In recent years, Hepatitis B virus (HBV) persistent infection mouse model with recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome (rAAV8-1.3HBV) is concerned. We studied and compared the efficacy among HBV persistent infection mice models by other serotypes except AAV8. First, we prepared and purified five viruses: rAAV1-1.3HBV, rAAV2-1.3HBV, rAAV5-1.3HBV, rAAV8-1.3HBV and rAAV9-1.3HBV. Then we injected each virus into 3 C57BL/6J mice with the dose of lx 1011 vg (Viral genome, vg) per mouse. We detected HBsAg and HBeAg in sera by enzyme-linked immunosorbent assay (ELISA) at different time points post injection. We killed mice 8 weeks post injection and took blood and livers for assay. We detected copies of HBV DNA by real-time quantitative PCR in sera and livers. Meantime, we detected HBcAg in the livers of mice by immunohistochemistry and further performed pathology analysis of these livers. The five groups of mice, HBeAg and HBsAg expression sustained 8 weeks in serological detection and HBV DNA was both detected in sera and livers at the time of 8 weeks post injection. HBeAg, HBsAg, HBV DNA copies expression levels in descending order were AAV8>AAV9>AAV1>AAV5>AAV2. HBcAg expression was detected in livers as well. Varied degrees of liver damage were shown in five groups of mice. This study provides more alternative AAV vector species to establish a persistent infection with hepatitis B model.
Animals ; Dependovirus ; classification ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; Genome, Viral ; Hepatitis B ; virology ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; Mice ; Mice, Inbred C57BL ; Serogroup ; Virus Replication

<b>BACKGROUNDb>Cirrhosis is a common complication of chronic hepatitis B. It remains unclear if viral and biochemical parameters at baseline affect virological response to entecavir and therefore warrant investigation. In the present study, we aimed to evaluate the efficacy of entecavir therapy by monitoring virological response at the end of the 3 rd month of treatment and try to figure out whether baseline factors could help predict it in a cohort of hepatitis B virus (HBV) compensated cirrhosis patients and to determine the cut-off value of a predicting parameter.

<b>METHODSb>A total of 91 nucleos(t)ide-naïve patients with HBV induced cirrhosis (compensatory stage) were enrolled in a prospective cohort. HBV DNA and alanine aminotransferase (ALT) were tested at baseline and monitored every 3-6 months after starting therapy.

<b>RESULTSb>Of all 91 patients, the median follow-up time was 12 (9-24) months. Overall, 64 patients (70.3%) achieved virological response in the 3 rd month. Univariate analysis showed that the 3 rd month virological response can be predicted by baseline HBV DNA levels (P < 0.001, odds ratio [OR]: 2.13, 95% confidence interval [CI]: 1.44-3.15), ALT value (P = 0.023, OR: 1.01, 95% CI: 1.00-1.01) and hepatitis B e antigen (HBeAg) negativity (P = 0.016, OR: 0.30, 95% CI: 0.11-0.80). Multiple regression analysis showed baseline HBV DNA level was the only parameter related to full virological response. Higher baseline HBV DNA strata indicated a higher probability that HBV DNA remains detectable at the 3 rd month (P = 0.001). Area under receiver operating characteristic curve for determining the 3 rd month virological response by baseline HBV DNA was 77.6% (95% CI: 66.7-85.2%), with a best cut-off value of 5.8 log 10 .

<b>CONCLUSIONSb>Baseline HBV DNA, HBeAg negativity, and ALT were independent factors contributing to virological response at the 3 rd month. Further, multiple regression showed that HBV DNA level was the only parameter predicting full virological response as early as the 3 rd month, in this cirrhosis cohort.

Adolescent ; Adult ; Aged ; Alanine Transaminase ; metabolism ; Antiviral Agents ; therapeutic use ; DNA, Viral ; genetics ; Female ; Guanine ; analogs & derivatives ; therapeutic use ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; metabolism ; pathogenicity ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Liver Cirrhosis ; drug therapy ; virology ; Male ; Middle Aged ; Prospective Studies ; Regression Analysis ; Retrospective Studies ; Young Adult

<b>OBJECTIVEb>To explore the effect of the cytoplasmic DNA sensor DAI on replication of hepatitis B virus (HBV) and its possible mechanism.

<b>METHODSb>The hepatocyte-derived cell line HepG2 was co-transfected with DAI siRNA and the HBV1.3 replicative plasmid PHY106, and the cells were divided into two experimental groups. Six hours later, total RNA was extracted from the first group of cells and expression of IFIT1 and IL-6 were detected by real-time RT-PCR. The second group of cells was incubated for 4 days, after which the cell supernatant was collected and the HBV surface antigen (HBsAg) and envelope antigen (HBeAg) were detected by ELISA. In addition, HBV core particles were extracted and applied to southern blot assay to detect the intracellular HBV replication intermediates (rcDNA, dlDNA and ssDNA). Next, the HepG2 cells were triple transfected with siRNA targeting the type I interferon pathway molecule TBK1 and DAI simultaneously and HBV1.3, after which HBV viral proteins were detected. Two-group comparisons were made using the independent sample t-test, and more-than-2-group comparisons were made using ANOVA.

<b>RESULTSb>DAI gene expression was down-regulated in response to DAI siRNA transfection. Cells with down-regulated DAI showed inhibited HBV replication (in a dose-dependent manner), accompanied by reduced levels of HBsAg (0.0195+/-0.0050 vs.

<b>CONTROLb>0.3150+/-0.0200, P less than 0.05, t = 14.77) and HBeAg (0.0140+/-0.0040 vs.

<b>CONTROLb>0.01235+/-0.0135, P less than 0.05, t = 7.777). No effect of down-regulated DAI was observed for the expression of IFIT1 of IL-6. siRNA-mediated down-regulation of TBK1 and DAI simultaneously led to reduced expression of HBsAg and HBeAg.

<b>CONCLUSIONb>Down-regulation of DAI gene expression inhibited HBV replication and HBV protein expression, but the underlying mechanism was not related to the type I interferon or NF-kB signaling pathway.

Carrier Proteins ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Down-Regulation ; Gene Expression Regulation ; Hep G2 Cells ; Hepatitis B Surface Antigens ; isolation & purification ; Hepatitis B e Antigens ; isolation & purification ; Hepatitis B virus ; physiology ; Humans ; Interleukin-6 ; metabolism ; NF-kappa B ; metabolism ; Plasmids ; RNA, Small Interfering ; genetics ; Signal Transduction ; Transfection ; Virus Replication

BACKGROUND/AIMS: We investigated the efficacy and safety of tenofovir disoproxil fumarate (TDF)-based treatment in chronic hepatitis B (CHB) patients who failed previous antiviral therapies. METHODS: Seventeen patients who failed to achieve virological responses during sequential antiviral treatments were included. The patients were treated with TDF monotherapy (four patients) or a combination of TDF and lamivudine (13 patients) for a median of 42 months. Hepatitis B virus (HBV) DNA and hepatitis B e antigen (HBeAg) were measured, and renal function was also monitored. RESULTS: Prior to TDF therapy, 180 M, 204 I/V/S, 181 T/V, 236 T, and 184 L mutations were detected. After TDF therapy, the median HBV DNA level decreased from 4.6 log10 IU/mL to 2.0 log10 IU/mL and to 1.6 log10 IU/mL at 12 and 24 months, respectively. HBV DNA became undetectable (< or =20 IU/mL) in 14.3%, 41.7%, and 100% of patients after 12, 24, and 48 months of treatment, respectively. HBeAg loss was observed in two patients. Viral breakthrough occurred in five patients who had skipped their medication. No significant changes in renal function were observed. CONCLUSIONS: TDF-based rescue treatment is effective in reducing HBV DNA levels and is safe for patients with CHB who failed prior antiviral treatments. Patients' adherence to medication is related to viral rebound.
Adenine/*analogs & derivatives/therapeutic use ; Adult ; Biological Markers/metabolism ; DNA, Viral/blood ; Drug Resistance, Viral/drug effects ; Drug Therapy, Combination ; Female ; Hepatitis B e Antigens/blood ; Hepatitis B, Chronic/*drug therapy ; Humans ; Lamivudine/*therapeutic use ; Male ; Middle Aged ; Organophosphonates/*therapeutic use ; Reverse Transcriptase Inhibitors/*therapeutic use ; Treatment Outcome

MicroRNA polymorphisms may be associated with carcinogenesis or immunopathogenesis of infection. We evaluated whether the mircoRNA-604 (miR-604) polymorphism can affect the persistence of hepatitis B virus (HBV) infection, and the development to hepatocellular carcinoma (HCC) in patients with chronic HBV infection. A total of 1,439 subjects, who have either past or present HBV infection, were enrolled and divided into four groups (spontaneous recovery, chronic HBV carrier without cirrhosis, liver cirrhosis and HCC). We genotyped the precursor miR-604 genome region polymorphism. The CC genotype of miR-604 rs2368392 was most frequently observed and T allele frequency was 0.326 in all study subjects. The HBV persistence after infection was higher in those subjects with miR-604 T allele (P=0.05 in a co-dominant and dominant model), which implied that the patients with miR-604 T allele may have a higher risk for HBV chronicity. In contrast, there was a higher rate of the miR-604 T allele in the chronic carrier without HCC patients, compared to those of the HCC patients (P=0.03 in a co-dominant model, P=0.02 in a recessive model). The T allele at miR-604 rs2368392 may be a risk allele for the chronicity of HBV infection, but may be a protective allele for the progression to HCC in chronic HBV carriers.
Adult ; Aged ; Aged, 80 and over ; Base Sequence ; Carcinoma, Hepatocellular/etiology/*genetics/pathology ; Case-Control Studies ; Demography ; Female ; Gene Frequency ; *Genetic Predisposition to Disease ; Genotype ; Hepatitis B Antibodies/blood ; Hepatitis B Surface Antigens/blood ; Hepatitis B e Antigens/blood ; Hepatitis B virus/metabolism ; Hepatitis B, Chronic/complications/*genetics/virology ; Humans ; Liver Neoplasms/etiology/*genetics/pathology ; Male ; MicroRNAs/*genetics/metabolism ; Middle Aged ; Polymorphism, Single Nucleotide ; Risk Factors

<b>BACKGROUNDb>Some hepatitis B extracellular antigen (HBeAg)-positive chronic hepatitis B (CHB) patients in their immune active phase can clear the virus spontaneously and enter into an inactive hepatitis B virus (HBV) carrier state, indicating a benign prognosis. In this study, the association between cytokine-inducible SRC homology 2 domain protein (CISH) gene polymorphisms at -292 (rs414171) and the spontaneous clearance of HBV in HBeAg-positive CHB patients in immune the active phase was investigated.

<b>METHODSb>Seventy HBeAg-positive CHB patients in the immune active phase were followed up for 76 weeks without antiviral therapy. The alanine transaminase, aspartate transaminase, HBV DNA, HBeAg and hepatitis B extracellular antibody levels were tested regularly. At week 76, 27 patients were classified into group A (HBV DNA level below 2 104 IU/ml and the value of HBeAg declined below 10% of the baseline at week 76), and 43 patients were classified into group B (HBV DNA level higher than 2×10(4) IU/ml or the value of HBeAg did not decline substantially at week 76). CISH (rs414171) polymorphisms were also tested using the iPLEX system.

<b>RESULTSb>The HBV DNA levels at week 12 were significantly greater in group B compared with group A (group A: (6.87±1.40) log10IU/ml; group B: (7.61±1.38) log10IU/ml, P = 0.034) and the HBeAg values were greater in group B at week 28 compared with group A (P = 0.001). The differences in HBV DNA and HBeAg values increased between the groups over time. Sixteen patients in group A and 11 in group B were genotype AA. Those with genotype AT or TT included 11 in group A and 31 in group B (AA vs. AT and TT, odds ratio 4.10 (95% confidence interval: 1.462-11.491), P = 0.006).

<b>CONCLUSIONb>CISH gene polymorphisms at -292 (rs414171) are associated with HBV clearance in HBeAg-positive CHB patients in the immune active phase, and AA is a favorable genotype for this effect.

Adolescent ; Adult ; Aged ; Female ; Genotype ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; immunology ; Hepatitis B, Chronic ; immunology ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; genetics ; Suppressor of Cytokine Signaling Proteins ; genetics ; Young Adult

<b>OBJECTIVEb>To study the role of tumor necrosis factor-alpha (TNFalpha) in the anti-replication effects of tetracycline (Tet) on hepatitis B virus (HBV).

<b>METHODSb>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.

<b>RESULTSb>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.

<b>CONCLUSIONb>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.

DNA, Viral ; biosynthesis ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; drug effects ; physiology ; Humans ; Tetracycline ; pharmacology ; Transfection ; Tumor Necrosis Factor-alpha ; genetics ; Virus Replication

BACKGROUND/AIMS: Carnitine and vitamin complex (Godex(R)) is widely used in patients with chronic liver disease who show elevated liver enzyme in South Korea. The purpose of this study is to identify the efficacy and safety of carnitine from entecavir combination therapy in Alanine aminotransferase (ALT) elevated Chronic Hepatitis B (CHB) patients. METHODS: 130 treatment-naive patients with CHB were enrolled from 13 sites. The patients were randomly selected to the entecavir and the complex of entecavir and carnitine. The primary endpoint of the study is ALT normalization level after 12 months. RESULTS: Among the 130 patients, 119 patients completed the study treatment. The ALT normalization at 3 months was 58.9% for the monotherapy and 95.2% for the combination therapy (P<0.0001). ALT normalization rate at 12 months was 85.7% for the monotherapy and 100% for the combination group (P=0.0019). The rate of less than HBV DNA 300 copies/mL at 12 months was not statistically significant (P=0.5318) 75.9% for the monotherapy, 70.7% for the combination and it was. Quantification of HBsAg level was not different from the monotherapy to combination at 12 months. Changes of ELISPOT value to evaluate the INF-gamma secretion by HBsAg showed the increasing trend of combination therapy compare to mono-treatment. CONCLUSIONS: ALT normalization rate was higher in carnitine complex combination group than entecavir group in CHB. Combination group was faster than entecavir mono-treatment group on ALT normalization rate. HBV DNA normalization rate and the serum HBV-DNA level were not changed by carnitine complex treatment.
Adult ; Alanine Transaminase/blood ; Antiviral Agents/*therapeutic use ; Carnitine/*therapeutic use ; DNA, Viral/analysis ; Drug Therapy, Combination ; Enzyme-Linked Immunospot Assay ; Female ; Guanine/*analogs & derivatives/therapeutic use ; Hepatitis B Surface Antigens/blood ; Hepatitis B e Antigens/blood ; Hepatitis B virus/genetics ; Hepatitis B, Chronic/*drug therapy ; Humans ; Interferon-gamma/metabolism ; Male ; Middle Aged ; Mitochondria/physiology ; Treatment Outcome ; Vitamin B Complex/*therapeutic use

<b>OBJECTIVEb>To investigate whether hepatitis B e antigen (HBeAg) can modulate the ability of dendritic cells (DCs) to produce inflammatory cytokines (IL-12/IL-6) upon stimulation in vitro.

<b>METHODSb>Purified adherent mononuclear cells isolated by Ficoll-hypaque density gradient centrifugation were cultured in complete medium containing granulocyte macrophage colony-stimulating factor plus interleukin (IL)-4 to generate immature (i)DCs. Microscopic analysis and flow cytometry were performed to define the phenotypic characteristics of the iDCs. Then, different concentrations (1, 2 and 5 mug/ml) of HBeAg were added to the culture medium and for 24 hrs of incubation. To induce iDCs' maturation, the various groups of cells were incubated for 24 hrs in differentiation culture with lipopolysaccharide (LPS). Effects on secreted inflammatory cytokines were determined by enzyme-linked immunosorbent assay of the cells' supernatants.

<b>RESULTSb>All concentrations of HBeAg led to significant reductions in IL-6 (all P less than 0.05). Similar significant reduction trends were seen for IL-12 at the HBeAg concentrations of 2 and 5 mug/ml (both P less than 0.05), but not at the 1 mug/ml concentration.

<b>CONCLUSIONb>HBeAg may suppress the production of cytokines from DCs; this mechanism may contribute to the immune escape of HBV that supports persistent infection.

Cells, Cultured ; Dendritic Cells ; immunology ; metabolism ; Hepatitis B e Antigens ; immunology ; Humans ; Interleukin-12 ; metabolism ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; adverse effects

<b>OBJECTIVEb>To study the immunoregulatory effect of hepatitis B virus (HBV) e antigen (HBeAg) on peripheral blood monocytes (PBMCs).

<b>METHODSb>PBMCs were isolated from patients with chronic hepatitis B (CHB; both HBeAg- and HBeAg+) and healthy controls, and cultured with recombinant HBeAg. The HBeAg-induced changes in expression of PD-1/PD-L1 were measured by flow cytometry of the cells and in secreted cytokines were measured by enzyme-linked immunosorbent assay of the supernatants. Comparisons between two groups were made by the independent-samples t-test; the relationship between PD-1/B7-H1 level and HBV DNA copy number was evaluated by Spearman's correlation analysis.

<b>RESULTSb>Exposure to HBeAg led to a significant decrease in CD3+CD4+ T lymphocyte-specific expression of IFNa for both the CHB patients' and healthy controls' samples (t = 2.382 and -4.190 respectively, P less than 0.01). For the HBeAg- CHB patients' and healthy controls' samples, the HBeAg exposure led to increased levels of secreted cytokines IL-6, IL-10 and TNFa (t = 2.504, 3.583 and 4.324, P less than 0.01 and t = 3.542, 6.246 and 5.273, P less than 0.01 respectively) and of CD14+ PBMC-specific expression of PD-L1 (t = 4.815 and 3.454, P less than 0.05 respectively). Compared to the HBeAg-negative CHB patients' and healthy controls' samples, the HBeAg+ CHB patients' samples had significantly lower CD3+CD4+ T cell-specific expression of IFNa (t = -3.177 and -4.541, P less than 0.01 respectively), but significantly higher levels of secreted IL-4 (t = 3.382 and 4.393, P less than 0.01 respectively), of CD3+ T cells-specific expression of PD-1/PD-L1 (t = 4.755, 2.942 and 4.518, 4.595, P less than 0.01 respectively), and of CD14+ T cells-specific expression of PD-L1 (t = 5.092 and 5.473, P less than 0.01 respectively). The CD3+ T cells-specific expression of PD-L1 was significantly higher in the samples from HBeAg- CHB patients than from the healthy controls (t = 3.214, P less than 0.01).

<b>CONCLUSIONb>HBeAg was able to down-regulate the production of Th1-type cytokines (IFNgamma), and up-regulate the secretion of Th2-type cytokines (IL-6, IL-10) and the expression of PD-1/PD-L1on monocytes. These changes are conducive to the formation of immune tolerance to HBV. Therefore, HBeAg may play an important role in immune tolerance to chronic HBV infection.

Adult ; Case-Control Studies ; Cells, Cultured ; Female ; Hepatitis B e Antigens ; genetics ; immunology ; Hepatitis B, Chronic ; blood ; immunology ; Humans ; Interferon-gamma ; immunology ; Interleukin-10 ; immunology ; Interleukin-6 ; immunology ; Leukocytes, Mononuclear ; immunology ; metabolism ; Male ; Middle Aged ; Recombinant Proteins ; immunology ; Th1 Cells ; immunology ; Th1-Th2 Balance ; Th2 Cells ; immunology