A human monoclonal antibody against small envelope protein of hepatitis B virus with potent neutralization effect

Hepatitis B virus (HBV) produces large (L), middle (M), and small (S) envelope proteins, alternatively referred to as hepatitis B surface antigen (HBsAg). Currently, yeast-derived S protein serves as the preventive vaccine, while hepatitis B immune globulin (HBIG) concentrated from pooled plasma of vaccine recipients is employed for post-exposure prophylaxis. However, only a small proportion of the antibodies in HBIG are HBV specific. In the present study, a human monoclonal anti-S antibody (G12) was developed, produced under GLP conditions, and subjected to a panel of functional assays. In vitro results demonstrated high affinity of G12 for the S protein (K_D = 7.56 nM). It reacted with envelope proteins of all 7 HBV genotypes tested (A-F, H) by immunofluorescent staining, and more than 97% of HBsAg-positive patient serum samples by enzyme-linked immunosorbent assay. G12 recognized a conformational epitope, although the exact sequence remains unknown. Strikingly, G12 was at least 1,000-fold more potent than HBIG in neutralizing HBV infectivity in both HepaRG cell line and HepG2 cells reconstituted with the HBV receptor. In a transgenic mouse model of HBV persistence, a single peritoneal injection of G12 markedly diminished serum HBsAg titers in all 7 mice, which was sustained for the observation period of 144 d in mice with low pre-treatment levels. While the therapeutic potential of G12 warrants further investigation using a large number of animals, G12 is a potent neutralizing human monoclonal antibody and a promising candidate to replace or supplement HBIG in the prevention of HBV infection.     

Hepatitis B surface antigen levels and sequences of natural hepatitis B virus variants influence the assembly and secretion of hepatitis d virus

Various domains of hepatitis B surface antigen (HBsAg) are essential for the assembly and secretion of hepatitis D virus (HDV). This study investigated the influences of the levels and sequences of HBsAg of naturally occurring HBV variants on the assembly and secretion of HDV. Six hepatitis B virus (HBV)-producing plasmids (three genotype B and three genotype C) and six HBsAg expression plasmids that expressed various HBsAg levels were constructed from the sera of HDV-infected patients. These plasmids were cotransfected with six expression plasmids of HDV of genotype 1, 2, or 4 into the Huh-7 hepatoma cell line. Serum HBsAg and HBV DNA levels were correlated with HDV RNA levels and outcomes of chronic hepatitis D (CHD) patients. The secretion of genotype 1, 2, or 4 HDV generally correlated with HBsAg levels but not with HBV genotypes or HBV DNA levels. Swapping and residue mutagenesis experiments of HBsAg-coding sequences revealed that the residue Pro-62 in the cytosolic domain-I affects the assembly and secretion of genotype 2 and 4 HDV and not those of genotype 1. The pre-S2 N-terminal deletion HBV mutant adversely affects secretion of the three HDV genotypes. In patients, serum HDV RNA levels correlated with HBsAg levels but not with HBV DNA levels. Viremia of HDV or HBV correlated with poor outcomes. In conclusion, the assembly and secretion of HDV were influenced by the amounts and sequences of HBsAg. For an effective treatment of CHD, reduction of HBsAg production in addition to the suppression of HBV and HDV replication might be crucial.     

Tumor necrosis factor alpha negatively regulates hepatitis B virus gene expression in transgenic mice.

It is well known that several inflammatory cytokines can modulate hepatocellular gene expression in a complex physiological process known as the hepatic acute-phase response. Since hepatitis B virus (HBV) characteristically induces a vigorous lymphomononuclear inflammatory response in the liver during acute and chronic hepatitis, it is possible that hepatocellular HBV gene expression may also be modulated by one or more of the cytokines produced by these cells. Using bacterial lipopolysaccharide (LPS) as a surrogate inducer of inflammatory cytokines in vivo, we have tested this hypothesis in a transgenic mouse model system. In experiments with two independent transgenic mouse lineages that express the HBV envelope region under the control of either HBV or cellular promoters, we observed a 50 to 80% reduction in the hepatic steady-state content of a 2.1-kb HBV mRNA following administration of a single intraperitoneal dose of LPS. The regulatory influence of several inflammatory cytokines known to be induced by LPS was also examined in this system. The negative regulatory effect of LPS was consistently reproduced by the administration of a single nontoxic dose of tumor necrosis factor alpha, and it was occasionally observed following the administration of high doses of alpha interferon and interleukin-6, while no effect was detectable in response to high-dose interleukin-1 alpha or to gamma interferon. These observations suggest that tumor necrosis factor alpha and perhaps other cytokines may activate a heretofore unsuspected intracellular pathway that negatively regulates HBV gene expression. The intracellular mechanism(s) responsible for this effect and its pathophysiologic relevance remain to be elucidated.     

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.     

Cytokine expression during chronic versus occult hepatitis B virus infection in HIV co-infected individuals

Chronic hepatitis B virus infection is characterized by persistent detectable levels of hepatitis B surface antigen (HBsAg) and HBV DNA in the serum. In contrast, HBsAg is not detectable during occult HBV infection, despite the presence of HBV DNA. An altered host immune response could play a role in the development of occult HBV infection; however, potential differences in immune responses among chronic and occult HBV-infected patients have not been evaluated in vivo. In the current study, we evaluated serum levels of regulatory, apoptotic, and fibrotic/anti-fibrotic cytokines/markers as indicators of immune responses in 25 chronic and 12 occult HBV-infected patients. More than half of the patients in both chronic and occult HBV infection groups had IL-2, IL-4, IL-13, and IFN-γ levels below detectable limits. In contrast, most patients had detectable levels of IL-8, IL-10, IP-10, sFas, sFasL, and TGF-β1. Of these, only sFas was significantly different between the two groups, with lower levels observed during occult compared to chronic HBV infection (p = 0.01). As a surrogate marker of apoptotic inhibition, decreased sFas during occult HBV infection suggests that apoptosis occurs at different rates in occult compared to chronic HBV infection and therefore, may contribute to persistence of occult HBV infection.     

Algorithm of serologic screening and assessment of prevalence of serologically meaningful mutations of HBsAg in hepatitis B virus carriers

Algorithm of serologic screening for HBsAg-mutants in hepatitis B virus (HBV) carriers with high level of HBsAg was developed which is based on the detection of defects of interactions of serum HBsAg with monoclonal anti-HBs realizing as a decrease of ELISA sensitivity in 10 times or more during serial 10-fold dilutions. During 1st stage commercial test-systems based on monoclonal antibodies was used to select serum samples with discrepancy of test results. During 2nd stage HBsAg contained in selected sera was analyzed by the panel of monoclonal and polyclonal anti-HBs conjugates using decrease in ELISA sensitivity as a criterion. Serum samples from 2510 chronic carriers of HBV with high level of HBsAg were studied. 19 samples with discrepant results were found. Subsequent characterization of HBsAg with panel of 11 monoclonal and 1 polyclonal conjugates allowed to distinguish groups of sera with specific serologic "portraits". Atypical features of HBsAg were confirmed by genotyping 9 of 19 samples. Analysis of primary nucleotide sequence revealed serologically meaningful mutations in S-gene of HBV in all 9 isolates: 3 of them contained substitution mutation G145R, 5--S143L, and one--T143M. Distribution of mutations in HBsAg corresponded with specific serologic "portraits". Prevalence of HBsAg mutations in HBV carriers with high level of HBsAg was assessed for the first time: prevalence of G145R, S143L/T143M mutations, and all serologically atypical variants was 0.12%, 0.24%, and 0.76% respectively. Developed algorithm was proposed for epidemiologic monitoring of HBsAg-mutants of HBVand control of diagnostic test-systems.     

Interaction of interferon-alpha with interleukin-1 beta or tumor necrosis factor-alpha on hepatitis B virus enhancer activity.

The interaction of IFN-alpha with IL-1 beta or TNF-alpha on hepatitis B surface antigen (HBsAg) expression was analysed in hepatitis B virus (HBV)-DNA integrated PLC/PRF/5 and non-integrated HuH-7 human hepatoma cells. Secretion of HBsAg in PLC/PRF/5 cells was reduced by IFN-alpha, IL-1 beta or TNF-alpha, and synergistically depressed when IFN-alpha was used in combination with IL-1 beta or TNF-alpha. By Northern blot analysis, the levels of HBsAg mRNA were suppressed by IFN-alpha in combination with IL-1 beta or TNF-alpha. In the chloramphenicol acetyltransferase plasmid transfection assay, IFN-alpha in combination with IL-1 beta or TNF-alpha caused a much greater suppression of HBV enhancer activity than IFN-alpha, IL-1 beta or TNF-alpha alone in both hepatoma cells. These findings suggest that the interaction of IFN-alpha with IL-1 beta or TNF-alpha synergistically represses HBV enhancer activity, resulting in depressed expression of HBsAg.     

Overcoming tolerance in hepatitis B virus transgenic mice: a possible involvement of regulatory T cells

The hepatitis B virus (HBV) transgenic mouse (Tg) 50-4 strain is immunologically tolerant to HBV antigens. Various vaccination strategies have been attempted but failed to break the tolerance in the mouse. Although the tolerance to HBV antigen is maintained, this mouse strain develops spontaneous liver disease beginning at the age of about 3 months. We attempted to induce immune responses to HBV surface antigen (HBsAg) in the Tg by immunization with recombinant vaccinia virus expressing HBsAg (vvHBV), and observed different immunological responsiveness between 2-month-old and 5-month-old Tg. In contrast to the unbreakable tolerance reported previously, we could induce both the cytotoxic T lymphocyte (CTL) and the antibody response against HBsAg by the vvHBV immunization. The cytokine expression pattern indicated that T helper 1 type immune response was induced. However, interestingly, these immune responses were observed only in the 5-month-old Tg, but not in the 2-month-old Tg. Furthermore, CD4+ T cells from 2-month-old mice, but not those from 5-month-old mice, inhibited CTL response to HBV antigen when adoptively transferred to C57BL/6. These results suggest the possible involvement of regulatory T cell function in the HBV Tg for maintaining tolerance. This study would contribute to a better understanding of immune status of the HBV Tg as a model of human chronic hepatitis and to the search for new therapeutic targets for chronic viral infections.     

Seroepidemiologic study of adult T-cell leukemia virus (ATLV) and hepatitis B virus infection in Okinawa, Japan

A total of 2,283 serum samples were collected from healthy subjects in three islands of the Yaeyama district of Okinawa, Japan. These sera were tested for the presence of hepatitis B surface antigen (HBsAg), for antibody to hepatitis B core antigen (anti-HBc), and for antibody to adult T-cell leukemia-associated antigen (anti-ATLA). Correlation between hepatitis B virus infection and adult T-cell leukemia virus (ATLV) infection was determined by using the prevalence rates for three virus markers. Overall prevalence of HBsAg, anti-HBc and anti-ATLA was 6.5%, 57.4%, and 17.9%, respectively. Age-specific prevalence of anti-HBc and anti-ATLA increased with age, but that of HBsAg did not. Sex-specific prevalence of HBsAg was significantly higher in males than in females, but that of anti-ATLA was significantly higher in females than in males. Statistical analysis revealed that prevalence of anti-ATLA was significantly higher in HBsAg-positive persons and HBsAg-negative/anti-HBc-positive persons than in those negative for HBsAg and anti-HBc. These data suggest that hepatitis B virus-infected persons have a significantly higher chance of adult T-cell leukemia virus infection than those without hepatitis B virus infection in the area studied.     

Direct injection of hepatitis B virus DNA into liver induced hepatitis in adult rats

Hepatitis B virus (HBV) surface antigen (HBsAg) genes were injected directly into the liver of adult rats with non-histone chromosomal protein high mobility group 1 by the hemagglutinating B virus of Japan (Sendai virus)-liposome method (Kato, K., Nakanishi, M., Kaneda, Y., Uchida, T., and Okada, Y. (1991) J. Biol. Chem. 266, 3361-3364). Immunohistochemical analysis showed that HBV surface antigen was expressed by the hepatocytes in vivo. On successive injections of the HBsAg genes, the antibody to HBV surface polypeptides was produced in the rats, and characteristic pathological changes of lymphocytic focal necrosis and denaturation of hepatic cells were observed in the liver of all the rats. We conclude that hepatitis is caused by the direct injection of HBsAg genes.     

Intracellular inhibition of hepatitis B virus S gene expression by chimeric DNA-RNA phosphorothioate minimized ribozyme

Chronic hepatitis B virus (HBV) infection is a major problem in Asia. Current therapies for chronic hepatitis B have limited efficacy. The successful use of ribozymes for intracellular inhibition of HBV gene expression was recently reported. As an alternative to ribozymes, the use of DNA-containing, phosphorothioate-modified, minimized hammerhead ribozymes (minizymes) to inhibit hepatitis B surface antigen (HBsAg) expression and viral replication was investigated. Such molecules can be synthesized and supplied exogenously. Two conserved sites within the HBsAg open reading frame (ORF) were targeted. PLC/PRF5 cells or 2.2.15 cells were treated with minizymes or antisense oligomers to assess the effects on cell viability, HBsAg expression, and viral DNA production. Treatment with the minizyme, MZPS1, resulted in >80% inhibition of HBsAg expression in PLC/PRF5 cells. MZPS1 had more inhibitory effect than the antisense oligonucletoide target at the same region, whereas the control minizyme had little effect. Another gene-specific minizyme, MZPS2, did not show any effect. Treated cells remained fully viable. Treatment of 2.2.15 cells with MZPS1 also led to decreased HBsAg expression. In addition, a 2.3-fold decrease in viral production was observed. Our data showed that minizymes can inhibit HBV gene expression and may potentially be useful for clinical therapy against chronic HBV infection.     

Stable inhibition of hepatitis B virus expression and replication by expressed siRNA

RNA interference might be an efficient antiviral therapy for some obstinate illness. Here, we studied the effects of hepatitis B virus (HBV)-specific 21-nt small interfering RNAs (siRNA) on HBV gene expression and replication in 2.2.15 cells. Seven vectors expressing specific hairpin siRNA driven by the RNA polymerase II-promoter were constructed and transfected into 2.2.15 cells. In the cell strain that can stably express functional siRNA, the HBV surface antigen (HBsAg) and the HBV e antigen (HBeAg) secretion into culture media was inhibited by 86% and 91%, respectively, as shown by an enzyme-linked immunosorbent assay. Immunofluorescence and Western blot indicated similar results. HBV DNA was markedly restrained by 3.28-fold, as assessed by the fluorescent quantitation PCR. Moreover, the HBV mRNA was significantly reduced by 80% based on semiquantitative RT-PCR. In conclusion, the specific siRNA can knock down the HBV gene expression and replication in vitro, and the silence effects have no relationship with interferon response.