fibrosis varies widely from one patient to the other. Many factors influencing the pro100

The role of various host-related factors such as immune status, age and alcohol as cofactors associated with the progression of liver disease has been shown but is poorly understood. Recent studies showed that liver graft recipients generally have a more progressive liver disease than immunocompetent patients with

Management of hepatitis C

higher incidence of cirrhosis, which might be responsible for an increased mortality (26,27). In such patients, high levels of serum HCV RNA have been demonstrated and direct cytopathic mechanisms of HCV can not be ruled out. It is believed that the more rapid progression of fibrosis is related to immunosuppressive therapy and, although the rationale is not clearly demonstrated, it is generally recommended to decrease immunosuppressive therapy as much as possible. In another population of immunocompromised patients, such as patients with HIV coinfection, progression to cirrhosis is more rapid than in HIV-negative patients, although the severity of the liver disease varies considerably between patients (28). In this population, cofactors such as drugs used against HIV or alcohol might play an important role. Another viral coinfection, with HBV, aggravates chronic hepatitis C and increases the risk of development of hepatocellular carcinoma. The detrimental role of alcohol in accelerating the evolution of chronic hepatitis C is well recognized. Several studies have shown that alcohol consumption accelerates the progression of fibrosis and the development of cirrhosis (29-31). In addition, heavy alcohol consumption is probably associated with a higher risk of hepatocellular carcinoma (32). The mechanisms may involve alterations of the immune response and direct toxicity of alcohol. A relationship was shown between the alcohol intake and serum HCV RNA levels; however, the significance of this observation is not clear (30). In addition, heavy alcohol consumption decreases the response to interferon therapy (33). These observations justify the recommendation of alcohol abstinence in patients with chronic hepatitis C. Many studies have emphasized the role of the age in the outcome of chronic hepatitis C. Patients infected at a higher age have more severe histologic lesions and a more rapid evolution to cirrhosis (34,35). Indeed, age at the time of infection seems the most significant known factor influencing the outcome of chronic hepatitis C. It appears to be crucial to clarify the mechanisms responsible for this relationship, in particular with regard to immune response and fibrogenesis, to understand the pathogenesis of the disease. Poynard et al. have proposed a model predicting the evolution of liver fibrosis according to three factors (age, gender and alcohol) (29). This schematic model predicts an evolution to cirrhosis with a mean delay of 13 yr in men infected after the age of 40 yr and 42 yr in women infected before the age of 40 yr who do not drink alcohol. Although this model illustrates the influence of age, gender and alcohol, it is clear that these factors account only partially for the various outcomes of chronic hepa-

titis C. Furthermore, the progression of HCV-related liver disease is probably not linear. More accurate models taking into account other potential factors need to be elaborated from longitudinal cohort studies. Hormonal factors may also play a role since several studies have reported more severe and progressive liver diseases in males (29,3436). Interestingly, chronic hepatitis generally has a benign course in young women (37) and normalization of serum ALT levels associated with a significant increase of serum HCV RNA levels are usually observed in pregnant women (38). Recent studies suggested the role of genetic factors showing an association between MHC class II alleles and different outcomes of hepatitis C (39,40).

Diagnosis
Cloning of the HCV genome and sequence analysis has led to the development of a variety of antigens and synthetic peptides that have been successfully used in immunoassays to detect antibodies to HCV Third generation enzyme linked immunosorbent assays (ELISA) and recombinant immunoblot assays (RIBA) have considerably improved in sensitivity and specificity as compared with prior first and second generation assays (41,42). Techniques for HCV-RNA detection and quantification, such as the branched DNA (bDNA) and polymerase chain reaction (PCR), are more standardized (41,42) and the sensitivity has been improved. Furthermore, genotyping the virus has important clinical implications for the therapeutic management of the patients. New techniques characterizing the viral diversity of specific regions of the genome might be useful in the future.
Hepatitis C virus antibody testing

The detection of antibodies to HCV is commonly the most practical means of screening and diagnosing hepatitis C. Third generation ELISAs have a high sensitivity and specificity. However, even the more recent tests have shortcomings. In acute HCV infection, anti-HCV are not detectable in all patients at the time of the peak of serum ALT levels and anti-HCV may become detectable several weeks later (43). Furthermore, immunosuppressed patients, such as graft recipients or patients coinfected with HIV, may have chronic HCV infection with low or even undetectable anti-HCV (44). Although the residual risk of HCV transmission, due to the anti-HCV window period, is estimated at present to be extremely low (about 1 in 100 000 transfusions of cellular products), given industrial developments, nucleic acid testing (NAT) of individual blood donations may become available very soon (45). In the screening of low-risk subjects, such as blood
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N. Bayer & P. Marcellin

donors, false positive results of ELISAs are prevalent and supplemental or confirmatory tests are still recommended (46). The most commonly used is the RlBA, which is more specific than the ELISA. However, in subjects with increased serum ALT levels or in high-risk subjects, the confirmatory test appears to be unnecessary and the direct detection of HCV RNA in the serum by PCR should be preferred to confirm the presence of active HCV infection (46).
Detection of serum HCV RNA

The detection of HCV RNA in the serum by reverse transcriptase PCR is nowadays considered the gold standard for the diagnosis of HCV infection and for assessing the antiviral response to therapy. PCR assays have a high sensitivity (in the order of 100 copies per ml) and a good reliability. A French multicenter quality control study performed in nine routine laboratories between 1991 and 1993 showed an overall improvement in sensitivity and specificity compatible with clinical use (47). Detection of serum HCV RNA by PCR is the best test for early diagnosis of acute HCV infection since it is positive as soon as 1 week after exposure (this may allow early diagnosis after needlestick injury) and it is positive at the acute phase of hepatitis when anti-HCV may still be undetectable (43). PCR is necessary to distinguish patients with chronic HCV infection with normal serum ALT levels from those with past resolved hepatitis C who remain anti-HCV positive for decades. In addition, PCR should be considered mandatory to confirm HCV infection in patients with increased serum ALT levels before indicating liver biopsy and before discussing antiviral therapy to prevent misdiagnosis with other causes of chronic liver diseases (46). During antiviral therapy, loss of detectable serum HCV RNA is nowadays the gold standard for the definition of the end of treatment and of the sustained response since as many as lo-20% of patients may show discrepancies between the biochemical response (normal serum ALT levels) and the virological response. In particular, some patients, for unknown reasons, may have a virological response during therapy despite the persistence of elevated serum ALT levels. Only the sustained virological response, defined by undetectable serum HCV RNA, 6 months after discontinuation of therapy, has been clearly shown to be associated with long-term virological, biochemical response and marked and progressive histologic improvement (48,49). In those patients with long-term response, HCV RNA has not been found to be detectable in the liver of the 27 patients studied, l-5 yr after interferon therapy (49). However, more studies with a 102

longer follow-up are crucial to confirm the eradication of HCV infection. In most studies, but not all, the results of the detection of HCV RNA in the liver were consistent with the results in the serum. However, such studies should be performed again with more sensitive PCR methods now available to rule out the possibility of very low replication persisting in the liver in the absence of detectable viral genomes in the serum, as already demonstrated in chronic hepatitis B even after HBsAg seroconversion (50). Unfortunately, no standardized techniques are available so far for in situ hybridization and in situ PCR in liver tissue and further studies are warranted to establish such techniques. Detection of HCV antigens in the liver by immunochemistry is also an interesting approach but only a few studies have shown convincing results (51,52) and there is a need for reliable and standardized techniques.
Quantljication of HCV RNA

Quantitative assessment of serum HCV RNA levels by signal amplification or quantitative PCR is a very useful tool in the clinical management of patients before therapy and in the assessment of antivirals. New assays currently under evaluation combine a reliable quantification and a high sensitivity (in the range of 100 copies per ml). In addition, recent studies showed that the standardization between the different assays has been considerably improved. Thus, results obtained with different commercial assays are now well correlated. Quantification with first generation assays was dependent on the HCV genotypes; new assays give a more reliable quantification independent of the genotypes. Thus it was possible to confirm serum HCV RNA level as a predictor of sustained response to interferon therapy or to interferon-ribavirin combination therapy, independently of the genotype (53-55). Quantification of serum HCV RNA level therefore appears to be useful before therapy to assess the chance of response. Quantitative assays have made it possible to understand the early viral kinetics under interferon administration and to elaborate mathematical models for HCV turnover (12,13). Furthermore, the early decay of serum HCV RNA during the first hours or days of interferon therapy is a good predictor of response. Further studies should determine the clinical relevance and the optimal time for serum HCV RNA quantification after initiation of therapy and its potential applications for tailoring treatment schedule.
HCV genotypes

Comparison of the genomes of different HCV isolates showed important variations leading to the classifi-

Management of hepatitis C

cation of HCV into types and subtypes (56). For nomenclature, according to sequence homology, HCV was classified into 6 types (1 to 6) each including subtypes (a, b, c...). HCV genotypes are distributed differently depending on geography and etiology (57). HCV genotype is not useful for the assessment of the prognosis but it is useful for therapeutic management, since it is a predictor of sustained response. In addition, the optimal duration of interferon-ribavirin therapy is dependent on genotype. Most methods for HCV genotyping are based on amplification of subgenomic viral fragments by PCR. Sequence motifs specific for each HCV type can be distinguished using type specific primers or probes or restriction fragment length polymorphisms (RFLP). HCV types can also be determined by serological methods (serotyping), which are simpler to use but are a little less sensitive and specific (5-10% of samples can be genotyped but not serotyped) (58). Furthermore, subtyping is still not possible with the existing serotyping assays. However, for clinical management, subtyping (in particular between subtypes la and lb) is not necessary since the response to therapy does not seem to be different in these two subtypes.
Quasispecies

what is the clinical relevance of measuring quasispecies heterogeneity for the prognosis and the therapeutic decision? Studies on the dynamics of the quasispecies under therapy with a characterization of viral populations selected could be very important to understand the mechanisms of viral resistance. In addition, recent studies have suggested that the quasispecies circulating in the plasma are different from those found in the liver or in lymphocytes (64). This compartmentalization of viral populations might have important implications in the persistence of HCV infection and the resistance to therapy.
Liver histology

There are both direct and indirect methods of detecting and quantifiying quasispecies within an individual patient. The gold standard is sequencing after cloning. However, this method is labor-intensive and simpler and indirect methods have been developed. The most used is single-strand conformation polymorphism (SSCP) (59). In SSCP analysis, products of PCR are subjected to electrophoretic analysis under denaturing conditions, such that single-stranded DNA is obtained. Single nucleotide polymorphisms result in different mobilities of the single-stranded fragments of the most prevalent HCV variants, reflecting the quasispecies diversity. This method allowed investigations of relatively large series of patients. Almost all studies showed a correlation between a high quasispesies heterogeneity and a more severe liver disease and a poor response to interferon therapy (60-62). Of note is the lower heterogeneity of quasispecies we found in patients with chronic hepatitis C with normal ALT levels as compared with patients with increased serum ALT levels, consistent with a lower immune pressure in the former (63). However, further studies should clarify the significance of the heterogeneity of quasispecies: is it the consequence or one of the mechanisms implicated in the pathogenesis of the liver disease? Furthermore,

Liver biopsy remains the best way to assess the severity of chronic hepatitis C, to determine the prognosis and to evaluate the indication of therapy. Indeed, the risk of developing cirrhosis depends on the stage (degree of fibrosis) and the grade (degree of inflammation and necrosis) observed on the initial liver biopsy. Clinical symptoms and biochemical markers (mainly serum ALT levels) are poorly correlated with liver lesions (65). Serum markers of fibrosis, such as procollagenIII, hyaluronic acid or laminin are non-specific, they depend on the degree of inflammation and are therefore not reliable. More accurate noninvasive ways to assess the severity of the liver disease (in particular the progression of fibrosis) are needed. In patients with chronic hepatitis C and normal serum ALT levels, liver histology in about 80% of cases shows normal liver, non-specific changes or mild hepatitis (2). In patients with chronic hepatitis and increased serum ALT levels, liver histology shows moderate or severe chronic hepatitis C in about 50% of cases (2). Therefore, a liver biopsy is recommended in the latter patients but not in the former (46). Since the scoring system proposed in 1981 by Knodel et al. (66) other scoring systems have been proposed to improve the accuracy or the reproducibility of histologic assessment (67-69) The recent scoring system proposed by the METAVIR group gives a better intraand interobserver consistency that may be helpful for studies of cohorts or histologic assessment in therapeutic trials (69). However, the predictive value of the liver biopsy for the prognosis is imperfect and a minority of patients with mild chronic hepatitis may eventually develop more progressive liver disease. Indeed. since the factors influencing the progression of liver disease, which is probably not linear, are not known, a second liver biopsy should be proposed 3-5 yr later to reassess the evolution and to re-discuss the indication for therapy (46). 103

N. Boyer & P. Marcellin (49). A comparison of liver histologic findings before and l-6.2 yr after alpha interferon treatment showed a clear improvement in 94% of patients; in 62% of patients, the last biopsy done showed normal or nearly normal histologic findings. Further follow-up of these sustained responders did not show any case of relapse (unpublished). Sustained response is associated with improvement in the quality of life (71). Thus, interferon therapy appears to be beneficial in 15 MU 1 7 Days Fig. 1. Time course of serum HCV RNA during alpha interferon therapy. Eight hours ajter theJirst injection, a rapid decline is observed. Then, after 24 h, the decline is much slower. The slope of the$rst phase and of the second phase depends on the dose of interferon administered (5 MU, 10 MU or 15 MU). (Adaptedfrom Neumann et al. (12)). 14
I

5MU

-- r_l l

sustained responders. However, the eradication of HCV infection is not proven and the benefit on survival is probable but has not been clearly demonstrated. In relapsers or non responders, a partial short-term biochemical or histological improvement can be observed in a proportion of patients. Furthermore, a short-term anti-fibrogenesis effect of alpha interferon has been shown (72,73). However, the long-term benefit of therapy in these patients has not been proven. Therapeutic schedule Since the first pilot study by Hoofnagle et al. in 1986 (3), many controlled trials confirmed the efficacy of alpha interferon therapy in a minority of patients with chronic hepatitis C and established the schedule of 3 million units (MU), three times a week, for 12 months, as a standard (74). Higher doses of interferon, ranging from 5 MU to 10 MU or longer duration for 18-24 months did not increase markedly the efficacy and were associated with a poor tolerability (75,76). The stan-

Treatment
The objective of therapy in patients with chronic hepatitis C is to inhibit viral replication to decrease the activity of the liver disease, which is believed to be associated with a decreased risk of occurrence and hepatocellular carcinoma. of cirrhosis

Effects of alpha interferon In chronic hepatitis C, the anti-viral effect of alpha interferon is well demonstrated, with a rapid decrease of serum HCV RNA within the first weeks of therapy, with a parallel decrease of serum ALT (70). Recent studies on viral kinetics under alpha interferon therapy showed a rapid decline in viral load within the first day, followed by a second phase viral decline that is much slower (Fig. 1) (12,13). In the first phase, which is due to the direct antiviral effect of interferon, the slope is dependent on the dose of interferon administered. In the second phase, which is related to the clearance of infected cells, the slope is less dose-dependent and might be influenced by the quality of the immune response. While the first phase decline is observed in almost all patients treated, the second phase decline is not observed in non responders. Sustained responders with persistently normal ALT and undetectable serum HCV RNA 6 months after treatment usually sustain a biochemical and virological response (48,49). In a study of 80 patients with a sustained response, with a follow-up of 1-7.6 yr (mean?SD, 4.022.0 yr) after alpha interferon treatment, 93% of patients had persistently normal ALT and serum HCV RNA remained undetectable in 96%

Fig. 2. End-of-treatment (solid) and sustained virologic (white bars) response rates wYth alpha interferon (IFN) alone for 24 weeks or 48 nseeks or combination of alpha interjeron and ribavirin for 24 Itleeks or 48 weeks. (Adapted from Poynard et al. (54) and McHutchison et al. (55)).

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dard schedule gives roughly a 20% sustained response, a 15% end-of-treatment response followed by relapse and a 65% non response rate (70,75,76). The different types of alpha interferon (recombinant interferon, alpha 2a or alpha 2b, lymphoblastoid or consensus) give comparable sustained response rates (77,78). The lower rates of virological response observed in the more recent trials, as compared with the previous studies, are related to the increased sensitivity of the assays used for the detection of serum HCV RNA. With assays currently used, it is possible to detect about 100 viral genomes per ml. Combination of alpha interferon with ribavirin Ribavirin is a guanosine-like nucleoside analog which has a broad spectrum of antiviral activity against several viruses. Reichard et al. first showed that ribavirin administration caused a significant decrease in serum ALT in patients with chronic hepatitis C (79). However, further studies showed a small or non-significant effect on serum HCV RNA levels, suggesting that ribavirin acted more as an immunomodulator than as an antiviral (80). Despite the significant effect of ribavirin therapy on serum ALT levels, controlled trials of ribavirin administered alone did not show a significant effect on liver histology (81,82). Brillanti et al. first showed that combination therapy of alpha interferon with ribavirin could induce a sustained response in some patients who had responded and then relapsed after a first course of alpha interferon (83). Furthermore, small trials showed higher rates of sustained response with the combination of alpha interferon and ribavirin than with interferon alone in patients who had never been treated (naive patients) (84,85). Two recent large controlled trials confirmed that combination therapy was more effective than interferon alone (54,55). These studies showed that combination therapy for 24 or 48 weeks gives overall sustained virological response rates of 33% and 41%, respectively versus 6% and 16% with interferon alone

(Fig. 2) (86). These results led to the consideration of combination therapy as the reference treatment for chronic hepatitis C, as stated at the EASL International Consensus Conference on Hepatitis C (46). The mechanisms responsible for the improved efficacy of interferon in combination with ribavirin are not known. Treatment of relapsers In patients who responded and then relapsed after interferon therapy, interferon retreatment may be effective if a higher dose and/or a longer duration is used (87,88). Combination therapy is particularly effective in this group of patients, with a 4449% sustained response (89,90). Treatment of non-responders Interferon retreatment is ineffective in non-responders to interferon therapy (87,88). Combination therapy is slightly effective: preliminary studies showed 5-20% sustained response rates (91-93). A recent small study suggested that triple therapy combining interferonribavirin and amantadine may induce a sustained response in some interferon non responders (3 of 10 patients developed sustained virological response) (94). These promising results obviously need to be confirmed in large controlled trials. Treatment of patients with cirrhosis In patients with cirrhosis, alpha interferon therapy is poorly effective, with about a 9% sustained biochemical response and less than a 5% sustained virological response (95,96). In these patients, interferon therapy is associated with more frequent side effects. There are very few data on the treatment of patients with cirrhosis with the combination of alpha interferon with ribavirin. A meta-analysis suggests that combination therapy is more effective than interferon alone, especially in the patients with genotype 2 or 3 (Table 1) (93). In patients with cirrhosis, combination therapy should be monitored with particular care since

TABLE

1
of sustained ALT and HCV RNA response and 95% Confidence Intervals in non-responders, by genotype and stage of

Estimated* percentage liver disease Patient

type

Interferon-ribavirin No cirrhosis Cirrhosis

Interferon No cirrhosis Cirrhosis

Non-responder Genotype 1 Genotype 2, 3 *Derived from multivariate analysis,

5 (l-17)

8 (2-25)

18 (343)
intention-to-treat analysis

29 (1 l-58)
(11) of 114 patients,

0.5 (0.1-3) 1 (0.1-6) including 114 responders.

0.7 (0.14) 2 (0.3-13)

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N. Boyer & P. Marcellin

these patients are more likely to develop side effects. Recent studies have suggested that alpha interferon therapy might decrease the risk of complications or the incidence of hepatocellular carcinoma (97,98). This hypothesis remains controversial and other studies have not found a significant difference between treated and untreated patients (96,99). A recent study, performed on a large cohort of patients, showed that risk of hepatocellular carcinoma is decreased in tients who have either a sustained virological or a chemical response after interferon therapy, while potential benefit is much less clear in the the pabiothe

Treatment of special groups

In patients with hemophilia, kidney disease requiring hemodialysis, mixed cryoglobulinemia or HIV infection, the decision to treat should be taken on an individual basis. Current data and clinical experience demonstrate that alpha interferon therapy may be considered in these patients (70,103-105). In patients undergoing renal allograft transplantation, alpha interferon therapy is contraindicated because of the risk of rejection. In liver graft recipients, alpha interferon therapy is ineffective. Preliminary studies in liver graft recipients showed that combination therapy is more effective than interferon alone and is not associated with graft rejection (106). However, most patients with HCV recurrence after liver transplantation have a slowly progressive liver disease and further studies are necessary to better define the indication of combination therapy in these patients.

non-re-

sponders (100). Controlled prospective studies are needed to evaluate the long-term benefit of alpha interferon therapy in patients with cirrhosis.

Treatment of patients with normal ALT

In small, uncontrolled trials of alpha interferon in this group of patients, the rates of response were not different from those reported in patients with abnormal ALT levels (101). Importantly, in most studies, serum ALT levels became abnormal during therapy in approximately half of patients. These findings suggest that alpha interferon therapy is not usually beneficial and may be harmful in these patients. The efficacy of combination therapy has not been evaluated in this group of patients.

Predictors of response
The probability of sustained response to therapy depends mainly on genotype and serum HCV RNA level. Young age, female gender and mild or absent fibrosis have a weaker predictive value. Unfortunately, genotype 1 (la or lb), which is associated with a sponse to therapy, is the predominant genotype ope and USA, ranging from 60% to 80% of the different countries (57,107,108). Genotypes 3, which are associated with a good response poor rein Eurcases in 2 and to ther-

Acute hepatitis C
Several studies have shown that interferon therapy given to patients with acute hepatitis C decreases the risk of evolution to chronicity (75,102). One may estimate that the treatment decreases the risk of chronicity from 85% to about 50%. However, the long-term outcome of treated patients is unknown and whether the treatment is more effective at an early phase of acute infection is not clear. Moreover, the optimal interferon schedule (dose and duration) in this particular situation is not known. No information is available on the efficacy of combination therapy in this group of patients.

apy, are in the minority, ranging from 15% to 35%. Furthermore, about half of the patients infected with genotype 1 have a relatively high viral load (serum HCV RNA above 2X lo6 Eq genome/ml), which further decreases the chance of sustained response. Finally, one may estimate that at least one third of the patients have genotype 1 with high viral load and have therefore a very low chance of sustained response. With interferon therapy, one may estimate that the overall sustained response rate is about 30% in patients with HCV genotype 2 or 3 and 5% in those with genotype 1 (la or lb) (53). With combination therapy, the overall sustained response rate is roughly doubled,

TABLE Sustained

2 virologic response according to HCV genotype IFN+placebo (24 weeks) 25% 11% 4% 0.8% et al. (55). and pretreatment serum HCV RNA level IFN+ribavirin (24 weeks) 61% 62% 32% lo %, IFN+ribavirin (48 weeks) 64% 60/;, 33% 27 %,

Genotype

HCV-RNA (copies/ml) 52x >2x %2X >2x 106 106 106 106

IFN+placebo (48 weeks) 36 %1 26% 25% 3%

Non- 1 Non-l 1 1 Adapted from Poynard

et al. (54) and McHutchison

106

Management of hepatitis C

with 60% in patients with HCV genotype 2 or 3, whatever the baseline viral load is, and 35% in patients with genotype 1 and low viral load (Table 2) (54,55). In patients with genotype 1 and high viral load, the sustained response rate depends on the duration of therapy. By contrast, in patients with genotype 1 and low viral load and in patients with genotype 2 or 3, the rates of sustained response are not different with 12 months or 6 months of therapy. The early loss of detectable serum HCV RNA during interferon therapy is predictive of sustained response (109). Therefore therapy may be discontinued if serum HCV RNA remains detectable after 3 months of therapy. With interferon-ribavirin combination therapy, late virological response may be observed and a minority (5-10%) of the patients with detectable serum HCV RNA at 3 months may still develop a sustained response if therapy is continued (86). The characteristics of the patients who benefit from continuing therapy need to be precisely determined. As previously mentioned, preliminary studies suggest that more sophisticated virological markers such as the heterogeneity of quasispecies might be useful. However, these observations have to be confirmed and simple assays should be available. Recent studies showed that a specific sequence in the NSSA region of the viral genome is associated with resistance to interferon (interferon sensitive dependent region, ISDR) (110). The NSSA protein binds and inhibits the function of the PKR protein kinase induced by interferon, allowing the viral replication to proceed (111). This is the first demonstration of a specific mechanism by which HCV has the capability to resist to interferon. More molecular studies on resistant HCV strains are crucial to better understand the molecular strategies developed by HCV to escape therapy.

Indications of treatment
In patients with acute hepatitis C, interferon therapy is indicated since it significantly decreases the risk of chronicity. In patients with chronic hepatitis C, the decision to treat is a complex issue which must take into consideration numerous variables: age of the patient, general state of health, risk of cirrhosis, likelihood of response, other medical conditions that may decrease life expectancy or contraindicate the use of interferon or ribavirin. Also, impairment of quality of life during treatment has to be taken into account (71). The indication is mainly based on the result of the liver biopsy. While the benefit/risk ratio of the treatment appears to be positive in patients with moderate or severe chronic hepatitis C, this has not been proven for patients with mild chronic hepatitis C. Genotype

and viral load are useful to assess the probability of sustained response to therapy. However, the virological characteristics should not be used as a reason to deny treatment since the decision to treat is mainly based on liver histology. Naive patients should be treated with combination therapy (interferon at the dose of 3 MU three times weekly, ribavirin at the dose of 1000-1200 mg daily). The duration of treatment should be 12 months in patients with genotype 1 and high viral load and 6 months for the others (46). Some authors recommend a 12-month treatment in those patients with genotype 1 and low viral load who have bad predictors of response such as male gender, old age, or bridging fibrosis or cirrhosis. However, the small increase expected in the sustained response rate should be weighed against increased side effects: roughly, the frequency of severe side effects necessitating discontinuation of therapy is 10% with 6 months and 20% with 12 months of treatment (54,55,71). In relapsers after interferon therapy, retreatment is indicated: interferon (higher dose for 12 months) or combination of interferon with ribavirin for 6 months (46). In relapsers with genotype 1 and high viral load, although there is no controlled study demonstrating the superiority of a 12-month treatment to a 6-month treatment, after taking into account the results in naive patients, it seems to be justified to propose 12 months of therapy. In non-responders to interferon therapy, it is important to distinguish the patients who did not respond at all (no decrease of serum ALT) from those who had a significant decrease of serum ALT (more than 50% of decrease) and from those who responded and experienced a breakthrough. While the chance of sustained response with a retreatment is very low in the former, it is not negligible in the second and it is high in the latter. In practice, the decision for retreatment in nonresponders should mainly be based on the severity of the liver disease. While it is logical to try combination therapy in a non-responder patient with a rapidly progressing disease, a patient with moderate or mild disease may wait for new advances in the treatment of hepatitis C.

New strategies
Despite significant advances in the treatment of chronic hepatitis C, roughly two thirds of the patients still do not have a sustained response after combination therapy with interferon and ribavirin. Thus, improved therapy is needed. New strategies under evaluation mainly include induction therapy and pegylated interferon. 107

N.

Boyer & P. Marcellin understanding of the pathogenesis ment of hepatitis C. The first challenge is to obtain 1 ing of the natural history better define the prognostic and in the managea better understand-

100%

80% 60% 40% 20% 0%

3tdJ 5MU 10 MU

of HCV infection and to factors. For this purpose

large cohort studies are needed. Ideally, procedures less invasive than the liver biopsy should be developed to assess the severity of the liver disease. In particular, more accurate markers of fibrosis would be useful. The influence of cofactors such as age, gender and alcohol need to be better understood and other unknown potentially important factors remain to be found. One of the most pressing challenges is to understand the mechanisms involved in the fibrogenesis associated with chronic hepatitis C. The second and most important challenge is the improvement of the efficacy of therapy. Although interferon-ribavirin combination is undoubtedly an important advance, the efficacy remains unsatisfactory, with roughly 60% non responders. Furthermore, the benefit of combination therapy needs to be assessed in many special populations (Table 3). Moreover, current therapy is poorly tolerated and is associated with many adverse effects, some of which are severe. Future treatments should be aimed at improving the efficacy/tolerability ratio. The mechanisms by which interferon inhibits HCV replication and the mechanisms by which ribavirin increases the efficacy of alpha interferon need to be understood. Ribavirin might act by direct inhibition of viral replication (by interfering with the RNA polymerase) or by modulating the immune response (by affecting the balance of Thl and Th2 cell responses) or by direct cytoprotection (80).

Fig. 3. Serum HCV RNA levels (percentage of initial level) 24 h and 48 h after a single injection of 3 MU, 5 MU or 10 MU. (Adaptedfrom Lam et al. (112)).

As already mentioned, new information on HCV viral kinetics clearly showed that HCV is replicating rapidly and that the antiviral effect of alpha interferon is occurring very quickly within the first day and is dose-dependent (Fig. 1). Furthermore, 48 h after the injection, the viral load is increasing again (Fig. 3) (112). Therefore, the standard schedule of administration of 3 MU three times a week may not be the most appropriate. Induction therapy, using a daily dose higher than 3 MU, induces a more rapid and more complete inhibition of viral replication. However, preliminary studies are disappointing (113) and a better efficacy in terms of sustained response remains to be demonstrated. Conjugation of alpha interferon with polyethylene glycol (pegylated interferon) increases the half-life (90 h) and improves the pharmacodynamics of the molecule. Therefore, a single injection a week is sufficient and the antiviral effect seems significantly improved as compared with standard interferon. A preliminary study showed 36% of sustained response with 180 ,ug of pegylated interferon for 48 weeks as compared with 5% with standard interferon therapy (114). Current trials are assessing the efficacy of pegylated interferon combined with ribavirin. If there is an additional improved efficacy related to pegylated interferon on the one hand and to ribavirin on the other hand, combination therapy of pegylated interferon with ribavirin could give a more than 50% sustained response. This optimistic view, however, needs to be confirmed.

TABLE

3 therapy for the treatment of hepatitis C: unresolved

Combination issues

Benefit of combination ~ Children ~ Elderly

therapy

in other populations:

Benefit of combination therapy at other stages of the liver disease: Acute hepatitis Chronic hepatitis with normal ALT Mild chronic hepatitis - Compensated cirrhosis Non-responders Benefit of combination therapy HIV coinfection Extrahepatic manifestations - Graft recipients ~ Hemophiliacs ~ Hemodialysis Benefit of maintenance ~ Non-responders Cirrhosis therapy in special populations

Future perspectives
Despite very significant progress in the field of hepatitis C in the last 10 yr, many challenges remain in the 108

in:

Management of hepatitis C

Better use of the drugs presently available is unlikely to achieve much better results. New drugs are needed. It is envisioned that inhibitors of viral enzymes (protease, helicase or polymerase) could be used as therapeutic agents. The three-dimensional structures of protease (115, I 16) and of helicase (117) have been solved, providing significant insights into their functions. This information is an important first step towards identifying inhibitors and it should be only a matter of time before candidate compounds are available for clinical use. As in HIV infection, the optimal use of these inhibitors would probably be in combination with other antivirals. An innovative approach to treatment involves antisense oligonucleotides designed to bind specifically to viral RNAs, therefore inhibiting HCV replication (118). Another molecular approach involves the use of ribozymes (ribonucleic acid enzyme), which are RNA molecules that selectively cleave RNA in a sequencedependent manner. Elimination of HCV RNA from infected hepatocytes by ribozymes has recently been demonstrated (118,119). However, major problems with antisense and ribozyme strategies are related to the delivery of stable nucleic acid molecules to the infected hepatocytes. Cytokines and cytokine modulators which may affect the life cycle of HCV also deserve evaluation. Among them, IL-12 has interesting immunomodulatory properties: it induces differentiation of CD4 cells in Thl effector cells and enhances antigen-induced Thl immune response. This cytokine is currently under clinical evaluation. IL- 10, which has anti-inflammatory, antifibrogenic, and anti-immune effects, seems to be a promising agent. Of particular importance is the capacity of IL-10 to downregulate the production of proinflammatory cytokines such as TNF-a, IL-l, IFNy and IL-2 from T cells. Due to these interesting immunomodulating characteristics, IL- 10 is currently undergoing investigation in patients with chronic hepatitis C who did not respond to antiviral therapy. Finally, the most important challenge is the development of vaccines. Several major practical and scientific problems arise in designing HCV vaccine (120). First, viral proteins have a very high variability. Second, the only animal model that can be infected by HCV, the chimpanzee, is limited and the virus does not replicate efficiently in vitro. Thus, there is a need to develop relevant animal models and in vitro culture models for HCV replication. Candidates for protective vaccines seem to be still far away. However, some significant progress has been made recently with the production of recombinant envelope proteins which can elicit antibodies and in-

flammatory CD4+ T cells (121). Also, nucleic acidbased vaccines appear to be a promising approach (122). Indeed, in the short term, therapeutic vaccine might be a more achievable goal than protective vaccine. In conclusion, hepatitis C is a considerable present and future health burden. An increased incidence of HCV-related cirrhosis and hepatocellular carcinoma is expected in the next 10 yr. Large-scale screening and effective therapy are needed to slow down the progression of the liver disease in infected subjects. This means that major efforts are necessary to improve the efficacy and to reduce the cost of therapy for the best management of the largest number of patients all over the world.

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