The hepatitis B virus JONATHAN L. BROWN WILLIAM F. CARMAN HOWARD C. THOMAS INTRODUCTION
On a global scale, the hepatitis B virus (HBV) is the most significant of the hepatotropic viruses in terms of the number of people chronically infected and the severity of the complications of infection. It has been estimated that 40% of chronic HBV carriers will die as a consequence of their infection. In this chapter we aim to provide an overview of the current understanding of the epidemiology and biology of HBV, and the pathology, prevention and treatment of disease. EPIDEMIOLOGY Developing world
Most chronic carriers live in the developing world and have acquired the infection from their mothers at birth or from other close contacts during early childhood. The age of acquisition is different in Africa and Asia (Botha et al, 1984). In the former, children become infected after birth, probably through spread of secretions, including breast milk, saliva and serous exudates, by passive vectors such as bedbugs and rituals such as scarification. In Asia, infants usually become infected at birth, especially if the mother is hepatitis B e antigen (HBeAg) positive, or very early in life. The child is at particular risk if the mother has had acute hepatitis during the last 6 months of pregnancy or the first 2 months of the postpartum period. In utero infection seems to be unusual. Adults usually become infected during sexual activity or from blood transfusions and, unlike children, develop acute disease and often clear the virus. A study from the island of Nauru in the Western Pacific region (Speed et al, 1989) found that less than 25% of infected children were born to hepatitis B surface antigen (HBsAg) carrier mothers, yet about 70% of the total population had evidence of past or present HBV infection (17% of the population tested were HBsAg carriers). On this island, therefore, infection usually occurred after infancy, often between the ages of 5 and 15 years. Baillidre’s Clinical GastroenterologyVol. 4, No. 3, September 1990 ISBN O-702&1471-0
721 Copyright 0 1990, by Baillike All rights of reproduction in any form
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Horizontal infection at this aie occurs mainly from contact with other children and may be from fluid oozing from open skin lesions (Tibbs, 1987). A review of the evidence for horizontal transmission has been published recently (Davis et al, 1989). Developed world
In the developed world, infection is acquired by sexual contact, both heterosexual and homosexual, via non-medical intravenous drug use and from occupational exposure. Expatriates on contract in countries with a high carriage rate are particularly likely to acquire the infection sexually. Although family contacts of an adult carrier are at increased risk, adopted children from countries with a high prevalence of carriers pose a greater risk to their new families. In one study, the risk to other children in such a situation was 26%) while the risk from exposure to fathers or other infected adults was insignificant (Franks et al, 1989). Since the introduction of screening for HBsAg, the incidence of infection acquired from medical products in the developed world has become extremely low. The incidence of this mode of transmission could be further reduced by testing for antihepatitis B core (anti-HBc) as infection acquired from transfusion of HBsAg-negative but anti-HBc-positive blood has been described. Ultimately, the screening of blood products with polymerase chain reaction methods (see later) will eradicate this means of transmission. BIOLOGY Viral structure
Particles are easily seen in electron micrographs of the serum of infective carriers. The large particles, 42nm in size, are the complete virus. They consist of a central core containing DNA, the polymerase, a DNA-linked protein and a capsid, surrounded by the envelope. The capsid is composed of multiple copies of nucleocapsid protein, termed ~21. This nomenclature defines the type of protein (p is unglycosylated protein, gp is glycoprotein) and the molecular weight, measured in kilo-Daltons. In most enveloped viruses a matrix protein links the nucleocapsid to the envelope (this may have a role in the budding of particles from the cell). There is no evidence that such a protein exists in HBV. The envelope consists of a host-derived phospholipid bilayer membrane in which three types of glycoprotein derived from the same open reading frame (each with HBsAg at the carboxyl terminus) are embedded. The large envelope protein (p39/gp42) consists of pre-Sl, pre-S2 and HBsAg; the middle envelope protein p33/gp36) has pre-S2 and HBsAg; the major is composed of HBsAg alone (p24/gp27). Gerken et al (1989) have recently studied the distribution of these proteins and have determined that there is a preponderance of the major protein, although some middle and a small quantity of the large protein are also found in the complete viral particle. The large protein is not
secreted in non-viraemic carriers, but found in the hepatocyte. In viraemic patients, it is associated with the virion. PreS2 and S proteins are found in low amounts in the liver of individuals with replicating virus. Pre-Sl is found in large amounts in the livers of these patients and in healthy carriers. The molecular weight of pre-Sl has been found to be different in the serum and in the liver. On electron microscopy, other forms of the virus are seen which do not contain HBV DNA. There are spherical and tubular forms, both 22 nm in diameter. The tubular forms consist largely of aggregates of the major and the large proteins with some middle, whilst the spherical forms contain very little of the large surface protein. The function of these particles is unknown, but they may represent an overactivity of transcription or translation of the surface gene in the hepatocyte. The HBV DNA is partially double-stranded, one molecule per virus. It is incomplete in the circulating virion, having a large gap in the plus strand and a nick in the minus strand. The economy of the coding strategy is unique in the viral world. The complete genome is only 3.2kb long, the smallest genome of any double-stranded virus infecting man, and every gene overlaps with another. Some protein-coding regions also act as transcription controlling areas and are important in replication. There are four known genes. The first encodes the surface proteins, as detailed above. From the second are derived ~21 (HBcAg) and HBeAg. The latter is translated from an in-frame start codon 29 amino acids before the beginning of the core protein. These extra amino acids act as a signal peptide, allowing secretion of HBeAg from the cell. The two proteins therefore have identical amino acid sequence for most of their length and share common epitopes, although there are other epitopes that are unique to HBeAg. The antigenic differences are conformational, since the treatment of HBcAg with detergent reveals these extra sites. Furthermore, inoculating chimpanzees with HBcAg elicits an immune response in which both anti-HBc and anti-HBe activity can be detected (Iwarson et al, 1985a; Murray et al, 1987). HBeAg is not found in viral particles, but the antigen circulates freely in serum and is found on the surface of the hepatocyte (Schlicht and Schaller, 1989). Its function, as for the excess HBsAg particles, is not known, but it may play a role in modulation of the immune response. The third open reading frame encodes for a large protein which has a DNA polymerase, a reverse transcriptase and an RNAase H function. The gene has been cloned and expressed in a baculovirus system (L. Scully, personal communication). Finally there is the Xprotein, a transactivator, which probably plays a role in the replication of the virus and the interaction between the host and the viral genes. Replication
HBV enters the cell after binding to an as yet undefined receptor on the hepatocyte membrane. The virus infects a limited number of cells in vivo, for example hepatocytes, white blood cells (both CD4- and CDS- positive T-cells) and spleen cells, but is incapable of infecting cells in vitro in any
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system yet studied. The viral DNA can be transfected into a number of hepatocyte derived cell lines and whole viral particles will be produced and extruded into the culture medium. It would thus seem that viral tropism is limited and that cellular attachment and entry may be more selective than that seen with other viruses that can grow in a wide variety of cell lines. The amino-terminal 120 amino acids of the large protein (pre-Sl) are hydrophilic and glycosylated. The 21-47 amino acid section of this protein is capable of binding to the hepatocyte membrane (Neurath et al, 1986) and may, therefore, be involved in trans-membrane passage of the virus. Others believe that HBsAg is also required for this process. Whether the virus gains entry via endocytosis or by envelope-cell membrane fusion is not known. Once inside the cell, the DNA is liberated and passes to the nucleus, where the partially double-stranded form is completed and a complete RNA copy of the plus strand of the DNA is synthesized using host-derived enzymes. This plays two roles, that of a messenger RNA from which viral proteins can be translated and that of a template to produce more DNA strands, using virus-derived reverse transcriptase, to generate new particles (Summers and Mason, 1982). The particles are assembled in the cytoplasm and liberated, probably by budding through cell membranes, during which process the viral-derived envelope proteins are inserted into the newly the excess envelope acquired host-derived envelope. Simultaneously, proteins are extruded and the HBeAg is secreted into serum. In addition, particles may be liberated into the endoplasmic reticulum, as cells with viral-filled sacs can be seen in electron micrographs. Detection of viraemia
The quantification of HBV DNA in serum permits an assessment of viral activity, and on occasions may be the only marker of infection (HBV DNA polymerase activity was used in the past for this purpose). In patients who are responding to interferon therapy, HBV DNA levels usually become negative before loss of HBeAg. Dot-blot hybridization is the most widely used method of detection (Weller et al, 1982a), with a sensitivity of about a picogram (lo-l2 g) of HBV DNA. A review of the subject has been published recently by Krogsgaard (1988). Recent innovations include the use of non-radioactive labels (Saldanha et al, 1987; Casacuberta et al, 1988), and hybridization in solution rather than to a solid phase (Kuhns et al, 1989). However, the technique is unable to detect low levels of replication, such as in those patients who are both HBsAg- and anti-HBe-positive and yet have viral DNA in liver samples. The polymerase chain reaction (PCR) method for the amplification of nucleic acid sequences provides a far more sensitive method for the detection of HBV DNA in the serum. Synthetic oligonucleotide primers can be synthesized to match conserved regions because considerable sequence data is available. Methods have been developed for the use of PCR on serum samples, either directly or after extraction of DNA. There are a variety of standard methods for the detection of the PCR products. In the most simple
(termed PCR-EB), the amplified DNA is run out directly on an ethidium bromide stained gel and visualized with ultra-violet light. Alternatively, the PCR products can be detected after a hybridization step, either Southern blot, dot-blot or solution hybridization; these methods (termed PCR-SB) have similar sensitivities, about 1000 times that of PCR-EB. The third method (termed nested, double or PCR-PCR) is to subject an aliquot of the PCR products to a further PCR using primers internal to those used for the first PCR. In practice this is the simplest method and has a sensitivity equal to PCR-SB. Using PCR methods it has been established that all HBeAg-positive patients have circulating HBV DNA, although a small percentage of these are negative with standard dot-blot assays. The majority of HBsAg carriers with anti-HBe are negative on dot-blot hybridization but positive by PCRsometimes with PCR-EB, the least sensitive method (Carman et al, 1989). The presence of raised transaminases is correlated with a positive PCR in these patients (Baker et al, 1990). In patients who clear HBsAg, the findings are dependent on the clinical situation. In healthy people HBV DNA is not found in the serum by PCR, but about 25% of patients with chronic hepatitis have a positive reaction (Kaneko et al, 1989; Baker et al, 1990). However, in the study by Baker et al (1990) all of the patients became negative within 6 months. Other studies have tested sera from healthy patients with anti-HBc alone (Sumazaki et al, 1989; Donea and Vaira, 1990). About 50% of the patients were found to be HBV DNA positive by PCR. This technology may be used in the future for the elimination of infected blood products from donor panels or for the diagnosis of HBV infection in HBsAg-negative patients with abnormal liver biochemistry. Although most transfusion-associated HBV infection from HBsAg-negative blood can be avoided by the use of anti-HBc assays, a small percentage of patients can be positive by PCR in the absence of both these markers. Since HBV DNA detection by PCR is more sensitive than chimpanzee inoculation (Ulrich et al, 1989), samples that are PCR negative can be considered to be noninfectious. Viral variants HBV may be classified into immunological subtypes, each with an associated geographical distribution. This immunological variation is caused by single base changes in the surface gene. However, all known HBV isolates have the ‘a’ determinant, which is thought to consist of two highly hydrophilic loops between amino acids 124 and 147, generated by intramolecular disulphide bonds (Figure 1). Some variation in the first loop can occur, though the use of a monoclonal antibody to amino acids 124-137 in agammaglobulinaemic HBV carriers controls viraemia and does not result in the emergence of escape mutants. The second loop is highly conserved. Over the years, a number of examples of infection have been found in which viral variants have been implicated. Some cases of non-A, non-B hepatitis have been attributed to these, but the new diagnostic test for
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hepatitis C virus infection should help in their analysis. Some patients with chronic hepatitis have HBV DNA in their liver, yet sera are negative for all markers of infection except for very low amounts of HBsAg, detected by highly sensitive techniques (Brechot et al, 1985). In these cases, however, infection need not be attributable to a novel variant of HBV as a negative test can occur if the epitopes are concealed in immune complexes. In one patient without serum markers of disease, sequencing of the HBsAg region has revealed a single amino acid change at the conserved 3’ end of the gene (Thiers et al, 1988). In addition, an analysis of restriction enzyme profiles of PCR products from Israeli patients with cirrhosis and no HBV markers has revealed patterns different from all known HBV subtypes (J. Wands, personal communication). A study using the chimpanzee model detected isolates that could infect previously vaccinated animals (Wands et al, 1986). One isolate came from a patient without any markers of HBV infection except a very low titre of HBsAg. PCR was not available at that time. The implication was that the isolate had little homology to existing isolates and that the vaccine-induced immune response was not protective. It should be noted that few of the isolates mentioned above have been characterized at both the clinical and the molecular levels and thus their relevance is not known. HBV2
Some patients (mainly from Senegal, Spain and Taiwan) have had an HBV infection characterized by absence of anti-HBc or anti-HBe responses (Coursaget et al, 1987). It has been suggested that this infection is caused by a variant termed HBV2. A pair of monoclonals against the pre-S2 region and the ‘a’ determinant recognized HBsAg, which was the only marker of infection in these patients. HBV DNA was not found in serum and the patients did not become ill. Importantly, a number of these patients had responded successfully to vaccination, implying that this novel strain was very different in the protective region of the HBsAg. It has since been shown that infection of a chimpanzee with one of these strains gives rise to classical disease with the characteristic markers of infection, suggesting that the unusual serological profile in the patients was due to an aberrant immune response (Gallagher et al, 1990).
The protective antibody generated by vaccination is directed against epitopes of HBV in the surface region, in particular the ‘a’ determinant. Some adults and children successfully vaccinated (as judged by a satisfactory anti-HBs response) during trials in southern Italy have subsequently developed HBsAg and anti-HBc (Carman et al, 1990a). In one of these cases, an infant born to a carrier mother, sequencing of the DNA encoding the ‘a’ determinant revealed a single amino acid substitution in the second loop (Figure 2). This change was found in the child at the age of 11 months -‘a’
and 5 years later, yet the normal sequence was found in the mother at the time of delivery. It is postulated that this represented an escape mutant that developed in response to the immune pressure, but it could have been a novel strain selected as a result of the high local vaccination rate. The same amino acid substitution has been found in an isolate from a liver transplant recipient treated with monoclonal anti-‘a’ reactive with the amino acid region 131-148 (McMahon et al, 1990). The normal virus was present before transplantation. In some of the known sequences of HBV, this mutation would result not only in the amino acid change in the surface protein but in a translational stop codon in the polymerase gene, a change predicted to result in a virus that is unable to replicate. This may help to explain why this change has not been found in other areas of the world that have been involved in vaccination trials. Pre-core mutants
There are patients in the Mediterranean and the Far East that remain viraemic after seroconversion to anti-HBe because they carry a virus that has a mutation in the pre-core gene, giving rise to a novel translational stop codon (Figure 3; Brunetto et al, 1989a; Carman et al, 1989). As translation of the pre-core gene is known to be unnecessary for the formation of viral particles but essential for the production of HBeAg (Schlicht et al, 1987), this finding explains the’serological paradox. It is not known whether this mutation directly affects viral pathogenicity. Since HBeAg has been thought to down-regulate cell mediated immunity, more aggressive cytotoxic T cell
activity could be predicted against infected hepatocytes in these cases. Sequencing of serial isolates from patients with acute hepatitis has shown that this mutation arises from the normal strain of the virus at the time of anti-HBe seroconversion (Carman et al, 1990b). This does occur in British patients but is almost invariable among Greeks. Japanese patients do not seem to develop the mutation during the acute disease, but develop it during the anti-HBe seroconversion phase of the chronic illness (Okamoto et al, 1990). HBsAg-positive Greek patients with anti-HBe, yet without liver disease, have both the normal and the mutant strains co-circulating (Carman et al, 1989). The presence of both strains may be a transition phase or represent an equilibrium. The anti-HBe, HBV DNA positive form of chronic illness described above is not found in northern Europe. HBeAg positive strain (wild type) TAG
“,~.T-s...“..y* w------j t
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variable cleavage site
fixed cleavage site +
HBeAg negative strain (pre-core mutant) TAG
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Figure 3. Novel
in the pre-core
rise to the ‘pre-core’
Studies of Greek and British patients with fulminant hepatitis have been performed because of the epidemiological links between this form of the disease and anti-HBe-positive carriers. The results show that Greek patients with fulminant hepatitis have an incidence of the variant similar to that seen in acute patients and that British patients with fulminant disease have the variant only when the patient is anti-HBe-positive (Carman et al, 1990b). In the British patients there was a perfect correlation between the presence or absence of the mutant and anti-HBe or HBeAg positivity. Interestingly, those fulminants with anti-HBe had a much improved survival. The mutation may merely be a molecular explanation for seroconversion to anti-HBe, a development known to be related to viral clearance. This explanation would tally with the emergence of the mutant in patients who have lost HBeAg during the recovery phase of either acute or chronic hepatitis. The clinical differences between populations probably reflect their immunogenetic differences, although the exact mechanisms for the emergence of the variant and why the variant causes such severe disease in certain geographical areas remains unclear. PATHOLOGY
Recovery from HBV infection is dependent on the integrated activity of host cellular and humoral immunity, in combination with an intact interferon system. Chronic HBV infections probably arise because of defects within these defences. Acute hepatitis B infection
Few hepadnaviruses (partial double-stranded hepatotropic DNA viruses with a reverse-transcriptase replication step) are cytopathic. Liver damage is caused by immune lysis of infected hepatocytes, an essential part of the recovery process. This typically begins between 6 weeks and 6 months after exposure to the virus, and follows a prodrome of general malaise attributable to rising interferon levels and circulating HBs and HBc immune complexes. The hepatic lesions consist of necrosis and autolysis initially with a centrilobular distribution. The reticulin framework is preserved and the cellular infiltrate is most intense around the portal tracts. Analysis of the inflammatory infiltrate has demonstrated the presence of NK and cytotoxic T-cells (Eggink et al, 1982). Viral antigens appear on the hepatocyte surface (Gudat et al, 1975) and these, in association with class I major histocompatibility complex (MHC) proteins, promote cytotoxic T-cell lysis (Doherty and Zinkernagel, 1975). Studies in patients with chronic HBV infection suggest that the nucleocapsid antigens (core and e antigen) are the principal target (Eddleston et al, 1982; Pignatelli et al, 1987). Normal hepatocytes express little MHC class I glycoprotein (Thomas et al, 1982) but, in the early stages of acute HBV infection (under the influence of a-interferon), MHC expression increases and coincidentally the transaminases rise. There is some
evidence that the X gene product may activate cellular genes, including those for MHC class I proteins. In addition, interferon activates the 2-5 A oligoadenylate synthetaseendonuclease system, which leads to inhibition of viral protein synthesis and destruction of viral mRNA. This induces an antiviral state in uninfected regenerating liver cells, preventing re-infection following the lysis of infected hepatocytes. Serological response
The earliest indications of infection are the detection HBsAg and HBeAg in the serum (Krugman et al, 1979). HBV DNA may also be detected at this time. As the surface and ‘e’ antigens are cleared, anti-HBc and anti-HBe antibodies appear. There is often a short period when the detection of anti-HBc is the only marker for the disease (Hoofnagle et al, 1974). There are some distinctions between the nature of the humoral response to HBcAg and HBeAg probably because of differences in T cell dependence (Sallberg et al, 1989). HBcAg causes a strong IgM and IgA response and HBeAg a low level IgG (mainly IgGr) response. HBcAg has a single B-cell epitope (Ferns and Tedder, 1986; Waters et al, 1986) resulting in a homogeneous humoral response, but the T-cell response is less restricted (Milich and M cL achl an, 1986) and unable to discriminate between HBcAg and HBeAg. The IgM component disappears within a few months of recovery (alongside declining titres of IgGr and IgGs), at the time when the anti-HBs antibody becomes detectable, but the IgG4 component persists for life. Anti-HBc antibodies are not neutralizing, and persisting high titres in the absence of anti-HBs suggest continued viral replication (Hoofnagle et al, 1978).
In chronic hepatitis, IgM antibodies to human serum albumin (HSA) have been detected during HBe antigenaemia (Hellstrom and Sylvan, 1989), and their disappearance preceded anti-HBe seroconversion. IgA antibodies remained after seroconversion except in one case that subsequently underwent anti-HBs seroconversion. The authors concluded that albumin-HBs and albumin-HBe complexes elicited the immune response to HSA, preventing the development of traditional anti-HBs and anti-HBe, and it was the regression of these antibodies that permitted full sensitization to HBs and HBe. The virus neutralizing antibodies are directed against epitopes on the large, middle and major envelope proteins, and appear 1-4 months after the onset of symptoms in those that make a complete recovery. The immunogenetics of the response to pre-Sl, pre-S2 and HBsAg have been well defined in a murine model (Milich et al, 1987). In humans, antibodies to pre-Sl and pre-S2 regions appear before antibodies to the HBs region (Neurath et al, 1985; Petit et al, 1986). In vitro studies of peripheral blood mononuclear cells (PBMC) at the time of anti-HBs seroconversion demonstrate an anti-pre-S2 and anti-HBs response following pre-S2 stimulation, and an anti-HBs response alone following HBsAg stimulation (Cupps et al, 1990). Following this initial stage, no further IgG reactivity can be detected
for about a year, but later, anti-HBs appears again after HBsAg or pre-S2 stimulation, but the anti-preS2 response does not return. For most hepadna virus infections, seroconversion from HBeAg positivity to anti-HBe positivity is accompanied by the biochemical and histological resolution of hepatitis, and the disappearance of viral replication detectable by traditional methods (Realdi et al, 1980; Hoofnagle et al, 1981). In most of these cases HBV DNA remains detectable in the serum by PCR. Hepatocytes that only contain integrated HBVsequences avoid immune lysis because they do not express HBV proteins encoded from the 3.4 kb large transcript (HBc, HB polymerase and HBX); the preferred site of integration of the viral genome is within the regulatory region for this protein (Fowler et al, 1986). The destruction of hepatocytes supporting productive HBV infection results in a reduction in hepatocyte mass, and the remaining hepatocytes, including those containing integrated HBV sequences, proliferate to restore liver function. Many years after recovery the anti-HBs neutralizing antibody titre begins to fall. At this stage, the detection of anti-HBc (IgG4) may be the only marker of previous virus exposure. Chronic hepatitis B infection
Persistence of HBsAg for greater than 6 months occurs in 5-10% of infected adults, and defines the carrier state. This is more common in males and patients with natural or acquired immunodeficiencies. Most chronic carriers, however, develop as a result of vertical transmission or childhood infection. The chronic carrier state exists in several different forms. Chronic HBe antigenaemic patients
This is the first phase of chronic infection and may arise after exposure at any stage of life. Chronic HBV infection following neonatal exposure. Most babies born to HBeAg-positive mothers become infected and develop a chronic carrier state (Beasley et al, 1981). The reasons for failure to clear the virus are unknown but likely to relate to the immaturity of the neonatal immune system (Nash, 1985). HBeAg passes across the placenta and may suppress the development of the cellular immune response to the nucleocapsid proteins essential for hepatocyte lysis. Fulminant hepatitis following neonatal exposure. Rarely, children born to HBsAg- and anti-HBe-positive mothers develop fulminant hepatitis. The mechanism for this is unknown, but one hypothesis is that maternal antiHBe and anti-HBc pass across the placenta and hinder the cytotoxic lysis of infected hepatocytes (Pignatelli et al, 1987). The virus could spread throughout the liver and, as the titre of maternal antibodies declined at 3-6 months, cytotoxic T-cells sensitized to HBe and HBc would destroy the
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many infected cells, resulting in hepatic failure. An alternative hypothesis is that the pre-core variant is transmitted at birth, and in these cases a more severe disease follows. Chronic HBV infection acquired after the neonatal period. In contrast to the situation at birth, only 5-10% of subjects infected after this period develop chronic infection. There are several causes for the development of chronic infection at this stage of life. One defect is the production of subnormal quantities of cx-interferon by PBMC (Tolentino et al, 1985; Abb et al, 1985; Ikeda et al, 1986a). Some patients also demonstrate poor hepatocyte activation by interferon. The concentration of hepatic 2-SA-synthetase becomes only minimally elevated (Ikeda et al, 1986b), and MHC class I proteins are only expressed in low density on infected hepatocytes (Montano et al, 1982). The degree of 2-SA-synthetase activity and MHC class I display is usually increased in the peripheral blood lymphocytes of chronic HBV carriers (Poitrine et al, 1985). These data suggest activation of peripheral blood lymphocytes but not of infected hepatocytes and raise the possibility that HBV within the hepatocyte has selectively down-regulated the interferon response of the hepatocyte. Transfection of HBV genomes into tissue culture cells makes them specifically non-responsive to interferon; they remain susceptible to lysis by Sindbis virus and MHC induction by interferon does not occur (Onji et al, 1988). This raises the possibility that, during HBV replication, cytotoxic T cells cannot lyse infected cells because the virus suppresses the expression of MHC class I proteins. Anti-HBe-positive
Low level replication (PCR positive). The sera of these patients do not contain virus particles detectable by dot-blot hybridization. The HBsAg is encoded by integrated HBV DNA sequences, and in the presence of normal liver histology this condition carries little risk for the patient. These patients are not considered infectious for normal domestic life but they should be excluded from blood transfusion donor panels. When PCR techniques are used to amplify HBV DNA from serum, sequence data reveal the presence of a mixture of wild type and pre-core mutant viruses (1896 G+C). (dot-blot positive). These patients are infected with the pre-core mutant (1896 G-+C) which has undergone a second mutation (1899 G+C), and the wild-type is absent. Infection by this pre-core HBV mutant causes considerable viraemia in the absence of circulating HBeAg and these patients have significant liver disease. As discussed in the section on HBV variants, the clinical course in patients infected with this virus appears to be dissimilar in different races. The Mediterranean patients tend to have lower HBV DNA levels in their sera, and a cytoplasmic rather than nuclear distribution of HBcAg. The source of their infection is usually unknown, and they present with longer-standing and more advanced disease.
High level replication
These tumours are common in the areas of the world where hepatitis B carriage rate is high. In 80% of patients there is a background of cirrhosis with chronic HBs antigenaemia (Song et al, 1983). The risk of developing hepatocellular carcinoma (HCC) in a chronic HBsAg carrier with normal liver histology is almost negligible. Integrated HBV DNA sequences have been found in most tumours (Rogler et al, 1985), and it has been suggested that during cell division these sequences rearrange (Shafritz et al, 1982) and cause malignant transformation of the hepatocyte. The ability of the duck and woodchuck hepadnaviruses to cause liver cell cancer, and the production of HBsAg in tissue culture by cell lines derived from human HCCs provides further evidence in favour of this hypothesis. Even in cases with negative serological markers of HBV infection (anti-HBc and anti-HBs), HBV DNA has been detected within tumour DNA using PCR (Paterlini et al, 1989) with primers for the pre-S, S and X genes. Wang et al (1990) recently demonstrated the integration of HBV sequences within the human cyclin A gene (a protein involved in the control of cell division) of a well-differentiated HCC. Although the HBVX gene encodes a trans-activating factor (Wollersheim et al, 1988), not all HCCs maintain this sequence. The preS2iS region of integrated HBV DNA has recently been shown to have trans-activating activity of both an artificial SV40 promoter-chloramphenicol acetyl transferase reporter plasmid (pSV2CAT) and the promoter of the human c-myc oncogene (Kekule et al, 1990). In a transgenic mouse model containing the large envelope protein sequence under the transcriptional control of the albumin promoter (Chisari et al, 1989) hepatic inflammation and regenerative hyperplasia occurred in the absence of core gene products, and the mice developed hepatocellular carcinomas. Interrelationship
between HBV and HIV
There are structural and functional similarities between human immunodeficiency virus (HIV) and HBV. They both have an RNA and a DNA stage during replication, although the infective stage is DNA for HBV and RNA for HIV. In addition, they both have a reverse transcriptase, with large regions of partial homology. Shared genomic features include the direct repeated sequences at the ends of the DNA, with their complementarity enabling self-binding during a critical stage of replication, and the presence of four major genes; in HBV these are the core, polymerase, envelope and X corresponding in HIV with the gag, pal, ey1vand tat. Both can infect white blood cells. HBV seems to be the major factor in the causation of hepatocellular carcinoma, and although HIV is not directly oncogenic, other retroviruses have this potential. The HBV X protein is known to be able to transactivate the enhancer region of the long terminal repeat (LTR) of the HIV genome (Siddiqui et al, 1989; Twu et al, 1989), but the tat protein of HIV does not have a similar effect on HBV.
As the epidemiology of the two viruses is so similar, many cases of dual infection occur. Patients with chronic hepatitis tend to improve histologically and clinically when infected with HIV, probably because of the decreased cellular immune response against infected hepatocytes (Bodsworth et al, 1989). For the same reason, the concentration of HBV DNA in serum and the serum DNA polymerase levels both increase. Hepatocytes in these patients tend to contain greater amounts of HBc- and HBe-related proteins. The consequences of this in terms of risk for development of hepatocellular carcinoma or the degree of infectivity are not known. There is no evidence that the rate of progression of HIV infection to AIDS is accelerated by concomitant HBV infection. PREVENTION
The antibodies found in convalescent serum bind predominantly to the epitopes of the HBs gene encoded region, with the two hydrophilic loops (amino acids 124-147) contributing most (>80%) of the antigen-binding capacity (Brown et al, 1984). Using monoclonal antibodies, it has been possible to show that administration of antibody to the region 124-136 to chimpanzees prevents infection (Iwarson et al, 1985b). Antibodies to this region, as well as to other epitopes on the S gene encoded polypeptide, are present in the serum of patients convalescent from HBV infection and in normal subjects immunized with plasma-derived and recombinant HBs vaccines (Thomas et al, 1987). Pre-exposure prophylaxis Active immunization is recommended for health care personnel, particularly those working in emergency, haemodialysis and coagulopathy units, and laboratories involved in blood and secretion analysis. Vaccination should also be offered to persons in certain other non-medical high risk occupations, e.g. the police and those responsible for the care of the mentally handicapped (Zuckerman, 1984). The first successful vaccines were licensed in 1981, and consisted of 22-nm spherical HBsAg aggregates purified from the serum of chronic carriers (Szmuness et al, 1980). The high production costs and the unjustified anxiety concerning the safety of these vaccines resulted in the development of yeast-derived recombinant vaccines, the first of which were licensed in 1986 (Goilav et al, 1988; Tilzey et al, 1988). The vaccine is usually given intramuscularly (20 pg into the deltoid) in a three-dose series with delays of 1 and 6 months between the doses (a gap of only one month between the second and third dose is equally effective). The recombinant vaccines are probably as effective in the general population as the plasma-derived ones, are simpler to prepare and will become cheaper as the target population expands. However, there are still some doubts about their long-term efficacy. A comparison of the two types of vaccine given to medical students by the intradermal route revealed that the anti-HBs levels after recombinant
vaccine were about 25% of the levels obtained with the serum-derived vaccine (Kurtz et al, 1989). The plasma-derived vaccines appear to be superior in patients with chronic renal failure (Seaworth et al, 1988). lntradermal vaccination is effective if given correctly and is cheaper as the required dose is smaller. However, current recommendations are to give the vaccine intramuscularly (only 10 kg for children, although 2 kg doses are probably adequate). The protective level of antibody established empirically is 10 sample ratio units (SRU) as determined by radio-immunoassay; W--97% of normal adults will respond. The question of how to manage the non-responders and when to boost the responders remains unresolved. Hadler et al (1986) found that 15% of vaccine recipients had undetectable antibody at 5 years and that 27% had levels less than 10 SRU at 5 years. The peak antibody titre after vaccination predicted the duration of the protective effect. For the nonresponders, a second vaccine course brought a moderate antibody response in 50% of the recipients. The recommendation of the Center for Disease Control (CDC) is to re-test and boost if necessary after 7 years, but there is little clinical evidence to support this. Laboratory evidence predicts that this is unnecessary as in vitro anti-HBs production following natural infection can be stimulated even after anti-HBs disappears from the serum (Cupps et al, 1990). Although patients who have recovered from HBV or have been successfully vaccinated with HBs antigen are protected from further infection, there remains the question of whether the middle pre-S2 bearing and large pre-Sl bearing polypeptides should be added to existing vaccines. Because there is evidence that pre-Sl is the region of the envelope of the virus that binds to the hepatocyte during infection (Neurath et al, 1986), antibodies to this region may be neutralizing and, therefore, inclusion of the pre-S region in a vaccine could be desirable. Furthermore, 15% of normal individuals do not respond to HBs small polypeptide and, at least in mice, inclusion of pre-S2 facilitates, by recruiting additional T helper cells, the development of antibody to the HBs epitopes (Milich et al, 1985). In haemophiliacs, following vaccination with HBsAg and pre-S2, a serological response to the pre-S2 component was accompanied by an anti-HBs titre 3 1000 international milliunits per millilitre in 87% of recipients, and a negative anti-pre-S2 response was associated with high anti-HBs titres in only 50% (Zanetti et al, 1989). More recent data indicate that the inclusion of HBc antigen with HBs antigen further enhances the humoral response to HBs epitopes (Milich et al, 1987). The MHC haplotype of vaccine recipients has been shown to influence vaccine response. Carriers of HLA-B8, SCOl, DR3 in particular are poor responders (Alper et al, 1989). There are some doubts about the efficacy of the vaccine in homosexual men, with or without HIV infection. In one study, five out of seven uninfected men responded to the recombinant vaccine, whereas none of 13 HIV-infected men responded. However, the vaccine did not have any deleterious effects on the HIV infection; CD4positive cells declined by an equal amount in the infected, vaccinated group as in an infected control group (Hess et al, 1989).
Transmission may occur whenever infected blood or secretions have been inoculated or have come into contact with the mucous membranes or conjunctivae of a susceptible person. Although the administration of purified sera from patients with high titre anti-HBs antibody appears to confer temporary passive immunity to the disease, the mechanism of action of hepatitis B immune globulin (HBIg) seems to be prolongation of the incubation period (Grady et al, 1978) and attenuation of clinical illness in order to prevent the chronic carrier state (Hoofnagle et al, 1979). In this respect HBIg does not confer true passive immunity, and it is recommended for use within 48 h of a single acute exposure to the virus (preferably within 12 h for the prevention of perinatal transmission in combination with active immunization. The dose, established empirically, is 250-500 i.u. given twice, 30 days apart (Deinhardt and Zuckerman, 1985). Combined active/passive immunization has been shown to reduce the risk of infection from 33% (passive immunization only) to 4% in renal dialysis staff following infectious needlestick injuries (Mitsui et al, 1989), and the risk of neonatal transmission by infectious mothers from 90% with no prophylaxis to 5%) with a transmission rate of 25% following either active or passive immunization alone (Beasley et al, 1983). There is evidence in chimpanzees that HBIg is not necessary if active immunization is given in three or four doses in rapid succession, but such a study has not been undertaken in humans. For the prevention of neonatal infection, children born to carrier mothers should be targeted. Even though the age of infection in Africa and Asia tends to be different, it is still recommended that vaccination is introduced at birth, when the population is available to the health service. DTP, BCG, poliovirus and HBV vaccines can all be given concurrently. In China, locally produced vaccine is only US$l per dose, and in a number of countries the cost has fallen below US$5 per dose. However, for the poorest countries, the cost of vaccination, without support from the richer nations, still remains prohibitive. Chemotherapy
of chronic HBV
There is no place in the treatment of chronic HBV infection for regimens involving solely anti-inflammatory or immunosuppressive drugs (Scullard et al, 1981; Weller et al, 1982). A variety of synthetic and natural antiviral compounds have been developed and are being evaluated in clinical trials. Indications for antiviral chemotherapy
Chemotherapy is indicated for patients with progressive liver disease (histological evidence of chronic active hepatitis) and for infectious patients with HBe antigen and HBV DNA positivity. Decompensated cirrhosis is a contraindication for therapy. Currently interferons are the most satisfactory therapy, but less than 50% of patients respond and the side-effects of interferon are substantial.
Responses to antiviral
Several investigations have shown improvement in transaminase levels following clearance of HBeAg and HBV DNA from the serum. A review of 50 treated and 25 untreated control patients has shown a reduction in inflammatory necrosis of liver cells and a diminished rate of development of cirrhosis in patients responding to therapy with loss of serological markers of HBV replication (Brook et al, 1989a). The responses to antiviral therapy may be of three types, as follows. Transient response. This is the inhibition of HBV replication, with loss of HBV DNA polymerase activity during therapy but return of this marker on cessation of therapy. response. This is characterized by the sustained inhibition of HBV replication, with the disappearance of HBV DNA (by dot-blot) and HBV DNA polymerase activity persisting after cessation of therapy. A seroconversion from HBeAg to anti-HBe accompanies this, but HBs antigenaemia remains due to the translation of integrated HBV DNA sequences. The majority of these patients still have HBV DNA detectable in the serum by PCR, however (Carman et al, 1990~).
Complete response. This is characterized by the sustained inhibition of HBV replication, with loss of HBV DNA (by dot-blot) and continuation of HBV DNA polymerase activity after cessation of therapy, and permanent seroconversion from HBeAg and HBsAg to anti-HBe and anti-HBs. However, HBV DNA can still be detected by PCR in the serum and liver of these patients (Carman et al, 1990~; Marcellin et al, 1990). Nucleoside analogue therapies
These agents inhibit HBV DNA polymerase. Most trials conducted have demonstrated diminished viral activity during therapy, but a return to pre-treatment activity afterwards. Some patients, however, have seroconverted as a result of viral inhibition. The future will see the development of new drugs in this class, more suited for long-term therapy because of their activity by the oral route and because they have few side-effects. arabinoside and adenine arabinoside monophosphate. The first nucleoside analogue to be used successfully in hepatitis B infection was adenine arabinoside (ARA-A), a potent inhibitor of HBV DNA polymerase. The clinical value of ARA-A was limited because of poor aqueous solubility, which necessitated administration by intravenous (i.v.) infusion. A controlled study of i.v. ARA-A demonstrated that a lo-day course produced HBe to anti-HBe seroconversion rates of 40% (Bassendine et al, 1981). Most clinical trials on ARA-A have involved the use of a water-soluble derivative, adenine arabinoside monophosphate (ARA-AMP), which can be given by intramuscular injection. The major disadvantage of ARA-AMP was toxicity,
J. L. BROWN
with many patients developing painful sensory peripheral neuropathies and myalgias. Some of these side-effects have caused long-standing disability, resulting in the withdrawal of ARA-AMP for use as a single agent in chronic hepatitis B infection. Acyclovir has also been used in chronic hepatitis B infection. A reduction in DNA polymerase (Smith et al, 1981) and HBV DNA levels (Smith et al, 1981; Weller et al, 1983) have been described following a 7-day iv. treatment period, but few controlled studies in patients with chronic hepatitis B infection have been performed (Alexander et al, 1987).
It is not surprising that azidothymidine (AZT) has been studied in HBV infection, since dual infection with HIV is common and reverse transcription is part of the HBV replication cycle. Although AZT inhibits HBV DNA polymerase in vitro, it has shown no clinical activity against the duck hepadnavirus (Haritani et al, 1989). Human studies have failed to show a reduction in HBV DNA concentrations using this drug, and in some cases the levels have risen (Farraye et al, 1989). However, because of the possible synergistic effect of the two agents, trials are underway using AZT with interferon in patients with chronic HBV infection.
DNA polymerase inhibitors
Foscavir (Foscarnet [trisodium phosphonoformate]) inhibits viral-specific DNA polymerases and is particularly active against the herpesviruses. The drug has been used in acute hepatic failure in eight patients with HBV infection (four with concomitant 6 infection), and six of the patients made a complete recovery (Hedin et al, 1988). A recent study in eight patients with chronic HBV infection (Bain et al, 1989) demonstrated a fall in HBV DNA levels in all patients, but within a month of stopping therapy HBV DNA levels had returned to pre-treatment values in seven of the eight cases (the other seroconverted). The place of this drug in the treatment of chronic HBV infection remains unclear. Cytokine therapies
Leucocyte (x-, l3- and y-interferon and interleukin-2 have all been investigated for the treatment of chronic HBV infection, with disappointing results. Lymphoblastoid and recombinant a-interferons have had the most promising results. The mechanism of action of interferons in chronic hepatitis B infection is unknown, but several changes in the immune system and in the infected hepatocyte are believed to be important. An increase in MHC class I protein display (Pignatelli et al, 1986) and viral protein expression (Thomas and Scully, 1986) in the hepatocyte occur within 24 h of starting therapy. A rise in the helper/suppressor T lymphocyte cell ratio occurs at 6-8 weeks in those subjects who successfully undergo HBe to anti-HBe seroconversion. Changes in humoral immunity also occur. Patients who respond to
therapy either have IgM anti-HBc present before treatment or develop it during the course of therapy (Chen et al, 1989). Inhibition of viral replication, associated with HBe antigen/antibody seroconversion, is usually long-lasting and accompanied, after a transient exacerbation, by amelioration of the inflammatory liver disease (Brook et al, 1989b, c). Reactivation may occur particularly in homosexual patients with or without HIV infection (Davis and Hoofnagle, 1985). Patients treated early in the course of the disease will lose HBs antigen from their serum if termination of HBV replication (as judged by dot-blot) is achieved. If integration of HBV sequences into the host cell genome has occurred before initiating treatment, then HBs antigenaemia will continue after cessation of detectable HBV replication. However, inflammatory liver disease will ameliorate in these patients if long-term inhibition of HBV replication is achieved. Lymphoblastoid a-interferon. Several controlled studies of human lymphoblastoid or-interferon in chronic hepatitis B infection have been undertaken. A multicentre study from Italy has demonstrated an overall seroconversion rate of 50% (Mazzella et al, 1988). Six months of therapy is no better (and tolerated worse) than 3 months of therapy (Scully et al, 1987), and thrice weekly injections for 3 months are more effective and better tolerated than daily injections for 1 month. The ideal dose seems to be 5-10 million units/M’, three times a week. A review of the factors that appear to predict a seroconversion response indicate that Chinese chronic carriers (presumably infected from birth) have a very poor response, whereas European heterosexual carriers (who usually acquire their infection in adulthood) have the best response (Thomas and Scully, 1986). Other factors that are predictive of a seroconversion are high serum transaminases (Brook et al, 1989b), marked hepatic inflammatory activity (Brook et al, 1989a), the presence of serum IgM anti-HBc (Chen et al, 1989) and low serum HBV DNA (McDonald et al, 1987). Recombinant a-interferon can also induce Recombinant winterferon. complete remission in certain HBsAg carriers (Hoofnagle et al, 1988). Homosexual males seem less responsive than heterosexuals, particularly those who are anti-HIV antibody-positive (McDonald et al, 1987). Poor results have been obtained in Chinese adults (Lok et al, 1988) and children (Lai et al, 1987). In a study of interferon therapy for infection with the pre-core mutant, 67% of the patients made an initial response, but 88% of these subsequently relapsed (Brunetto et al, 1989b). Development of interferon antibodies during recombinant a-interferon therapy occurs in about 25% of patients (Porres et al, 1989) and may reduce the chances of successful seroconversion. This is most likely if they appear while the patient remains HBV DNA positive. Combination
It is believed that following
a short course of steroid therapy a rebound
immune stimulation occurs. This phenomenon, in combination with antiviral and cytokine therapies, has provided a new rationale for therapy in chronic carriers who acquired their infection at birth. The effects of natural rebound immunostimulation have been documented recently in a study from Taiwan examining HBV DNA levels and HBeAg in chronic HBeAgpositive female carriers postpartum and non-pregnant female carrier controls (Lin et al, 1989). In the postpartum group, 15% lost HBV DNA, a further 15% lost HBeAg and 3% developed anti-HBe antibodies, most frequently after l-2 months. There were no changes in the control group. Prednisolone and interferon. The results of Perillo et al (1988) suggest slightly higher seroconversion rates in patients with combination therapy than with interferon alone. In this study the interferon was given daily, whereas in most other studies interferon is administered thrice weekly. An interesting aspect of this study was that three anti-HIV antibody-positive patients were treated and two seroconverted. Both of these patients had normal CD4ICD8 ratios, indicating that anti-HIV seropositivity should not be used as a predictor for treatment failure. Encouraging preliminary results have also been obtained in a study from Taiwan, where the combination of prednisolone pre-treatment followed by lymphoblastoid interferon is being compared with placebo and interferon alone (Liaw et al, 1988). Long-term results of this study are awaited with interest. and interferon. A pilot uncontrolled study has shown that this combination may be better than either agent alone (Schalm et al, 1985). One small controlled study of acyclovir with interferon in chronic hepatitis B infection has been performed (de Man et al, 1988). In this study, 40% of 18 patients treated with lymphoblastoid interferon and oral deoxyacyclovir for 4 months seroconverted from HBe-positive to anti-e-positive, compared with 0% of 18 controls, but no comparison was made with patients treated for an identical treatment period with interferon alone. A further casereport from the Netherlands has suggested that the combination of acyclovir and interferon may be useful in HBV-associated glomerulonephritis (de Man et al, 1989).
Of all the hepatotropic viruses, HBV is associated with the greatest worldwide morbidity and mortality. This is because of the ease of transmission and the potential for progression to a chronic infective carrier state, with the complications of cirrhosis and hepatocellular carcinoma. The use of PCR has shown that some of the earlier concepts concerning the interpretation of serological data were inaccurate. Many patients with anti-HBe and anti-HBs have viral DNA detectable by PCR, and some hepatocellular carcinoma patients have detectable HBV DNA in their livers in the absence of all serological markers of HBV disease. The clearance of HBV infected cells from the liver is dependent on the
interplay between the interferon system and the cellular limb of the host immune response. The importance of the nucleocapsid proteins as targets for sensitized cytotoxic T cells has been established for chronic HBV infection. The importance of pre-S sequences as inducers and targets of the virus-neutralizing humoral immune response is becoming established, but their precise role must await the development of in vitro models of hepadnavirus infection and a greater understanding of the mechanisms of viral uptake. The epidemiology and clinical course of the disease can be modified by immunization, immune stimulation and antiviral chemotherapy. For the developing world, a programme of immunization at birth would be the most effective way of eliminating this disease, but at present the cost is prohibitive. For the developed world, immunization is realistic for the at-risk population, and anti-viral and immunostimulatory therapy available for those already infected. In adult acquired chronic HBV infection c-w-interferon produces HBe antigen clearance in 40-60% of cases and is followed by resolution of the hepatic inflammation. Results in neonatally acquired infection are less impressive and prednisolone priming followed by interferon may be needed. The presence of a mutation in the pre-core region of some virus isolates has recently been described. Hepatocytes infected with this virus cannot produce EIBe antigen and the course of the liver disease is fairly rapid. Whether this mutant causes liver damage in the same way as the wild virus or is directly cytopathic remains unclear, and its relationship to fulminant hepatitis is under investigation. REFERENCES Abb
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