A case of repeated seroconversion to HBe antibody due to co-infection with Mutant- and wild-type hepatitis B virus clones

It is reported that co-infection with different hepatitis B virus (HBV) clones in a patient with chronic hepatitis B induces rare serotypes (adywr) or abnormal laboratory data such as negative HBs antigen, in the presence of positive HBV DNA. In this study, we experienced a case of repeated seroconversion to HBe antibody in a patient with chronic hepatitis B. Since seroconversion is considered to be related to genetic mutations, we investigated the HBV genes in this male patient in his 30's. We amplified and cloned parts of the HBV genes by the polymerase chain reaction (PCR), and sequenced the PCR products. As a result, mutated HBV genes were found in the serum of each specified period. By DNA sequencing we confirmed the coexistence of different HBV clones (wild-type clone and pre-S deletion mutant) and that both clones had the same genotype C. These clones took turns to be dominant; when the wild-type clone was dominant, HBe antigen was positive, and when the mutant clone was dominant, HBe antibody was positive. These findings demonstrated that repeated seroconversion of HBe antigen to HBe antibody was induced by co infection with mutant- and wild-type HBV clones. It is interesting that increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was noted at the time of the change from positive wild-type HBV clone to positive mutant clone.     

Analysis of the complete hepatitis B virus genome in patients with genotype C chronic hepatitis in relation to HBeAg and anti-HBe

To investigate the relationship between viral factors and the development of chronic hepatitis B, the entire hepatitis B virus (HBV) genome of chronic carriers at different disease stages were analyzed. Eighty genotype C HBV carriers including 12 hepatitis B e antigen (HBeAg) positive asymptomatic carriers (Group A), 49 HBeAg positive patients with chronic liver diseases (Group B) and 19 anti-HBe positive patients with chronic liver diseases (Group C) were studied. HBV nucleic acid from serum samples was sequenced directly and compared with GenBank reference sequences HBV X01587 and M12906. On phylogenetic analysis, 76 cases were genotype C2. Of the 76 genotype C2 cases, the nucleotide and amino acid substitution rates in the precore/core region were significantly higher in Groups B and C than in Group A, also in Group C than in Group B. The nucleotide substitution rates in the full genome and the core promoter region were significantly higher in Group C than in Group A, also in group C than in Group B. The nucleotide and amino acid substitution rates in the X region were significantly higher in Group C than in Group A. The amino acid substitution rate in the pre-S2 region was significantly higher in Group C than in Group B. Deletion mutations were found mainly in Groups B and C. This whole genome analysis of HBV chronic carriers suggested that the nucleotide substitutions and deletions in HBV were closely associated with the pathogenesis of chronic HBV infection.     

Genotypic differences in the hepatitis B virus core promoter and precore sequences during seroconversion from HBeAg to anti-HBe

Hepatitis B virus (HBV) strains from anti-HBe positive patients often show specific mutations in the precore gene, the core promoter region, or both. The dynamics of seroconversion in relation to the appearance of these mutations has not been studied and compared between defined HBV genotypes. Samples from patients followed during seroconversion from HBeAg to anti-HBe were amplified by polymerase chain reaction (PCR), sequenced and genotyped. Among 16 sets of samples, 6 belonged to genotype A, 6 to genotype D, 2 to genotype B, 1 to genotype C, and 1 to genotype E. Whereas strains from genotypes B, C and E showed changes in the core promoter, precore codon 28 or both, genotype A and D strains displayed a different pattern. In 4 of 6 anti-HBe positive samples from genotype A, the precore had a wild-type sequence while the core promoter sequence showed a specific TGA mutation. In another genotype A strain a precore stop mutation was preceded by a mutation in codon 15, thus conserving base-pairing at the pregenomic RNA level in this region. In contrast, all genotype D strains showed wild-type sequences in both the core promoter and precore codon 28 in pre- and post-seroconversion samples. Thus, in 8 patients with a mean follow-up time of 17 months, wild-type sequences in both the core promoter and precore codon 28 were found after seroconversion to anti-HBe. This study also confirmed, for genotype D, that HBeAg seroconversion often occurs earlier than genomic conversion.     

Natural seroconversion from hepatitis Be antigen to antibody among hepatitis B virus carriers in Okinawa Islands

In the Okinawa Islands, the great majority of hepatitis B surface antigen (HBsAg) carriers have already acquired antibody to hepatitis Be antigen (anti-HBe) by the age of 30 years (preliminary cross-sectional data). To elucidate natural seroconversion from hepatitis Be antigen (HBeAg) to anti-HBe among HBsAg carriers found in the islands of Okinawa Prefecture, 34 HBeAg-positive HBsAg carriers were followed for 1-6 years with serial measurements of aminotransferase levels, HBeAg, and anti-HBe. The 34 subjects included 24 patients with chronic hepatitis (group 1) and ten asymptomatic HBsAg carriers (group 2). During the follow-up period, HBeAg disappeared from 14 subjects in group 1 with the cumulative clearance rate of HBeAg of 56.3% within the first 2 years and with 10 of the 14 subsequently developing anti-HBe. Moreover, the aminotransferases in 12 of the 14 spontaneously seroconverted fell into the normal range. The annual clearance rates of HBeAg among group 1 and group 2 were 25.6% and 9.3%, respectively. The tendency for early disappearance of HBeAg during a carrier's life time or in the course of chronic hepatitis may lead to the low death rate from hepatocellular carcinoma (HCC) particularly HCC associated with hepatitis B virus infection in this area.     

Genetic heterogeneity in the precore region of hepatitis B virus in hepatitis B e antigen-negative chronic hepatitis B Patients: Spontaneous seroconversion and interferon-induced seroconversion

To elucidate the relationship between the clinical severity of chronic liver disease and the precore mutations in hepatitis B e antigen (HBeAg)-nega-tive hepatitis B virus (HBV) carriers, mutations were investigated in the precore region of HBV DNA in 20 chronic hepatitis B patients who sero-converted either spontaneously or after the administration of α-interferon (IFN), and 5 asymptomatic carriers. The precore mutation with a stop codon at nucleotide 1896 was found in all patients, irrespective of the histology and in all asymptomatic carriers. The second mutation at nucleotide 1899 was found in 40% of cases studied but always followed by the first mutation at nucleotide 1896. The mixed viral infection of precore mutant and wild-type HBV virus was found in 40% of seroconverted cases after IFN treatment and in sera of HBV carriers obtained within a year after the spontaneous Seroconversion. These data suggest that the precore mutants prevail over wild-type HBV in all HBeAg-negative HBV carriers within several years after the sero-conversion, but their prevalence could not confine the clinical severity of chronic liver disease. © 1995 Wiley-Liss, Inc.     

Re-examination and further characterization of a monoclonal antibody to hepatitis B e antigen (anti-HBe)

A purification procedure for serum hepatitis B e antigen (HBeAg) was developed to immunize mice for monoclonal anti-HBe production. Two monoclonal anti-HBe secreting hybridomas were identified. Immunoglobulin G (IgG2a) was isolated from each hybridoma and labeled with either 125I or horseradish peroxidase. Each label was used as a probe in solid phase immunoassays for HBeAg and anti-HBe detection. Both monoclonal antibodies recognized the beta epitope on HBeAg, but one consistently performed better as a probe. When this monoclonal probe was compared to commercially available polyclonal assays, it showed equivalent sensitivity and specificity.     

Virus-like particles in the liver of a patient with fulminant hepatitis and antibody to hepatitis E virus

In earlier studies, hepatitis E virus (HEV) particles were detected in the stools of patients with enterically transmitted non-A, non-B (ENANB) hepatitis, and HEV was etiologically associated with this disease. Such particles have not been observed in the liver, however. We describe the pathological findings in the liver of a young pregnant woman from Nepal who died as a result of fulminant NANB hepatitis. IgM antibody to HEV was detected in the patient's serum by immune electron microscopy, suggesting that she was acutely infected with that virus. On light microscopic examination of the liver we observed cholestatic hepatitis with proliferation of bile ductules and pseudoglandular arrangement of hepatocytes around distended bile canaliculi. Three types of virus-like particles were detected by electron microscopy. The most frequently observed particles were in cells lining small bile ductules; they measured 32-37 nm and were enclosed by a membrane. Particles of a second type were seen in clusters in the sinusoidal cells; they were uniform in size, without a membrane, and measured about 32 nm in diameter. Particles of a third type (65 nm) were found in epithelial cells of the small bile ductules. Among the particles we detected, the 32 nm particles most closely resembled those of HEV.     

Analysis of HCV co-infection with occult hepatitis B virus in patients undergoing IFN therapy.

BACKGROUND/AIM: Occult hepatitis B virus (HBV) infection is characterized by the presence of HBV DNA in the absence of hepatitis B surface antigen (HBsAg) in the patient serum. Although such infections have been identified in patients with chronic hepatitis C, the clinical significance of those co-infections is still not understood. Our aim was, therefore, to assess the prevalence and clinical consequences of occult HBV infection in chronic hepatitis C patients undergoing antiviral therapy. METHODS: The study population consisted of 53 HBsAg-negative patients with chronic hepatitis C treated with IFN/ribavirin or IFN/ribavirin/amantadine. Nine patients experienced a viral breakthrough (BT), 30 were non-responders (NR) and 14 were responders (R). HBV-DNA detection by PCR was performed using primers specific for the S region of the HBV genome and HCV-RNA detection by PCR with primers localised in both the 5'NC and core region of HCV genome, before, during and after treatment. Viral genome sequences were also studied. RESULTS: Occult HBV genomes were found in the serum of four of 53 (7.5%) patients, unrelated to anti-HBc status. No significant differences in biochemical, virological, or histological markers, age, duration of infection, were observed in patients with or without HBV DNA. There was an inverse correlation in the evolution of HBV DNA and HCV RNA levels. Direct sequencing showed that S gene of occult HBV presented mutations in the "a" determinant while no specific mutation in the core region of HCV was observed. None of the four patients co-infected with HBV and HCV were responders to anti-HCV therapy. CONCLUSION: In our clinical setting, the prevalence of occult HBV co-infection among patients with chronic hepatitis C was low and independent of the presence of markers of previous HBV infection. Further studies in larger cohort of patients are warranted to determine if occult HBV co-infection may be involved in HCV resistance to combination therapy.     

Cytotoxic T cell responses in patients with chronic hepatitis B virus infection undergoing HBe antigen/antibody seroconversion

Cytotoxic T cells have been identified in the peripheral blood of patients with acute hepatitis B virus infections for a short period after clinical presentation. However, in patients in whom the virus persists these have been difficult to demonstrate. In the chronic infection during HBe antigen clearance, when there has been an exacerbation of the disease, we have been able to demonstrate MHC class I-restricted cytolytic response directed against the nucleocapsid antigens. In an HLA-A2 patient this was induced in vitro with the peptide pl8-27, previously described as an HLA-A2-restricted T cell epitope. In patients of other HLA types, recombinant core antigen was used to induce antigen-specific lysis: statistical analysis of the cytolytic responses of chronically infected patients demonstrated a nucleocapsid antigen-specific lysis in patients who were seroconverting. Removal of CD4⁺ cells reduced non-MHC-restricted cytolysis, allowing an MHC class I-restricted cytolytic component to be demonstrated.     

Lipomatous pseudohypertrophy of the pancreas in a patient with cirrhosis due to chronic hepatitis B

Lipomatous pseudohypertrophy of the pancreas was found at autopsy In a 52-year-old Japanese woman with cirrhosis due to chronic hepatitis B. Clinically, there were no clear symptoms of pancreatic Insufficiency during the entire course. Marked atrophy and fat deposition of the pancreas had already been detected by computed tomography (CT) at least 6 years before her death. She died of hepatic failure due to decompensated cirrhosis. Autopsy revealed uniform enlargement of the pancreas due to massive fat replacement (lipomatous pseudohypertrophy): the exocrine glandular elements showed marked atrophy and loss, while the islets of Langerhans were preserved. Although the etiology and pathogenesls of lipomatous pseudohypertrophy Is still unclear, this case suggests that this condition Is causally related to chronic hepatitis B or other chronic advanced hepatic lesions.     

Elimination of circulatory IgM anti-HSA precedes anti-HBe seroconversion in patients with CAH type B

The presence of IgM and IgA antibodies with specificity for human serum albumin (HSA) were consecutively analyzed in serum samples from four patients with biopsy verified CAH type B during seroconversion in the HBe/anti-HBe antigen system. Circulatory IgM anti-HSA antibodies were present during HBe antigenemia. The antibody titers fluctuated, decreased, and were finally lost from the circulation. After the disappearance of IgM anti-HSA antibodies, seroconversion to anti-HBe reactivity occurred and a quiescent phase of the disease was established, as judged by normalization of transaminases and absence of circulatory HBV-DNA. IgA anti-HSA antibodies persisted in the circulation after the elimination of IgM anti-HSA and seroconversion to anti-HBe reactivity. For one of the patients, a dramatic increase in titers was followed by elimination of IgA anti-HSA and seroconversion to anti-HBs. The data indicate that the host "self"-component HSA, when associated with "foreign" HBe or HBs antigenic structures, elicit immune responses to HSA, preventing the adequate development of anti-HBe and anti-HBs. The cessation of anti-HSA reactivity, however, seemed to permit subsequent sensitization to HBe and HBs antigenic determinants, as detected by the presence of circulatory antibodies.     

Toward a complete cure for chronic hepatitis B: Novel therapeutic targets for hepatitis B virus

Hepatitis B virus (HBV) affects approximately 250 million patients worldwide, resulting in the progression to cirrhosis and hepatocellular carcinoma, which are serious public health problems. Although universal vaccination programs exist, they are only prophylactic and not curative. In the HBV life cycle, HBV forms covalently closed circular DNA (cccDNA), which is the viral minichromosome, in the nuclei of human hepatocytes and makes it difficult to achieve a complete cure with the current nucleos(t)ide analogs and interferon therapies. Current antiviral therapies rarely eliminate cccDNA; therefore, lifelong antiviral treatment is necessary. Recent trials for antiviral treatment of chronic hepatitis B have been focused on establishing a functional cure, defined by either the loss of hepatitis B surface antigen, undetectable serum HBV DNA levels, and/or seroconversion to hepatitis B surface antibody. Novel therapeutic targets and molecules are in the pipeline for early clinical trials aiming to cure HBV infection. The ideal strategy for achieving a long-lasting functional or complete cure might be using combination therapies targeting different steps of the HBV life cycle and immunomodulators. This review summarizes the current knowledge about novel treatments and combination treatments for a complete HBV cure.     

Complete nucleotide sequences of hepatitis B virus genomes associated with epidemic fulminant hepatitis

Pre-core/core mutants are frequently observed in patients with fulminant hepatitis. To investigate the extent of molecular characteristics of hepatitis B virus (HBV) genomes implicated in the development of fulminant hepatitis, full-length HBV genomes were sequenced directly from sera of two patients with epidemic fatal fulminant hepatitis, after amplification by the polymerase chain reaction. These two genomes, of 3215 nucleotides, were 99.6% identical, indicating that a common source of HBV potentially caused fulminant hepatitis. Thirty unique nucleotide mutations were commonly found in the two entire HBV genomes. Three were located in the stem-loop structure, changing this element to a more stable structure. Twenty-five unique amino acid substitutions were found in each open reading frame, except for the X and pre-surface 2 genes. One was located in the pre-surface 1 gene; two were in the surface gene; three were in the pre-core gene, including codons 28 (tryptophan to stop codon) and 29 (glycine to aspartic acid); eight were in the core gene; and 11 were in the polymerase gene. The pre-core mutations at codons 28 and 29 were common to the two HBV strains reported previously in patients with epidemic fulminant hepatitis. Thus, HBV genomes associated with epidemic fatal fulminant hepatitis have numerous unique mutations, located mainly in the polymerase gene, as well as the pre-core/core gene, including mutations in the stem-loop structure of the pregenome encapsidation signal sequence. These mutations may be associated with the development of fulminant hepatitis. © 1996 Wiley-Liss, Inc.