Immune response to HBsAg, HBcAg and e-antigen in patients with acute hepatitis and HBsAg carriers with and without liver diseases

Humoral and/or cell-mediated (CMI) immune responses to HBAg components, human and rabbit liver specific proteins (HLP and RLP) and tuberculin were tested in patients with acute virus B and non-B-hepatitis, asymptomatic HBsAg carriers and HBsAg positive chronic active hepatitis (CAH). Furthermore, the presence of HBsAg, HBcAg and/or "e"-antigen has been studied in patients with sera and/or liver tissue. Asymptomatic HBsAg carriers are characterized by a status of immunological tolerance against HBsAg. HBcAg in liver nuclei could not be detected. All sera were positive for anti-HBc, some had anti "e". - Patients with uneventful acute virus-B-hepatitis developed CMI against HBsAg 4-6 weeks and anti-HBs 4-6 months after onset of the disease. Acute virus hepatitis without detectable HBsAg are defined as non-B-hepatitis by negative humoral and cell-mediated immune reaction against HBsAg 1-12 months after onset of the disease. - Patients with type B chronic active hepatitis are characterized by inadequate CMI against HBsAg without immune elimination of virus and virusantigens. Acute and chronic type-B-hepatitis showed temporary or constant CMI against HLP. These findings suggest an alteration or a carrier function of membrane antigens of virus infected hepatocytes or an induction of new membrane antigens by a virus. The results indicate that recovery from type B-hepatitis is associated with the ability to elicit a specific immune response to HBsAg. Furthermore immune responses to virus, virus antigens and virusinfected hepatocytes seemed to be the pathogenic principle of virus induced acute and chronic liver diseases.     

Functional analysis of HBV genomes from patients with fulminant hepatitis

Two previous case reports suggest that hepatitis B virus (HBV) core promoter variants with a high replication competence contribute to the pathogenesis of fulminant hepatitis B (FHB). We recently found in HBV genomes from patients with FHB an accumulation of mutations within the core promoter region. Therefore, the aim of this study was to investigate the phenotype of these HBV variants. Replication competence and expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) of viral genomes from seven patients with FHB and one patient with fulminant recurrent hepatitis after liver transplantation were analyzed by transfection experiments in human hepatoma cells. Compared with wild-type virus, the HBV variants from the seven patients with FHB produced similar or slightly lower levels of intracellular replicative intermediates and extracellular viral particles. In contrast, the HBV genomes from the patient with fulminant recurrent hepatitis synthesized and secreted significantly more HBV DNA. All genomes tested expressed similar or even higher levels of HBeAg compared with wild-type virus, except for those from four patients with a precore stop codon mutation in the respective dominant viral populations. The level of HBsAg produced by all variant genomes was similar or reduced compared with wild-type virus. These data indicate that in some cases HBV variants with enhanced replication competence and/or a defect in HBeAg expression may contribute to the development of FHB. However, neither phenotype is an essential prerequisite; thus, an additional role of other viral or host factors in the pathogenesis of FHB is suggested.     

Use of pharmacological agents in elucidating the mechanism of insulin action

Hepatitis B virus (HBV) mutants have recently been identified in patients with acute or fulminant as well as chronic infections. Naturally occurring mutations have been identified in all viral genes and regulatory elements. Mutations in the gene coding for the hepatitis B surface antigen (HBsAg) may result in infection or viral persistence despite the presence of antibodies against HBsAg (anti-HBs) ("vaccine escape" or "immune escape"). Mutations in the gene encoding the pre-core/core protein (pre-core stop codon mutant) result in a loss of hepatitis B e antigen (HBeAg) and sero-conversion to antibodies to HBeAg (anti-HBe) with persistence of HBV replication (HBeAg minus mutant). Mutations in the core gene may lead among others to an immune escape due to a T cell receptor antagonism. Mutations in the polymerase gene can be associated with viral persistence or resistance to nucleoside analogues. Thus, HBV mutations may affect the natural course of infection, viral clearance and response to antiviral therapy. The exact contribution of specific mutations to diagnosis and therapy of HBV infection as well as patient management in clinical practice remain to be established.     

Fibrosing cholestatic hepatitis in a transplant recipient with hepatitis B virus precore mutant

A patient with hepatitis B virus (HBV) precore mutant (seropositive for hepatitis B surface antigen [HBsAg], anti-hepatitis B e antigen [HBeAg], and HBV DNA) who underwent orthotopic liver transplantation for end-stage liver disease is described. Sequencing of the HBV precore region of the pretransplant serum sample confirmed the presence of the precore stop-codon mutant (G-->A mutation in codon 1896) only. The patient received HBV immunoglobulin prophylaxis for 6 months but HBV recurred thereafter with a mild hepatitic flare, and he remained seropositive for HBsAg, anti-HBe, and HBV DNA. The initial hepatitic illness resolved in 3 months. The patient remained well for another 16 months before presenting with fibrosing cholestatic hepatitis (FCH). During his entire initial hepatitic flare, quiescent period, and final FCH phase, he remained seropositive for HBsAg, anti-HBe, and HBV DNA. Moreover, sequencing of the serum HBV DNA in final FCH phase showed the presence of the identical HBV precore mutant. Immunohistochemical staining showed extensive expression of HBsAg/pre-S1, pre-S2, and hepatitis B core antigen, but HBeAg was scarcely detectable. This case illustrates that (1) recurrence of HBV precore mutant infection can occur in liver; (2) it can give rise to FCH; and (3) hepatic accumulation of HBeAg is not essential for the development of FCH.     

High degree of conservation in the hepatitis B virus core gene during the immune tolerant phase in perinatally acquired chronic hepatitis B virus infection

BACKGROUND: Mutations in the hepatitis B virus genome have been implicated in the persistence of hepatitis B virus infection and the pathogenesis of hepatitis B virus related liver disease. In view of the heterogeneity in published sequences, data from cross-sectional studies of unrelated subjects cannot differentiate true mutations from infections with variant sequences. AIMS/METHODS: We compared the hepatitis B virus core gene sequences of 42 HBsAg positive subjects from 11 Chinese families with those of the index patients (maternal carriers) to determine the frequency and rate of true hepatitis B virus core gene mutations in patients with chronic hepatitis B virus infection. RESULTS: Completely identical nucleotide sequences were present in all the family members and index patients in two families, suggesting that the hepatitis B virus core gene can be conserved for more than 20 years. The high degree of sequence conservation in these families is related to the young age of the subjects (mean 19.2+/-8.9 years), the fact that they were all HBeAg positive and that 75% of them had persistently normal aminotransferase levels. Longitudinal studies confirmed that mutations were rare in those who remained HBeAg positive with normal aminotransferase levels (immune tolerant phase), but significantly more common in HBeAg positive subjects who had elevated aminotransferase levels and in those who cleared HBeAg (immune clearance phase), the rates of nucleotide and amino acid changes were respectively: 0.28+/-0.12 vs 1.30+/-0.26/10(3) nt position/yr and 0.04+/-0.01 vs 0.18+/-0.5/10(2) codon/yr. CONCLUSIONS: Identical nucleotide differences could be found in the sequences of all the subjects in some families. These differences were more likely to be due to intra-familial transmission of stable variants. Sequence analysis based on comparisons with published sequences would have led to over-reporting of mutations. The hepatitis B virus core gene can remain highly conserved for more than two decades during the immune tolerant phase of perinatally acquired chronic hepatitis B virus infection. However, significant changes can occur within 2-3 years during the immune clearance phase.     

Detection of hepatitis B surface antigen in pregnant women attending a public hospital for delivery: implication for vaccination strategy in Bangladesh

Routine antenatal hepatitis B surface antigen (HBsAg) screening and immunization of risk babies is very effective in preventing perinatal transmission of hepatitis B virus (HBV). We studied 1,800 parturients attending a public hospital to assess the rationale for such vaccination in Bangladesh. In one in every 29 deliveries (63 of 1,800 or 3.5%), the mother was found to be HBsAg positive. All were asymptomatic and many (41 of 63 or 65%) without risk factors would remain undetected if HBsAg screening were performed on selected groups. Most of the HBsAg-positive mothers (54 of 63 or 85.7%) were found to be chronic carriers and 30.2% (19 of 63) were also hepatitis B e antigen (HBeAg) positive, indicating high infectivity. Although 23 cord blood were positive for HBsAg or HBeAg, none were positive for IgM antibody to hepatitis B core antigen (IgM anti-HBc), suggesting transplacental transmission of the antigens rather than intrauterine infection. These findings are discussed in relation to the cost-effectiveness of routine prenatal screening and immunization of risk babies compared with universal infant immunization.     

Nested polymerase chain reaction (PCR) and multiple cloned antibody capture PCR for the examination of serum hepatitis B virus DNA in negative hepatitis B surface antigen patients suffering from liver diseases

In order to find out rapidly the causes of the liver diseases suffered by patients with negative hepatitis B surface antigen (HBsAg), nested polymerase chain reaction (PCR) and multiple cloned antibody capture PCR techniques were established to examine serum hepatitis B virus (HBV) DNA. By using both techniques along with the examination of hepatitis C virus (HCV) infection, the causes of chronic liver diseases with negative HBsAg were studied. It is found that nested-PCR can increase the sensitivity of single PCR more than 1,000 fold and multiple cloned antibody capture-PCR can detect concentration of HBV DNA as low as 0.1-0.01 pg/L. HBV DNA positive patients were found in 45.5%, 30.8%, 13.3% and 100% respectively of the patients suffering from liver cirhosis with negative HBsAg (group A, 22 cases), chronic hepatitis with negative HBsAg (group B, 13 cases), normal subjects with negative HBsAg and positive hepatitis B core antibody (HBcAb, group C, 30 cases) and liver cirhosis with positive HBsAg and negative HBeAg (group D, 12 cases). HBV DNA can be also found in the serum of HBsAb positive patients and subjects supposed to be healthy, 81.8% and 53.8% of the patients were infected with HBV and/or HCV in group A and group B respectively. All these results suggest that nested-PCR and multiple cloned antibody capture-PCR are rapid and highly sensitive methods for detection of serum HBV DNA. HBV infection is an important cause of chronic liver diseases in patients with negative HBsAg. The causes of most of the HBsAg-negative chronic liver diseases are related with infection of viruses. The clinical significance of serum HBsAb in naturally infected patients should be reconsidered.     

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The clinical importance of hepatitis B virus (HBV) genome variability has been reported recently. One example is the occurrence of hepatitis B virus pre-core mutants, which arise during spontaneous or interferon-induced seroconversion from HBeAg to anti-HBe and are thought to be selected by immune pressure. A survey of HBV pre-core mutants and viral genotypes in 35 HBeAg negative patients during interferon therapy was carried out to understand viral pathogenesis in this form of chronic hepatitis B. Seventeen patients responded to interferon therapy as assessed by the sustained normalization of serum ALT levels and the significant decrease of viremia levels. The response rate to interferon was independent of both initial serum viral DNA level and interferon doses. During interferon therapy, a significant decrease of M0 (wild-type pre-core sequence at pos. 1887-1908), M1 (TGG to TAG at pos. 1896) or M2 (TGG to TAG at pos. 1896, and GGC to GAC at pos. 1899) positive viral genomes was found in 48%, 42%, and 33% of patients, respectively. A higher response rate to interferon therapy was observed in patients infected with HBV genotype A (70%) or M0 positive strains (75%) as compared to patients infected with genotype D/E (40%) or M1/M2 positive strains (44%). The data support the hypothesis that pre-core defective HBV represent viral mutants with an increased capacity to resist exogenous alpha interferon. These findings emphasize that characterization of HBV genome variability prior to interferon therapy may help to predict antiviral response in HBeAg negative patients.     

Point mutations in the S and pre-S2 genes observed in two hepatitis B virus carriers positive for antibody to hepatitis B surface antigen

Two hepatitis B virus (HBV) carriers who had antibodies to HBV surface antigen (anti-HBs) were studied. Case 1 was a 47 year old woman positive for hepatitis B e antigen (HBeAg), and case 2 was a 61 year old man positive for antibody to HBeAg (anti-HBe) and DNA-polymerase (DNA-p). Neither case had received the HBV vaccine. The nucleotide sequences of the HBV-DNA extracted from the patients' sera were determined within the pre-S2 and S genes. Seven out of nine S gene clones from case 1 and six out of nine S gene clones from case 2 had an amino acid replacement from Thr or Ile to Ser at codon 126 in the alpha-determinant of the S gene. Amino acid substitution of codon 145 of the S gene previously reported was not observed. Although two previous reports on HBV escape mutant carriers with both anti-HBs and HBeAg described some deletions in the pre-S2 gene, our cases did not show these deletions. Our analysis indicated that carriers with the HBV escape mutant did not always have pre-S2 gene deletions. We found two HBV escape mutant carriers who had amino acid substitutions at codon 126 in the S gene due to point mutation without any deletions in the pre-S2 gene.     

The intrahepatic expression of the hepatitis B virus in chronic delta hepatitis

In order to evaluate the interference of hepatitis delta virus (HDV) in hepatitis B viral particle (HBsAg, HBcAg) expression in the liver of chronic HDV patients, 39 and 81 liver biopsies of HBsAg carriers seropositive for anti-HDV and anti-HDV negative controls, respectively, were studied. HBcAg was positive in 16.7% of the HBeAg-positive patients with HDAg in the liver and in 91,4% of controls. In contrast, in HBeAg- and anti-HDV negative patients the intrahepatic expression of HBcAg was detected in 32.6%. In anti-HDV negative patients the HBcAg liver expression correlated significantly with the HBeAg in serum (p < 0.00001). The distribution of HBcAg was exclusively cytoplasmatic in 30% of HDV-infected patients but mixed nuclear and cytoplasmic in 38.3% of the controls. The nuclear expression of HBcAg was decreased in chronic HDV infection. HBsAg was positive in 70.3% of patients who were anti-HDV positive and in 82.3% of controls. The membranous expression of HBsAg was detected less frequently in HDV-infected patients (p < 0.05) than in controls, while associated with HBeAg in serum of HBV carriers without HDV superinfection (p < 0.00001). The prevalence and the HBsAg cytoplasmic expression was not different for the chronic HDV infection or controls. Our results show: 1) decreased intrahepatic expression of HBcAg and membranous HBsAg in HBV carriers superinfected with HDV, suggesting decreased HBV replication in the liver of these patients. 2) the changing of HBcAg and HBsAg expression in the liver of HDV-infected patients, suggest not so much a decrease but rather a modulation in HBV replication.     

Lymphocytotoxins in leprosy and in asymptomatic hepatitis B virus infection

Serum lymphocytotoxic antibodies (LCAs) were detected in 67% of Papua New Guinean lepromatous leprosy patients who were persistent carriers of hepatitis B surface antigen (HBsAg). Lymphocytotoxins were not associated with asymptomatic HBsAg in either healthy controls or tuberculoid leprosy patients. It was apparent that, although HBsAg itself is a poor indicator of in vitro lymphocytotoxicity, when the antigen occurred in a host with impaired immune response, lymphocytotoxicity, when the antigen occurred in a host with impaired immune response, lymphocytotoxicity was enhanced. In contrast to this finding, lepromatous leprosy patients without HBsAg had significantly depressed LCA production in comparison with tuberculoid patients and controls. The interaction between leprosy and hepatitis B virus was highly significant (P = 0.001) in an analysis of variance of cytotoxicity scores. It is proposed that the previously reported equivocal results regarding autoantibodies in leprosy patients may be explained by this unusual interaction between lepromatous leprosy and hepatitis B virus infection.     

Hepatitis B virus infection in dental surgical practice

Sixty-one dental surgeons at King's College Hospital were interviewed to establish the incidence of attacks of viral hepatitis and to relate this to environmental risk factors. Six (10%) had a history of hepatitis, in one case due to infection with the hepatitis B virus. Screening blood for HBsAg by radioimmunoassay showed no carriers of the antigen, but transient antigenaemia was observed in one dentist. Antibody to HBsAg, tested by radioimmunoassay, was detected in four dentists (7%), only one of whom had had clinical hepatitis. Dental surgeons may be more at risk from infection with the hepatitis B virus than the general population, although this should be minimised in hospital practice, where the most infected patients will already have been identified and appropriate precautions can be taken. The risk of transmission from an antigen-positive dentist to his patients is probably much smaller, and there is no evidence to restrict his clinical activities.     

Inhibitory effect of 9-(2-phosphonylmethoxyethyl)-adenine (PMEA) on human and duck hepatitis B virus infection

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) was evaluated for its inhibitory effect on hepadnavirus replication in three different cell systems, i.e., human hepatoma cell lines HepG2 2.2.15 and HB611 (transfected with human hepatitis B virus (HBV)) and primary cultures of duck hepatocytes infected with duck hepatitis B virus (DHBV). PMEA inhibited HBV release from HepG2 2.2.15 cells and HB611 cells at a 50% inhibitory concentration (IC50) of 0.7 and 1.2 microM, respectively. Intracellular viral DNA synthesis was inhibited at concentrations equivalent to those required to inhibit virus release from the cells. DHBV secretion from duck hepatocytes was inhibited by PMEA at an IC50 of 0.2 microM. HBsAg secretion was inhibited by PMEA in a concentration-dependent manner in HB611 cells and DHBV-infected duck hepatocytes but not HepG2 2.2.15 cells. The 50% cytotoxic concentration, as measured by inhibition of [3H-methyl]deoxythymidine incorporation was 150 microM for the two human hepatoma cell lines and 40 microM for the duck hepatocyte cultures. In a pilot experiment PMEA was found to reduce the amounts of DHBV DNA in the serum of Pekin ducks.     

Factors associated with intrafamilial transmission of hepatitis B virus infection in Korea

Epidemiologic and serologic data on 137 household contacts of 51 chronic carriers of HBsAg and 111 household contacts of 38 controls who were negative for serologic markers of hepatitis B virus (HBV) were obtained from March 1990 to August 1991. Using this data, possible routes of intrafamilial transmission of hepatitis B virus among household contacts of chronic carriers of hepatitis B surface antigen (HBsAg) were evaluated and analyzed. The HBsAg prevalence among the household contacts of carriers was 14. 1% (95% CI 7.8-24.0) compared to 0.0% (95% CI 0.0-7.0) among those of controls (P < 0.01). The offspring of carriers showed significantly higher risk of HBV infection(relative risk; 6.6). Sharing of towels and handkerchieves, and drinking vessels was associated with an increased risk of HBV infection via intrafamilial transmission in Korea (relative risk 11.5 for towel and handkerchief, 12.1 for drinking vessels).     

Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis

The molecular and cellular mechanisms responsible for cytotoxic T lymphocyte (CTL)-induced immunopathology are not well defined. Using a model in which hepatitis B surface antigen (HBsAg)-specific CTL cause an acute necroinflammatory liver disease in HBsAg transgenic mice, we demonstrate that class I-restricted disease pathogenesis is an orderly, multistep process that involves direct as well as indirect consequences of CTL activation. It begins (step 1) almost immediately as a direct antigen-specific CTL-target cell interaction that triggers the HBsAg-positive hepatocyte to undergo programmed cell death (apoptosis). It progresses (step 2) within hours to a focal inflammatory response in which antigen-nonspecific lymphocytes and neutrophils amplify the local cytopathic effect of the CTL. The most destructive pathogenetic function of the CTL, however, is to secrete interferon gamma when they encounter antigen in vivo, thereby activating the intrahepatic macrophage and inducing a delayed-type hypersensitivity response (step 3) that destroys the liver and kills the mouse. We propose that the principles illustrated in this study are generally applicable to other models of class I-restricted, CTL-induced immunopathology, and we suggest that they contribute to the immunopathogenesis of viral hepatitis during hepatitis B virus infection in humans.     

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Co-Cinobufotalin Oral Liquor (CCOL) was studied for its ability to inhibit hepatitis B virus DNA replication, HBsAg and HBeAg expression in a HBV-transfected cell line (2.2.15 cell). The result showed that ID50 (the drug concentration that inhibits HBsAg or HBeAg secretion by 50%) was 0.08 mg/ml and 0.07 mg/ml on HBsAg and HBeAg respectively. CD50 (the drug concentration that reduces cell growth by 50%) was 2.5 mg/ml. TI (therapeutic index) was 31.3 and 35.7 respectively. The present data suggest that CCOL could exert a potent antiviral activity against HBV in vitro. Southern blot showed that CCOL inhibited HBV-DNA repication in a dose-dependent manner.     

Monitoring of antiviral therapy with quantitative evaluation of HBeAg: a comparison with HBV DNA testing

The serological endpoint of response in the treatment of chronic hepatitis B is the loss of hepatitis B virus DNA and HBeAg. Because the quantitative measurement of hepatitis B virus DNA in serum has been shown to be useful for monitoring and predicting response to interferon-alpha therapy, we decided to evaluate whether changes in HBeAg concentration could also be used in this manner. Twenty-nine patients who were initially positive for HBeAg and HBV DNA were serially evaluated for HBeAg concentration with a microparticle-capture enzyme immunoassay. HBeAg levels in serum were calculated by means of comparison with a standard curve of fluorescence rate vs. HBeAg concentration. The results, expressed in milliunits per milliliter, were compared with hepatitis B virus DNA levels determined by means of solution hybridization. The baseline HBeAg concentration proved to be the best independent predictor of response on stepwise Cox regression analysis (p = 0.026). Similar disappearance curves were observed for the two markers, although hepatitis B virus DNA became undetectable at an earlier interval in 13 of 16 cases (81%). In the 16 responders, a decline in HBeAg concentration of more than 90% was observed by wk 12 of therapy (mean +/- S.D., 95% +/- 13%). Nonresponders did not demonstrate such steep declines in HBeAg values by wk 12 (mean +/- S.D., 45% +/- 27%), and levels tended to increase at subsequent time points. We conclude that serial monitoring of HBeAg concentration with a technique that should be readily adaptable to clinical laboratories may be useful in the initial evaluation and monitoring of patients undergoing antiviral therapy.     

GB virus-C/hepatitis G virus infection in an area endemic for viral hepatitis, chronic liver disease, and liver cancer

BACKGROUND & AIMS: GB virus-C/hepatitis G virus (GBV-C/HGV) is a newly identified flavivirus, and little is known about its clinical significance. GBV-C/HGV was investigated in different populations, and its coinfection was investigated in patients with liver disease in Taiwan where hepatitis B and C are endemic. METHODS: Viral RNA was studied in 70 high-risk individuals, 20 patients with chronic non-B, non-C hepatitis, 13 with non-A-E fulminant hepatitis, 100 with asymptomatic hepatitis B surface antigen carriage, 120 with hepatitis B surface antigen-positive chronic liver disease and hepatocellular carcinoma, 100 patients with chronic hepatitis C, and 100 healthy adults. RESULTS: GBV-C/HGV infection was more frequent in high-risk groups (15%-30%) and hepatitis C virus carriers (10%) than in healthy adults (1%) and hepatitis B virus carriers (3.2%). Eighty-three percent of those infected had undergone blood transfusions previously. The prevalence in hepatitis B virus carriers increased with the severity of liver disease, being 1% in asymptomatic carriers and 10% in hepatocellular carcinoma. In chronic hepatitis C, clinical and virological data were comparable between those with and without coinfection. CONCLUSIONS: In Taiwan, GBV-C/HGV infection is common in high-risk groups, and its coinfection seems to not aggravate the course of chronic hepatitis B or C.