Hepatitis B Surface Antigen Expression Research Articles

Dynamic correlation between induction of the expression of heme oxygenase-1 and hepatitis B viral replication.

Heme oxygenase‑1 (HO‑1) possesses significant potential as a drug target for hepatitis B, which may be transferable to patient therapy. The aim of the present study was to clarify the dynamic correlation between the hepatitis B virus (HBV) and HO‑1. The levels of HBV replication and expression of HO‑1 were investigated in HepG2.2.15 hepatoma cells following exposure to 5‑50µMhemin for 1‑6days. The mRNA expression levels of HO‑1 were then detected using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). HBV replication levels were determined by enzyme‑immunoassay and a PCR‑fluorescence quantitation assay. The results of the present study demonstrated that the mRNA expression levels of HO‑1 increased in a dose‑dependent manner in the HepG2.2.15 cells, following exposure to 5‑50µM hemin. The mRNA expression levels of HO‑1 reached a peak following exposure of the cells to hemin for threedays, subsequently the expression of HO‑1 decreased. Following exposure to hemin at an optimal concentration of 50µM for 1‑6days, the levels of the hepatitisB surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the cells were significantly reduced. This marked reduction in the expression of HBsAg and HBeAg reached its peak on the first day, following which the inhibition weakened as the duration of exposure increased. In addition, the inhibition of HBV DNA replication increased with the a longer duration of exposure. Furthermore, HBV DNA levels were significantly decreased following exposure to hemin for 3‑6days. In conclusion, the present study demonstrated that induced expression of HO‑1 interfered with HBV replication in a dose and time‑dependent manner, implying that a reduction of the HBV viral load may contribute to upregulation in the expression of HO‑1.

Correlations Among Serum Hepatitis B Surface Antigen and Hepatitis B e Antigen Titers and Viral Load in Chinese Patients with Chronic Hepatitis B.

This study aimed to provide a detailed and comprehensive analysis of serum hepatitis B surface antigen (HBsAg), HBeAg, and serum hepatitis B virus (HBV) DNA in Chinese patients with chronic hepatitis B (CHB), predominantly genotypes B and C. HBV serological markers, HBsAg titer, HBeAg titer, and HBV DNA were detected and genotyped in 129 Chinese patients with CHB. HBeAg-positive CHB patients were younger, had higher serum alanine aminotransferase, aspartate transaminase, and HBV DNA levels and were more likely to be infected with HBV genotype C. The median HBsAg titer was significantly higher in HBeAg-positive compared with HBeAg-negative CHB patients (3.73 versus 2.93; p < 0.01), and the median HBsAg titer was significantly higher in patients with genotype C. The correlation between HBsAg titer and HBV DNA was stronger in HBeAg-positive CHB patients (r = 0.56; p < 0.01). HBeAg titer was positively correlated with serum HBsAg titer (r = 0.77; p < 0.01) and serum HBV DNA (r = 0.72; p < 0.01). This study detected key differences in HBsAg expression between HBeAg-positive and HBeAg-negative CHB patients, which may have practical implications for the use of quantitative serological clinical biomarkers.

S-phase arrest after vincristine treatment may promote hepatitis B virus replication.

To observe the effect of vincristine on hepatitis B virus (HBV) replication in vitro and to study its possible mechanisms. Vincristine was added to the cultures of two cell lines stably expressing HBV. Then, the levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core antigen (HBcAg) in the supernatants or cytoplasm were examined using by enzyme-linked immunosorbent assay and Western blot. The HBV pregenome RNA (pgRNA) was detected using reverse transcription-PCR and real-time fluorescent quantitative PCR (RT-qPCR), and viral DNA was detected using Southern blot and RT-qPCR. Cell proliferation after drug treatment was detected using the BrdU incorporation test and the trypan blue exclusion assay. Cell cycle and cell apoptosis were examined using flow cytometry and Western blot. Vincristine up-regulated HBV replication directly in vitro in a dose-dependent manner, and 24-h exposure to 0.1 μmol/L vincristine induced more than 4-fold and 3-fold increases in intracellular HBV DNA and the secretion of viral DNA, respectively. The expression of HBV pgRNA, intracellular HBsAg and HBcAg, and the secretion of HBeAg were also increased significantly after drug treatment. Most importantly, vincristine promoted the cell excretion of HBV nucleocapsids instead of HBV Dane particles, and the nucleocapsids are closely related to the HBV pathogenesis. Furthermore, vincristine inhibited the proliferation of cells stably expressing HBV. The higher the concentration of the drug, the more significant the inhibition of the cell proliferation and the stronger the HBV replication ability in cells. Flow cytometry indicated that cell cycle arrest at S-phase was responsible for the cell proliferation inhibition. Vincristine has a strong stimulatory effect on HBV replication and induces cell cycle arrest, and cell proliferation inhibition may be conducive to viral replication.

Two-phase fed-batch modification for 48 hour peak expression of hepatitis B surface antigen in Pichia pastoris shake flask system

Abstract A study of the Mut+ phenotype for the expression of recombinant hepatitis B surface antigen (HBsAg) in Pichia pastoris strain GS115 using the pPIC3.5K vector with a two-phase fed-batch protocol in a shake flask system is described. Expression levels of HBsAg protein of 6.74 g L−1 Dry Cell Weight (DCW) and 26.07 mg L−1 of HBsAg concentration were achieved 48 h from the induction point which added to a 120 h reduction in production period in comparison with MutS expression (168 h). The use of the pPIC3.5K-HBsAg plasmid in the Mut+ phenotype enhanced the expression of HBsAg by a nearly 13 times higher volumetric productivity in the first 24 h and 35 times higher at peak production concentration. Comparison of AOX expression cassettes relative to the HBsAg gene in the role of mRNA secondary structure during translation initiation revealed that HBsAg possesses lower folding stability with AOX1 Mut+ phenotype. The results from this study demonstrated that expression of HBsAg with Mut+ AOX1 promoter is adequate as an alternative for the production of HBsAg. In addition, the AOX promoter of the Mut+ phenotype was observed to be better suited for HBsAg expression, which correlates with the ease of translation initiation under shake flask conditions.

Tapasin modification on the intracellular epitope HBcAg18–27 enhances HBV-specific CTL immune response and inhibits hepatitis B virus replication in vivo

HBV-specific cytotoxic T-lymphocyte (CTL) activity has a very important role in hepatitis B virus clearance. Present studies suggest that Tapasin, a endoplasmic reticulum (ER) chaperone, stabilizes the peptide-receptive MHC I conformation, allowing peptide exchange and increasing more peptides to be translocated into the ER. We have previously testified that cytoplasmic transduction peptide (CTP)-HBcAg18–27-Tapasin fusion protein could enter cytoplasm of dendritic cells, and enhance T cells' response to generate specific CTLs efficiently in vitro. In the present study, we evaluated specific immune responses of CTP-HBcAg18–27-Tapasin fusion protein in HLA-A2 transgenic mice (H-2Kb) and anti-viral ability in HBV transgenic mice, and explored the mechanisms probably involved in. The studies showed that CTP-HBcAg18–27-Tapasin not only increased production of cytokine IFN-γ and interleukin-2 (IL-2), compared with CTP-HBcAg18–27, HBcAg18–27-Tapasin, and PBS, but also significantly induced the higher percentages of IFN-γ+CD8+ T cells and specific CTL responses in HLA-A2 transgenic mice. Moreover, enhancement of specific CTL activity induced by the fusion protein reduced HBV DNA and hepatitis B surface antigen (HBsAg) levels and decreased the expression of HBsAg and hepatitis B core antigen (HBcAg) in liver tissue of HBV transgenic mice. In addition, CTP-HBcAg18–27-Tapasin could upregulate the expression of JAK2, Tyk2, STAT1, and STAT4 in T lymphocytes in HLA-A2 transgenic mice splenocytes. However, there was no significant difference on the expressions of JAK1, JAK3, and STAT6 between each group. In conclusion, CTP-HBcAg18–27-Tapasin fusion protein could enhance not only the percentages of CTLs but also induce robust specific CTL activity and inhibits hepatitis B virus replication in vivo, which was associated with activation of the JAK/STAT signaling pathway.

Role of tumor necrosis factor-alpha in the anti-HBV activity of tetracycline

To study the role of tumor necrosis factor-alpha (TNFalpha) in the anti-replication effects of tetracycline (Tet) on hepatitis B virus (HBV). The Tet-dependent regulatory fragment (TO) was PCR amplified from the pcDNA4TM/TO vector, inserted into the pUC118 cloning vector, and verified by sequencing. The counterpart fragment in the pVITRO3 expression vector, which contains two multiple cloning sites (MCSs), was replaced with the confirmed TO to generate a pVITRO3-TO vector. The Tet repressor (TR) gene from the pcDNA6/TR regulatory vector was incorporated into one MCS of pVITRO3-TO and the TNFalpha gene was subsequently incorporated into the other MCS. The resultant vector, pVITRO3-TOTR-TNFalpha, was transiently transfected into HepG2 cells. TNFalpha expression from the vector was induced by exposure to various concentrations of Tet and measured by enzyme-linked immunosorbent assay to determine the appropriate Tet concentration for experimentation. To investigate whether Tet inhibits TNFalpha expression as a mechanism of its anti-replication activity against HBV, the HepG2.2.15 cell line stably transfected with pVITRO3-TOTR-TNFalpha was used as an HBV replication model. Levels of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) were detected by immunoassay. HBV DNA level was detected by fluorescence quantitative PCR. The TNFalpha expression from the newly constructed pVITRO3-TOTR-TNFalpha vector was Tet-controllable in the eukaryotic cells examined. The optimal concentration of Tet for the experimental system was 1.0 mug/ml. HBsAg and HBeAg expression was down-regulated in the HepG2.2.15 cells stably transfected with the pVITRO3-TO-TR-TNFalpha vector. After incubation with Tet for 1, 3 and 5 days, the inhibition rate of HBsAg was 2%, 1.1% and 0, compared to 14.8%, 11.5% and 28.4% in the non-Tet control group. The corresponding inhibition rates of HBeAg were 50.0%, 26.7% and 47.9%, compared to 0.3%, 1.6% and 0.0%, in the control group. HBV DNA levels in the cells and the cell culture supernatants exposed to Tet were decreased by 70.3% and 79.9%, respectively. TNFalpha inhibited production of HBsAg mRNA. A Tet-dependent regulatory fragment double-expressing TNFalpha single vector system was constructed successfully, achieving controllable TNFalpha expression in both transiently transfected eukaryotic cells and stable cell lines. In this HBV cell model system, Tet-induced overexpression of human TNFalpha inhibited HBV DNA replication and reduced HBsAg and HBeAg expression. Inhibition of HBV transcription may be a key role of TNFalpha against HBV replication.

Anti-HBV Activities of Xanthones From Swertia Punicea Hemsl

We studied the effects of two xanthones compounds isolated from Swertia punicea Hemsl (from Geutianaceae), swertianolin (I) and bellidifolin (II), on Hepatitis B surface antigen (HBsAg) and e antigen (HBeAg) in cultured human hepatocellular carcinoma cell line (HepG 2 ). The HepG 2 cells were first cultured for 24h, various concentrations of these two xanthones were then added to the culture medium. The culture medium containing the two xanthones was exchanged once every 4 days. After 8 days, the cytotoxic activities of these two xanthones were assessed by cytopathic effect. The HepG 2 cells were then treated with the two compounds at a concentration of swertianolin (1.6, 3.1, 6.2, 12.5, 25 m g/ml) and bellidifolin (2.0, 3.9, 7.8, 25.5, 31.2 m g/ml). Four or eight days later, the culture medium was collected and the expression of HBsAg and HBeAg were determined by radioimmunoassay. Our results show that swertianolin can suppress the expression of HBeAg with IC 50 of 8.0 m g/ml, while bellidifolin can inhibit the expression of HBsAg with IC 50 of 13 m g/ml at the eighth days. The Therapeutic Index for swertianolin and bellidifolin are 6.2 and 6.8, respectively. Our findings suggest that swertianolin and bellidifolin have anti-HBV activities in vitro.

The fusion protein of cytoplasmic transduction peptide (CTP)-HBcAg18-27-Tapasin enhances specific immune response to hepatitis B virus and inhibits viral replication in transgenic mice

To investigate the effect of protein transduction domain-hepatitis B virus core antigen (CTP-HBcAg18-27)-Tapasin fusion protein-induced specific cytotoxic T lymphocyte (CTL) response on hepatitis B virus (HBV) replication in HBV transgenic mice. Twenty HBV-transgenic mice were randomly divided into two groups for a 3-week course of once weekly subcutaneous immunizations with either CTP-HBcAg18-27-Tapasin fusion protein or CTP-HBcAg18-27. Mice administered isotonic saline served as blank controls. Expressions of cytokines in splenocytes were analyzed by flow cytometry. Serum levels of hepatitis B surface antigen (HBsAg) and HBV DNA were determined by microparticle enzyme immunoassay and real-time fluorescent PCR assay, respectively. Expression of HBsAg in hepatic tissues was detected by immunohistochemistry. Immunization with 100 mug of CTP-HBcAg18-27-Tapasin fusion protein led to a significant increase in proportions of CTLs in spleen (2.70%+/-0.20% vs. 50 mug of CTP-HBcAg18-27-Tapasin: 1.66%+/-0.53%, 50 mug of CTP-HBcAg18-27: 1.26%+/-0.56%, and blank controls: 0.75%+/-0.71%; F = 741.45, P = 0.000) and up-regulation of inflammatory cells in hepatic tissue. In addition, both immunizations of CTP-HBcAg18-27-Tapasin led to significant decreases in serum HBsAg and HBV DNA levels compared to those in the CTP-HBcAg18-27 group. HBV-related modification of the expression of the molecular chaperone Tapasin may affect its interaction with intracellular antigen peptides, thereby leading to increases the number of specific CTLs in the spleen, decreases in serum HBsAg and HBV DNA levels, and down-regulation of HBsAg expression in hepatic tissue. These results obtained in HBV-transgenic mice suggest that the CTP-HBcAg18-27-Tapasin fusion protein has anti-HBV activity.

A modified murine model based on hydrodynamic injection for the analysis of chronic human hepatitis B virus infection

Hepatitis B virus (HBV) is a persistent pathogen that causes acute and chronic necroinflammatory liver disease and is attributable to ~1 million deaths per year. In the present study, a conventional murine model was introduced based on the hydrodynamic injection of engineered replication‑competent HBV DNA into the tail veins of C57BL/6 mice. In a previous study, nine in‑frame ATG (start) codons in the S open reading frame (S1‑S9) were analyzed. The highly conserved ATG S5 was mutated to ACG by T378C, which led to the substitution sM75T and inhibition of the production and secretion of the hepatitis B surface antigen (HBsAg), and subsequent inhibition of HBV replication. In the present study, T378C was introduced into the pAAV‑HBV1.3 plasmid and was confirmed to affect HBsAg production and secretion, and HBV replication in vivo, which was in agreement with the previous in vitro results. Furthermore, the murine model was improved by co‑injection of the replication‑competent HBV plasmid DNA with Lipofectamine 2000 (LP). In this model, LP not only significantly enhanced HBV replication in mice, but also upregulated the expression of HBsAg and the hepatitis B core antigen. The current modified murine model was superior to the conventional murine HBV model based on HBV challenge by hydrodynamic injection.

Study on the antiviral activity of San Huang Yi Gan Capsule against hepatitis B virus with seropharmacological method

BackgroundSeropharmacology arising recently is a novel method of in vitro pharmacological study on Chinese herb using drug-containing animal serum. As seropharmacology possesses the advantages of experiments in vitro and in vivo, it is increasingly applied in pharmacological research on Chinese medicine. However, some issues of seropharmacology remain controversial and need to be clearly defined. San Huang Yi Gan Capsule (SHYGC) is a Chinese herbal formula with antiviral property against hepatitis B virus (HBV), but little is known about the mechanism underlying its anti-HBV activity. The aim of the present study was to elucidate the action mechanism of SHYGC using seropharmacological method and systematically address the methodology of preparing drug-containing serum.MethodsNew Zealand rabbits were orally administrated SHYGC with various regimens, followed by preparation of SHYGC-containing rabbit sera with a variety of methods. After HBV-producing HepG2 2.2.15 cells were treated with SHYGC-containing sera or entecavir for 9 days, the levels of hepatitis B surface antigen (HBsAg) and HBV DNA and the activity of DNA Polymerase were determined in HepG2 2.2.15 cells-conditioned media.ResultsAn optimally standardized method of preparing drug-containing serum was raised for seropharmacology, with which SHYGC was demonstrated to suppress HBsAg expression, HBV DNA replication and DNA Polymerase activity in a dose-dependent fashion.ConclusionsThis seropharmacological study shows SHYGC is a potentially powerful anti-HBV agent. Additionally, seropharmacology is a promising pharmacological method with a broad range of advantages, and it can be widely used in biomedical research, if combined with pharmacokinetics.

Clinical significance and evolution of hepatic HBsAg expression in HBeAg-positive patients receiving interferon therapy

Serum hepatitis B surface antigen (HBsAg) level is important in the management of chronic hepatitis B (CHB). However, it is unclear whether serum HBsAg reflects its expression in liver and the hepatic HBsAg evolution following interferon therapy. Forty-five HBeAg-positive CHB patients receiving interferon-based therapy within a randomized, controlled, multicenter study during 1998-1999 were included. The hepatic HBsAg expressions were categorized into cytoplasmic, inclusion, marginal and negative patterns by immunohistochemical staining. The HBsAg-positive hepatocytes were quantified by image-based cytometry and correlated to HBV serological and virological profiles for clinical implications. The evolution of hepatic HBsAg levels was analyzed among 22 patients with paired liver biopsies before and after interferon therapy, sequentially. There was a positive correlation between pretreatment serum HBsAg and hepatic HBsAg levels (r=0.67, P<0.0001). The hepatic HBsAg expression pattern significantly evolved from cytoplasmic/inclusion pattern to marginal/negative pattern after interferon treatment. The serum HBV-DNA, HBsAg and hepatic HBsAg levels all decreased significantly after interferon therapy. Among 36% patients with HBeAg loss after therapy, pretreatment hepatic HBsAg levels were significantly lower compared with those without HBeAg loss. After multivariate analysis, low pretreatment hepatic HBsAg levels rather than serum HBsAg titers were associated with a higher rate of HBeAg loss (OR: 4.97, 95% CI: 1.12-22.00, P=0.035). The serum HBsAg level positively reflects the HBsAg level in liver which evolves significantly after interferon therapy. A lower hepatic HBsAg level is associated with HBeAg loss after interferon treatment. Hepatic HBsAg may have clinical significance in CHB patients receiving interferon treatment.

Correlation between Vertical Transmission of Hepatitis B Virus and the Expression of HBsAg in Ovarian Follicles and Placenta

BackgroundThe aim of this study was to investigate the correlation between the expression of hepatitis B surface antigen (HBsAg) in human ovary and placenta and the vertical transmission of hepatitis B virus (HBV).Methodology/Principal FidningsOvarian and placental tissue specimens of pregnant women infected with HBV were collected during cesarean section and immunostained for HBsAg. The sera of the corresponding newborns were tested for HBV markers and HBV DNA. HBsAg was detected in 15 out of 33 (45%) placental tissues and was further detected in capillary endothelial cells in 4 specimens (26%), of which 3 (75%) corresponding infants were infected with HBV in utero. Out of the 33 ovarian tissues, 7 (21%) were positive for HBsAg, of which 2 (28%) showed HBsAg in ovarian follicles and the 2 corresponding infants (100%) had intrauterine HBV infection.Conclusions/SignificanceHBsAg expression in cells of the ovarian follicle or placental capillary endothelium signal a higher risk for intrauterine HBV infection.

Inhibition of Hepatitis B Virus Replication and Expression in Vitro and in Vivo by the Hammerhead Ribozymes Targeted Different Sites

Abstract Objective To develop an effective and specific medicine targeting hepatitis B virus (HBV) pregenome. Based on the identified accessible target sites for hammerhead ribozyme in our previous researches, a recombinant hepatitis delta virus (HDV) ribozyme was chosen and used to demonstrate the effective cleavage in vitro and in vivo. Methods Three hammerhead ribozymes for potential target sites (S, X and C genes) and co-expression plasmid (pTr-dB, pTdδ-dB, pTrX-dB and pTrC-dB) as well as four HDV-ribozyme chimera constructs with HBV (pTdXX, pTdXC, pTdSX and pTdSC) were severally chosen to validate the inhibition of the replication and expression of HBV. The co-expression plasmids (pTdδ and pTr-Db) in physiological saline were hydrodynamically injected to mice by tail vein. Results Compared with the group injected with pTr-dB in Huh-7 cell, hepatitis B surface antigen (HBsAg) was reduced by 31% in the group injected with pTdδ-dB, by 54%, 26%, 72% and 97% in the group injected with recombinant-ribozymes pTdSX, pTdSC, pTdXC and pTdXX, respectively. The inhibiting effects of endogenous ribozymes RzX and RzC on the HBsAg expression were 66% and 57%, respectively. Compared with the positive control, the amount of HBsAg was decreased in mice injected with pTdXX through tail vein by 88% and 96% on the second day and the third day, respectively. HBsAg was undetectable on the 6th day and could not primitively be detected on the 9th day in the sera from all mice. HBV DNA was not detected in the sera of BALB/c mice injected with pTdXX-dB, pTrX-dB or replicating-defective plasmid pHBV, while HBV DNA replication in control group could be detected on the 6th day. While HBcAg could not be detected in liver tissues of mice injected with plasmid pTdXX-dB on the 3rd day. Conclusions Encoding regions of HBV S, C and X gene were the effective cleavage sites for hammerhead ribozyme in vitro and in vivo, which provides basis for further construction of therapeutic recombinant HDV and the development of targeting antiviral gene therapy.

The response to interferon is influenced by hepatitis B virus genotype in vitro and in vivo

PurposeTo investigate the effectiveness of an interferon administration on different genotypes of hepatitis B virus (HBV) in vitro and in vivo. MethodsIn vitro, we transfected plasmids carrying different HBV genotypes including recently identified new genotype I into HepG2 and HuH7 cells, then treated with standard interferon alpha (IFN-α); in vivo, we treated mice with pegylated interferon alpha (Peg-IFN-α) after injection with HBV DNA of different genotypes. The culture supernatants from cell culture and sera from mice were collected and used in hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) assays by ELISA and HBV DNA measurement by PCR. ResultsBoth in cell culture and in mouse model, it was observed that HBV genotypes A and B exhibited significantly better response to IFN-α2a or Peg-IFN-α2a in terms of reduced expression of HBsAg, HBeAg and the HBV DNA level as compared to HBV genotypes C and D. Moreover, the inhibitory effect of IFN-α2a or Peg-IFN-α2a on HBV genotype I was greater than on genotype C or D, but less than genotype A. However, there was no significant response difference between genotypes A and B, C and D, B and I, respectively. ConclusionThe effectiveness of IFN/Peg-IFN to suppress HBV replication is dependent on different HBV genotypes. IFN/Peg-IFN is more effective on HBV genotype A or B than on genotype C, D or I. Treatment regimens are suggested to be adapted to HBV genotype.

Dynamic expression profile of HBsAg according to hepatic parenchyma cells' volume at different liver fibrosis stages in the immune clearance phase

The aim of this study was to determine the dynamic expression profile of hepatitis B surface antigen (HBsAg) according to hepatic parenchyma cells' volume at different stages of liver fibrosis during the immune clearance phase. Eighty-nine patients with HBeAg-positive chronic hepatitis B (CHB) in the immune clearance stage were recruited for study. Each patient's serum HBsAg levels were detected by electrochemiluminescence. The serum HBsAg levels were apportioned according to hepatic parenchyma cells' volume at liver fibrosis stages 1, 2, 3, and 4 and compared by ANOVA. The unapportioned serum HBsAg levels (IU/mL) at liver fibrosis stages 1 (227.2+/-237.7), 2 (211.0+/-131.4), 3(300.1+/-144.6), and 4 (278.7+/-148.8) were not significantly different (all comparisons, P range: 0.061 to 0.759). However, when the serum HBsAg levels were apportioned by the same hepatic parenchyma cells' volume at liver fibrosis stages 1 (343.9+/-359.8), 2 (336.4+/-209.5), 3 (508.7+/-245.1), and 4 (525.2+/-274.8), the levels were significantly different (all comparisons, F = 3.045 and P = 0.033; stage 1 vs. 3, P = 0.041; stage 1 vs. 4, P = 0.046; stage 2 vs. 3, P = 0.028; stage 2 vs. 4, P = 0.034). During the immune clearance phase of chronic hepatitis B, increased HBsAg expression is associated with increased hepatic parenchyma cells' volume and progressive liver fibrosis stage.

Inhibition of the HCV Core Protein on the Immune Response to HBV Surface Antigen and on HBV Gene Expression and Replication In Vivo

The hepatitis C virus (HCV) core protein is a multifunctional protein that can interfere with the induction of an immune response. It has been reported that the HCV core protein inhibits HBV replication in vitro. In this study, we test the effect of the HCV core gene on the priming of the immune response to hepatitis B surface antigen (HBsAg) and on the replication of HBV in vivo. Our results showed that the full-length HCV core gene inhibits the induction of an immune response to the heterogeneous antigen, HBsAg, at the site of inoculation when HCV core (pC191) and HBsAg (pHBsAg) expression plasmids are co-administered as DNA vaccines into BALB/c mice. The observed interference effect of the HCV core occurs in the priming stage and is limited to the DNA form of the HBsAg antigen, but not to the protein form. The HCV core reduces the protective effect of the HBsAg when the HBsAg and the HCV core are co-administered as vaccines in an HBV hydrodynamic mouse model because the HCV core induces immune tolerance to the heterogeneous HBsAg DNA antigen. These results suggest that HCV core may play an important role in viral persistence by the attenuation of host immune responses to different antigens. We further tested whether the HCV core interfered with the priming of the immune response in hepatocytes via the hydrodynamic co-injection of an HBV replication-competent plasmid and an HCV core plasmid. The HCV core inhibited HBV replication and antigen expression in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, the mouse models of acute and chronic hepatitis B virus infections. Thus, the HCV core inhibits the induction of a specific immune response to an HBsAg DNA vaccine. However, HCV C also interferes with HBV gene expression and replication in vivo, as observed in patients with coinfection.

Mutations associated with occult hepatitis B virus infection result in decreased surface antigen expression in vitro

Occult hepatitis B virus (HBV) infection is characterized by the absence of detectable hepatitis B surface antigen (HBsAg) in the serum, despite detectable HBV DNA. Investigations of the mechanisms underlying the development of occult HBV infection are lacking in the current literature, although viral mutations in the surface region, resulting in decreased HBsAg expression or secretion, represent one potential mechanism. Wild-type HBsAg expression vectors were constructed from genotype-matched chronic HBV sequences. Site-directed mutagenesis was then utilized to introduce three genotype A mutations - M103I, K122R and G145A - associated with occult HBV infection in vivo, alone and in combination, into the wild-type HBsAg vectors. Transfection of Huh7 and HepG2 cell lines was performed, and cell culture supernatants and cell lysates were collected over 7 days to assess the effects of these mutations on extracellular and intracellular HBsAg levels. The G145A mutation resulted in significantly decreased extracellular and intracellular HBsAg expression in vitro. The most pronounced reduction in HBsAg expression was observed when all three mutations were present. The mutations evaluated in vitro in the current study resulted in decreased HBsAg expression and potentially increased hepatic retention and/or decreased hepatic secretion of synthesized HBsAg, which could explain the lack of HBsAg detection that is characteristic of occult HBV infection in vivo.

Short hairpin RNAs with a 2- or 3-base mismatch inhibit HBV expression and replication in HepG2 cells.

To assess the functions of mismatched short hairpin RNAs (shRNAs) that inhibit replication and the expression of hepatitis B virus (HBV), two shRNAs possessing a 2- or 3-base mismatch that targeted HBV were studied. shRNAs and pHY106-HBV were cotransfected into HepG2 cells. The culture supernatants were collected and used in hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) assays. The levels of HBsAg and HBcAg mRNA were detected by reverse-transcriptase PCR (RT-PCR). HBV DNA replication intermediates were extracted for Southern blot hybridization. The results demonstrate that mismatched shRNA-458 and shRNA-635 can significantly inhibit HBsAg and HBeAg protein expression, and the maximal inhibition ratio for both proteins was found at 72h after cotransfection: 80 and 50%, respectively. Similar inhibitory effects were found on HBsAg and HBcAg mRNA levels and HBV DNA replication intermediates at 72h after cotransfection, and the inhibition ratio was found to be approximately 70 and 90%, respectively. Despite the 2- or 3-base mismatch between the shRNAs and the HBV target sequences, shRNA-458 and shRNA-635 exerted a significant inhibitory effect on HBsAg and HBeAg expression and HBV replication. This indicates that mismatched shRNAs could be a promising therapy for HBV.

High hepatitis B virus infection in B-cell lymphoma tissue and its potential clinical relevance

Our previous studies found that patients with B-cell non-Hodgkin lymphoma (NHL) had a higher incidence of hepatitis B virus (HBV) infection in serum than patients with T-cell NHL or other cancers. We sought to identify a possible role of HBV infection in B-cell NHL tumorigenesis and to understand its underlying clinical relevance. Fresh and paraffin-embedded primary tumor tissue from patients with NHL as well as from those with other lymphatic system diseases were investigated by PCR and immunohistochemistry. Many more patients with B-cell lymphoma whose serum was positive for hepatitis B surface antigen (HBsAg) were also positive for HBV-DNA than were those with T-cell NHL or other lymphatic system diseases whose serum was positive for HBsAg, in both fresh (55 vs. 15.4%) and paraffin-embedded (38.3 vs. 11.8%) tissue. Positive expression of the HBV-associated proteins HBsAg and hepatitis B core antigen was found in B-cell NHL lymphocytes and endothelial cells. Only 8.3% of patients with B-cell NHL who were negative for HBsAg but positive for other HBV markers were positive for HBV-DNA in tumor tissue. These results suggest that chronic HBV infection in lymph nodes could be associated with B-cell lymphoma.

Large Fragment Pre-S Deletion and High Viral Load Independently Predict Hepatitis B Relapse after Liver Transplantation

Hepatitis B virus (HBV) associated end-stage liver diseases are the leading causes of liver transplantation (LT) in Taiwan. Relapse of hepatitis B occurs after LT, raising the risk of graft failure and reducing patient survival. Although several oral antiviral agents have been approved for anti-HBV treatment, lamivudine (LAM) remained to be the most widely used preventive regimen in Taiwan. While several clinical predictors have been identified for hepatitis B relapse, the predictive roles of the histopathological characteristics in liver explants as well as the genotypic features of the viruses in pre-LT serum samples have not been assessed. Between September 2002 and August 2009, 150 consecutive hepatitis B surface antigen (HBsAg) positive patients undergoing LT were included for outcome analysis following assessment of the clinicopathological and virological factors prior to LT. Kaplan-Meier analyses discovered that pre-operative LAM treatment ≤3 months; membranous distribution and higher expression of tissue HBsAg in liver explants; preoperative viral load ≧106 copies/ml; and presence of large fragment (>100 base pairs) pre-S deletion (LFpreSDel) correlated significantly with hepatitis B relapse. Multivariate Cox regression analysis showed that the presence of LFpreSDel (P = 0.001) and viral load ≧106 copies/mL (P = 0.023) were independent predictors for hepatitis B relapse. In conclusion, besides high viral load, LFpreSDel mutation is an important independent predictor for hepatitis B relapse after LT. More aggressive preventive strategies should be applied for patients carrying these risk factors.