Hepatitis B Virus

Chinese Version

Updated September, 2011

Stéphanie Villet, Ph.D.,  David Durantel, Ph.D.,  Fabien Zoulim, M.D., Ph.D.

GENERAL DESCRIPTION

Virology Guided Medline Search

                Hepatitis, or inflammation of the liver, denotes an organ-specific designation for a clinical entity of widely diverse etiology and pathogenesis. Over the last 35 years, viruses belonging to at least 6 different families have been identified and linked to most hepatitis found in man (243). Hepatitis B virus (HBV) is the most common amongst those that cause chronic infections in human.

                HBV is small and spherical, 42 nm in diameter, enveloped DNA virus and represents the prototype virus of the Hepadnaviridae family (112). Infectious virion, also termed Dane particle, contains a relaxed circular, partially double-stranded genome of approximately 3200 base pairs that is encapsidated by the core (C) protein to form the nucleocapsid. This genome consists of four overlapping open reading frame (ORF): S, for the surface, or envelope gene; C, for the core gene; X, for the X gene; and P, for the polymerase gene (Figure 1). The S and C genes have upstream regions termed pre-S (pre-S1 and pre-S2) and pre-C (Figure 1). A mutation in one viral gene often results in a mutation in an overlapping ORF, thereby restricting the number of viable mutant viruses (Figure 1). However, HBV variants do occur and their presence might alter the clinical presentation and disease course (30, 125, 254) . A copy of the viral polymerase (P) protein is covalently attached to the 5' extremity of the negative strand of DNA in the nucleocapsid. This enzyme carries several activities responsible for the priming of DNA synthesis (terminal protein), the synthesis of the first DNA strand (reverse transcriptase activity), the destruction of the RNA template (RNase H activity), and the synthesis of the second strand (DNA-dependant DNA polymerase activity). Other proteins, mainly host derived, have been found in minute amount in the nucleocapsid and are thought to be involved either in the morphogenesis of the particles or in events happening in the early stage of the infection. Surrounding the nucleocapsid is the lipid envelope that contains small (S), medium (pre-S2) and large (pre-S1) viral surface glycoproteins (HBsAg). The pre-S1 and pre-S2 represent two of the more immunogenic portions of HBsAg (147). The development of humoral and cellular response to HBsAg is protective, and recombinant HBsAg provides the basis for the HBV vaccines currently available. In addition to Dane particles, two other types of particles are found in excess in the bloodstream of HBV carriers, the spheres (22 nm) and the rods (22 nm width and variable length) particles which contain HBsAg and host-derived lipids, but no nucleic acid. These particles are noninfectious and might play a protective role by absorbing neutralizing antibody and thus hiding virions from host defenses.

                 Little is known about the earliest events in the viral life cycle due to the lack, until recently, of a fully infectable cell line (123). Enveloped virions bind to the cell via contact between, most likely, its pre-S proteins and one (or several) host receptor(s) (76, 122, 163, 285, 332) and enter the cell most likely by a pH-independent fusion mechanism (133, 164). After endocytic entry, a translocation mechanism allowing viral particle exit from the endosome has been recently suggested (316). Then, the virus is uncoated, and the viral genome translocated to the nucleus where it is converted to covalently closed circular DNA (ccc-DNA) most likely by host DNA polymerase (296) (Figure 2). The host RNA polymerase II uses the ccc-DNA as template for transcription (296). Four (possibly 5) different transcripts of 0.7, 2.1, 2.4, and 3.5-kb respectively, are produced from internal hepatic-specific promoters and are processed, 5 capped and 3 polyadenylated, by host machinery. The greater-than-genome-length pre-genomic (pgRNA) transcript is translated into three different products, the nucleocapsid or core protein, a secreted protein also called the "e antigen" (HBeAg) as well as the polymerase protein (P). This 3.5-kb pgRNA is also used as a template for the viral RNA-dependent DNA polymerase (P) to produce the minus DNA strand that will be subsequently used as template, by the same enzyme (DNA-dependent DNA polymerase activity of P), to produce the partially double-stranded progeny genomes (251). The reverse transcription takes place within subviral core particles (321), thereby implying that encapsidation of the genomic RNA template must represent the initial step in the genomic replication pathway. The large surface protein is produced via the 2.4-kb pre-S1 transcript, and the medium and the small surface proteins are produced via the 2.1-kb pre-S2/ S transcript. A fourth 0.7-kb transcript is translated into the X protein (HBx). The function of HBx is yet not completely elucidated, but appears to be complex (40, 244). HBx is required for mammalian Hepadenavirus infection in vivo and might function as a weak transcriptional activator and as a stimulator of cytoplasmic signal transduction pathways (40, 244, 384).

                 Viral assembly occurs at the level of intracellular membranes where viral surface glycoproteins are expressed, folded and retained. The nucleocapsid is thought to bud from the endoplasmic reticulum (ER) or an ER-derived vesicle. Virions are then transported through the secretory pathway, traversing the Golgi apparatus where their carbohydrates are processed, and released in the extracellular compartment.

                More details on the biology of the virus and the life cycle (Figure 2) can be found in several general reviews published in journals (111, 194, 251, 296) and a book (112).

Epidemiology Guided Medline Search

Incidence of Chronic Hepatitis B Worldwide

Despite the availability of efficient vaccines (196), HBV continues to pose a serious global health problem. The world health organization (WHO) estimates that hepatitis B is the ninth leading cause of death worldwide (336). Of the 2 billion people who have been infected with HBV worldwide, more than 350 millions are chronically infected. These chronic carriers are exposed to a high risk of life-threatening progressive chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (26, 218). The distribution of HBV infection varies greatly throughout the world. HBV incidence remains particularly high in developing countries of Asia, Africa, the Amazon, the Middle East, the Eastern Europe, and the Pacific Islands (5, 48, 221, 305, 323) where vaccination programs are either not available, being installed or too recently introduced to measure their impact, and where perinatal or child to child transmission is still important (18, 43, 48, 305). Infection is less common in Western Europe and North America where less than 1% of the population is infected (339). For instance, in the United States there are approximately 1.25 million HBV carriers and the number of new infections per year has declined from an average of 450,000 in the 80's to about 78,000 in 2001 (Center for Disease Control data). However, worldwide, it is estimated that more than 50 million new infections with HBV occur yearly, and as many as 1 million deaths annually can be attributed to the effects of this infection (142, 232).

Review Article:  Hoffman C, Thio C. Clinical Implications of HIV and Hepatitis B Co-infection in Asia and Africa. The LANCET Infectious Diseases 2007; Vol.7, Issue 6, 402-409.

Classification

              HBV has been divided into 8 genotypes, A through H. Genotype A is found in Western and Northern Europe as well as in Africa. Some studies suggest that it may be associated with a better response rate to IFN therapy for chronic hepatitis (155). Genotypes B and C are found in Asia. Clinical studies have suggested an association of genotype C with a more severe chronic liver disease and a poorer response rate to IFN therapy (157, 372). Genotype D is observed mainly in the Mediterranean bassin and is associated with a higher prevalence of pre-C mutants in chronically infected individuals (120) as well as a lower response rate to IFN (155). Genotype E is mainly observed in Africa, while genotype F has been identified in South-America and Polynesia. Finally, genotype H has been recently described in Amerindians from Central America.

Transmission

HBV is present in many body fluids of infected individuals. As viral load is very high and may reach 1010 copies per ml of serum, the rate of transmission may be very high after exposure, as compared to other viruses such as HIV and HCV. The main routes of infection are perinatal transmission, blood and percutaneous transmission and sexual transmission (194).

Neonatal infections: The greatest sources of new infections worldwide have been from infected mothers to the newborn, or among very young children. About 90% of infants infected at birth or during their first year of life, and 30 to 50% of children infected between 1 to 4 years of age, develop a chronic infection. This risk of chronic infection declines to ~ 5% for older children and adults. The risk of death from HBV-related cirrhosis or liver cancer is estimated at 25% for persons who become chronically infected during childhood. The risk of vertical transmission varies depending on geographical regions. In North America, Western Europe and Africa, the risk of vertical transmission from chronically infected mothers is approximately 10%. This is expected from the low incidence of HBeAg serum positivity among carriers in these areas, indicating that the infected mothers usually have a low viral load. Furthermore, in many countries pregnant women are screened for HBV markers and newborns are vaccinated to reduce the risk of chronic infection. The high rate of chronicity in many parts of Africa is attributed to horizontal spread to young children from playmates, and adults involved in their care.

               In Asia, the perinatal rate of transmission is as high as 90%, because most of the pregnant women who are chronically infected are HBeAg serum positive and have high titers of circulating HBV. In infants who are infected by HBV, the rate of chronicity of viral infection can reach 90% (314). Studies performed in mice suggest that HBeAg may cross the placenta during pregnancy and induce an HBV specific immune tolerance in the fetus to epitopes of the viral core protein (241). This finding was interpreted to indicate that when the newborn is exposed to HBV at birth, the risk of chronic infection becomes very high because the potential to react to a major viral antigen has been reduced. Some data from humans suggest that HBeAg may indeed cross the placenta (353), though evidence that this causes immune tolerance and facilitates chronic infection has not yet been obtained. Injection of hyper-immune globulin to HBsAg (HBIG) and vaccination of the newborns at birth can reduce the risk of chronic infection from HBeAg positive mothers by >90% (17, 213, 315).

                Intra-uterine infection is thought to be uncommon and may be related to very high viremia levels in the mother. This may explain in part some of the failures of passive-active immunization at birth. Other causes of prophylaxis failure have been identified in the newborns. They include host genetic factors of non response to the vaccine (35) and the selection of surface gene mutants that may escape both HBIG and the anti-HBs response induced by the vaccine (38, 241).

Blood transfusion, intravenous drug use, sexual transmission and nosocomial infections: The risk of HBV transmission by blood transfusion has decreased dramatically since the early 1970s, because of the exclusion of paid donors and the introduction of serological screening of volunteer blood donors for serum HBsAg and anti-HBc immunoglobulins. In the US, the risk of HBV transmission via blood products is now one out of 63,000 transfusions, down from 15% in the 1960s (4, 119). The current incidence may be attributed to the failure to identify infected blood donors because of the serological window during the incubation period following infection, the presence of some rare variants escaping the serologic assay for HBsAg, particularly when concurrent testing for anti-HBc is not performed, and the problem of occult HBV infections, in which neither HBsAg or anti-HBc are detected. HBV genome detection by PCR in blood donors may decrease this risk but the cost-effectiveness of this strategy remains to be evaluated.

                Percutaneous infection remains a major mode of HBV transmission, and this includes intravenous drug use, tattoos, acupuncture, ear piercing, sharing razors, etc... Sexual transmission still represents 40% of the new cases of acute hepatitis B in many developed countries (4, 119), while the role of intravenous drug use seems to be decreasing with time representing now only 6-10% of new cases. HBV can be transmitted by accidental needle stick in the healthcare setting (4, 119). The risk of nocosomial transmission is estimated to be 30% from highly viremic patients. On the other hand, transmission from healthcare worker to patient although rare, may occur occasionally as has been reported, for example, with the transmission of HBV from infected surgeons (136). Other cases of nosocomial transmission have been reported in hemodialysis centers and in the setting of organ transplantation even from donors who only have anti-HBc antibody, which when found by itself is usually a marker of a past infection from which an individual has recovered. For instance, de novo HBV infection in the liver graft recipient can be observed in more than 50% of cases when the donor has antibody to HBcAg but no other serological marker of infection, implying a transient infection at some time in the past, with survival of residual virus. Horizontal transmission can be observed among children or in institutionalized persons via close bodily contact leading to HBV infection through minor skin breaks and mucous membranes (339).

                In summary, high-risk groups for HBV infection have been identified. They include healthcare workers, especially surgeons and physicians working in haemodialysis, oncology, or AIDS units; laboratory workers in contact with blood or human fluids; institutionalized handicapped persons, their attendants and family; patients requiring frequent blood product transfusions ; patients on haemodialysis; patients with organ transplantation; intravenous drug users; men who have sex with men; promiscuous heterosexuals.

Carroll MB, Bond MI.  Use of Tumor Necrosis Factor-alpha Inhibitors in Patients with Chronic Hepatitis B Infection.  Semin Arthritis Rheum 2008 Jan 24 [Epub ahead of print].

Clinical Manifestations Guided Medline Search

                HBV is a highly infectious agent and infection occurs frequently after exposure (275). The clinical course of primary infection can be acute or chronic, and the outcome is frequently age dependent (91). In adults, HBV infection is usually self-limited and resolves in less than 6 month. In two-third of adults infections are sub-clinical; only one-third of infected adults develops clinical hepatitis (236). Fulminant hepatitis occurs in less than 1% of acute infection and results in the death of patients most of the time. A small number of HBV-infected adults develop chronic hepatitis (209). In contrast, HBV infection in the first few months of life seldom causes clinical illness and almost always leads to persistent infection (236, 266).

                During acute symptomatic HBV infection, patients may have fever, malaise, fatigue, weakness, anorexia, abdominal discomfort, conditions that may be due to the high production of IFNs, TNF-α, and other inflammatory cytokines. When massive liver cell necrosis occurs over a short period of time, patients may also present with jaundice. The diagnosis of acute HBV infection is based on history, ALT profiles (up to 100-fold increase), and HBV antigen-antibody or/and serum HBV DNA detection. After an incubation period of 4 to 24 weeks, HBsAg can be detected in blood. HBeAg and HBV DNA occurring at the same time indicate active viral replication. Loss of HBeAg is usually associated with marked decrease in HBV DNA. Some patients seroconvert to HBeAg but remain HBV DNA positive with active liver disease. These patients have the "e" mutant variant or pre-core mutant of the virus (30, 262, 324) (Figure 1). IgM specific anti-HBcAg antibody develops early during infection and is followed by anti-HBs antibody as the patient improves. Most patients recover within 6 months of illness onset, and the ALT level return to normal.

                Persistence of HBsAg 6 months after the onset of hepatitis illness indicates that the HBV infection has become chronic. This happens in approximately 5% of adults infected (209). In wild-type HBV chronic infections, three stages characterize the progression of liver disease in immunocompetent individuals, possibly leading to advanced liver disease with cirrhosis and its complications.

                The first stage, lasting from one to 15 years, is characterized by an immunotolerant state with high HBV replication levels and low-grade inflammatory lesions in the liver. Often, in the early stages of infection, infected hepatocytes do not stimulate a strong immune response, with the result that serum transaminase levels remain near normal and the liver continues to shed high titers of virus. This is especially true in the case of vertical transmission of HBV. These patients are defined serologically by HBsAg and HBeAg positivity, high HBV DNA levels (usually higher than 108 copies per ml), and strictly normal serum ALT levels. In this situation, treatment is not indicated because patients usually have no liver damage or very minimal liver disease at liver biopsy examination. The risk of disease progression is minimal so long as ALT levels remain within the normal range. Furthermore, the results of clinical trials of IFN-α, or nucleoside analogue inhibitors of the viral DNA polymerase, showed that patients with high HBV DNA load and normal ALT levels have almost no chance of undergoing HBeAg sero-conversion as a result of treatment.

                The second stage, lasting from a few weeks to months, is characterized by clearance of infected hepatocytes with an increase of hepatocyte necrosis and serum aminotransferase levels and decrease of serum HBV DNA level. Loss of serum HBV DNA (by non-PCR-based methods) may occur spontaneously in about 5 to 10% of patients yearly or as a result of antiviral therapy. If antiviral therapy is not carried out during this immunoactive stage, clinical symptoms may appear. If this stage persists due to the inability of the immune system to control viral infection, chronic hepatitis with cumulative liver damage may occur.

                 In many chronically infected patients, HBeAg sero-conversion occurs, in which HBeAg antigen becomes undetectable, and anti-HBe antibodies appear, while only low levels of viral DNA (<104 copies per ml) persist in the serum. Biological markers of hepatic function normalize. This is considered a good clinical outcome, and is indicative of immunological control of the infection in the liver. In this phase, patients are considered inactive carriers of HBV (56). In this situation, liver histology usually reveals remission of liver disease activity, and the risk of progressing liver disease is considered to be minimal so long as viral load remains low and ALT levels normal. As viral ccc-DNA persists in the liver, episodes of viral reactivation may however occur either spontaneously or as a result of immune suppression (358).

                Although, the liver remains the main locus for HBV-induced diseases, some extrahepatic diseases are caused by immune complexes of hepatitis B antigens and antibodies. These include serum sickness syndrome, polyarteritis nodosa, membranoproliferative glomerulonephretis, and cryoglobulemia (194). The formation of these complexes is a consequence of active viral replication and usually begins in the early stages of an infection, particularly in children. This is the reason why treatment of HBV associated periarteritis nodosa or glomerulonephritis relies on the use of antiviral therapy. This may be combined initially with plasmapheresis and a short course of corticosteroids to decrease the consequences of the deposition of immune complexes on the artery walls (276, 333). Several studies have shown the clinical benefit of anti-HBV agents including vidarabine (adenine arabinoside), IFN-α, lamivudine, famciclovir, and adefovir (92, 171, 220, 354, 359). Indeed, the control of viral replication is accompanied by a decrease of immune complex formation and the improvement of the clinical signs of vasculitis. Clearance of HBsAg and concurrent cure of the vasculitis have been observed by several investigators. In patients presenting with a life threatening vasculitis, therapy may combine prednisone to decrease the consequences of vascular inflammation, plasmapheresis to decrease the circulating immune complexes and their consequences, and antivirals. After a few weeks, when the clinical situation is under control, prednisone and plasmapheresis can be stopped while antiviral therapy is continued until HBe or HBs sero-conversion occurs.

Laboratory Diagnosis Guided Medline Search

                The aim of the following section is to briefly review the markers and tests used in the diagnosis of acute and chronic hepatitis B.

Serologic Diagnosis of HBV Infections: The serological markers of HBV infection vary depending on whether the infection is acute or chronic (Table 1). The first serologic marker of HBV infection is HBsAg, which can be detected from 2 to 24 weeks after contamination with HBV. The presence of HBsAg often antedates symptoms or abnormalities of hepatic biochemistry (e.g. raised ALT) by 6-8 weeks. In patients who recover, HBsAg disappears from the serum in 2-6 months, and protective anti-HBs antibody appears after a variable window period. Persistence of HBsAg beyond 6 month after the onset of the infection is widely accepted as a sign of chronic infection. Detection of HBsAg is the most widely used diagnostic test for HBV infection. An improved version of the previously developed sandwich enzyme-linked immunoabsorbent assay (ELISA), which includes microparticles as solid phase and computerized instrumentation, is currently used for the detection of HBsAg (77). These microparticle enzyme immunoassays (MEIAs) can rapidly (45 min) detect 100-200 pg of HBsAg per ml of serum, corresponding to an approximate titer of 3.107 particles (virion + 22nm particles) per ml. Antibody to hepatitis B surface antigen (anti-HBs) becomes detectable during the recovery from acute HBV infection in patients who do not progress to a chronic infection. The disappearance of the HBsAg occurs a few weeks before the advent of the anti-HBs. The presence of anti-HBs after acute infection indicates recovery from the infection and generally lifelong immunity from reinfection. A measurable anti-HBs response, which can vary among subjects, is as well induced in most recipients of HBV vaccine. An other interesting marker of acute HBV infection is the anti-HBc antibodies of the IgM class which appear shortly after HBsAg and remain detectable up to 6 month after the onset of the acute hepatitis. The detection of the IgM anti-HBc is of interest in diagnosing an acute infection in patients with HBsAg concentrations that are below the sensitivity threshold of the diagnostic assay. Moreover a high titer of this marker is generally accepted as an indicator for acute infection. Anti-HBc IgM may also become detectable in chronic HBV infection in case of viral reactivation and acute exacerbation of the liver disease. A third very useful hepatitis serological marker is HBeAg, which usually becomes detectable in the serum when HBsAg first appears. The presence of HBeAg in serum correlates with the presence of viral replication in the liver. During a typical acute infection, HBeAg is detectable early in the course of disease and disappears within several weeks as hepatitis resolves. In chronic hepatitis B, HBeAg is an important marker to monitor as it usually remains detectable for many months or years, indicating active replication of the virus and potential ongoing liver injury. Moreover, the loss of HBeAg and acquisition of anti-HBe (seroconversion) by chronic carriers tend to be associated with biochemical and histochemical improvement, with the notable exception of pre-core mutants. HBeAg in serum can be detected by a sandwich immunoassay format similar to that for HBsAg. The above mentioned points occur in HBV infection with typical course (Table 1). Sometimes, the immunologic pattern is different. This can be due to an error in the evaluation using immunological techniques or to an atypical pattern, whose can include i) isolated anti-HBc positivity, ii) simultaneous HBsAg and anti-HBs. To avoid misdiagnosis, additional tests involving detection of circulating HBV DNA have been implemented in routine laboratory analysis.

Molecular Diagnosis of HBV Infections: Determination of HBV DNA levels in serum is now accepted as part of the serological profile for screening of blood donors and for monitoring the ongoing viral replication in patients with diagnosed chronic hepatitis B (even when HBeAg is not detectable), or the response to therapy. It is also useful as a prognostic index regarding response to interferon therapy. For instance, patients with HBV DNA concentrations <200 pg/ml are more likely to have a successful therapeutic outcome than those with higher HBV DNA concentrations. The currently commercialized tests are of three types: quantitative hybridization-based assays (e.g. Digene Hybrid-Capture from Murex Diagnostics), branched-DNA assays (e.g. Versant bDNA version 3.0, Bayer Diagnostics), polymerase chain reaction (PCR) assays (e.g. Amplicor HBV monitor from Roche), and real time PCR assays (Roche Diagnostics and Abbott). All the techniques have reasonable specificity and reproducibility, but have different range of linear quantification (11, 160, 264). The PCR-based assays have a good sensitive with a threshold of detection at 400 genomes/mL (~ 1 fg/ mL). The real time PCR based assays have the best range of detection and sensitivity limit of approximatively 50 copies/ml. Caution is necessary when using different commercial kits as their internal standards are not equivalent. To resolve this problem, the European Expert Group on Viral Hepatitis have formulated a consensus preparation of an HBV DNA-positive plasma and assigned to it a concentration in Eurohep Units (EU), with 1 EU being equivalent to about 1 to 2 molecules of HBV DNA (373). The use of this international standard facilitates comparisons and allows harmonization in clinical and laboratory practice.

                Since the introduction of antiviral agents (e. g. lamivudine) for the treatment of patients with chronic hepatitis B, the accumulation of a variety of mutations in the HBV polymerase gene has been observed (Figure 3). The detection of these variants is of clinical importance, as they may be associated to nonresponsiveness and treatment failure. Innogenetics has developed a line probe assay (INNO-Lipa HBV polymerase drug resistance) for monitoring drug resistance in HBV-infected patients before and during antiviral therapy. This assay is based on nitrocellulose strips on which are fixed different probes corresponding to different known mutations in the catalytic domain (C domain), that includes the YMDD motif and in the B domain. The viral DNA, purified from patient’s serum, is first amplified by PCR then hybridized to the strips in order to determine the local genetic pattern of the patient (319). An alternative to this assay is to use PCR amplification and sequencing of the polymerase gene. But this technique is less common in routine laboratory diagnosis.

FDA: FDA Approves First Nucleic Acid Test to Screen for Additional Types of HIV In Donated Blood and Tissues. December, 2008. http://www.fda.gov/bbs/topics/NEWS/2008/NEW01936.html

(Printable Version of Laboratory Diagnosis for Hepatitis B Virus)

Pathogenesis Guided Medline Search

                The pathogenesis of hepatitis B virus infections has been abundantly reviewed elsewhere (111, 126, 142, 194, 251, 278, 296), and only a very brief summary will be given here. HBV is generally considered to be noncytopathic, meaning that its replication does not induce directly cellular damage. Instead, liver cell damage is self-inflicted and occurs when the immune system either clears the infection in the case of resolving acute HBV infection or repetitively attempts to clear the infection in the case of chronic viral infection. The effectors that mediate this immune response responsible for liver injury are diverse. Earliest intervention of the host response likely involves the non-specific innate immune system that includes macrophages, neutrophils, natural killer cells as well as diverse cytokines (278). The effectors of the specific adaptive immune response are of two kinds. They are antibodies that reduce viral load and prevent infection of non-infected cells. More importantly in respect with pathogenesis, CD8+ T cells that induce necroinflammatory or apoptotic destruction of virus-infected cells or, alternatively, may suppress viral replication in infected cells by the production of TH1 cytokines independently of cell lysis. Necroinflammatory damage may be virus-specific or non-specific and may involve participation of cells of the non-specific immune system. Most often, the interplay of the immune mechanisms brings about elimination of the virus over varying periods of time. In chronic infection the immune system may be too weak to clear infected-cells, but sufficient to induce the lysis of a part of them. Alternatively persistence might be the result of a particularly efficient virus with enhanced or modified replication properties. Altogether, the repetitive immune attack by CD8-positive cytotoxic T lymphocytes and other NK-T cells is probably responsible for the progressive liver injury.

                The most serious pathologic consequences of chronic hepatitis B are cirrhosis of the liver, and hepatocellular carcinoma (HCC). Approximately 10 to 25% of chronic carriers die from either cirrhosis or hepatocellular carcinoma (194, 296). The origin of cirrhosis, that results from long-term inflammation cell death, and replacement with non-parenchymal (fibrotic) tissues, is well defined and understood. But, the mechanism and role of the virus in the induction of hepatocellular carcinoma is still a complex issue. Hepatocellular carcinoma is among the most common cancers in the world and one of the rare human cancer to show sero-epidemiological association with viral infection. The role of HBV as a major etiological agent of hepatocellular carcinoma has been firmly established, and the increased risk of developing hepatocellular carcinoma has been estimated to be 100 fold for chronic HBV carriers as compared with noninfected populations (18, 19). Although the details of this process are still not understood, the field has been driven by two hypotheses, one positing a direct effect of the virus in cancer development, the other predicting that hepatocellular carcinoma formation is a consequence of persistent liver injury caused by the immune response against infected hepatocytes and hence, attributing an indirect role to the virus.

                Evidence for a direct mechanism was fueled by the observation that DNA from hepatocellular carcinoma contained integrated HBV DNA and that tumors were clonal with respect to the viral integration site (93). This discovery was consistent with the model of insertional mutagenesis coined by retrovirologists to describe the activation of proto-oncogenes by retroviruses (138, 265). However efforts to identify candidate genes adjacent to integrated HBV DNA were not met with success save for a few isolated cases, where integration sites were found adjacent to or within coding regions of candidate proto-oncogenes including cyclin A, erb-A or retinoic acid receptor beta (78, 79, 352). Alternative models consistent with a direct mechanism posit that one or several viral gene products are oncogenic. Although the long latency period observed for hepatocellular carcinoma development indicates that HBV gene products cannot transform hepatocytes in a single hit event, the hypothesis gained momentum with the discovery that transgenic mice expressing HBx developed hepatocellular carcinoma (161). However, it became rapidly clear that the success of this model depended on the selection of the mouse strain, CD1, known to develop spontaneous hepatomas at an increased rate compared to other mouse strains, and high expression levels of the transgene that are not observed during natural infections (165). However, it appears that HBx transgenic mice are more susceptible to hepatocellular carcinoma induction by diethylnitrosamine (308), as are mice expressing HBx and c-myc (327). These and other observations invoke the possibility that HBx could, at best, contribute to a multistep transformation process by either activating certain signal transduction pathways or by binding to host proteins, such as the DNA repair protein UVDDB or the proteasome subunit XAPC7 (149, 193). Also, it is important to consider that HBx is not always expressed in hepatocellular carcinoma (320). In addition, as stated earlier, tumors are normally not permissive for HBV infection. Therefore, it is unlikely that the protein acts as an oncogene that is required to maintain the transformed phenotype. The discovery of the transactivation function of truncated M envelope proteins rekindled the viral oncogene hypothesis, in particular since the mutant genes were occasionally found in hepatocellular carcinomas (293). Transgenic mice over-expressing the HBV L-protein develop hepatocellular carcinoma (54).

                Models explaining hepatocellular carcinoma development by indirect mechanisms take into consideration a hallmark of HBV infections, cell death and regeneration of hepatocytes. While this process is inconsequential on a sporadic basis, it can cause substantial fibrosis and cirrhosis of the liver under persistent condition such as those observed in chronic alcohol abuse, storage disease and chronic HBV or HCV infections. Independent of the cause, chronic liver disease is associated with hepatocellular carcinoma. It was suggested that a contributing factor in emergence of hepatocellular carcinoma was free radicals generated by the large number of macrophages activated in response to the high rate of apoptosis, leading to extensive hepatocyte DNA damage. A possibility is that DNA damage is the underlying cause of hepatocellular carcinoma and that hepatocyte proliferation in response to cell death facilitates emergence of mutated cells. Recent cohort studies performed in Asia demonstrated a link between the level of viral load and the subsequent risk of developing hepatocellular carcinoma (49).

SUSCEPTIBILITY IN VITRO AND IN VIVO  OF HBV TO ANTIVIRALS Guided Medline Search In Vitro and In Vivo

               HBV belongs to the Hepadnaviridae, a virus family that includes the woodchuck hepatitis virus (WHV (322), the duck hepatitis virus (DHBV) (231), the ground squirrel hepatitis virus (GSHV) (295), and the more recently described viruses, heron hepatitis virus (HHV) (313), snow goose hepatitis virus (SGHV) (44) and woolly monkey hepatitis virus (WMHV) (183). All members of this family share homology in their genome organization, viral particle structure and strategy of replication. They offer opportunities for in vivo study and in vitro studies when cell lines are available. Antiviral agents have been studied extensively in the DHBV and the WHV systems. The duck system offers the advantages of being relatively inexpensive and readily available, in comparison to the woodchuck model. However, the woodchuck model is particularly useful because it is a mammalian virus, thus closer to HBV, and chronic infection in the woodchuck leads to hepatocellular carcinoma within 2 to 3 years (226).

               Efficacy of HBV-infection in primary human hepatocytes cultures (108) is low and primary liver cells become non-permissive for infection within days after plating. Moreover, unlike animal tissues, access to human liver is limited and clearly human hepatocytes cannot be used for routine screening of anti-HBV agents. Only recently, a hepatoma cell line was shown to be susceptible to HBV infection under specific conditions, but the full potential of this cell line is still under investigation (123). These difficulties are circumvented by several in cell culture systems, which have proven very useful for the screening of anti-HBV compounds and study of their mode of action. They are i) stably transfected hepatoma lines HepG 2.2.15 or HepAD38, ii) transiently transfected hepatoma cell lines (HepG2 or Huh-7 human cells for HBV and LMH avian cells for DHBV), and iii) infected hepatoma cell lines via recombinant HBV-baculovirus that allow full viral replication including ccc-DNA formation. Furthermore, determination of the mode of action may be investigated i) in primary cultures of hepatocytes taken from the well-characterized animals models of HBV, the duck and the woodchuck, ii) on endogenous viral DNA and RNA dependant DNA polymerase which can be assayed in purified nucleocapsid or iii) in a tube reconstituted assay for both priming and reverse transcription of the viral genome. Each of these different in vitro systems presents different advantages, inconveniences and limitations that have been discussed extensively (135, 381).

                All these in vitro and animal models contributed to the molecular characterization of the replication cycle of HBV and consequently to the identification of viral targets for antiviral therapy of HBV infection (Figure 2): i) neutralization of the virus-cell interaction with anti-envelope antibodies or peptides, (ii) cytokine and/or antisense inhibition of viral transcription and viral gene expression, (iii) packaging inhibition with peptides, (iv) RNA or DNA dependent DNA polymerase activity inhibition by nucleoside analogs or antisense and (v) viral secretion with glucosidase inhibitors.

                Antiviral therapy has to face the problem of viral persistence and resistance to nucleoside analogs and thus since several years the field of drug discovery continues to be extremely active to find new agents (75, 95, 124, 134, 303, 382, 385).

Single Drugs (Table 2)

Nucleoside/ Nucleotide Analogues

Cytidine analogues

Lamivudine: Lamivudine or 3TC (beta-L-2', 3'-dideoxy-3'-thiacytidine) is a potent in vitro inhibitor of both duck (302) and human hepadnaviruses (Table 2) (41, 89), acts as chain terminator of the reverse transcription (302), and has no mitochondrial toxicity (70, 156). Interestingly, it was shown, using an HBV-baculovirus system infecting HepG2 cells, that lamivudine has also an inhibitory activity on ccc-DNA synthesis and accumulation (82). Lamivudine is a very effective inhibitor of DHBV in ducks (190), and HBV in chimpanzees, but only a moderate inhibitor of WHV in woodchucks (230). Consistently with the latter observation, it was observed that a prolonged 3TC treatment on primary woodchuck hepatocytes infected by WHV does not allow viral eradication (242). Treatment with lamivudine is as well associated with viral resistance (47, 210, 330). Variants presenting mutations within the catalytic domain (C domain), which includes the YMDD motif, (e. g. YVDD and YIDD), and within the B domain (e. g. L180M, frequently associated with YVDD) have been identified and studied to determine their biological features. It was found that these mutants have a reduced replicative capacity compared to the wild-type virus (101, 239, 299). To find an alternative treatment for 3TC resistant patients, many works on the susceptibility of these mutants to others drugs were performed in vitro and animal models (28, 53, 90, 104, 259, 260, 299, 371). Two categories of drug have been characterized; those which present a cross-resistance pattern with 3TC, such as the pyrimidine analogues emtricitabine, beta-L-Fd4C, or clevudine and those which are active against 3TC-resistant mutants, such as the purine analogues such as adefovir, ganciclovir, entecavir or amdoxovir. Details on these drugs follow.

Emtricitabine: Emtricitabine or FTC (beta-L-2',3'-dideoxy-5'-fluoro-3'-thiacytidine) is a 5-fluoro oxathiolane derivative, closely related to lamivudine. It was found to be a potent inhibitor of HBV replication in the HepG 2.2.15 (107), in primary human hepatocytes, and in vivo in nude mice (65). In the woodchuck animal model, FTC reduced WHB DNA significantly in a dose dependent manner, showing antiviral activity levels similar to those obtained with lamivudine (73, 170). Cross-resistance between lamivudine and FTC has been reported, thus precluding its use in the treatment of patients with lamivudine-resistant variants (101, 173).

Elvucitabine: Elvucitabine or Beta-L-Fd4C (beta-L-2',3'-dideoxy-2'3'-didehydro-5-fluorocytidine) inhibits HBV replication in the HepG 2.2.15 cell line, without inducing significant mitochondrial DNA toxicity in CEM cells (50, 307). Evaluation in the DHBV infection model showed that beta-L-Fd4C is a more potent inhibitor of the DHBV RT than 3TC and other cytidine analog triphosphates and inhibits viral DNA synthesis in primary duck hepatocyte cultures. It was also demonstrated that early administration of beta-L-Fd4C after experimental infection of ducklings dramatically inhibits viral replication but does not prevent the progression to chronic infection (190). Furthermore, in woodchucks chronically infected with WHV, although beta-L-Fd4C activity was more potent than 3TC, it did not allow clearance of viral ccc-DNA and infected cells from the liver (191). Lamivudine-resistance mutations also confer resistance to elvucitabine (28).

Thymidine analogues

Clevudine: Clevudine or L-FMAU (2'-Fluoro-5-Methyl-beta-L-Arabinofuranosyluracil) is a beta-L-nucleoside analogue derived from deoxythymidine (TTP) which was found to be a potent inhibitor of hepatitis B virus (HBV) replication in HepG 2.2.15 and to have a low in vitro cytotoxicity (55). In this cell line, it was further demonstrated that L-FMAU inhibits HBV without affecting the host DNA synthetic machinery (12). By contrast to D-FMAU and to D-FIAU, L-FMAU does not decrease mitochondrial DNA content, does not affect mitochondrial function, and is not incorporated in cellular DNA (12). Moreover, Aguesse-germon et al. (1) showed that L-FMAU exhibits antiviral activity in vivo in experimentally infected ducklings and primary duck hepatocytes. Interestingly, L-FMAU is a weak inhibitor of the reverse transcriptase activity, but an inhibitor of the DNA-dependent DNA polymerase activity (301). Lamivudine-resistance mutations also confer resistance to L-FMAU (28). L-FMAU may have additional modes of action, such as immunological modulation, since more than 50% of patients under treatment with L-FMAU for 3 months show a slow viral rebound for at least a 6 months period after treatment withdrawal (225). Despite a potent inhibition of viral replication when used alone or in combination with emtricitabine in chronically infected woodchucks, experimental studies showed the persistence of viral ccc-DNA (154, 377).

Telbivudine: Telbivudine or LdT (beta-L-2’-deoxythymidine), is a compound of the novel class of the beta-L-nucleosides with potent, selective and specific activity against hepadnavirus (274). In vitro studies have shown that this compound have marked effects on HBV replication (13). In woodchuck chronically infected with woodchuck HBV, up to 28 days of LdT treatment produced consistent, multilog reductions in circulating serum woodchuck HBV DNA levels. Toxicology strudies conducted with rats and monkeys revealed no clinical abnormalities (31).

Adenosine analogues

Adefovir Dipivoxil: Adefovir dipivoxil or PMEA [9-(2-phosphonolmethoxyethyl) adenine] is an acyclic phosphonate nucleotide analogue of adenosine monophosphate, which, unlike nucleoside analogues, does not require the first of three phosphorylation steps for conversion to the active triphosphate form. In vitro, adefovir diphosphate, the active metabolite of adefovir is a potent inhibitor of viral replication in human hepatoma cells lines stably transfected with HBV and in primary duck hepatocytes infected with DHBV (140). The antihepadnaviral activity of adefovir dipivoxil was also demonstrated in vivo, in the duck model (139) and in woodchucks chronically infected with the woodchuck hepatitis virus (WHV) (72). Although, adefovir dipivoxil is a more potent inhibitor of DHBV replication than lamivudine in experimentally infected ducklings and primary duck hepatocytes, it does not prevent ccc-DNA synthesis and accumulation (83). The most important feature of adefovir dipivoxil resides on its capacity to inhibit the replication of lamivudine-resistant (259, 363), but several resistant variants that are inert to PMEA treatment have been detected according to recent reports (7, 346).

Tenofovir: Tenofovir or PMPA belongs to a class of acyclic phosphonate nucleotide analogues which have demonstrated clinical utility against a broad spectrum of viral infections. PMPA has selective activity against retroviruses and hepadnaviruses and is currently approved for the treatment of HIV-1 as the bis-alkoxyester prodrug tenofovir disoproxil fumarate. PMPA was shown to be active against DHBV and HBV in vitro (140), and more recently against WHBV in vivo (240). PMPA has demonstrated full activity against lamivudine-resistant variants, as well as against multiple drug resistant strains in vitro and clinically (28, 80, 338, 348, 365).

Guanosine analogues

Entecavir: Entecavir, or Baraclude, is a cyclopentyl guanosine analogue. A detailed molecular investigation of the mechanism of inhibition of the viral polymerase activity showed that entecavir triphosphate: (1) inhibits all three steps of viral genome replication, i.e. priming of reverse transcription, minus strand-and plus strand-DNA synthesis; (2) is a competitive inhibitor of the natural dGTP substrate incorporation within nascent viral DNA; (3) binds to the viral polymerase with a high affinity. Furthermore, its template-dependent incorporation in viral DNA in place of deoxyguanosine led to chain termination two or three nucleotides later (298). Studies were performed in the animal models of hepadnavirus replication , including the woodchuck and duck, to gain insight in the in vivo effect of entecavir and the kinetics of viral clearance. First, entecavir was administrated orally in WHV chronically infected woodchucks for 1-3 months in comparison with lamivudine. After 1 month of administration, viremia declined by 3 log 10 copies/mL. At month 3, serum viral load dropped by 7-8 log 10 copies/mL down to 100-1000 copies/mL (113). This was followed by a prolonged study of entecavir administration for 14-36 months which showed a prolonged suppression of viral replication including suppression of viral load, a decline in ccc-DNA levels and expression of viral antigens in the liver of infected animals (62). A delayed emergence of hepatocellular carcinoma was also observed although some animals developed liver cancer despite viral suppression. Studies performed in duck model confirmed these results (103). In vitro studies showed its activity against several multiple drug resistant strains (28, 348). Entecavir resistant mutants have been described; they require a lamivudine resistance mutation backbone to which additional specific entecavir resistance mutants are added (326, 347). These strains remain sensitive to adefovir and tenofovir (347).

                FLG (2',3’-Dideoxy-3’-Fluoroguanosine) is a synthetic deoxyguanosine analogue originally developed to inhibit HIV replication (137). The functional similarity of the HBV and HIV DNA polymerases has led to the examination of FLG as an inhibitor of human and duck HBV polymerase activity. In vitro, FLG was shown to be an efficient inhibitor of DHBV and HBV replication (294). In contrast to entecavir, FLG inhibits weakly the priming of the reverse transcription, but is a competitive inhibitor of dGTP incorporation and a DNA chain terminator (153). It inhibits similarly the replication of wild-type, lamivudine-resistant, adefovir-resistant, and lamivudine+adefovir resistant HBV mutants (153). In vivo, FLG showed a favorable safety profile and inhibited DHBV replication in DHBV-infected ducks with a significant reduction of serum DHBV DNA levels (215).

Other Molecular Drugs

                Beside the nucleoside/nucleotide analogs, a number of other molecules with anti-HBV properties have been discovered and studied in vitro and in animal models.

Antisense Oligodeoxynucleotides: Antisense oligodeoxynucleotides (ODN) are synthetic DNA molecules that can inhibit gene expression within cells by binding to complementary mRNA sequences, thus preventing translation (350). Indeed, use of antisense oligonucleotides specific to the hepadenaviral genome showed its efficacy by stimulation of RNAseH activity and degradation of viral RNA, inhibition of ribosomal assembly, modification of RNA conformation and inactivation of RNA sequences involved in viral replication (169, 257, 349, 351). In the cell culture system, antisense DNA directed to HBV polyadenylation signal (248), and X gene (99) are particularly effective. Korba and Gerin examined the ability of 56 single-strand ODN, which target several HBV specific functions, to inhibit HBV replication in the HepG2.2.15 cell line. The oligomers directed against the HBV encapsidation signal/structure (epsilon) showed the most effective antiviral activity against HBV (168). Similar experiments in DHBV-infected ducks or avian cells in culture yielded promising results (282, 310). ODNs containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are known as potent activators of the immune system and inducers of several Th1-associated immunomodulatory cytokines. Some results have shown that CpG ODN can inhibit indirectly HBV replication in vitro via activating the immune cells, and could contribute to the development of an immunoregulator against HBV infection (202). Another class of antisense agents, named PNA (peptide nucleic acids), also showed an efficacity against HBV replication by targeting the HBV encapsidation signal (283). Small interference RNA: The transfer of short interfering double-stranded RNA (siRNA), first discovered in C.elegans (100), has proven a powerful tool for interfering with gene expression through a process known as RNA interference. Eventually, this mechanism results in degradation of the respective target RNA (238). Several investigators succeeded in down-regulating HBV RNA and therefore core protein (235, 370), HBe and HBs antigen expression (162) in cell culture or in mice using siRNA or integrating viral vectors expressing shRNA (short hairpin RNA) to prolonge siRNA expression (279).

 Ribozymes: Ribozymes (ribonucleic acid enzymes) are naturally occurring RNA molecules that catalyse RNA sequence-specific cleavage and splicing (96). RNA cleavage specificity is mediated by the ribozyme sequence. The hammerhead and the hairpin ribozymes have been analyzed in a cell culture system. The targeted sequences include poly (A) signal sequence (97), the encapsidation signal in pgRNA (20, 250), the HBc RNA (98, 201, 203), X RNA (118, 203, 357) and HBs RNA (250). Presently a chemically modified ribozyme, targeting HBV mRNA, is at the clinical development stage.

Aptamers: Many aptamers are designed to block protein functions. Aptamer, a small nucleotide can bind to its ligand (protein, ion, antibiotic, etc...) with high affinity and high specificity. Their binding is due to their 3-D conformation, there is no sequence complementation between an aptamer and its ligand. Peptide aptamers which specifically bound to the HBV or DHBV core protein have been used in cell culture to inhibit HBV replication by blocking viral capsid formation (34, 331).

Peptides: Peptide may be used as a therapeutic antigen by interfering with the interaction between HBV particles and the cell surface or the viral replication and maturation. Recently a myristoylated Pre-S peptide, comprising the pre-S1 region essential for HBV infectivity, efficiently targeted and inactivated a receptor at the hepatocyte surface, preventing HBV infection (122).

HAP (Heteroaryldihydropyrimidines): HAP were discovered as highly potent non-nucleosidic inhibitors of HBV replication in vitro and in vivo (356). HAP, like Bay41-4109 (84) (Table 2) or HAP-1 (317), prevent the proper formation of viral core particles (nucleocapsids) which are the site of viral DNA replication. The clinical efficacy of this treatment modality of HBV infection will now need to be demonstrated.

Chimeric core proteins: The restriction of HBV genome replication to the nucleocapsid makes this nucleoprotein particle an attractive target for intervention. Dominant negative (DN) core protein variants have been shown to interfere with nucleocapsid assembly and to inhibit HBV replication effectively (369).

Helioxanthin analogues: Helioxanthin analogues not only inhibit viral DNA synthesis but also decrease the amount of viral RNA and viral protein in HBV-harbouring cells. (51, 204). The detailed mechanism of action of this class of compounds is under further exploration.

ER alpha-glucosidase Inhibitors: One function of N-linked glycans is to assist in the folding of glycoproteins by mediating interactions of the lectin-like chaperone proteins calnexin and calreticulin with nascent glycoproteins. These interactions can be prevented with inhibitors of the alpha-glucosidases. Treatment of woodchucks with the N-nonyl-DNJ alpha-glucosidase inhibitor shows a significant decrease of WHV levels, as a result of an inhibition of the endoplasmic reticulum alpha-glucosidases, at concentrations that do not appear to be toxic for the animal (25).

Single chain antibody: Studies using cloned single chain Fv (sFv) fragment directed against HBsAg or HBV pre-S1 showed that this antibody fragment could reduce extracellular HBsAg or have a high neutralizing activity against pre-S1 or HBV virion binding to liver cell line (263).

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Review Article: Lok ASF, McMahon BJ. Chronic Hepatitis B: Update 2009.  AASLD Practice Guidelines. Hepatology 2009:50;1-36.

Review Article: Keeffe EB, et al.  A Treatment Algorithm for the Management of Chronic Hepatitis B Virus Infection in the United States: 2008 Update. Clin Gastroenterol Hepatol. 2008;6:1315-1341.

                Hepatitis B virus infection can be acute or chronic, asymptomatic to fatal. Most acute HBV infections clear completely and leave no evidence of long-term hepatic injury. Therefore the primary target for antiviral therapy is chronic infection. The main aims of antiviral therapy are to clear the replicating virus and thereby, if administered early enough in the disease process, to prevent or delay the development of life-threatening complications. Several studies have shown that patients who lose HBV DNA and seroconvert HBeAg, with eventual loss of hepatitis surface antigen (HBsAg), in response to therapy have improved clinical outcomes.

                Many agents, belonging to different classes of antiviral therapeutics (e. g. immunomodulators and HBV DNA polymerase inhibitors), have been evaluated for the treatment of chronic hepatitis B. Most have been found to be ineffective or too toxic at effective doses in vivo. Others are still under evaluation (Table 2). Six therapeutic agents have been approved by the FDA and are currently licensed as therapy in several countries worldwide: standard and pegylated interferon-alpha 2a, lamivudine, adefovir dipivoxil, entecavir and telbivudine (379).

Interferon-alpha (standard and pegylated)

                Interferon-alpha (IFN-alpha) belongs to a family of natural occurring proteins that have antiviral and immunomodulatory actions. They bind to cellular receptors and activate secondary messengers so as to initiate production of multiple proteins critical to the defense of the cell against viruses (141). The antiviral effects include degradation of viral mRNA, inhibition of viral protein synthesis, and prevention of the viral infection of cells. The immunomodulating effects include enhancement of foreign antigen presentation by HLA I and II to the immune system, activation of natural killer (NK) cells and other immune cells, and increased cytokine production. IFN-alpha is obtained by two main methods. It can be either purified from human lymphoblastoid cells, which have been stimulated by Sendai virus or produced using recombinant molecular biology in Esherichia coli. In the later case, the gene encoding for one of the human subtypes (beta-2a or beta-2b) of interferon is cloned into bacterial expression vector and used for the production.

Standard interferon

Efficacy: The efficacy of both lymphoblastoid and recombinant IFN-alpha for the treatment of chronic hepatitis B has been demonstrated in numerous placebo-controlled trials (2, 14, 27, 146, 217, 233, 268, 269, 284, 360). The dose and duration of IFN-alpha treatment varied in these studies. The dose of IFN-alpha ranged from 1 to 10 million units daily or three times weekly for 3 to 6 months. Patients were usually followed for 6 to 12 months after completion of interferon therapy. The best-designed studies were summarized in several meta-analysis (68). In average, a four-month course of treatment results in a 25-40% virological response with significant reduction of serum HBV DNA concentration, normalization of ALT level, and loss of HBeAg. Seroconversion to anti-HBe positively occurs in 19-34%. The data show that a high dose of IFN-alpha is more effective than a low dose, and the 3-to 4-month interferon treatment protocol is as effective as 6 months of therapy (362). In 60-70% of responders, approximately 2 months after the onset of treatment, a rise in ALT level is observed, which is thought to reflect the immune-mediated clearance of HBV-infected hepatocytes. This level returns to normal as HBV DNA or HBeAg is cleared. In nonresponders, such changes are not normally observed. The intensity of this are seldom aggravates the underlying liver status. Indeed, if there is a striking increase in ALT, a rise in bilirubin, or new signs or symptoms of hepatic decompensation, then the IFN-alpha therapy should be reduced or withheld and the patient should be closely followed.

Indications: Patients with chronic hepatitis who present an increased serum ALT level for more than 6 months and have serologic evidence of active viral replication should be considered for interferon therapy provided they have no contraindications that exclude them from therapy (117). The main contraindications are summarized in Table 3.

Dosage: The recommended dose of IFN-alpha is a daily injection of 5 million international units (5 MU) or a thrice-weekly injection of 9-10 MU (329, 362), self-injected, subcutaneously for 16 weeks. A monitoring of a complete blood count, prothrombin time, total bilirubin, ALT, AST, HBsAg, anti-HBs, HBeAg, anti-HBe, and quantitative HBV DNA should be done at 2, 4, 8, 12, and 16 weeks.

Prognosis for interferon therapy and adverse effects are detailed below.

Pegylated interferon

                Standard, unpegylated interferons exhibit rapid onset of activity, short half-lives, and rapid renal clearance, when administrated subcutaneously in typical dosing regimens. Because unpegylated interferons display substantial peaks and troughs in their serum concentrations, they are ideal molecules to undergo pegylation modification (267). Pegylation is the attachment of a polyethylene glycol (PEG) moiety to a molecule to slow the renal elimination and improve tissue drug distribution, thereby enhancing clinical activity of the therapeutic agent (267, 376).

Efficacy: The efficacy of PEG IFNs has been recently assessed in the treatment of chronic hepatitis B. A first randomized controlled study of PEG IFN alpha 2a has been performed in patients with HbeAg-positive chronic hepatitis B (67). Treatment duration and follow-up were each 24 weeks. At the end of follow-up, treatment response defined by loss of HbeAg with serum HBV DNA level below 500 000 copies/mL with normal ALT was observed in 19-28% of patients receiving PEG IFN alpha 2a (at a dose of 90 µg, 180 µg or 271 µg per week) versus 12% of patients who received standard IFN 2a. This study does not prove the superiority of PEG IFN alpha 2a as compared with standard IFN 2a particularly since the dose of IFN 2a used was relatively low (4.5 million units, three times a week). However a retrospective analysis showed that the rates of response were higher with PEG IFN for the patients who were the most difficult to treat (with high HBV DNA level or low ALT levels) (187). Therefore this study strongly suggests that PEG IFN alpha 2a is more effective than standard IFN 2a for the treatment of chronic hepatitis B. Several large randomized controlled trial have clearly confirmed the efficacy of PEG IFN alpha 2a or 2b for the treatment of HBe Ag-positive or negative chronic hepatitis B (155, 187, 224).

Prognosis and indications for interferon therapy: Favorable prognostic indices for a successful outcome with standard or pegylated IFN-alpha therapy have been evaluated and are listed in Table 4. These indicators do not provide an absolute guarantee of success with interferon therapy, but suggest a higher likelihood of success and may assist in patient selection for interferon therapy. A low concentration of HBV DNA and a raised ALT level are the best indices of predicting a successful treatment outcome (59, 255, 329, 362). One study suggested that genotypes A and B infected patients responded better than those infected by genotypes D and C respectively, in terms of HBeAg seroconversion and HBsAg loss (155).

Dosage: The recommended prescription for PEG IFN alpha 2a is once-weekly subcutaneous administration at 180 µg for 48 weeks.

Adverse effects: Side effects associated with PEG IFN were comparable to those observed with standard interferon (67). They both have numerous adverse effects that tend to be dose dependent. Flu-like symptoms, fevers, rigors, fatigue, myalgia, arthralgia, and headaches are very common immediately after injection, but are more frequent with pegylated than with standard IFN. Acetaminophen (or nonsteroidal anti-inflammatory drugs) helps relieve these symptoms, and these early adverse effects tend to subside or improve after the first or second week of treatment. A reduction of the count of the platelets and/or white cells also often occurs and requires a modification of the dosage. A granulocyte count of < 7.5 × 108/L or a platelet count < 4 × 1010/L necessitates cessation of therapy. A reversible moderate alopecia can manifest as well as depression with insomnia or an inability to concentrate. Hypnotics and mild anti-depressants may be required. Weight loss, impotence and vitreous hemorrhages have also been noted to occur. About 50% of patients receiving IFN-alpha therapy for 16 weeks or longer develop antinuclear antibodies, smooth muscle antibodies, and thyroid antibodies. Autoimmune disorders such as thrombocytopenic purpura, hemolytic anemia, vasculitis, or type-1 diabetes can manifest. These usually resolve after the cessation of interferon therapy. However, about 3% of patients receiving IFN-alpha develop a permanent hypothyroid state requiring lifelong thyroid replacement therapy. In all cases, a close monitoring and supervision are necessary during treatment.

(Printable Version of Interferon-alpha for Hepatitis B Virus)

Hoa PT, Huy NT, et al. Randomized Controlled Study Investigating Viral Suppression and Serological Response Following Pre-S1/Pre-S2/S Vaccine Therapy Combined with Lamivudine Treatment in HBeAg-Positive Patients with Chronic Hepatitis B. Antimicrob Agents Chemother. 2009 Dec;53:5134-40.

Alternative Therapy: Nucleoside/Nucleotide Analogs

                Many nucleoside/ nucleotide analogues, which are inhibitors of the HBV DNA polymerase (reverse transcriptase), have been evaluated for the treatment of HBV infection. Since 1998 four of them are FDA approved and used in clinical practice: lamivudine FDA-approved in 1998, adefovir dipivoxil in 2002, entecavir in 2005, and telbivudine in 2006. Some, like the tenofovir, or emtricitabine are still under evaluation (Table 2). After the tragedy that occurred during the clinical evaluation of the fialuridine (FIAU), which caused the death of patients because of mitochondrial toxicity (70, 71, 200, 234), more vigilance was necessary. For instance, and despite promising results obtained in early phase I/II clinical trials, studies on famciclovir (penciclovir) and lobucavir have been stopped because of efficacy and toxicity (e. g. development of hepatocellular carcinomas in rats) problems respectively.

Lamivudine

Efficacy: The efficacy and safety of lamivudine for the treatment of chronic hepatitis B has been demonstrated in numerous placebo-controlled trials (86-88, 174, 175, 253). Lamivudine administration at a daily dose of 100mg is associated with a significant drop of HBV DNA (3-log decrease), HBeAg, and HBsAg in the serum of virtually all patients treated, including those who did not respond to IFN-alpha. During phase III placebo-controlled trials, including more than 600 patients with HBeAg and elevated transaminases (88, 175), a course of 12 months of lamivudine therapy resulted in i) decrease of HBV DNA to undetectable level in most of the patients, ii) anti-HBe seroconversion in 16 to 17 % of the patients compared with 4 to 6 % in the placebo groups, iii) a normalization of serum ALT level in 41 to 72% versus 7 to 24% in the placebo groups, and liver histology improvement in 52 to 56% of the patients versus 25% in the placebo group (Table 5).

                However, viral breakthrough, due to the apparition of a lamivudine-resistant variant (see below), was observed from the 6th month of therapy and became progressively more frequent until the end of 12 months therapy where it reached 14% to 32% of the patients (88, 175).

               The effect of extended lamivudine therapy was analyzed in subgroups of patients who were initially enrolled in the 12 months course of lamivudine (88, 175, 186, 198, 207). An increase in anti-HBe seroconversion rate was observed over time: 17-22% at 1 year, 27-29% at 2 years, and 40% at 3 years. This suggests that long-term lamivudine therapy is required in the majority of the patients to obtain anti-HBe seroconversion. However, the incidence of viral breakthrough increases as well with the length of the treatment (Figure 4), indicating that lamivudine should be used with caution (see "resistance of HBV to lamivudine" section).

               Lamivudine-induced anti-HBe seroconversion is usually durable provided that the treatment lasts at least 6 month after the onset of seroconversion. For instance, in an Asian study (309) it was shown that HBeAg seroconversion was not durable in endemic areas of HBV infection, with anti-HBe loss in 37.5% and 49.2% at 1 year and 2 years of follow-up respectively, and reappearance of HBeAg in 81% and elevation of serum ALT in 94% of these relapsers. However, in this Korean study, lamivudine was discontinued 2 to 4 months after HBeAg disappearance. As the duration of additional lamivudine therapy after HBeAg seroconversion was one independent predictive factor for post-treatment relapse, it is therefore suggested to maintain antiviral treatment for at least 6 months after anti-HBe seroconversion.

                Moreover, few studies showed that the risk of relapse after anti-HBe seroconversion is minimal when both serum HBV DNA level decreases below 104 copies/ml and no pre-core mutant is selected. This suggests that monitoring of antiviral therapy should rely on quantitative and sensitive assays for HBV DNA detection and mutant detection assays.

Prognosis For Lamivudine: Studies have shown that patients with pre-therapy ALT level higher than 5 times the normal had a much higher anti-HBe seroconversion rate up to 65% at 1 year (52, 290) and 80% at two years (207), suggesting that lamivudine is more effective in patients who have mounted an ongoing endogenous immune response to HBV. Among patients with level of ALT higher than two times the normal, anti-HBe seroconversion increases from 17-22% to 37.5-38% at year one, 27-29% to 42-47.5% at year two, and 40% to 65% at year three (52, 198, 207). Moreover anti-HBe seroconversion is sustained in approximately 75% of these patients. This also indicates that anti-HBe seroconversion is unlikely in patients with normal ALT level at baseline.

Indications: Patients with chronic hepatitis who present an increased serum ALT for more than 6 months and have serologic evidence of active viral replication, in particular high HBV DNA level which represents an unfavorable situation for IFN-alpha treatment, may be treated by lamivudine. Patient selection is not influenced by factors such age, ethnicity, hepatic function, prior IFN-alpha therapy, or the presence of pre-core mutant HBV (87, 175, 291, 325, 367). Lamivudine is also effective at suppressing HBV replication both before and after liver transplantation (121, 271, 272), and provides clinical benefit to those with liver decompensation (312, 341, 345).

Dosage: The recommended dose of lamivudine is 100 mg per day. From the results of clinical trials, mathematical models were applied to determine the kinetics of viral clearance. These studies demonstrated that long-term lamivudine treatment is required to control or eradicate HBV infection (256). The antiviral treatment should be maintain for at least 6 months after anti-HBe seroconversion to prevent relapse. Then, this places the patient at risk of developing resistance to lamivudine by the selection of polymerase mutants that are resistant to this nucleoside analog (386). It remains therefore to determine the risk-benefit for those patients who failed to seroconvert and require long-term treatment.

Adverse Effects: Lamivudine is well tolerated in most patients (291). Its safety has been established in four international, multicentre, controlled phase III trials that included 967 patients among whose 558 received lamivudine at 100 mg/day (175, 290). In these studies, patient age, sex, presumed route of infection, and the presence or absence of cirrhosis had no influence on the incidence of adverse reactions. Adverse effects were mild and included headache, nausea, gastrointestinal upset, musculoskeletal discomfort fatigue, and slight increase of serum amylase. Moreover, liver enzyme elevations and heamatological toxicity did not differ significantly in patients receiving lamivudine or placebo. In contrast to IFN-alpha, lamivudine was associated with only modest, asymptomatic, and transient ALT elevations during treatment. In lamivudine treated patients, 20% of patients with an ALT elevation exhibited HBeAg seroconversion, in contrast to 10% of placebo-treated patients, indicating that in these cases, ALT are likely was the reflect of the anti-HBV immune response. No increase in clinically significant post-treatment ALT exacerbation was noted in patients receiving lamivudine by comparison with the other treatment groups. Very importantly, no mitochondrial toxicity was observed in prolonged use (144).

Resistance of HBV to Lamivudine: Development of mutants resistant to effective antiviral agent is a significant concern. As previously observed for HIV and anticipated in in vitro studies, HBV develops resistance to lamivudine. Results of clinical trials and cohort studies have shown that approximately 20% of patients develop lamivudine resistance each year ; a plateau of 70% of patients with resistance is reached after 4 years of therapy (176, 216, 383) (Figure 4), suggesting that approximately 30% of patients present a long term response to lamivudine. The main polymerase gene mutations conferring resistance to lamivudine have been characterized by in vitro phenotypic studies. Main lamivudine resistance mutants harbor a M204V or I substitution in the YMDD motif of the C domain of the reverse transcriptase domain (3) (Figure 3). It has been hypothesized that these mutations affect lamivudine triphosphate efficacy by a mechanism of steric hindrance in the cataclytic site of the viral enzyme (16, 74). As these substitutions affect the enzymatic activity and the replication capacity, compensatory mutations restoring some levels of replication capacity have been described, i.e. V173L and L180M (3, 81). After lamivudine cessation the wild-type virus tends to re-emerge (290). Recently, lamivudine failure was also associated to the emergence of A181V and A181T mutations in reverse transcriptase (114, 368)

                The management of patients with YMDD variants is challenging, but some nucleoside analogues proved their efficay on lamivudine-resistant variants. The susceptibility of lamivudine-resistant HBV to eleven nucleoside analogues in various stages of clinical development was assessed (104, 259). The results of these studies indicate that lamivudine-resistant HBV remain sensitive to acyclic phosphonate nucleotides (adefovir, tenofovir, and alamifovir), have reduced susceptibility to entecavir, and have high-level cross-resistance to all L-nucleosides tested including emtricitabine, telbivudine, clevudine, and torcitabine. These findings are of clinical relevance for the management of antiviral treatment failure in patients and the future design of new combination therapy trials.

               Indeed, clinical relapse may occur in individuals who develop a significant population of YMDD variants during treatment and may be associated with a deterioration of liver disease (205). However, in most patients with YMDD variants serum ALT and HBV DNA remain below baseline values initially when lamivudine therapy is continued. In these patient the likelihood of the progression of liver disease is reduced on the shirt term (366). Moreover, in the Liaw et al . (205) study, it was observed that 27% of these YMDD-variant-carrier patients achieved anti-HBe seroconversion. Recently long term data showed that lamivudine resistance is associated with the progression of liver disease (208). These data suggest that antiviral therapy should be modified as soon as viral breakthrough is detected, to prevent disease progression (182, 379).

                Lamivudine-resistant mutants occur more frequently and rapidly in patients receiving lamivudine to suppress HBV in liver transplantation than in those receiving lamivudine for chronic HBV.

Adefovir Dipivoxil

Efficacy: Adefovir dipivoxil (bis-POM PMEA) is broad-spectrum antiviral nucleoside analogue that is active against HBV and HIV, by acting as chain terminator. A large randomized, placebo-controlled study enrolled 515 Hbe Ag-positive patients randomized to receive adefovir dipivoxil 10 mg daily (n=172), adefovir 30 mg daily (n=173), or placebo (n=170) for 48 weeks (223). There was a rapid decrease in the median serum HBV DNA level in patients treated with adefovir dipivoxil, with statistically significant differences with placebo (Table 6). At week 48, the median change from baseline in serum HBV DNA was -3.5 log10 for adefovir (10 mg) compared with -0.5 log10 copies/mL for placebo. Significantly more patients had undetectable serum HBV DNA levels (below 400 copies/mL) (21% vs 0%), (P<0.001). HbeAg seroconversion was observed in 12% of the patients receiving adefovir 10 mg compared with 6% in the placebo group (p<0.05). ALT normalization was achieved in 48% compared with 16% (p<0.001). A significantly higher proportion of patients receiving adefovir 10 mg showed improvement in liver histology at week 48 compared with those receiving placebo (53% versus 25%; p<0.001). A recent study on 480 chineese subjects confirmed the safety and effectiveness of adefovir to treat HBeAg positive CHB patients. This study is continuing for an additional 4 years (375). These results were confirmed in HBe Ag-negative patients during a 240 weeks extended period, with an improvement of liver histology which was correlated with the control of viral load (129-131).

                The efficacy and safety of adding adefovir to lamivudine in patients with YMDD mutants were also tested. Clinical trials to date show that virological and biochemical improvement are observed (273) (Table 6), and that early add-on therapy is recommended in patients with lamivudine resistance. Moreover, virological breakthrough was observed in 21% of patients treated with adefovir monotherapy during 18 months following lamivudine failure against 0% of patients treated with lamivudine+adefovir therapy (277).

Dosage: The decrease of Adefovir dipivoxil tolerability at a dose of 30 mg in some patients, led to choosing the 10 mg dose as the best dose with regard to the benefit/risk ratio, and the 10 mg dose is the dose registered (223). The durability of the response after withdrawal of treatment is now becoming known. Of 60 patients undergoing HBe sero-conversion on adefovir, 91% were still HBe antigen negative after one year of follow-up (10). All the patients who relapsed were genotype C. However, cessation of therapy in some patients without HBe seroconversion or HBe negative patients was associated with relapse (129). Therefore, maintenance therapy is recommended. Preliminary results suggest that the antiviral effect is maintained and the rates of virological response with HBe seroconversion increases with the duration of therapy. However, a longer follow-up is planned (up to 5 years) in order to answer the question of the durability of the response under treatment and of the possible increased efficacy of long-term treatment.

Adverse Effects: The tolerability and safety profile of adefovir at the dose of 10 mg was similar to that of the placebo. Adefovir at the dose of 30 mg was associated with increase in creatinine level in some patients. This increase was moderate, occured after 24 weeks of treatment, and resolved in all cases after withdrawal of the drug.

Resistance of HBV to adefovir: Genotypic resistance to adefovir dipivoxil in phase III trials shows a slower rate than lamivudine resistance, starting during the second year of therapy (129) to reach 29% after 5 years of administration in patients treated for HBeAg negative chronic hepatitis B (127) (Figure 4). Adefovir resistance mutants harbor a N236T and/or A181V/T amino acid substitution in the D and B domains of the viral polymerase respectively (7, 195, 346).

Entecavir

Efficacy: Entecavir has been evaluated in several clinical trials including patients with HBeAg positive or HBeAg negative chronic hepatitis B patients, patients with lamivudine failure, HIV-HBV coinfected patients, and liver transplanted patients. More than 1600 patients were enrolled in phase III trials whose results led to the US FDA approval for the treatment of adult patients with chronic hepatitis B with evidence of viral replication and either evidence of persistent elevation of serum ALT levels or histologically active liver disease. In 1 randomized study involving nucleoside naive patients (HBeAg positive), entecavir administered 0.5 mg orally once daily for 52 weeks was superior to lamivudine (100 mg orally once daily for 52 weeks) on the primary efficacy endpoint of histological improvement (72% vs 62%) and on secondary endpoints, such as the reduction in viral load (-6.98 vs -5.46 log 10) and normalization of ALT (68% vs 60%) (45, 115) (Table 7). Results of entecavir administration for two years are also available in HBeAg positive patients with chronic hepatitis B. After 2 years of treatment, 81% of patients receiving entecavir had a viral load below 300 copies / mL versus only 39% of patients receiving lamivudine, 31% seronconverted to anti-HBe versus 26% in the lamivudine group, and 5% showed a clearance of HBsAg versus 3% in lamivudine treated patients (115). These results were also confirmed in HBe Ag-negative patients with chronic hepatitis (179). As in vitro studies had shown a partial cross-resistance of entecavir with lamivudine resistance mutations (199, 326), a randomized dose-ranging phase II study evaluating the efficacy of the switch to entecavir was performed in 182 lamivudine resistant patients (46). Entecavir administered at 1 mg once daily induced a significant viral load reduction and histological improvement, by comparison with the control group treated with lamivudine (46) (Table 7). Recently, another phase III, double-blind trial was performed on HBeAg positive patients who were refractory to lamivudine therapy (persistent viremia or documented YMDD mutations while receiving lamivudine). One hundred and forty one patients were switched to entecavir 1mg daily and 145 continue lamivudine 100mg daily for a minimum of 52 weeks. Histologic improvement occured in 55% of entecavir-treated vs 28% of lamivudine-treated patients. Mean change from baseline in HBV DNA was -5.11 log10 copies/mL for entecavir-treated patients and -0.48 log10 copies/mL for lamivudine-treated patients (306). Moreover, the results of several in vitro studies showed that entecavir inhibits the replication of adefovir resistant strains. These studies were performed with either cloned HBV genomes from clinical strains (346), or HBV genomes harboring a single point mutation introduced by site directed mutagenesis (28), and showed that the N236T polymerase mutant is as sensitive to entecavir as wild-type HBV. In agreement with these in vitro findings, a clinical report of eight patients found to have adefovir resistant HBV polymerase gene mutations showed that in two patients who received entecavir as a rescue therapy a decline of viral load by 4.3 and 5.5 log 10 copies/mL was observed (105).

Woo G, et al. Tenofovir and Entecavir are the Most Effective Antiviral Agents for Chronic Hepatitis B: a Systematic Review and Bayesian Meta-Analyses. Gastroenterology. 2010 Oct;139:1218-29.

Hsin-Yun Sun, M.D.  Entecavir Should Not Be Used for HBV Patients Co-Infected with HIV. 2008

Fung J et al. Entecavir Monotherapy is Effective in Suppressing Hepatitis B Virus After Liver Transplantation. Gastroenterology. 2011 Jul: [Epub ahead of print].

Dosage: The recommended dose of entecavir is 0.5 mg once daily for nucleoside-naive CHB patients and 1.0 mg once daily in patients with lamivudine resistance or persistent HBV viremia during lamivudine therapy. As with the other nucleoside analogues, the suppression of HBV DNA is not durable after discontinuation of entecavir.

Resistance of HBV to entecavir: The phase II study performed on the 182 lamivudine resistant patients revealed that known entecavir resistance mutations were noted in 6 patients at baseline and emerged in two patients during entecavir therapy while only one patient experienced a virological rebound during the study period. In recent and larger phase III trial of entecavir for lamivudine failure, the emergence of entecavir resistance was observed in 10% patients after 2 years of therapy (64). The molecular characterization of entecavir resistance mutations showed that (1) lamivudine resistance mutations confer some level of cross-resistance to entecavir; (2) additional mutations are required for entecavir resistance; (3) the latter are not sufficient to confer entecavir resistance when the lamivudine resistance mutations are absent (326). Several patterns of mutations were shown to confer entecavir resistance on a genetic background of lamivudine resistance: (1) substitutions I169T and M250V, (2) substitutions T184G and S202I (326), or (3) S202G (347) (Figure 3). In lamivudine naïve patients, the rate of resistance to entecavir seems to be low after 2 years of treatment (63).

Telbivudine

Efficacy: The safety, antiviral activity, and pharmacokinetics of telbivudine have been assessed in 43 adults with hepatitis B e antigen-positive chronic hepatitis B (178). This placebo-controlled dose-escalation trial investigated 6 telbivudine daily dosing levels (25, 50, 100, 200, 400, and 800 mg/d); treatment was given for 4 weeks. Telbivudine was well tolerated at all dosing levels, with no dose-related or treatment-related clinical or laboratory adverse events. Antiviral activity was dose-dependent, with a maximum at telbivudine doses of 400 mg/d or more. In the 800 mg/d cohort, the mean HBV DNA reduction was 3.75 log10 copies/mL at week 4, comprising a 99.98% reduction in serum viral load. Subsequently, large phase III studies have shown the superority of telbivudine compared to lamivudine in the suppression of viral load (by 6.5 log10 versus 5.5 log10) and improvement of liver histology (177).

Resistance of HBV to telbivudine: Telbivudine resistance was observed in approximately 5% of patients after one year of therapy and associated with a M204I mutation in the viral polymerase (177).

Emtricitabine

Efficacy: In a randomized, double blind study, 98 asian patients (77 HBeAg-positive and 21 HBeAg-negative) were randomized to receive 25, 100 or 200 mg of emtricitabine daily for 48 weeks (197). At 48 weeks, the median decreases in viral load were 2.6 log10, 3.1 log10 and 2.9 log10 copies/mL for the 3 doses respectively. The proportions of patients with undetectable HBV DNA (below 4700 copies/mL) were 38%, 42% and 61% for the 3 doses respectively. HBeAg loss was observed in a high proportion (40%) of the HBeAg positive patients (ranging from 32 to 50% depending on the dose). The results of this study suggest that the optimal dose of emtricitabine is 200 mg once daily. Patients were then treated with open-label 200mg emtricitabine for an additional year at the end of the randomized phase (116). After two years, 53% of the patients had serum HBV DNA below 4,700 copies/ml, 33% seroconverted to anti-HBe and 85% had normal ALT. However, the role of emtricitabine as a monotherapy may be limited by its structural similarity to lamivudine with the risk of development of drug resistance.

Dosage: Emtricitabine was recently licensed for the treatment of HIV-disease at a dose of 200 mg daily, which seems the effective dose also for HBV (116).

Resistance of HBV to emtricitabine: Genotypic analysis performed at week 8 of the previously described phase II clinical trial showed that 12% of patients treated with 100 mg of emtricitabine and 6% of those treated with 200 mg developed drug-resistant HBV. However, although emtricitabine exhibits the same mutation-profile as lamivudine, after 48 weeks only 12.6% of the 165 patients enrolled in the emtricitabine arm of phase III clinical trial developed YMDD mutation, and 18% at year 2 (116).

Tenofovir

Efficacy: In a pilot, nonrandomized study on 53 lamivudine-resistant HBV infected patients, tenofovir showed an earlier and stronger efficacy in suppressing HBV DNA than adefovir: at week 48, only 44% of the 35 adefovir patients had HBV DNA levels below 105 copies/mL in contrast to 100% of the 18 tenofovir-treated patients (337). The anti-HBV efficacy of tenofovir was confirmed in several other studies performed in HIV/HBV coinfected patients (22, 172).

Dosage: Tenofovir is a nucleotide analogue which inhibits both HIV and HBV replication at a dose of 300 mg given once daily. It is licensed for the treatment of HIV infection.

Resistance of HBV to tenofovir: No evidence of phenotypic viral resistance could be demonstrated in the tenofovir-treated patients in the long-term follow-up (up to 130 weeks), although a controversy has emerged for one mutation (80, 304).

Woo G, et al. Tenofovir and Entecavir are the Most Effective Antiviral Agents for Chronic Hepatitis B: a Systematic Review and Bayesian Meta-Analyses. Gastroenterology. 2010 Oct;139:1218-29.

Marcellin P, et al.  Tenofovir disoproxil fumarate versus aadefovir dipivoxil for chronic hepatitis B.  NEJM 2008;359:2442-2455.

Combinations of Antiviral Agents (Table 8)

                Monotherapy is not sufficient to eradicate HBV in the majority of patients who are chronically infected, as observed during clinical trials or in clinical practice. Combined chemotherapy, including concurrent or sequential treatment using more than one drug, provides in theory several advantages over monotherapy; this has been recently demonstrated in practice for HIV chemotherapy and for HBV (214, 303). Several types of combination have been or are being tested in clinical trials for the treatment of chronic hepatitis B.

Combination of IFN with lamivudine

               For historic reasons, the first combination to be tested was between IFN and lamivudine. Previous studies on the combination of IFN and lamivudine suggested that this combination could be more effective than lamivudine monotherapy (290). However, the results of different studies were discordant which could be due to different treatment regimens which could not be optimal.

               In a large randomized controlled study, 307 patients with HBeAg-positive chronic hepatitis B were randomized to receive either the combination of PEG IFN alpha 2b 100 µg per week for 52 weeks then 50 µg for 20 weeks and lamivudine 100 mg per day or PEG IFN alpha 2b at the same dose with placebo (155). At the end of the 26-week post treatment follow-up, there was no difference in response rates between the two treatment groups: serum HBV DNA was undetectable by PCR (below 400 copies per mL) in 7% and 9%; HBeAg seroconversion was both observed in 29%; normal ALT was obtained in 32% and 35%) in the PEG IFN monotherapy and the PEG IFN with lamivudine combination therapy groups. This study shows that in patients with HBeAg-positive chronic hepatitis B, 26 weeks after therapy, the combination of PEG IFN alpha 2b with lamivudine (with the simultaneous regimen used) is not superior to PEG IFN alpha 2b used in monotherapy. Main predictors of response were HBV genotype and pretreatment ALT level. Response was 34% for those with ALT levels under 3 times the upper limit of normal (3xULN) and 50% of those with ALT levels above 5xULN. Response was 60% for genotype A versus 42% for genotype B, 32% for genotype C and 28% for genotype D.

               Lau et al. (2005) recently reported the results of a phase III study evaluating pegylated IFN alpha 2a in 814 patients with HBeAg-positive chronic hepatitis B(187). They compared three treatment groups: pegylated IFN alpha 2a 180 mg once weekly plus placebo, pegylated IFN alpha 2a 180 mg plus lamivudine 100 mg daily, and lamivudine alone. The duration of therapy was 48 weeks, with a 24-week follow-up. After 24 weeks of follow-up, significantly more patients who received peginterferon alpha 2a monotherapy or peginterferon alpha 2a plus lamivudine than those who received lamivudine monotherapy had HBeAg seroconversion (32% vs. 19% (P<0.001) and 27% vs. 19% (P=0.02), respectively) or HBV DNA levels below 100 000 copies per mL (32% vs. 22% (P=0.01) and 34% vs. 22% (P=0.003), respectively). However, there was no benefit of combination therapy in comparison with peginterferon alpha 2a monotherapy. The same was true in the study of HBe Ag-negative patients (224)

Combination of Adefovir with Lamivudine in Lamivudine-Resistant Patients

               The concept of improving the efficacy by combining two analogues is based on the hypothesis that the combination would maximise the viral suppression and would decrease the occurence of viral resistance.

               One randomized study evaluated the efficacy of the combination of adefovir with lamivudine as compared to lamivudine alone or adefovir alone in 59 patients with HBeAg-positive chronic hepatitis B with lamivudine resistant HBV (273). In contrast to lamivudine monotherapy group, there was no significant difference in median serum HBV DNA reduction (-3.59 and -4.04log copies/mL), rates of ALT normalization (53% and 47%) and HBeAg loss (3 patients in each group) between the adefovir-lamivudine combination group and the adefovir monotherapy group. Noteworthy, serum HBV DNA level remained stable and there was no significant biochemical or serological change during the monotherapy.     

                Another study compared the efficacy of the combination of adefovir with lamivudine versus lamivudine used in monotherapy in 95 lamivudine-resistant (270). There was a significant difference in median serum HBV DNA reduction (-4.6 vs +0.30 log copies/mL), rates of undetectable HBV DNA with PCR (33% vs 4%) and HBeAg seroconversion (8% vs 2%) between the adefovir-lamivudine combination group and the lamivudine monotherapy group.

                A recent study showed the benefit of the long-term treatment with the combination of adefovir and lamivudine as compared to adefovir monotherapy in HBe-negative lamivudine resistant patients (277). Others studies also showed the benefit of early adefovir add-on with continuing lamivudine in patients with lamivudine resistance. These studies showed a benefit interest of limiting the risk of adefovir resistance and of controlling liver disease with the adefovir add-on strategy (106, 182, 277).

De Novo Combination Therapy

                The optimal antiviral regimen may combine drugs with a different mechanism of action on viral replication, lacking cross-resistance and exhibiting antiviral synergy. This theoretical type of combinations should reduce the risk of selection of drug resistant mutants because of the inhibition of viral replication and the selective pressure exerted on the different viral strains that compose the quasi-species. Up to now, combination of nucleoside analogues has not shown a synergistic antiviral effect in clinical trials, mainly because these antiviral agents inhibit a single target, i.e. the viral polymerase (see above). With the development and evaluation of newer drugs acting at different steps of the viral genome replication, combination relying on drugs with different mechanism of action can be evaluated using for instance drugs with potent anti-priming activity, drugs inhibiting viral minus strand DNA and others inhibiting plus strand DNA synthesis. The combination of such compounds was found to be either additive and more rarely synergistic in polymerase assays (reverse transcriptase activity) (300) as well as in tissue culture experiments (viral DNA synthesis) or in chronically infected woodchucks (166, 167). Although some of the combinations based on the inhibition of all three steps of viral genome replication inhibited more potently viral DNA synthesis than the single drugs, none were able to completely prevent the initial formation of viral ccc-DNA following de novo infection of hepatocytes, or to clear ccc-DNA once chronic infection of hepatocytes has been established in tissue culture (300). However, such combinations by inhibiting more potently viral DNA synthesis may delay the onset of viral resistance by limiting the chance of a given mutation to occur.

                A multitude of other combinations involving different immunomodulators and/or reverse transcriptase inhibitors has been tried in clinical setting (303). But so far, their benefit is not clearly established. The ideal combination will likely involve 2 or 3 drugs that have different mechanism of action (e. g. one or 2 inhibitor(s) of reverse transcriptase plus immunomodulator, or inhibitor(s) of RT plus inhibitor of morphogenesis). As new anti-HBV agents become available, the search for optimal combination of drugs without cross-resistance will continue (for review see Zoulim F. (381)).

(Printable Version of Alternative Therapy for Hepatitis B Virus)

Special Infections

Patients With Hepatitis B e Antigen ( HBeAg)-Negative Chronic Hepatitis B: This form of chronic hepatitis B is mostly associated to the presence of replication-competent pre-core mutants that, contrary to the wild-type HBV, are unable to produce HBeAg (37). These mutants are selected either spontaneously following natural anti-HBe seroconversion in one fourth to one third of patients (132) or during antiviral therapy. The natural history of HBeAg-negative chronic hepatitis B is characterized by a spontaneous fluctuation of ALT levels and HBV DNA load. Moreover, HBeAg-negative chronic hepatitis B usually has an aggressive course with rapid progression to cirrhosis and frequent development of hepatocellular carcinoma (262). Pre-core mutant chronic hepatitis B exists worldwide, but is particularly common in Mediterranean Europe and Asia where its represents a concern and a real therapeutic challenge.

                Interferon-alpha has been the treatment of choice for chronic hepatitis B during the last decade (145). Unfortunately, its efficacy in patients with HBeAg-negative chronic hepatitis is limited. Although, 40 to 90% of patients treated with IFN-alpha achieve remission at the end of 6 to 24 months therapy, the majority of them relapse soon after cessation of treatment (29, 180), a sustained virological and biochemical response being obtained in 20-25% of the cases and only with long treatment/re-treatment (222).

               Lamivudine administration has been evaluated in patients with HBeAg negative chronic hepatitis B in randomized trials and in cohort studies. Given at a dose of 100-150 mg daily for 52 weeks, lamivudine induces a marked suppression of serum HBV-DNA accompanied by normalization of ALT in approximately 80 % of the patients, and by liver histology improvement. However, with a few exceptions, treated patients do not clear HBsAg and are subject to disease reactivation after discontinuing therapy (325). Long-term therapy is therefore recommended. Unfortunately, prolonged lamivudine administration is hampered by the emergence of drug resistance. Long-term lamivudine studies have shown that after reaching a peak between 6 and 12 months of therapy, the response rate decreases because of virological breakthroughs associated with the emergence of lamivudine-resistant HBV mutants. In a study, the virological response diminished from 68% at month 12 and 24 to 52% and 41.6%, respectively at month 18 and 24 of therapy (128). Long term studies showed that the antiviral efficacy and histological improvement is progressively lost with time, as the prevalence of resistance mutations is increasing (281). After 3 to 4 years of therapy, the percentage of lamivudine resistance offsets the percentage of patients initially responding (33, 85, 261, 281). ALT levels increase progressively with the duration of infection with the YMDD mutants : no patient who developed lamivudine resistance mutation for 24 months had normal ALT levels (261, 280). In a retrospective nationwide analysis of lamivudine therapy in Italy, the development of clinically important events after virologic breakthroughs depended on the severity of the underlying liver disease; severe hepatitis flares at the emergence of YMDD were noted in patients with child B and C cirrhosis but not in patients with non-cirrhotic chronic hepatitis (85), in agreement with previous studies (128, 247). The rate of resistance observed in patients with HBeAg-negative chronic hepatitis was similar to that observed in HBe-positive chronic hepatitis (186, 261). As in HBeAg-positive patients, treatment by adefovir is generally effective in patients who develop lamivudine resistance.

                Adefovir dipivoxil administration given for 48 weeks in HBeAg negative patients (131) induced in 64% of patients an improvement of histologic liver abnormalities, compared with 33% of patients who received placebo (P<0.001) (Table 6). Serum HBV DNA levels were reduced to < 400 copies/mL in 51% of patients in the adefovir dipivoxil group (63 of 123) and in 0% in the placebo group (P<0.001). The median decrease in log-transformed HBV DNA levels was greater with adefovir dipivoxil treatment than with placebo (3.91 vs. 1.35 log copies/mL , P<0.001). ALT levels had normalized at week 48 in 72% of patients receiving adefovir dipivoxil (84 of 116), compared with 29% of those receiving placebo (17 of 59, P<0.001) (131). A longer duration study for 144 weeks showed a median decrease in serum HBV DNA of 3.47 log10 copies/ml at 96 weeks and 3.63 log10 copies/ml at week 144 (129). HBV DNA was below 1000 copies/ml in 71% and 79% patients after 96 and 144 weeks respectively. Interestingly, in the majority of patients who were switched from adefovir to placebo, the benefit of treatment was lost, indicating that antiviral therapy with nucleoside analogues has to be prolonged in this patient population to avoid viral reactivation and ALT flares. Resistance mutations rtN236T and rtA181V were identified in 3% and 5.9% of patients after 96 and 144 weeks respectively. Side effects after 144 weeks were similar to those observed at week 48. Recent studies showed the clinical response after 5 years of therapy : 70% of patients had a suppression of viral load below the limit of detection of PCR assay, which was accompanied by ALT normalization and histology improvement. Development of adefovir resistant mutations was observed in 29% of patients (127).

                Entecavir administration in 1 randomized study involving nucleoside naive patients (HBeAg negative), was superior to lamivudine (100 mg orally once daily for 52 weeks) on the primary efficacy endpoint of histological improvement (70% vs 61%) and on secondary endpoints, such as the reduction in viral load (-5 vs -4.5 log 10) and normalization of ALT (78% vs 71%) (179).

                Combination of pegIFN alpha 2a with lamivudine : A study evaluated the efficacy of a combination of pegIFN alpha 2a with lamivudine, in comparison with pegIFN alone and lamivudine alone (224). Treatment was administered for 48 weeks and end points were analyzed 24 weeks post-treatment. During therapy, there was a benefit in the combination by comparison with the single treatment in terms of viral load decline. The rate of lamivudine resistance was lower in patients who received the combination of lamivudine with pegIFN by comparison with lamivudine monotherapy. However, 24 weeks post-therapy, there was no difference in the rate of ALT normalization (approximately 60%) or virologic response (approximately 20% patients) between the groups who received pegIFN alone or in combination with lamivudine. The two groups of patients who received pegIFN had a better response rate 24 weeks post-therapy compared to the group who received lamivudine alone. In view of the fluctuating nature of HBeAg-negative disease, long-term follow-up studies are necessary to determine whether the response is indeed sustained.

Decompensated Cirrhosis: The prognosis of decompensated liver cirrhosis resulting from chronic hepatitis B is poor. Antiviral intervention has been evaluated in terms of risk-benefit with interferon-alpha and nucleoside analogues. Standard IFN-alpha regimens can be dangerous in this group of patients. It is thought that the immune lysis of infected hepatocytes, which follows successful IFN-alpha therapy, can cause a reduction in the functioning cell mass of the patient with end-stage disease, and result in fatal exacerbation of disease activity. Therefore, treatment with IFN-alpha , if chosen, must be performed only with a careful clinical monitoring. The starting dose of 0.5 million units 3 times weekly should be increases slowly according to patient's tolerance.

                The efficacy of lamivudine has been evaluated in several studies patients with decompensated cirrhosis. In the first study (345), 7 patients out of 33 underwent liver transplantation within 6 months after treatment initiation, 5 patients died, and 23 patients were treated for more than 6 months. In these latter patients, serum bilirubin decreased significantly, albumin levels increased and Child-Pugh score decreased significantly. Liver function improvement was slow but concomitant with the control of viral replication. Other studies including 13 and 18 patients respectively (158, 367), confirmed that lamivudine therapy appears highly effective in controlling viral replication and in improving liver functions in this serious clinical situation. In the last study, thirty patients with HBV-related decompensated and active viral replication were treated with lamivudine 100 mg daily for a median duration of 9 months. Among these patients, five patients died within 3 months. Two patients were lost to follow-up at week 8 and 9. One patient was treated for <6 months. Twenty-two patients were treated over 6 months. Univariate analysis revealed that the total bilirubin (P = 0.008), prothrombin time (P = 0.004), Child-Turcotte-Pugh score (P = 0.005), the model of efd-stage liver disease score (P = 0.004) and stage III hepatic encephalopathy (P = 0.001) were predictive factors of early mortality. Multivariate analysis revealed that the independent factor associated with early mortality was stage III encephalopathy. Among 22 patients, liver function improved markedly after lamivudine therapy. Of the nine hepatitis B e antigen (HBeAg)-positive patients, three had HBeAg seroconversion. Two patients had YMDD mutant and virological breakthrough at 41 and 46 weeks. One of the two had hepatocellular carcinoma and died of hepatic failure at week 125; the other received adefovir and is doing well. Lamivudine appeared to have benefits in viral suppression and significant improvement in liver function in patients with HBV-related decompensated. As noted in prior studies, poor baseline liver function is associated with a poor prognosis in patients with decompensated HBV cirrhosis treated with lamivudine (334).

                So, lamivudine therapy is associated with rapid viral suppression, improvement in Child-Pugh scores, and improved survival, but drug resistance is a major problem and is associated directly with a poor clinical outcome. Adefovir or entecavir is preferred in patients with decompensated cirrhosis who require long duration of treatment, due to the lower rate of development of resistance.

                The safety and efficacy of switching to adefovir monotherapy was examined in compensated and decompensated patients with liver cirrhosis. The clinical, biochemical and virological responses were compared between adefovir monotherapy in 18 cirrhotic patients and adefovir add-on lamivudine therapy in 10 comparable cirrhotic patients with lamivudine-resistant rtM204 I/V (206). After switching to adefovir monotherapy, Child-Pugh's score, serum alanine aminotransferase (ALT), bilirubin, albumin and HBV DNA levels improved significantly (P < 0.01). Serum HBV DNA response, defined as HBV DNA decreased to below 105 copies/mL or > or =2 log10 reduction form baseline, was achieved in all patients. A transient ALT flare without concurrent changes in serum bilirubin or prothrombin time was observed in only two patients (11%). In this study, the efficacy and safety profile was similar to those with adefovir add-on lamivudine therapy, but recent studies showed that adefovir add-on lamivudine therapy was more beneficial to chronically infected patients with lamivudine-resistant mutants than adefovir switch (181).

Underlying Diseases

Co-infection HBV and HIV: About 10% of people, who are infected with HIV-1, are co-infected with HBV. These patients have higher risk of HBV-related cirrhosis than HIV-negative HBV-infected patients, and co-infection have been associated to decrease survival in affected individuals (61).

                Lamivudine, administered at 100 mg/day or 300 mg/day (co-infection), inhibits HBV replication in more than 80% of infected patients with or without HIV-1 co-infection (21, 23, 291). However, the same problem of selection of drug resistant mutants during prolonged therapy is encountered for co-infected patients. Lamivudine-resistant HBV is found in about 15-32% of co-infected patients after 1 year of lamivudine therapy. It is anticipated that these patients who are receiving highly active antiretroviral therapy (HAART) might be more susceptible to progressive liver disease.

                To counteract lamivudine resistant HBV infection in these patients the use of adefovir dipivoxil was tested. In a recent study, 35 patients receiving 150 mg/day of lamivudine as part of their current anti-HIV regimen were treated with 10 mg/day of adefovir dipivoxil for 144 weeks (24). The mean decrease of the serum HBV DNA was of 5.08, 4.25 and 3.86 logs at week 48, 96 and 144 respectively, suggesting a good efficacy of adefovir dipivoxil against lamivudine-resistant HBV. No adefovir-associated resistance mutations in HBV DNA polymerase or HIV-1 reverse transcriptase were detected. So, long-term treatment with adefovir dipivoxil for 144 weeks was well tolerated and resulted in significant and sustained reductions in HBV DNA and ALT in HIV/HBV co-infected patients. Efficacy increased with treatment duration, with no loss of viral suppression. In these HIV/HBV coinfected patients, the superior efficacy of tenofovir over adefovir was also shown or suggested by several studies (22, 172, 337). The data on entecavir use in HBV/HIV co-infected individuals are conflicting. While the drug was not thought to be active against HIV (152), a recent report has highlighted that it can produce 1 log reduction in plasma HIV-RNA and occasionally select for mutation M184V in HIV (143, 237). However, another report has not confirmed this observation in an individual with HIV/HBV/HDV coinfection, in whom ETV did not cause any significant change in plasma HIV-RNA nor selected for the M184V mutation in HIV after more than 24 weeks of treatment (311). Therefore, further research is needed to elucidate the potential clinical relevance of any anti-HIV activity of ETV at the doses currently approved for treating HBV. In the mean time, a warning from the FDA has alerted against the use of ETV in HIV-coinfected patients in the absence of antiretroviral therapy. This recommendation, however, has been made in the absence of information about potential interactions of ETV with antiretrovirals, and specially with abacavir, which is another guanosine analog and might be subject to inhibitory competition phenomena (15).

Hsin-Yun Sun, M.D.  Entecavir Should Not Be Used for HBV Patients Co-Infected with HIV. 2008

Review Article:  Hoffman C, Thio C. Clinical Implications of HIV and Hepatitis B Co-infection in Asia and Africa. The LANCET Infectious Diseases 2007; Vol.7, Issue 6, 402-409.

Co-infection HBV and HCV: Coinfection with HBV and hepatitis C virus is associated with a more severe liver disease (380), increased frequency in the development of hepatocellular carcinoma, and, in respect with therapy, resistance to standard regimen of IFN-gamma (58). IFN has been the most studied agent in treatment of coinfected patients because of the wealth of experience with this agent in viral hepatitis and its proven activity against both viruses. One of the first case reports of successfull treatment with IFN of a coinfected patient was published by Burt et al. in 1993 (32). Utili et al. (335) studied a cohort of 32 HBV/HCV coinfected patints, 16 of whom received IFN treatment (5MU TIW for 12 months). They report an overall sustained virological response rate of 43.8% for HCV infection, and this rate was increased in patients who were HBeAg negative (66.7%). Loss of HBeAg occured in 2 of 13 patients (15.4%). Villa et al. (344) conducted a larger prospective randomized trial of 30 patients with HBV/HCV coinfection (HBsAg-positive, Anti-HCV-positive, HCV RNA-positive), in which patients received either 6 or 9 MU alpha-interferon three times a week for 6 months. This study found that higher dose IFN was more effective in inducing clearance of HCV RNA (31.2 vs 0%, p=0.045), and HBV DNA (100% vs 0%), as well as inducing a sustained biological response (37.5% vs 0%, p=0.019) compared to the lower dose.

                Several groups have published studies addressing treatment of coinfected patients with antiviral combination therapy with IFNα (2a or 2b) plus ribavirin (57, 151, 211). Results demonstrated the effectiveness of combined IFN and ribavirin in coinfected patients, with a rate of sustained virological and biological response comparable to HCV monoinfected patients (57, 211). In these studies, viral interaction was evident, in that coinfected patients who achieved a sustained virological response were less likely to achieve HBV DNA clearance, and more likely to have reactivation of HBV or HBV flares. Of the patients who cleared HBV DNA, most did not achieved a sustained virological response with undetectable HCV RNA following combination therapy (57).

                One study of lamivudine therapy in addition to IFN for coinfected patients has been published by Marrone et al. (228), suggesting that the addition of lamivudine to IFN may be effective in these coinfected patients.

Co-infection HBV and HDV: Patients persistently infected with both HBV and HDV develop chronic hepatitis with progressive liver disease more severe than that with chronic HBV infection alone (36). Cirrhosis with liver failure occurs in 25%, and hepatocellular carcinoma in 35% of these patients. Interferon-alpha in high dose monotherapy for 1 year remains the only therapeutic option for this group of patients, as lamivudine monotherapy (185) and combination of IFN and lamivudine (361) do not show significant efficacy (94).

Liver Transplantation

                Liver transplantation is a treatment option for end stage disease. However cirrhosis associated with chronic HBV infection, is a controversial indication for the liver transplantation because of the very high risk of reinfection rate of the hepatic allograft and rapid progressive liver disease in the allograft. Indeed, in patients positive for HBeAg or HBV DNA, the incidence of reinfection is over 80% (286, 288). With HBV reinfection the 1-and 3-years survival rates are 68% and 44% respectively (289). These rates are significantly below those for patients transplanted for other liver diseases. The clinical course of HBV infection in the hepatic graft is accelerated, and liver failure can occur in a short period time. An unusual form of rapidly progressive liver injury characterized by very high levels of hepatitis B antigen expression in hepatocytes, with marked cholestasis and brosis of the transplanted liver develops in 10-20% of patients. This condition, known as fibrosing cholestatic hepatitis or cytolytic hepatitis, is virtually 100% fatal (188, 219); patients do not respond to IFN therapy, and retransplantation is rarely of benefit (69). For these reasons, many liver transplant centers did not consider until recently patients with HBV-induced cirrhosis with ongoing viral replication to be suitable candidates for transplantation. Several options are now available to prevent reinfection of hepatic allograft after liver transplantation.

                Hence, the advent of long-term hepatitis B immune globulins (HBIg) administration as a prophylaxis of HBV recurrence has been a major advance in the management of liver transplantation for HBV infection. HBV recurrence has been reduced from over 80% to 20-35% in HBV cirrhotic patients with low level viral replication pre-transplant, but remained high, 30-80% in HBV cirrhotic patients with high HBV replication (184, 287, 289). The drawbacks of HBIg administration are the cost of the long-term administration, the need for a close monitoring to adapt the frequency of readministration depending on the anti-HBs levels, and high recurrence rate in patients with high HBV replication levels. Patients receive 100 ml of HBIg intravenously during the ahepatic phase, and 5 ml of HBIg daily for 1 week after liver transplantation. During the initial few months posttransplantation, anti-HBs titers should be maintained in the 300 to 500 IU/ml range. With this high level of anti-HBs, HBsAg often disappears from the blood. HBIg treatment has to be continued indefinitely, and 5 ml of HBIg should be given whenever the anti-HBs titer declines to 100 IU/ ml. HBIg treatment adds substantial cost to the liver transplantation procedure. HBsAg mutants can develop during maintenance HBIg treatment (328), and occur more commonly with monoclonal HBIg immunoprophylaxis. A recent study by Galun et al (2002) showed promising results using a mixture of two monoclonal antobodies in a phase I clinical study (109). Patients developed a rapid and significant decrease in HBV-DNA levels. Future studies are warranted, but this preliminary data suggests that monoclonal antibody preparations could replace the current polyclonal HBIg.

                As lamivudine is well tolerated and controls viral replication in cirrhotic patients, attempts to prevent HBV recurrence were made using lamivudine monotherapy pre-and post-transplant (121). However, an overall recurrence rate of only 40% was observed in a recent multicenter study (272), suggesting that lamivudine used alone was only partially effective. Moreover, lamivudine resistant mutants arise in 20% of liver transplant patients and may be associated with acute liver graft failure (245, 246). Other studies with lamivudine monotherapy have also been disappointing with high rates of HBV recurrence ranging from 23-50% of patients (246, 343).

                Several groups have developed a rationale strategy that combines lamivudine monotherapy pre-transplantation to decrease viral load and lamivudine plus HBIg post-transplantation. In these studies the HBV recurrence rate was less than 10% one to two years following transplantation (8, 110, 227, 229, 297), and the need for HBIg was reduced most likely by a decreased production of HBsAg. These studies clearly indicate that the latter approach is more effective in preventive HBV recurrence, but the optimal duration of prophylaxis remains to determine. Furthermore, by controlling viral replication with lamivudine pre-transplantation, it is now possible to transplant those cirrhotic patients with high HBV replication who were previously removed from the waiting list. An interesting study has shown that in patients with HBIg prophylaxis alone and low HBV replication, the substitution of HBIg by lamivudine administration is equally effective at one year (249). The long-term outcome of these patients needs to be studied and the optimal protocol of prophylaxis needs to be determined before changing the currently validated protocols.

                  Due to the high number of patients who develop resistance to lamivudine, several large studies have shown the utility of adefovir in the pre and post liver transplantation setting. The largest study by Schiff et al. showed that among patients with lamivudine-resistant HBV and who were pre-OLT, 81% achieved undetectable serum HBV DNA. Furthermore, serum ALT, albumin, bilirubin and prothrombin time normalized in 76%, 81%, 50% and 83% of these pre-OLT patients respectively. Unlike the use of lamivudine, no resistance was identified after 48 weeks of therapy in this population (292). Another study describing the use of prophylactic adefovir was from Lo et al.. They describe 16 patients who had developed YMDD mutations while on the waiting list for OLT. Eleven patients received adefovir for a median of 20 days before transplantation while 5 patients started the drug at the time of OLT. Lo’s cohort was divided in half:eight patients received HBIg (in addition to adefovir and lamivudine) for a median of 24 months whereas the other 8 patients received prophylaxis with adefovir and lamivudine alone. All 16 patients cleared the HBV DNA and had no evidence of recurrence; furthermore, all remained HBeAg negative. The graft survival was 94% at a median follow-up of 21 months. Lo concludes that add-on adefovir plus lamivudine should be the “preferred approach in those patients who have already developed resistance to lamivudine, so as to avoid the emergence of multiresistant viral strains” (212). But it is expected that the development of adefovir mutations may become a problem in the post-transplant setting. Villeneuve et al. recently reported the first case of a patient who developed sequential selection of lamivudine and adefovir resistant strains of HBV in a liver transplantation patient (346).

                Tenofovir was also tested for the treatment of patients with HBV recurrence following liver transplantation. In 2004, Neff et al reported 16 patients who developed a resistance to lamivudine following OLT (252). Half of these patients received tenofovir at a dose of 300mg/day 1-66 months after the development of resistance. Therapy was continued for 14-26 months . All the patients experienced viral DNA suppression with 7 having undetectable viral loads.

               It is expected that entecavir will be also efficacious in the prevention and treatment of recurrent HBV following liver transplantation.

               In the recent years, the development of new nucleoside analogs has clearly improved the management of liver transplantation for HBV liver disease. The use of antivirals pre-transplantation, followed by the combination of antivirals and HB Ig post-transplantation allowed to decrease significantly the risk of HBV recurrence on the liver graft, and to decrease dose of HB Ig to maintain high levels of antibodies.

Review Article: Lok ASF, McMahon BJ. Chronic Hepatitis B: Update 2009.  AASLD Practice Guidelines. Hepatology 2009:50;1-36.

Review Article: Sorrell MF, et al.  National Institutes of Health Consensus Development Conference Statement: Management of Hepatitis B.  Ann Intern Med 2009;150(2):104-10.

(Printable Version of Alternative Therapy for Hepatitis B Virus Special Infections/Undeerlying Diseases)

ENDPOINTS FOR MONITORING THERAPY Guided Medline Search

                Patients with chronic HBV infection commonly have progressive liver disease and are at risk of developing cirrhosis or hepatocellular carcinoma. The primary goal of antiviral therapy for chronic HBV infection is elimination of virus from all host cells, and the ideal response to therapy would be loss of all HBV markers with the appearance of anti-HBs. This has proven to be difficult but not an impossible challenge. An alternative and pragmatic approach is be to control HBV replication and prevent progression of liver disease. Antiviral therapy may result in loss of HBV DNA and HBeAg and normalization of transaminases without clearance of HBsAg. HBV DNA can integrate into the host cell DNA and may continue HBsAg production without producing infectious virus. Integrated HBV DNA is not likely to be eliminated by antiviral therapy, and may be one of the main factors involved in hepatocellular carcinoma development despite successfull therapy.

VACCINES Guided Medline Search

Indications

Pre-exposure Vaccination to Prevent HBV Infection: As infants who are exposed to HBV infection are also exposed to a high rate of chronicity of HBV infection, hepatitis B vaccination is recommended for all infants. Vaccination should start during the newborn period with a usual schedule.

                Special efforts should be made to ensure a complete vaccination schedule during the first year of life in highly endemic areas. All children and adolescents not previously vaccinated should be vaccinated with the appropriate dose of vaccine according to age. This is especially important as HBV is very efficiently transmitted via sexual route.

                People belonging to high-risk groups should be vaccinated with the age appropriate vaccine dose and schedule. They are i) sexually active heterosexuals with multiple partners or past-history of sexually transmitted diseases, ii) homosexuals and bisexuals, iii) household contacts and sexual partners of HBsAg carriers v) intra-venous drug users, v) people with occupational risk of HBV infection (health care workers in any field, public safety workers) vi) patients and staff of institutions for the disabled vii) hemodialysis patients, viii) patients who receive blood derived products such as clotting factors, ix) adoptees from highly endemic countries, x) international travelers xi) long-term inmates. Most of these persons should be tested for HBsAg prior to vaccination and should have post-vaccination test for anti-HBs antibody to ensure efficient prophylaxis.

Post-Exposure Prophylaxis to Prevent HBV Infection: Prevention of perinatal infection is a major goal for public health, as the rate of transmission and of chronicity of viral infection in newborn from chronically infected mothers reaches 90%. All pregnant women should be tested for HBsAg during prenatal visit in each pregnancy. Pooled serum from patients who have recovered spontaneously from acute HBV and who have significant anti-HBs titers can be used for passive immunization. These hepatitis B immune globulins (HBIg) are administered simultaneously with HBV vaccine to newborn infants whose mothers are HBsAg positive (6, 342, 378). Administration of HBIg vaccine therapy is most effective if given within 12 hours of birth (342). They should receive the complete schedule of vaccination and should be tested for HBsAg and anti-HBs following the completion of vaccination at approximately one year of age.

                In populations in which the screening of pregnant women for HBsAg is not feasible, all infants should receive the first dose of vaccine during the first 12 hours of life followed by a second dose at 1 month, and a third at 6 months. In this setting the use of HBIg is not indicated.

                People who have been exposed (e. g. accidental percutaneous or permucosal exposure), or potentially exposed (e. g. suspect sexual intercourse) to HBV, should be first tested for HBsAg and anti-HBs markers. If the exposed person is not vaccinated, he should receive HBIg at 0.06 mL/kg intramuscularly together with the first dose of vaccine. If the exposed person was vaccinated and known to be a responder to the vaccination, one may consider a booster vaccination, but no HBIg administration. If he was known to be a non-responder to the vaccine, one may consider either the administration of HBIg at double dose or HBIg plus vaccination.

Dose and Schedule

                The currently used HBV vaccines consist of the small envelope (S) protein, and the middle pre-S2 envelope (M) protein in European vaccines, assembled into 22 nm particles. Both the S and M proteins contain the HBsAg which consists of the common “a” determinant and several subtype determinants (GenHevac-B vaccine, Aventis-Pasteur). S vaccines are produced by processing of HBsAg purified from plasma of HBV carriers as well as from yeast cells (Saccharomyces cerevisiae) expressing recombinant DNA (Engerix-B, GSK and Recombivax-HB VAXII, Merck). The M/S vaccine is a recombinant vaccine prepared by expression in CHO cells.

                These vaccines are administered by the intramuscular route in the deltoid and are highly immunogenic, inducing a protective anti-HBs antibody titer (> 10 IUper ml) in more than 95% of healthy children or young adults (4, 189, 340). Two schedules of administration are approved: 1) 3 initial injections at one month intervals and a booster at month 12; 2) 2 initial injections one month apart followed by a booster at month 6 (Table 9). About 5% of those vaccinated fail to respond with development of antibodies to the vaccine. Several factors of non-response have been identified, including genetically determined non-responsiveness, age over 40 years, high body mass index, and immuno-suppression (4, 189, 340). The HBV vaccine is associated with rare side effects, most commonly pain or soreness at the injection site. Neurological disorders such as multiple sclerosis, Guillain-Barré syndrome, and transverse myelitis have not been causally linked to the HBV vaccine (9, 66).

                The efficacy of protection against HBV infection has been proven in large clinical studies of exposed populations such as homosexual men, healthcare workers, and infants born to HBsAg positive mothers (4, 189, 340). In these clinical studies, the protection was evident when anti-HBs Ab titers were above 10 IUper ml. Several years after vaccination, the anti-HBs Ab titer may decline to undetectable level but immunity against clinical disease persists for years, suggesting the existence of an immunological memory (4, 189, 340). Based on these results, a booster is not recommended in healthy individuals who are not exposed to a high risk of HBV infection. Protective levels are obtained in 95% of infants born from negative mothers, adolescents and adults (Table 10). In newborns from chronically infected mothers, the effectiveness of hepatitis B prophylaxis exceeds 75%. Immunity following vaccination is lasting and there is no recommendation by W. H. O. for routine booster revaccination. Indeed specific anti-HBs immunity persists based on immunologic memoric and capability for rapid anamnestic anti-envelope response, even when anti-HBs have disappeared long after vaccination.

               Vaccine escape mutants have been reported in follow-up studies of individuals receiving HBsAg vaccines composed of the S protein. Vaccine escape mutants can differ from wild-type by only a single nucleotide change in the “a” determinant. Vaccination probably does not totally block infection of the liver following exposure. Rather, spread of virus from infected hepatocytes is probably restricted by antibodies until the cellular immune response can destroy the infected cells. In immunocompetent individuals, spread of the rare vaccine escape variants in an inoculum would presumably be controlled by the cellular immune response as well as by antibodies to the altered S protein. Therefore, vaccine escape is still rare, most typically seen in patients who, if not vaccinated, would have been at high risk of developing chronic infection after exposure to HBV; that is, young children, or immunocompromised adults. It is rarer in healthy adults presumably because they are at low risk of developing chronic infection in any case. Vaccine escape mutants may fail to be detected by some diagnostic tests for HBsAg, the most common assay for an ongoing HBV infection (60). Most studies of vaccine escape have involved at risk children and infants who were either vaccinated or received vaccine plus HBIg, strongly reactive to the “a” determinant of HBsAg. During the 1980s, a follow-up study of childhood vaccination in Italy revealed vaccinees who became HBsAg positive, despite the presence of a strong antibody response to HBsAg. The incidence was rare, involving about 2% of the children of HBsAg positive mothers, or with HBsAg positive family contacts (38, 374). More detailed analysis of virus from one patient revealed a point mutation encoding a gly145arg substitution at amino acid 145 of the S protein; that is, within the “a” determinant (38). Analysis with monoclonal antibodies to the “a” determinant of wild-type HBV revealed a loss of binding to the mutant HBsAg (38, 355). Subsequent studies in other populations with high HBV endemicity confirmed the rare occurrence of vaccine escape mutants in children, including the glyc145arg substitution in “a”, other substitutions or insertions in “a”, and mutations within the external hydrophilic domain but outside of “a” (39, 102, 148, 150, 159, 192, 258, 364). “Vaccine escape” variants have also been detected in unvaccinated children and adults with circulating virus as well as anti-HBsAg (148, 364), possibly reflecting past or concurrent infection with wild-type and mutant virus. Approaches to deal with vaccine escape variants will be needed as individuals in whom these variants are predominant become an increasing fraction of the pool of HBV carriers (148).

PREVENTION OR INFECTION CONTROL MEASURES Guided Medline Search Smart search

                Even though a vaccine for hepatitis B has been available since the early 1980's, and WHO has made recommendation for large-scale vaccination program, there is still a need to generalize these programs to eradicate this ubiquitous infection. It was clearly shown that the initial vaccination program targeting only high-risk groups failed to affect the overall HBV carrier rate. As the risk of chronicity of viral infection is higher in young children and the efficacy of the hepatitis B vaccine is usually greater in children, it was therefore recommended by W. H. O. to target the population of newborns and children for mass vaccination.

                In Taiwan, a large-scale vaccination of all newborns regardless of the status of their mother, started in 1985 (42). In 10 years, this was program was able to decrease the prevalence of HBsAg carriage from 8% to 0.8% in Taiwanese children. Moreover, this was associated with the decrease in hepatocellular carcinoma incidence in children while the incidence of other cancers remained stable over time. The results of this study clearly demonstrated the benefit of HBV vaccination at the scale of a country.

                In Italy, a campaign for hepatitis B immunization of children and teenagers started in 1991. Its impact was determined by the incidence of hepatitis B. It was shown that the hepatitis B incidence declined from 10.4/100000 in 1987 to 2.9/100000 in 1997. The drop in hepatitis B incidence was even more significant after the introduction of mass vaccination (318).

                The results of these different studies confirm the prediction that universal immunization of the total world population could bring hepatitis B to a clinically non-relevant disease within a predictable time frame. Different models have also confirmed the cost-effectiveness of universal infant immunization against hepatitis B.

                Beside large-scale vaccination programs, infection control measures should include the implementation of routine screening for blood donors, pregnant women, health-care workers and people migrating from high endemic areas, and should rely on general population counseling to decrease exposure to HBV. Both non-carriers and HBV-carriers should be widely informed about the disease and its transmissions and therefore should be expected to have a responsible behavior (e. g. use of condoms) in respect with the spread of the infection. Finally, close contacts of HBV-carrier should be tested for HBV infection. If they have not developed HBV infection or immunity to HBV, they should receive HBIg and/or HBV vaccine.

Thompson ND, et al. Nonhospital Health Care-associated Hepatitis B and C Virus Transmission: United States, 1998-2008. Ann Intern Med. 2009. Jan 6;150(1):33-9.

TABLES AND FIGURES

TABLE 1. Natural History and Interpretation of Hepatitis B Markers

TABLE 2. Hepatitis B Therapies and Vaccines in Development or FDA-approved

TABLE 3. Contraindications for Interferon-α Therapy for Chronic Hepatitis B

TABLE 4. Factors Predictive of Responsiveness/ Nonresponsisveness of Chronic HBV Infection to IFN-α Treatment

TABLE 5. Biochemical and Virological Response During Lamivudine Therapy of Patients with Wild Type HBV Infection

TABLE 6. Biochemical and Virological Response During Adefovir Therapy of Patients with HBV Infection

TABLE 7. Biochemical and Virological Response During Entecavir Therapy of Patients with HBV Infection

TABLE 8. Biochemical and Virological Response During Combination Therapy of Patients with HBV Infection

TABLE 9. Dosage and Administration Schedules for Pre/Post Exposure HBV-Vaccination

Table 10. Performance of Hepatitis B Vaccine

Figure 1. HBV Genome and Main Mutants

Figure 2. The Virus Life Cycle and Its Inhibitors

Figure 3. Polymerase Domains and Main Antiviral Resistance Mutations

Figure 4. Emergence and Evolution of Antiviral Resistant Mutations

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