Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference

RNA interference (RNAi) mediated inhibition of virus-specific genes has emerged as a potential therapeutic strategy against virus induced diseases. Human hepatitis B virus (HBV) surface antigen (HBsAg) has proven to be a significant risk factor in HBV induced liver diseases, and an increasing number of mutations in HBsAg are known to enhance the difficulty in therapeutic interventions. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily based on the effectiveness and specificity of the RNAi targeting sequence. To explore the therapeutic potential of RNAi on HBV induced diseases in particular resulted from aberrant or persistent expression of HBsAg, we have especially screened and identified the most potent and specific RNAi targeting sequence that directly mediated inhibition of the HBsAg expression. Using an effective DNA vector-based shRNA expression system, we have screened 10 RNAi targeting sequences (HBsAg-1 to 10) that were chosen from HBsAg coding region, in particular the major S region, and have identified four targeting sequences that could mediate sequence specific inhibition of the HBsAg expression. Among these four shRNAs, an extremely potent and highly sequence specific HBsAg-3 shRNA was found to inhibit HBsAg expression in mouse HBV model. The inhibition was not only preventive in cotransfection experiments, but also had therapeutic effect as assessed by post-treatment protocols. Moreover, this HBsAg-3 shRNA also exhibited a great potency of inhibition in transgenic mice that constitutively expressed HBsAg. These results indicate that HBsAg-3 shRNA can be considered as a powerful therapeutic agent on HBsAg induced diseases.     

Atomic force microscopy detection of serological markers of viral hepatites B and C

The possibility of detection of serological markers, containing the hepatitis B surface antigen (HBsAg) and hepatitis C virus core-antigen (HCVcoreAg) in human serum, by a new atomic force microscopy (AFM)-based nanotechnological approach has been demonstrated. The antibodies against the hepatitis B virus surface antigen (anti-HBsAg) and the antibodies against the hepatitis C virus core antigen (anti-HCVcoreAg) were immobilized on an AFM-chip. It was shown that such approach enables to detect HBsAg, HCVcoreAg and the viral fragments containing these antigens in the serum. The comparative analysis of detection of HBsAg- and HCVcoreAg-containing particles by the AFM method versus traditional methods (ELISA, PCR) has demonstrated the 75% coincidence of results between the AFM and two other methods.     

Prognosis for the patients with chronic hepatitis B

The purpose of the research was to determine the influence of the hepatitis B virus on the progression of the chronic liver disease. In the present paper, 127 patients who were followed up for five years and who had histologically verified chronic liver disease, are described. Fifty two of them were carriers of HBsAg, 75 patients were HBsAg negative, but had other markers typical for a previous infection of HBV in the sera. All the patients were nonalcoholics and no drug addicts. In the sera of these 127 patients markers of HBV were prospectively followed up: HBsAg, HBeAg, anti-HBs, anti-HBc, anti-HBe, HBVDNA, antiHCV for C virus and anti-D for D virus. It was proved by these investigations that HBV provokes very severe chronic hepatitis: CAH (chronic active hepatitis) and CH (cirrhosis hepatis). It was also proved that HBV replicated in 44.20% patients, namely, HBVDNA was positive in the sera of those patients. In 26.08% of such patients the mutant form of HBV was present. In spite of progressive liver disease and without any antiviral therapy all the patients with chronic HBV cirrhosis hepatis were, after five year-follow-up, in Child-Pugh A grade. It was found that the patients who were HBsAg negative, but had one or more markers of HBV positive in the sera, had also a severe chronic hepatitis. That group of patients remains our object of further research. The five-years follow-up of all these patients demonstrates that it is necessary to find out an efficient medicament against HBV chronic hepatitis. Obligatory vaccination of the risk population against virus B remains the only prevention against this severe disease.     

Endocytosis of hepatitis B immune globulin into hepatocytes inhibits the secretion of hepatitis B virus surface antigen and virions

Hepatitis B immunoglobulin is used for prophylaxis against hepatitis B virus (HBV) and is thought to act by neutralization of virions and hepatitis B virus surface antigen (HBsAg)-containing particles in circulation. Using a panel of hepatocyte-derived cell lines, the present study investigated in vitro whether HBs-specific immunoglobulin G (IgG) is internalized in hepatocytes and whether it interacts with HBsAg in the cells. By immunoelectron microscopy and immunoblotting, human IgG and FcRn receptor for IgG were demonstrated on cellular membranes and in cytoplasmic extracts, irrespective of the HBsAg status of the cells. Furthermore, HBsAg and anti-HBs were shown to be colocalized in the same cellular compartment by two-color confocal microscopy. Endocytosis of HBs-specific IgG caused intracellular accumulation of HBsAg in a dose-dependent manner and inhibited the secretion of HBsAg and HBV virions from the cells. These effects were not observed with F(ab)(2) fragments or nonimmune IgG as controls. The specificity of intracellular HBsAg- anti-HBs interaction was further investigated in cells transfected with HBV genomes expressing wild-type HBsAg or immune escape HBsAg (with a G145R mutation). Monoclonal anti-HBs markedly reduced the secretion of wild-type HBsAg, while the secretion of mutant HBsAg was not affected. These results suggest that HBs-specific IgG binds to hepatocytes and interacts with HBsAg within the cells. This may be relevant for the selection of surface antibody escape mutations.     

Chronic Hepatitis Associated with Drug Abuse: Significance of Hepatitis B Virus

One hundred and seventy-seven former heroin addicts, consisting of 85 who were newly admitted to a methadone maintenance program and 92 who had received methadone for a mean period of 30 months, were prospectively studied for up to 2 years in order to determine: (1) the effect of heroin withdrawal on the hepatic abnormalities, and (2) the incidence of HBsAg, anti-HBs, and anti-HCc as indices of the frequency of hepatitis B virus infection. Our study indicates that (1) hepatic abnormalities persist when heroin is discontinued and are not temporally related to drug and/or needle usage, and (2) that 71% of subjects had either HBsAg or anti-HBs; anti-HBc was tested for in 16 patients and was present in 100%, although 9 of the 16 were both HBsAg- and anti-HBs-negative. This study suggests that hepatitis B is largely responsible for the liver dysfunction. It is proposed that an abnormality in immune function, induced by heroin, is responsible for the high incidence of chronic hepatitis. Attention is drawn to the similarity between former drug addicts and hemophiliacs, since both develop chronic hepatitis in spite of anti-HBs in the serum.     

HBsAg-positive chronic liver disease: inhibition of DNA polymerase activity by vidarabine.

Four patients who had chronic liver disease and were positive for hepatitis B surface antigen (HBsAg) were treated with vidarabine, a synthetic purine nucleoside that inhibits DNA polymerase activity in vitro and in vivo. Before treatment all had raised serum DNA polymerase concentrations. Three also had hepatitis B e (HBe) and were shown by electron microscopy to have hepatitis B virus (Dane) particles in their serum. In all patients 10 days'' treatment with vidarabine resulted in an immediate loss of DNA polymerase activity. In three patients the activity returned when treatment was stopped. In those three patients Dane particles and HBe antigen persisted during and after treatment; in the fourth patient, who remained negative for DNA polymerase, HBsAg titres fell. Although vidarabine inhibited virus replication, virus particles did not disappear from the blood in these patients, presumably because the particles were cleared only slowly. Similar results with interferon suggest that the virus disappears, and HBsAg titres fall, some weeks after the fall in DNA polymerase activity. Continued treatment may therefore have a sustained effect on viral replication. Whether vidarabine can permanently clear HBsAg and so arrest chronic liver disease remains to be seen, but at the very least it could reduce the spread of infection.     

Cellular and humoral immunity to hepatitis-B surface antigen in active chronic hepatitis.

The hepatitis-B surface antigen (HBsAG) may be persistently present in the serum in a few cases of active chronic hepatitis but the cause of the disease in most patients is unknown. In a study of 39 HBsAg-negative cases cell-mediated immunity to HBsAg was observed in 24 (62%), suggesting a high frequency of previous infection with the hepatitis-B virus. Hepatitis-B surface antibody was detectable by radioimmunoassay in six patients, in all of whom complexes of HBsAg were present in the serum on electron microscopy. Out of 12 patients with HBsAg-positive active chronic hepatitis who were also studied eight, including all those untreated at the time, showed a cellular response to the antigen. Evidence of sensitization to a liver-specific cell surface lipoprotein was found with similar frequency in the two groups. These results are consistent with the hypothesis that hepatitis-B virus infection is important in initiating the disease in many cases of active chronic hepatitis and that sensitization to the liver cell membrane antigen is the autoimmune process responsible for the perpetuation of the liver injury.     

Antinuclear antibody (anti-HBc) and liver pathology in acute and chronic virus B hepatitis]

HBsAg and anti-HBc, the antibody to core antigen of hepatitis B virion, were titrated by solid phase radioimmunoassay in 40 sera of HBsAg carriers with acute and chronic hepatitis and in 20 healthy subjects carrying anti-HBc alone or associated with anti-HBs. No correlation was found between HBsAg and anti-HBc titers in the single category of patients. In contrast, geometric mean titer of anti-HBc (ranging from 2(14) to 2(15)) of patients with chronic active hepatitis was significantly higher ( p = < 0.01) than that of patients with acute or chronic persistent hepatitis and healthy HBsAg carriers (ranging from 2(9) to 2(14)). Anti-HBc titer of 20 subjects without detectable HBsAg was less than 2(7). These data suggest that in subjects with persistent B virus infection, anti-HBc response is correlated with synthesis of viral genome rather than of surface antigens, so that a much higher titer of anti-HBc was detected only in patients with a more active liver disease.     

Antibody to hepatitis B core antigen in chronic active hepatitis.

Antibody to hepatitis B core antigen (anti-HBc), which has been assumed to be a more sensitive indicator of hepatitis B virus replication than hepatitis B surface antigen (HBsAg), was detected in the sera of 26 of our 65 patients with HBsAg-negative chronic active hepatitis. Thus despite the absence of HBsAg the liver disease could be the consequence of chronic infection with hepatitis B virus in these patients. They differed, however, from a group of 35 patients with HBsAg-positive hepatitis in being older on average and having less active liver lesions. The two groups could represent either two stages of chronic infection with hepatitis B virus or two types of response to it.     

Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope.

Hepatitis delta virus (HDV)-associated particles were purified from the serum of an experimentally infected chimpanzee by size chromatography and by density centrifugation. Hepatitis delta antigen (HDAg) was detected after mild detergent treatment at a column elution volume corresponding to 36-nm particles and banded at a density of 1.25 g/ml. The serum had an estimated titer of 10(9) to 10(10) HDV-associated particles and had only a 10-fold excess of hepatitis B surface antigen (HBsAg) not associated with HDAg. Therefore, HDV appears to be much more efficiently packed and secreted than is its helper virus, hepatitis B virus (HBV), which is usually accompanied by a 1,000-fold excess of HBsAg. The protein compositions of the HDAg-containing particles were analyzed by immunoblotting with HDAg-, HBsAg-, and hepatitis B core antigen-specific antisera and monoclonal antibodies to HBV surface gene products. The HBsAg envelope of HDAg contained approximately 95% P24/GP27s, 5% GP33/36s, and 1% P39/GP42s proteins. This protein composition was more similar to that of the 22-nm particles of HBsAg than to that of complete HBV. The significant amount of GP33/36s suggests that the HBsAg component of the HDV-associated particle carries the albumin receptor. Two proteins of 27 and 29 kilodaltons which specifically bound antibody to HDAg but not HBV-specific antibodies were detected in the interior of the 36-nm particle. Since these proteins were structural components of HDAg and were most likely coded for by HDV, they were designated P27d and P29d.     

The use of radioimmune assay in investigating reagents to be used in the immunocytochemical localization of hepatitis B surface antigen in immune complexes in the kidney of patients with membranous nephropathy and Australia antigenaemia

Radioimmune assay (RIA) was used to investigate the effect of fixatives on antigenicity of the hepatitis B surface antigen (HBsAg) and the effect of pronase on the elution of antibody (Ab) from the HBsAG-Ab complex. The effect of pronase on Ab elution was also tested on sections of kidney from a patient with the immune complex disease systemic lupus erythematosus (SLE). Immunoglobulin G (IgG) was located in pronase treated and untreated sections using the indirect immunoperoxidase technique. Glutaraldehyde was shown to be the fixative of choice for studies involving HbsAG. All fixatives were shown to have less effect on antigenicity at 4 degrees C than at room temperature. Osmium tetroxide reduced antigenicity to one-third, even at 4 degrees C. RIA and SLE kidney section studies showed that Ab was eluted from immune complexes by pronase. Pre-fixation of the antigen (Ag) by glutaraldehyde appears to have no effect on the final elution, although fixation after pronase treatment seemed to enhance the elution effects. The availability of an RIA kit with HBsAg- and AB-coated beads was of great assistance in evaluating reagents to be used in immunoperoxidase studies of HBsAg in immune complexes of patients with membranous nephropathy and Australia antigenaemia.     

蚓激酶在慢性乙型肝炎治疗中的潜在作用

纤连蛋白(FN)是参与乙型肝炎病毒感染和肝脏纤维化的重要分子。 蚓激酶(LK)是从赤子爱胜蚓(Eisenia foetide)中提取的一组蛋白水解同工酶,能水解纤维蛋白治疗凝血相关疾病。 从这组同工酶中分离纯化出单一活性成分,在体外可降解纤连蛋白,被命名为蚯蚓纤连蛋白水解酶(EFNase)。 然而,LK 能否预防乙型肝炎病毒感染以及缓解因乙型肝炎导致的肝脏损伤等问题还不清楚。本研究以人肝癌细胞系HepG2.2.15为细胞模型,观察LK 对乙型肝炎表面抗原(HBsAg)或 FN 水平的影响;以 C57BL / 6J-HBV 转基因小鼠为动物模型,探讨 LK 对小鼠血清中 HBsAg、FN、丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)水平及肝脏病理改变的影响。结果表明,LK 在体内外均能抑制HBsAg 的生成,降低血清和肝脏FN。与生理盐水处理组相比,LK 改善了肝脏的状态。这些数据为理解LK 作为治疗乙型肝炎的潜在有效药物的治疗作用提供了有价值的信息。     

The characteristics of the immune response during the development of hepatitis B against a background of chronic alcoholic liver lesions

In two groups of patients with viral hepatitis B (100 patients in each group) the comparative evaluation of immune response was carried out on the basis of the results obtained in the study of the duration of the presence of HBsAg in the blood, the time of the primary appearance of anti-HBsAg antibodies, cell-mediated and humoral immunity characteristics. The study revealed the aggravating influence of alcohol on the outcome of hepatitis B, manifested by the prolonged circulation of HBsAg, decreased antibody formation and T-immunosuppressive deficiency, which was linked with defective immune response in hepatitis B patients with disposition to the excessive use of alcohol.     

Importance of markers of hepatitis B virus in alcoholic liver disease.

To determine the importance of the presence of serological markers of hepatitis B virus infection in patients with alcohol related liver disease we compared cumulative alcohol intake and clinical and histological features in patients with markers of hepatitis B virus infection and in those without. Hepatitis B surface antigen (HBsAg) was detected in five (2%) out of 285 patients studied and antibody to HBsAg (anti-HBs) in 41 (14%); one patient had antibody to hepatitis B core antigen alone. The combined prevalence of markers of hepatitis B virus infection was similar in patients with alcoholic cirrhosis (18%) and precirrhotic liver disease (13%). Two patients positive for HBsAg had histological features of both alcoholic liver disease and chronic active hepatitis, with stainable HBsAg. Patients with anti-HBs were, however, histologically indistinguishable from patients without markers, and the mean cumulative alcohol intake of patients with anti-HBs was similar to or even higher than that of patients with liver disease of comparable severity who had no evidence of previous infection. The presence of markers of hepatitis B virus infection was related to former residence in countries with a high prevalence of the infection and to previous parenteral treatment and blood transfusions. Infection with hepatitis B virus does not enhance the development of chronic liver disease in heavy drinkers, except in the small number who remain positive for HBsAg.     

Vaccine- and hepatitis B immune globulin-induced escape mutations of hepatitis B virus surface antigen

Hepatitis B virus surface antigen (HBsAg) vaccination has been shown to be effective in preventing hepatitis B virus (HBV) infection. The protection is based on the induction of anti-HBs antibodies against a major cluster of antigenic epitopes of HBsAg, defined as the 'a' determinant region of small HBsAg. Prophylaxis of recurrent HBV infection in patients who have undergone liver transplantation for hepatitis B-related end-stage liver disease is achieved by the administration of hepatitis B immune globulins (HBIg) derived from HBsAg-vaccinated subjects. The anti-HBs-mediated immune pressure on HBV, however, seems to go along with the emergence and/or selection of immune escape HBV mutants that enable viral persistence in spite of adequate antibody titers. These HBsAg escape mutants harbor single or double point mutations that may significantly alter the immunological characteristics of HBsAg. Most escape mutations that influence HBsAg recognition by anti-HBs antibodies are located in the second 'a' determinant loop. Notably, HBsAg with an arginine replacement for glycine at amino acid 145 is considered the quintessential immune escape mutant because it has been isolated consistently in clinical samples of HBIg-treated individuals and vaccinated infants of chronically infected mothers. Direct binding studies with monoclonal antibodies demonstrated a more dramatic impact of this mutation on anti-HBs antibody recognition, compared with other point mutations in this antigenic domain. The clinical and epidemiological significance of these emerging HBsAg mutants will be a matter of research for years to come, especially as data available so far document that these mutants are viable and infectious strains. Strategies for vaccination programs and posttransplantation prophylaxis of recurrent hepatitis need to be developed that may prevent immune escape mutant HBV from spreading and to prevent these strains from becoming dominant during the next decennia.     

Prevalence of hepatitis B virus in chronic hepatitis B patients

The aim of the current study was to detect HBV by Real time - PCR in chronic hepatitis B patients. Fifty-eight sera of chronic hepatitis B patients were subjected during the period March 2009 to April 2010 in Ilam cities in West of Iran. Sera assayed by real-time PCR and ELISA methods. Twenty serum samples from healthy volunteers and non-hepatitis B patients and negative for hepatitis B seromarkers served as negative controls for the study. Among fifty-eight sera, ELISA showed fifty-five (94.8%) of the samples were positive for HBsAg and three (5.2%) negative results obtained while real-time PCR specified fifty-eight (100%) positive results in chronic hepatitis B patients. HBsAg status did not necessarily reflect HBV DNA level in the serum, as 5.2% of chronic Hepatitis B patients were positive for HBV DNA but negative for HBsAg. HBV DNA was not found to be positive amongst any of the negative controls. Real time - PCR is a sensitive and reproducible assay for HBV DNA quantization.     

Genotype and subtype analyses of hepatitis B virus (HBV) and possible co-infection of HBV and hepatitis C virus (HCV) or hepatitis D virus (HDV) in blood donors, patients with chronic liver disease and patients on hemodialysis in Surabaya, Indonesia

Four subtypes (adw, adr, ayw, and ayr ) and eight genotypes (A to H) of the hepatitis B virus (HBV) have been identified. They appear to be associated with particular geographic distribution, ethnicity, and possibly clinical outcomes. In this study, hepatitis B surface antigen (HBsAg) subtyping and HBV genotyping were carried out on sera obtained from HBsAg-positive HBV carriers, including healthy blood donors; patients with acute hepatitis, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma; and patients on hemodialysis all located in Surabaya, Indonesia. We report here that all HBV isolates tested in Surabaya belonged to genotype B, with more than 90% of them being classified into subtype adw. Our results also revealed that prevalence of hepatitis C virus (HCV) co-infection among HBV carriers in Surabaya was approximately 10% for healthy blood donors and patients with chronic liver disease, and approximately 60% for patients on maintenance hemodialysis. Interestingly, HBsAg titers were lower in HBV carriers with HCV co-infection than in those without HCV co-infection. We also found that prevalence of hepatitis D virus (HDV) co-infection was < 0.5% among HBV carriers in Surabaya.     

An epidemiological assessment of the chronic carriage of the hepatitis B virus surface antigen and other markers of this infection

On the basis of prolonged (up to 10 years) observations of chronic HBsAg carriers (87 persons) slowly progressing chronic hepatitis B virus infection was detected in most of them (57.5%). In these persons up to 6 negative results of the analysis for the presence of HBsAg were periodically obtained, with the subsequent renewal of HBs-antigenemia. More profound examination of 192 persons with chronic lesions of the liver and other digestive organs helped diagnose chronic persistent hepatitis and chronic active hepatitis, accompanied by HBs-antigenemia and the circulation of other markers of this infection, in 63.5% of the examinees.     

T cells from patients with chronic liver diseases: abnormalities in PHA-induced expression of HLA class II antigens and in autologous mixed-lymphocyte reactions

The expression of HLA Class II antigens by resting and phytohemagglutinin (PHA)-activated T cells and their functional properties in autologous mixed-lymphocyte reactions (MLR) were investigated in patients with chronic active hepatitis, with alcoholic cirrhosis, and with primary biliary cirrhosis. In all groups of patients the percentage of resting T cells expressing HLA Class II antigens was significantly higher than that in controls. The percentage of T cells which acquired HLA Class II antigens following PHA stimulation was reduced in patients with chronic active hepatitis, serum hepatitis B surface antigen (HBsAg) positive, and in those with alcoholic cirrhosis, HBsAg negative, although the level of [3H] thymidine incorporation was within normal limits. The degree of proliferation in autologous MLR with PHA-T cells was significantly reduced in patients with chronic active hepatitis, HBsAb positive, and in those with alcoholic cirrhosis, HBsAg positive. A reduced proliferation was also detected in autologous MLR with non-T cells, in patients with chronic active hepatitis, HBsAg positive. The abnormalities of autologous MLR are selective, since the proliferative and stimulatory activities of cells from patients with chronic liver diseases in allogeneic MLR were within normal ranges. The immunoregulatory role of HLA Class II antigens and of autologous MLR suggests that the abnormalities we have identified may play a role in the immunological dysfunctions underlying chronic liver diseases.     

Liver enzyme concentrations as measure of possible infectivity in chronic asymptomatic carriers of hepatitis B.

Fifty-two British-born blood donors who were chronic carriers of hepatitis B surface antigen (HBsAg) were tested for the presence of hepatitis B e antigen (HBeAg) and antibody to HBeAg by an immunoradiometric assay. The presence of HBeAg was closely associated with a slight rise in serum liver enzyme concentrations, a high HBsAg titre, and male sex. We suggest that the finding of persistently raised serum liver enzyme concentrations in an asymptomatic HBsAg carrier might be useful as a likely indicator of HBeAg and high infectivity.