Mechanism of hepatitis B virus infection in renal tissues with IgA nephropathy

AIM: To identify the pathogenesis of renal lesions induced by hepatitis B virus (HBV) infection in IgA nephropathy (IgAN). METHODS: Forty-eight renal biopsy tissues of IgAN were selected, and were divided into five grades from Ⅰ to Ⅴ according to the classified standard of Meadow. HBsAg and HBcAg in renal tissues were detected by immunohistochemistry methods of Envision. Eighteen renal tissues with IgAN among 48 renal biopsy tissues were selected randomly, and then HBV DNA in these tissues was detected by direct in site polymerase chain reaction (IS-PCR) method. RESULTS: Thirty-six (36/48, 75.00%) and twenty-one (21/39, 53.85%) cases were positive with HBcAg and HBsAg in 48 cases renal tissues with IgAN, respectively. The positive rate of HBV DNA in 18 cases with IgAN was 61.11% (11/18). The positive rate of HBcAg， HBsAg and HBV DNA in renal tissues were all no significance betwen every grade (P＞0.05), but the positive rate of HBcAg, HBsAg and HBV DNA in renal tubule were all higher than that in glomeruli (P＜0.05). CONCLUSIONS: HBV really takes part in the occurrence of IgAN. HBV maybe induces the renal injury by cell-mediated immunity or a series of cytokines but not by virus direct damage. The renal tubule epithelium may be the targeting cells of HBV infection.     

Effects of xeroderma pigmentosum group D and p53 on replication of HBV

AIM:To investigate the effects of xeroderma pigmentosum D (XPD) and p53 on the replication of hepatitis B virus (HBV). METHODS:Recombinant plasmid pEGFP-N2/XPD and vacant vector plasmid pEGFP-N2 were transfected into HepG2.2.15 cells by liposome. On the next day, these cells were incubated with pifithrin-α, a p53 inhibitor, at a concentration of 20 μmol/L for 24 h. The cells were divided into 5 groups: blank control group, pEGFP-N2 group, pEGFP-N2/XPD group, pEGFP-N2/XPD+pifithrin-α group and pifithrin-α group. The mRNA expression of XPD, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and hepatitis B virus X protein (HBx) was detected by RT-PCR. The content of HBsAg and HBeAg in the supernatants of culture medium was measured by ELISA. The content of HBV-DNA in the supernatants of culture medium was examined by fluorescence quantitative PCR. Using the method of bDNA, the content of HBV-DNA in the core particles was assessed. RESULTS:The expression of XPD mRNA was elevated by the transfection of recombinant plasmid pEGFP-N2/XPD. The increase in XPD expression significantly down-regulated the mRNA expression of HBsAg, HBeAg and HBx. The content of HBsAg and HBeAg in the supernatants of culture medium was significantly decreased by the increase in XPD expression. The results of fluorescence quantitative PCR showed that the content of HBV-DNA in the supernatants of culture medium was significantly down-regulated by the increase in XPD expression. bDNA results showed that the content of HBV-DNA in the core particles was significantly decreased by the increase in XPD expression.Pifithrin-α abolished the above-mentioned effects of XPD (all P<0.01). CONCLUSION: XPD inhibits the replication of HBV through p53 pathway. Therefore, XPD and p53 may be the targets for antiviral therapy of hepatitis B.     

HTPP-MDC inhibits replication and subcellular localization of hepatitis B virus in HepG2.2.15 cells

AIM：To investigate the antiviral effect of hepatocyte-targeting and cell-penetrating peptide and Musca domestica cecropin (HTPP-MDC) fusion polypeptide against hepatitis B virus (HBV) in vitro, and to observe the penetrating ability of HTPP-MDC in hepatocytes. METHODS： HepG2.2.15 cells and Chang liver cells were co-cultured in vitro with HTPP-MDC at different concentrations. MTT assay was used to detect the cytotoxicity of HTPP-MDC in vitro. The levels of HBsAg, HBeAg and HBV DNA in HepG2.2.15 cell culture supernatants were measured by ELISA and real-time fluorescence quantitative PCR. The penetrating ability of HTPP-MDC was detected by laser confocal microscopy. RESULTS：The levels of HBsAg, HBeAg and HBV DNA in the supernatants of HepG2.2.15 cells treated with HTPP-MDC were remarkably reduced. The inhibitory effect of HTPP-MDC depended on the dose and action time of the drug. FITC-labeled HTPP-MDC was observed inside the cells under laser confocal microscope. CONCLUSION：HTPP-MDC strongly inhibits HBV replication in HepG2.2.15 cells. The penetrating ability of HTPP-MDC into hepatocytes indicates that HTPP-MDC is useful in clinic therapy for chronic hepatitis B-related diseases in the future.     

Relationship between HBV genotype,PreS/S gene mutation and immunoprophylaxis failure to prevent HBV mother-to-child transmission

AIM：To investigate the relationship between hepatitis B virus (HBV) genotype, PreS/S gene mutation and immunoprophylaxis failure to prevent HBV mother-to-child transmission.
METHODS：Pregnant women with positive HBV surface antigen (HBsAg) and HBV DNA≥1×1010 IU/L were divided into case group (15 cases) and control group (45 cases) according to their neonates with immunoprophylaxis failure or not. The genotypes of HBV and the mutation rate and mutational hot spots in PreS/S gene were detected by PCR amplification technique in the two groups.
RESULTS：(1) Genotypes B and C of HBV were detected in both case and control groups, and the majority of HBV genotype was B in the two groups. Genotype distribution difference between case and control groups was not statistically significant (P>0.05). (2) There was no significant difference in the mutation rate of PreS/S gene between case and control groups (P>0.05). The mutation rates of PreS/S gene between genotypes B and C were significantly different (P<0.05), but when the HBV genotype was the same, the mutation rate of PreS/S gene had no significant difference between case and control groups. Homology tree model based on PreS2/S gene formed genotype B and genotype C clusters, and in each cluster, the sequences of case and control groups did not formed smaller different clusters further. (3) 529G-A, 530A-G, 826A-G1 and 166het-dupC were hot spots of mutation in PreS2/S gene and were found in 4 cases in case group, respectively. A530T (1 case), A530G (2 cases), T531C (3 cases) were found in control group.
CONCLUSION：(1) The mutation rates of PreS/S gene are different in various genotypes. (2) The mutation in PreS/S gene of HBV is prevalent, but not all of the mutations are related to immunoprophylaxis failure to prevent HBV mother-to-child transmission. To find mutational hot spots which are related to immunoprophylaxis failure is more important.     

Effect of hepatitis virus B protein on the functions of PBMCs isolated from patients with chronic HBV infection

AIM: To study the effect of hepatitis virus B proteins on peripheral blood mononuclear cells (PBMCs) from patients among various types of chronic hepatitis B virus (HBV) infection.METHODS: 80 patients of various types of chronic HBV infection were observed, including 40 HBeAg positive with abnormal alanine aminotransferase (ALT) (A group), 20 HBeAg positive with persistent normal ALT(B group), 20 HBeAg and HBV-DNA negative with persistent normal ALT level(C group). IL-10, IFN-γ in CD8+CD28+T cells, after stimulation with PHA, HBeAg and HBcAg for 48 h, were inspected respectively in PBMCs.RESULTS: IFN-γ was significantly lower in HBeAg positive patients. IL-10 was significantly higher in HBeAg positive with normal ALT. CD8+CD28+T were significantly lower than others. CONCLUSION: In HBeAg positive group, secretion of cytotoxic T lymphocyte (CTL) and Th1 type cellular immunologic reaction is decreased, Th2 type cellular immunologic reaction is enhanced.     

Inhibitory effect of HMGN2 protein on human hepatitis B viral HBeAg and HBsAg expression and DNA replication in HBV-transformed HepG2. 2.15 cell line

AIM: We previously demonstrated that HMGN2 is an antibacterial effector molecule in human LAK cells. This study was to examine the antiviral activity of HMGN2 against human hepatitis virus.METHODS: The purification and identify of HMGN2 proteins including preparative acid-urea polyacymide gel electrophoresis elution, reverse-phase high-performance liquid chromatography, mass spectrum, Western blotting and antimicrobial assay were conducted. The cellular toxicity of HMGN2 to the HepG2.2.15 cells was detected by MTT assay. HBeAg and HBsAg expressions were measured by ELISA. HBV DNA copies were determined by real time quantitative PCR.RESULTS: A bulk of HMGN2 was isolated and purified from the acid soluble proteins of human lymph node, and identified. The HBV-transfected HepG2.2.15 cell line was used in the in vitro assay system.In the range of testing 1-100 mg/L of HMGN2, no cytotoxicity to HepG2.2.15 cells was detected by MTT assay.When incubated with HMGN2 at 1-5 mg/L for 72 h or 144 h, a significant reduction in HBeAg and HBsAg expression and in HBV DNA copies was observed in the supernatant of HepG2.2.15 cells. CONCLUSION: HMGN2 protein markedly inhibits HBV expression and replication in vitro.     

Role of hepatitis B virus in intrahepatic TGF-β1/Smads signaling pathway

AIM:To explore the effects of hepatitis B virus (HBV) on intrahepatic expression of transforming growth factor β1（TGF-β1） and Smads. METHODS:The expression of intrahepatic TGF-β1, HBsAg and HBcAg in control group and chronic hepatitis B (CHB) group was detected by immunohistochemical method.The serum HBV DNA content was determined by real-time PCR. The role of HBV in the expression of TGF-β1, Smad3 and Smad7 in human hepatic stellate cell line LX-2 in vitro was observed by cell culture and Western blotting. RESULTS:The average score of intrahepatic TGF-β1 expression in CHB group was higher than that in control group. With the increase in serum HBV DNA content, intrahepatic TGF-β1 expression was also enhanced. In the HBcAg positive hepatic tissue, there was higher TGF-β1 expression than that in the liver tissue of HBcAg negative. Compared with control group and HBV+anti-TGF-β1 group, HBV caused increased expression of TGF-β1 and Smad3 in HBV group in vitro. No difference of Smad7 protein among control group, HBV group and HBV+anti-TGF-β1 group was observed. CONCLUSION: The expression of intrahepatic TGF-β1 is related to serum HBV DNA and hepatocellular HBcAg in the patients with CHB. HBV-induced liver fibrosis mainly relies on positive regulatory mechanisms of Smad3,and the negative regulation by Smad7 almost does not function.     

Two-unit ribozyme inhibit hepatitis B virus expressed in 2.2.15 cell

AIM: To observe the inhibition of HBc/HBeAg expression in the 2.2.15 cell transfected by two-unit ribozyme. METHODS: By use of subclone technique, two-unit ribozyme gene which was cutted from pGEM-Rz123 was ligated into the eukaryotic expression vector pBBS212. The recombinant plasmid was cotransfected into 2.2.15 cell using lipofectamine. Ribozyme was detected by dot-blot hybridization. The s and e/c antigen of HBV were detected by using ELISA, immunohistochemical technique, image analysis system and Western blot. RESULTS: After the transfected cell was selected two weeks by hygromycin B and G418. We found by dot-blot hybridization that ribozyme can express on 2.2.15 cell. HBeAg level can be reduced by 48.6% in the transfected 2.2.15 cell with two-ribozyme. Using immunohistochemical technique and image analysis system, Western blot, we observed that the level of HBcAg expressed in endocellular went down. CONCLUSION:Al these results strongly indicate that two-unit ribozyme can inhibit hepatitis B virus expression in the cell.     

Effect of Paecilomyces cicadae polysaccharide on duplication of hepatitis B virus in HepG2.2.15 cell strain

AIM: To investigative the inhibitory effects of Paecilomyces cicadae polysaccharide (PcPS) against HBV in vitro, and the effects on the Toll like receptor 4 (TLR4) mRNA expression in HepG2.2.15 cell strain. METHODS: HepG2.2.15 cell strain was co-cultured in vitro with PcPS in different concentrations, and lamivudine (LMV) was applied as positive control. MTT assay was employed to detect the cytotoxicities of PcPS in vitro, when the HepG2.2.15 cells was used as target cells. The effects of PcPS on the secretion of HBsAg and HBeAg were assayed by ELISA method. Fluorescence quantitative-PCR (FQ-PCR) was used to detect the inhibitory effects of PcPS on the content of HBV-DNA and TLR4 mRNA in HepG2.2.15 cells. RESULTS: The inhibitory effects of PcPS on the HBsAg and HBeAg were observed and the maximum inhibitory ratio up to 44.8% and 31.0%, respectively. The same inhibitory effects of PcPS on the HBV-DNA replication and TLR4 mRNA expression in HepG2.2.15 cells were also found. CONCLUSION: A certain concentration of PcPS significantly inhibits HBV replication in vitro.     

Expression of fractalkine and CX3CR1 in renal tissues of patients with WHO class IV lupus nephritis

AIM: To observe the expression of fractalkine, and its receptor, CX3CR1, in renal tissues of patients with diffuse proliferative lupus glomerulonephritis (WHO class IV), minimal glomerular abnormalities, and normal kidney. Meanwhile, the correlation among the expression of fractalkine, CX3CR1 and CD68-positive macrophages was investigated, and the role of fractalkine and CX3CR1 in the pathogenesis of lupus nephritis was discussed. METHODS: The expressions of fractalkine, CX3CR1 and CD68 were detected immunohistochemically in kidney tissue sections obtained from twenty-one patients with WHO class IV lupus nephritis, eighteen cases with minimal glomerular abnormalities, and eight normal kidneys which were no abnormality under light microscope. RESULTS: (1) Fractalkine was generally indistinguishable in tissue sections from normal kidney and minimal glomerular abnormalities. CX3CR1-positive cells and CD68-positive macrophages were sparsely detected in the glomeruli and in the cortical interstitium. (2) There were considerable CX3CR1-positive cells and macrophages in both the glomeruli and the interstitium in sections from class IV lupus nephritis. The number of CX3CR1-positive cells significantly correlated with the number of macrophages in the glomeruli and in the interstitium respectively (r=0.956, P<0.01 and r=0.965, P<0.01). (3) Significant expression of fractalkine was seen in the cortical renal tubules from class IV lupus nephritis. The percentage of fractalkine-positive tubules significantly correlated with the number of CX3CR1-positive cells and macrophages in the interstitium respectively (r=0.720, P<0.01 and r=0.770, P<0.01). CONCLUSION: These expression patterns show that fractalkine and CX3CR1 may play an important role in the pathogenesis of class IV lupus nephritis.     

HBx modulates apoptosis by activating JAK2/STAT3 signaling pathway in human renal proximal tubular epithelial cells

AIM: To investigate the correlation of hepatitis B virus X protein (HBx) with renal tubular epithelial cell apoptosis in hepatitis B virus-associated glomerulonephritis (HBVGN) and the possible signaling mechanism. METHODS: The activation of JAK2/STAT3 signal pathway and the expression of apoptosis-related proteins in human kindey proximal tubular epithelial cells (HK-2 cells) were determined by Western blotting after transfection with HBx eukaryotic expression vector. The cell proliferation was observed by CCK-8 assay. The cell apoptosis was analyzed by the imaging of HO33342 staining, transmission electron microscopy and flow cytometry with Annexin V/PI double staining. RESULTS: After transfection of the target gene HBx, the expression levels of both p-JAK2 and p-STAT3 were significantly increased. At the same time, the cell proliferation was obviously inhibited, and the apoptotic rate was increased. After incubation with AG490, the JAK2/STAT3 signal pathway was partially blocked, and the cell apoptosis induced by HBx was reduced. CONCLUSION：HBx up-regulates the activation of JAK2/STAT3 signal pathway to induce renal tubular epithelial cell apoptosis, which is possibly involved in the pathogenic mechanism that HBV directly damages nephridial tissue.     

Paired study on hepatitis B virus S gene mutation in immunoprophylaxis failure to prevent HBV vertical transmission

AIM：To explore the characteristics of hepatitis B virus S gene mutation in the vertical transmission after active and passive vaccination. METHODS：Fifteen cases of immunoprophylaxis failure were enrolled in the study. HBV S gene (including pres-S and S) from the mothers, newborns before active and passive vaccination and 7-month-old infants with immunoprophylaxis failure were detected by PCR amplification. The characteristics of HBV S gene mutation were compared among the 3 groups. RESULTS：The genotype of HBV in the newborns and the infants was the same as that in the mothers. The frequencies of mutation in the 2 fragments of the HBV S gene had no significant difference between the 3 groups. The homology tree model based on HBV S gene was analyzed in the 3 groups, in which every group had their own cluster. There were 15 different mutation sites between 7 pairs of mothers and newborns. There were 3 different mutation sites between 3 pairs of newborns and infants (nt273A→A/G, nt512C→C/T and nt1139C→A), among which the first 2 were located in the S gene region but not in the “a” determinant, and the latter was located in the overlap region of S and X genes. There were 25 different mutation sites between 9 pairs of mothers and infants, but only 1 case had a different mutation site between the mother, newborn and infant. CONCLUSION：The HBV species in newborns and infants with immunoprophylaxis failure were transmitted from the mothers. The mutations in the HBV S gene with immunoprophylaxis failure happened before and after active and passive vaccination, mainly before vaccination. The relationship between HBV S gene mutations and immunoprophylaxis failure should be further explored.     

Role of TGF-β1 and its signaling transduction molecule Smad4 in the development of glomerulosclerosis

AIM: To explore the role of TGF-β1 and signaling transduction molecule， Smad4， in the development of glomerulosclerosis. METHODS: Expression levels of TGF-β1, Smad4, collagen Ⅰ proteins were evaluated by immunohistochemistry in renal biopsies from 38 cases with a spectrum of glomerulonephritis, and compared with 20 normal kidney tissue with image analysis system. After stimulation with TGF-β1, expressions of endogenous Smad4 and collagen Ⅰ mRNA and proteins and its modulation by TGFβ1 were evaluated by RT-PCR and Western blotting analyses in cultured human mesangial cells. RESULTS: All types of proliferative and sclerotized glomerulonephritis showed an increased expression of TGF-β1, Smad4 and collagen Ⅰ ompared with the 20 normal kidney tissue in glomerular (P<0.05). The expression of TGF-β1 and Smad4 proteins were correlation with deposition of collagen Ⅰ (P<0.05). The expression of Smad4 mRNA and protein were elevated by TGF-β1 in a dose and time-dependent manner (P<0.05), and the expression of collagen Ⅰ mRNA and protein were also increased both in a dose and time-dependent manner (P<0.05). CONCLUSION: Our data indicate that TGF-β1 and its signaling transduction molecule (Smad4 proteins) may be involved in the excessive deposition of extracellular matrix in glomerular disease and play an important role in the genesis and progress of glomerulosclerosis.     

Effect of antiserum against complement C5b-9 complexes on nitric oxide(NO) synthesis and pathologic changes in renal tissue of ATSN rats

AIM: To explore the effect of antiserum against rat complement C5b-9 complexes on nitric oxide synthesis and pathologic changes in renal tissue of rats with mesangioproliferative glomerulonephritis. METHEDS: The rat model of mesangioproliferative glomerulonephritis,namely,anti-thymocyte serum nephritis(ATSN) was established and the rats with ATSN were treated with antiserum against complement C5b-9 complexes. Some parameters related to NO and pathologic changes of the rats were observed.RESULTS: Anti-C5b-9 serum not only reduced the expression of inducible NO synthase(iNOS) mRNA in renal tissue and urinary content of nitrate/nitrite(NO₃^-/ NO₂^-) of rats with ATSN, but also reduced renal pathologic changes. L-N G-nitro arginine methylester (L-NAME) also reduced the contents of urinary NO₃^-/NO₂^- and the renal pathologic changes of rats with ATSN. CONCLUSION: The antiserum against rat complement C5b-9 complexes inhibited glomerular NO synthesis and glomerular mesangial cell proliferation of rats with ATSN.     

Correlation between HBsAg level and HBV DNA titer during chronic hepatitis B treatment

AIM: Viral load is widely used as an indicator for the diagnosis and monitoring the treatment efficacy of chronic hepatitis B (CHB). Previous studies suggested that the quantity of hepatitis B surface antigen (HBsAg) in serum could be a surrogate marker of serum hepatitis B virus (HBV) DNA level. In this study, we aimed to investigate whether HBsAg level correlates with HBV DNA titer during CHB treatment. METHODS: Sera were collected from 47 CHB patients ［35 male, 12 female, mean age: (35±8) years］ treated for 48 weeks with a monotherapy (pegylated interferon alpha-2a, 18 patients; lamivudine, 15 patients) or a combination therapy (pegylated interferon alpha-2a and lamivudine, 14 patients). Serum samples were obtained at week 0 (just before the treatment), 4, 8, 24, 48 and week 72 (24 weeks after the treatment). HBV DNA was measured with real-time polymerase chain reaction (PCR). HBsAg was quantified with an automated chemiluminescent microparticle immunoassay. RESULTS: The titer of HBsAg correlated with the HBV DNA level in the 18 patients with monotherapy of pegylated interferon alpha-2a and the 14 patients with combination therapy (pegylated interferon alpha-2a and lamivudine). The significant correlation (canonical correlation=0.83) was found. However, no correlation in 15 patients with the monotherapy of lamivudine was observed. CONCLUSION: HBsAg titer correlates with HBV DNA level in CHB patients during the treatment with interferon or interferon and lamivudine, which can be a surrogate marker for monitoring the treatment efficacy.     

Relationship between precore/basal core promoter mutations of hepatitis B virus and therapeutic effect of peginterferon α-2b,and changes of mutations before and after treatment

AIM:To investigate the relationship between therapeutic effect of peginterferon α-2b (Peg-IFNα-2b) and precore (PC) region G1896A and basal core promoter (BCP) region A1762T/G1764A mutations of hepatitis B virus (HBV), and the changes of the mutations before and after treatment. METHODS:The patients with HBeAg-positive chronic hepatitis B (CHB) (n=69) were treated with Peg-IFNα-2b for 48 weeks and followed up for 24 weeks. The PC and BCP sequences at baseline and the 72th week were determined using polymerase chain reaction (PCR) and direct sequencing. Serum HBsAg, HBeAg, alanine aminotransferase (ALT) and HBV DNA was quantified in the samples taken at baseline (week 0), during the treatment period (weeks 4, 8, 12, 24, 36 and 48), and during follow-up (weeks 60 and 72). RESULTS:Within the total cohort, wild-type (WT) virus was detectable in only 14 patients (20.29%), and mutants were detected in 55 patients (79.71%). The serum HBeAg level in the patients with mutant virus was significantly lower than that in the patients with WT virus (P=0.024). The proportion of WT, PC mutant, BCP mutant and PC+BCP mutant was significantly changed at baseline and week 72 (P=0.004). No significant difference of HBeAg seroconversion and combined response between patients with WT virus or mutants (PC, BCP and PC+BCP) was observed. CONCLUSION:PC and BCP mutations have no effect on the response of HBeAg-positive CHB patients to Peg-IFNα-2b. The proportion of each mutation was significantly changed before and after treatment.     

Construction of the retroviral vector recombinant of HBV-S gene and its expression in antigen presenting cells

AIM: To investigate the effectiveness of recombinanted retrovirus vector in gene therapy. METHODS: The retroviral vector pLXSN-S was constructed and transferred into PA317 by means of electroporation, then HepG₂、RAW264.7 and EL₄ cells were infected with the pseudovirus produced from PA317, which highly expressed HBsAg. HBsAg expression was tested by RT-PCR and ELISA. RESULTS: HBsAg was expressed variously in the eukaryotic cells mentioned above. HBsAg ( A value) of the cell supernatants (48 hours) were 0.92,0.53,0.42, respectively. CONCLUSION: The vector used in this study was an effective vector to carry genes of interest to target cells and macrophage, and high level HBsAg was expressed in antigen presenting cell such as macrophage, It indicated that plasmid immunity can induce the B lymphocyte and T lymphocyte response to hepatitis B virus surface antigen by stimulating macrophage. As a vector, it may be useful in the test for gene immunity and gene therapy.     

Modulatory function of high-dose hepatitis B surface antigen vaccine to cellular immune responses in mice

AIM:To observe the effects of high-dose hepatitis B surface antigen (HBsAg) vaccine on cellular immune response in BALB/C mice.METHODS:The mice were immunilized separately with low-dose and high-dose HBsAg vaccine by intramuscular injection two times. The specific proliferative activities of T lymphocytes were measured by -[H³]TdR incorporation assay. IL-2 as well as IFN-γ levels in the culture supernatant of T cells and anti-HBs IgG2a lever in sera were detected by enzyme-linked immunoabsorbent assay.RESULTS:After first vaccination with high-dose HBsAg, the proliferative activities of T cells in the experimental group were significantly stronger, both levels of IL-2 and IFN-γ were markedly higher than that in the control group and the percentage of mice to produce serum anti-HBs IgG2a was significantly higher compared to that of mice immunilized by low-dose HBsAg. All data in experimental groups were further increased after second dose of vaccine.CONCLUSION:Vaccination of mice with high-dose HBsAg can induce cellular immune responses tended to Th1(T helper 1 subset) response.