Construction of a replication-competent hepatitis B virus vector carrying secreted luciferase transgene and establishment of new hepatitis B virus replication and expression cell lines.

BACKGROUND: Previously, we have successfully constructed replication-competent hepatitis B virus (HBV) vectors by uncoupling the P open reading frame (ORF) from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence. Consequently, the replication-competent HBV vectors carrying foreign genes, including pCH-BsdR, carrying blasticidin resistance gene (399 bp), and pCH-hrGFP, carrying humanized renilla green fluorescent protein gene (720 bp), were successfully obtained. However, the replication efficiency of the former is higher but it is tedious to use, while that of the latter is poor and cannot be quantified. Hence, we need to search for a new reporter gene that is convenient and quantifiable for further research. AIM: To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies. METHODS: We utilized the replication-competent HBV viral vectors constructed by our laboratory, combined with the secreted luciferase reporter gene, to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase (SecNluc). HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene. RESULTS: The replication-competent HBV vector carrying the SecNluc reporter gene pCH-sNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression. HBV replication intermediates could be produced from this vector. Via transfection with pTRE-sNLuc and selection by hygromycin, we obtained isolated cell clones, named HBV-NLuc-35 cells, which could secrete secNLuc recombinant viruses, and were sensitive to existing anti-HBV drugs. Using differentiated HepaRG cells, it was verified that recombinant HBV possessed infectivity. CONCLUSION: Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability, and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene. More importantly, the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.

Hepatitis B virus infection in an HBsAb-positive lymphoma patient who received chemotherapy: A case report.

RATIONALE: Tumor chemotherapy could weaken the immune system of patients, which might enhance the body sensitivities to the exogenous pathogens, among which the hepatitis B virus (HBV) infection should be concerned because of the higher chances of infection and the severe outcomes, especially in East Asia. The titer level of hepatitis B surface antibody (HBsAb) higher than 10 IU/L is considered to offer immunocompetent individuals adequate protection. However, whether this level is enough to the tumor patients during chemotherapy remains unclear. PATIENT CONCERNS: A 58-year-old female lymphoma patient was admitted to our hospital for asthenia, nausea, vomiting, and abnormal liver function lasting over 1 week and diagnosed as acute hepatitis B. The patient just finished a course of chemotherapy with CHOP regimen and had recent record (78.61 IU/L) of HBsAb positive. The only risk of infection we could found was that the patient had received blood transfusion shortly after chemotherapy from a donor who was recovering from an asymptomatic acute HBV infection. INTERVENTION: The patient was administered with entecavir and glycyrrhizic acid agent, and then the disease was resolved successfully with hepatitis B surface antigen serological conversion. LESSONS: Tumor chemotherapy might have weakened the immune system of the patient and enhanced the body sensitivities to hepatitis B virus, then led to the infection. We concluded that HBsAb-positive status, at least "weakly positive," might not enough to provide protection for tumor patients on chemotherapy though this level was enough for health individuals and donors recuperating from subclinical acute hepatitis B might be another potential risk of HBV infection.

B7-H4 overexpression is essential for early hepatocellular carcinoma progression and recurrence.

B7-H4, another member of costimulatory molecule, has been shown to be overexpressed in multiple types of tumors, including hepatocellular carcinoma (HCC). However, the specific biological role of B7-H4 in HCC still needs to be further explored. In this study, we observed that B7-H4 was highly overexpressed in HCC tissues and cells, and its overexpression strongly correlated with patient's TNM stage, overall survival and early recurrence. Downregulation of B7-H4 significantly suppressed cell growth, invasion, and stemness of HCC by inducing apoptosis in the in vitro experiment. In addition, depletion of B7-H4 could help restore CD8+ T anti-tumor immunity by elevating the expression and secretion levels of CD107a, granzyme A, granzyme B, perforin and IFN-γ. In a xenografted mouse model of HCC, stable depletion of B7-H4 resulted in significantly smaller mean tumor volume and less mean tumor weight after 30 days of growth, compared to the control group. Together, our results provide insights into the diverse functions of B7-H4 involved in the pathogenesis, recurrence and anti-tumor immunity of HCC, indicating B7-H4 as a novel and effective approach for future treatment strategies that benefits anticancer therapy.

Rapid Detection of Small Molecule Metabolites in Serum of Hepatocellular Carcinoma Patients Using Ultrafast Liquid Chromatography-Ion Trap-Time of Flight Tandem Mass Spectrometry.

A method was developed for analyzing broad spectrum small molecule metabolites in the serum of hepatocellular carcinoma (HCC) patients based on ultrafast liquid chromatography-ion trap-time of flight tandem mass spectrometry (UFLC-IT-TOF MS). Serum samples were collected from 80 HCC patients and healthy persons. After pretreatment process for protein precipitation, the supernatant was analyzed with the UFLC-IT-TOF MS to obtain information on the metabonomics of small molecules. The eight compounds of glycocholic acid, choline glycerophosphate, acetyl-L-phenylalanine, oleamide, tetradecanamide, acetylcarnitine, lysolecithin and glycochenodeoxycholic acid in the HCC group were identified with significant differences from those in the health group (P <0.01). By using multidimensional analysis of variation coefficient and principal component analysis for the repeatability and 48 h stability, the method was demonstrated to have good repeatability, excellent precision, and high stability, which can satisfy the metabonomics research requirement. The high throughput and practical usability of the method further shows perspective for metabonomic analysis of large-batch serum samples.

Serum hepatitis B core antibody titer use in screening for significant fibrosis in treatment-naïve patients with chronic hepatitis B.

BACKGROUND: Previous studies have revealed that hepatitis B core antibody (anti-HBc) levels vary throughout the different phases of treatment-naïve chronic hepatitis B (CHB) patients and can be used as a predictor of treatment response in both interferon-α and nucleoside analogue therapies. However, few data have been published regarding the relationship between quantitative anti-HBc (qAnti-HBc) levels and liver fibrosis in patients with CHB. RESULTS: A total of 489 HBeAg-positive (HBeAg (+)) and 135 HBeAg-negative (HBeAg (-)) patients were recruited. In both HBeAg (+) and HBeAg (-) groups, the S0-1/S0 subjects had significantly lower qAnti-HBc levels than the S2-4 subjects (p < 0.05). Multiple logistic regression analysis showed that the parameters for predicting significant fibrosis (S ≥ 2) included age, PLT and qAnti-HBc. In HBeAg (+) subjects, the AUROC of qAnti-HBc for predicting significant fibrosis was 0.734 (95% CI 0.689 to 0.778) and the optimal cut-off was 4.58 log10IU/mL, with a sensitivity of 63.08% and a specificity of 74.83%. In HBeAg (-) subjects, the AUROC was 0.707 (95% CI 0.612 to 0.801) and the optimal cut-off value was 4.37 log10IU/mL, with a sensitivity of 75.53% and a specificity of 56.10%. MATERIALS AND METHODS: From 2012 to 2015, we conducted a cross-sectional study of treatment-naïve CHB patients. Liver biochemistry, hepatitis B virus (HBV) serological markers, HBV DNA, hepatitis B surface antigen (HBsAg) titers and HBV genotype were determined using commercial assays, and serum qAnti-HBc levels were measured using double-sandwich immunoassay. Liver biopsies and serum samples were obtained on the same day. CONCLUSIONS: The present study showed an association between high serum qAnti-HBc levels and significant fibrosis (S ≥ 2) in treatment-naïve CHB patients. Furthermore, we described a serum qAnti-HBc cut-off for predicting significant fibrosis in CHB patients infected with HBV genotype B or C.

Tim-3 suppression combined with TLR3 activation enhances antiviral immune response in patients with chronic HCV infection.

OBJECTIVE: To investigate the regulation mechanism of T cell immunoglobulin and mucin domain-3 (Tim-3) combined with toll-like receptor 3 (TLR3) or TLR4 on antiviral immune and inflammatory response in patients with chronic hepatitis C virus (HCV) infection. METHODS: Patients with chronic HCV infection and healthy control subjects were recruited. Patients received interferon (IFN)-α based therapy. Plasma galectin-9 (Gal-9) was quantitated. Peripheral blood mononuclear cells (PBMCs) were cultured with TLR3 or TLR4 agonists, alone or in combination with Tim-3 antagonist. Levels of IFN-α, TNF-α, and 2'-5' oligoadenylate synthetase (2'-5'OAS), myxovirus resistance protein A (MxA) and suppressor of cytokine 1 (SOCS1) RNA in PBMC cultures were evaluated. RESULTS: Plasma Gal-9 levels were increased in patients (n = 52) compared with controls (n = 20) and significantly declined at treatment week 12 and 24 weeks post-treatment. IFN-α, 2'-5'OAS, MxA, TNF-α and SOCS1 were upregulated by TLR3 and TLR4 agonists. TNF-α and SOCS1 levels were suppressed by the addition of Tim-3 antagonist. CONCLUSIONS: Tim-3 blockade in combination with TLR activation induces the expression of antiviral molecules without a significant increase in TNF-α or SOCS1.

Individualized treatment strategies and predictors of virological response for chronic hepatitis C: a multicenter prospective study from China.

Combination therapy comprising pegylated interferon-alpha (PegIFNα) and ribavirin (RBV) has been the standard of care for the chronic hepatitis C patients for more than a decade. Recently, direct antiviral agents show better efficacy, tolerance, and shorter treatment duration. However, the prohibitive costs of the regimens limit their use in developing countries where most of the HCV infection exists. Optimizing the treatment and understanding the host- and virus-factors associated with viral clearance were necessary for individualizing therapy to maximize sustained virologic response. To explore individualized antiviral strategies with PegIFNα-2a/IFNα-2b plus ribavirin for CHC patients, and to clarify predictive factors for virological response. A cohort of 314 patients were included in this open-label, prospective clinical trial, which received individualized doses of PegIFNα-2a or IFNα-2b combined with RBV according to body weight, disease status and complications, with the duration of 44 weeks after HCV RNA undetectable. All the IL-28B (rs8099917), IL-17A (rs8193036), IL-17B (rs2275913) and PD-1.1 SNPs were genotyped using the TaqMan system. The sustained virological response (SVR) in PegIFNα-2a group was significantly higher than that in IFNα-2b (85.8% vs 75.0%, P = 0.034), especially in HCV genotype 1 (84.0% vs 64.3%, P = 0.022). However, no significant differences were found in rapid virological response (RVR), complete early virological response (cEVR) and SVR between PegIFNα-2a and IFNα-2b according to different doses, respectively. The genotype frequency of IL-28B TT in patients with cEVR, SVR was higher than that in non-responsed patients (93.8% vs 78.1%, χ(2) = 7.827, P = 0.005; 95.9% vs 80.4%, χ(2) = 9.394, P = 0.002). No significant correlation between the genotype distribution of IL-17A, IL-17B and PD-1.1 with virological response. Individualized regimens of PegIFNα-2a/RBV and IFNα-2b/RBV could achieve satisfied virological response in Chinese HCV patients. The IL-28B (rs8099917) TT genotype is a clinical usefully marker for cEVR and SVR.

B7-H3 promotes aggression and invasion of hepatocellular carcinoma by targeting epithelial-to-mesenchymal transition via JAK2/STAT3/Slug signaling pathway.

BACKGROUND: B7-homologue 3 (B7-H3), a recently identified immunoregulatory protein, has been shown to be overexpressed in human hepatocellular carcinoma (HCC). However, whether the dynamic expression pattern of B7-H3 contributes to early invasion of HCC is largely unknown. In addition, the biological roles of B7-H3 in HCC are still unclear. Herein, we are going to examine B7-H3 expression profile and its clinicopathological significance in primary and metastatic HCC, and further determine whether B7-H3 knockdown simulates different pathological states of HCC progression and metastasis. METHODS: Using immunohistochemistry, B7-H3 expression was studied on 116 HCC containing primary and metastatic HCCs. Survival curves and log-rank tests were used to test the association of B7-H3 expression with survival. HCC cells with B7-H3 depletion were established by RNA interference to investigate the effect of B7-H3 on cell proliferation, apoptosis, migration and invasion in vitro. RESULTS: Statistical analysis of clinical cases revealed that B7-H3 high expression group had inclinations towards late TNM stage, the presence of vascular invasion, lymph metastasis, and the formation of microsatellite tumors. Increased intensity of tumor B7-H3 staining was detected more significantly in metastatic HCC tumors. Consistently in experiments performed in vitro, B7-H3 was able to stimulate the wound healing, metastasis and invasion of hepatoma cells by targeting epithelial-to-mesenchymal transition (EMT) via JAK2/Stat3/Slug signaling pathway, while no obvious influence on cell growth and apoptosis. CONCLUSION: B7-H3 in the regulation of the metastatic capacity of HCC cells makes itself a promising therapeutic target for anti-metastasis therapy.

Interleukin-21 inhibits HBV replication in vitro.

BACKGROUND: Cytokines are crucial factors in the non-cytolytic antiviral process to inhibit HBV gene expression and replication. Interleukin (IL)-21 has been suggested to play an important role in HBV infection, but it remains unknown whether IL-21 can inhibit HBV replication or how it inhibits HBV replication. METHODS: In this study, we investigated the influence of IL-21 on HBV replication based on human hepatoma Huh7.93 cells co-cultured with human peripheral blood mononuclear cells (PBMCs) and the possible correlation among IL-21, interferon-γ, tumour necrosis factor-α and IL-10. RESULTS: We demonstrated that the decrease of IL-21 expression and the increase of IL-10 expression in PBMCs could promote HBV replication in vitro. We further revealed that IL-21 is not only able to effectively suppress HBV replication directly but also reduce HBV replication by inhibition of IL-10 secretion. CONCLUSIONS: Our results provide important evidence for the non-cytolytic antiviral mechanism mediated by cytokines and their interactions in chronic hepatitis B.

Hepatocellular carcinomas promote tumor-associated macrophage M2-polarization via increased B7-H3 expression.

B7 family members are aberrantly expressed on the human hepatocellular carcinoma (HCC) cell surface, and induce local and systemic immunosuppression. Tumor-associated macrophages (TAMs) are a significant immune cell subpopulation in HCC and may be induced to express co-inhibitory molecules including B7 homologue 3 (B7-H3). In the present study, 79.3% of the HCC tissue samples showed high expression of B7-H3 which was positively correlated with the number of TAMs in the evaluated cancer tissues. Furthermore, high levels of TAMs or B7-H3 were associated with a shorter survival time of the HCC patients. In vitro, B7-H3 expression was upregulated at both the mRNA and protein levels in phorbol 12-myristate 13-acetate (PMA)-induced THP-1 cells cocultured with HepG2 cells in a Transwell system. In addition, B7-H3 promoted PMA-induced THP-1 cells to differentiate into the M2 phenotype, with evidence of increases in arginase 1 (Arg1), vascular endothelial cell growth factor (VEGF) and macrophage-derived chemokine (CCL22) mRNA following coculture with HepG2 cells. However, this phenomenon was abrogated through knockdown of B7-H3 by RNA interference or by blocking the signal transducer and activator of trans-cription 3 (STAT3) signaling pathway. Overall, these results suggest that the B7-H3-mediated STAT3 signaling pathway is an important mechanism for inducing M2-type polarization of TAMs, which accelerates HCC development. Our findings may support the development of novel therapeutic strategies for HCC patients through the skewing of the TAM phenotype by targeting the B7-H3 signaling pathway.

Regulatory phenotype, PD-1 and TLR3 expression in T cells and monocytes from HCV patients undergoing antiviral therapy: a randomized clinical trial.

BACKGROUND & AIMS: The cellular immunity has a profound impact on the status of hepatitis C virus (HCV) infection. However, the response of cellular immunity on the virological response in patients with antiviral treatment remains largely unclear. We aimed to clarify the response of peripheral T cells and monocytes in chronic hepatitis C patients with antiviral treatment. METHODS: Patients with chronic hepatitis C were treated either with interferon alpha-2b plus ribavirin (n = 37) or with pegylated interferon alpha-2a plus ribavirin (n = 33) for up to 24 weeks. Frequencies of peripheral regulatory T-cells (Tregs), programmed death-1 (PD-1) expressing CD4+ T-cells or CD8+ T-cells and toll-like receptor (TLR) 3 expressing CD14+ monocytes were evaluated by flow cytometry in patients at baseline, 12 and 24 weeks following treatment and in 20 healthy controls. RESULTS: Frequencies of Tregs, PD-1 and TLR3 expressing cells were higher in patients than those in control subjects (P<0.05). Patients with complete early virological response (cEVR) showed lower Tregs, PD-1 expressing CD4+ or CD8+ T-cells than those without cEVR at 12 weeks (P<0.05). Patients with low TLR3 expressing CD14+ monocytes at baseline had a high rate of cEVR (P<0.05). CONCLUSIONS: Low peripheral TLR3 expressing CD14+ monocytes at baseline could serve as a predictor for cEVR of antiviral therapy in chronic HCV-infected patients. The cEVR rates were significantly increased in the patients with reduced circulating Tregs, PD-1 expressing CD4+ or CD8+ T-cells. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR10001090.

Tricistronic hepatitis C virus subgenomic replicon expressing double transgenes.

AIM: To construct a tricistronic hepatitis C virus (HCV) replicon with double internal ribosome entry sites (IRESes) of only 22 nucleotides for each, substituting the encephalomyocarditis virus (EMCV) IRESes, which are most often used as the translation initiation element to form HCV replicons. METHODS: The alternative 22-nucleotide IRES, RNA-binding motif protein 3 IRES (Rbm3 IRES), was used to form a tricistronic HCV replicon, to facilitate constructing HCV-harboring stable cell lines and successive antiviral screening using a luciferase marker. Briefly, two sequential Rbm3 IRESes were inserted into bicistronic pUC19-HCV plasmid, consequently forming a tricistronic HCV replicon (pHCV-rep-NeoR-hRluc), initiating the translation of humanized Renilla luciferase and HCV non-structural gene, along with HCV authentic IRES initiating the translation of neomycin resistance gene. The sH7 cell lines, in which the novel replicon RNA stably replicated, were constructed by neomycin and luciferase activity screening. The intracellular HCV replicon RNA, expression of inserted foreign genes and HCV non-structural gene, as well as response to anti-HCV agents, were measured in sH7 cells and cells transiently transfected with tricistronic replicon RNA. RESULTS: The intracellular HCV replicon RNA and expression of inserted foreign genes and HCV non-structural gene in sH7 cells and cells transiently transfected with tricistronic replicon RNA were comparable to those in cells stably or transiently transfected with traditional bicistronic HCV replicons. The average relative light unit in pHCV-rep-NeoR-hRluc group was approximately 2-fold of those in the pUC19-HCV-hRLuc and Tri-JFH1 groups (1.049 × 10(8) ± 2.747 × 10(7) vs 5.368 × 10(7) ± 1.016 × 10(7), P < 0.05; 1.049 × 10(8) ± 2.747 × 10(7) vs 5.243 × 10(7) ± 1.194 × 10(7), P < 0.05), suggesting that the translation initiation efficiency of the first Rbm3 IRES in the two sequential IRESes was stronger than the HCV authentic IRES and EMCV IRES. The fold changes of 72 h/4 h relative light units in the pHCV-rep-NeoR-hRluc and pUC19-HCV-hRLuc groups were similar (159.619 ± 9.083 vs 163.536 ± 24.031, P = 0.7707), and were both higher than the fold change in the Tri-JFH1 group 159.619± 9.083 vs 140.811 ± 9.882, P < 0.05; 163.536 ± 24.031 vs 140.811 ± 9.882, P < 0.05), suggesting that the replication potency of the Rbm3 IRES tricistronic replicon matched the replication of bicistronic replicon and exceeded the potency of EMCV IRES replicon. Replication of tricistronic replicons was suppressed by ribavirin, simvastatin, atorvastatin, telaprevir and boceprevir. Interferon-alpha 2b could not block replication of the novel replicon RNA in sH7 cells. After interferon stimulation, MxA mRNA and protein levels were lower in sH7 than in parental cells. CONCLUSION: Tricistronic HCV replicon with double Rbm3 IRESes could be applied to evaluate the replication inhibition efficacy of anti-HCV agents.

[Relation of IL-17 polymorphisms and serum levels in patients with chronic HCV infection].

OBJECTIVE: To investigate the association of single nucleotide polymorphisms (SNPs) in the interleukin 17 (IL-17) gene and serum protein levels in patients with chronic hepatitis C virus (HCV) infection. METHODS: A total of 228 patients with chronic HCV infection and 81 healthy controls were enrolled in the study. The frequencies of IL-17 rs8193036 and rs2275913 polymorphisms were detected by the TaqMan SNP genotyping assay. Serum levels of IL-17 protein were detected by ELISA. Pairwise comparisons were made by the Chi-square test, and the significance of between-group differences was assessed by the Student's t-test with P less than 0.05. RESULTS: The patients with chronic HCV infection and the healthy controls showed similar frequencies of the rs8193036 C/T allele (x2 = 1.428, P = 0.232) and the rs2275913 A/G allele (x2 = 0.106, P = 0.744). In addition, the two groups showed similar distribution of the rs8193036 CC (chronic HCV infection: 46.49% vs. healthy controls: 41.98%), CT (45.61% vs. 44.44%) and TT (7.89% vs. 13.58%) genotypes (x2 = 2.346, P = 0.309), and of the rs2275913 AA (16.23% vs. 13.58%), AG (48.25% vs. 50.62%) and GG (35.53% vs. 35.80%) genotypes (x2 = 0.340, P = 0.844). Subgroup analysis of chronic HCV infection patients stratified according to HCV genotypes 1 and 2 showed no differences in the distribution of rs8193036 and rs2275913 alleles (x2 = 1.127, P = 0.288; x2 = 1.088, P = 0.297) and genotypes (x2 = 2.825, P = 0.246; x2 = 0.970, P = 0.616). However, the chronic HCV infection group did show significantly higher levels of serum IL-17 than the controls (97.67+/-39.68 vs. 71.60+/-19.78 pg/ml, t = 2.414, P = 0.033). CONCLUSION: Chronic HCV infection is associated with increased serum IL-17; however, the IL-17 polymorphisms rs8193036 and rs2275913 were not associated with chronic HCV infection susceptibility in this study's Chinese cohort.

[Study of using an individualized treatment strategy to treat patients with chronic hepatitis C].

OBJECTIVE: To investigate the outcomes of chronic hepatitis C (CHC) patients treated with antiviral regimens of interferon (IFN) plus ribavirin (RBV) using individualized doses and durations. METHODS: This study was designed as an open-label, prospective clinical trial to analyze the virological responses of 169 CHC patients who received individualized dosages of IFNa-2b or pegylated (Peg)IFNa-2a combined with RBV based on their weight ( less than 60 kg or more than or equal to 60 kg), age (less than 65 years or 65-75 years), morbid state (liver cirrhosis or not), and complications (such as heart disease, diabetes, thyroid disorder). Treatment duration was calculated using the time required to induce HCV RNA negativity. The rates of virological response and adverse effects among the different groups were compared. RESULTS: The IFNa-2b treatment was given to 116 patients, and PegIFNa-2a was given to 53 patients. Compared to the IFNa-2b group, the PegIFNa-2a group showed significantly higher rates of complete early virological response (cEVR; 76.7% vs. 92.5%, P less than 0.05) and sustained virological response (SVR; 53.6% vs. 92.3%, P less than 0.05) among the patients who had completed their course of treatment; the rapid virological response (RVR) rate was also higher for the PegIFNa-2a group but the difference did not reach statistical significance (48.7% vs. 60.4%, P more than 0.05). Seventy-eight patients received the routine dose, and 91 patients received the low dose; there were no significant differences between these two groups for RVR (53.8% vs. 58.9%, P more than 0.05), cEVR (78.0% vs. 80.8%, P more than 0.05), or SVR (65.5% vs. 58.3%, P more than 0.05). CONCLUSION: Use of an individualized antiviral treatment strategy designed according to the patient's baseline condition, early viral kinetics, and tolerability to adverse reactions can achieve a high rate of SVR, as well as improve the safety, prognosis, and cost-effectiveness associated with treating CHC patients.

[Stable cell line for secretion of replication-defective hepatitis B virus vector expressing blasticidin resistant gene].

OBJECTIVE: To construct a stable cell line with permanent secretion of recombinant hepatitis B virus (HBV) vector, which express blasticidin resistant gene. METHODS: Replication-defective HBV vector, pCH-BsdR, which express blasticidin resistance gene was constructed by deleting the HBV genes and inserting the blasticidin resistance gene into the S region. The G418-resistant, the packaging signal deleted HBV helper plasmid, pcDNA3.1-CH3142, and the HBV vector pCH-BsdR were cotransfected into HepG2 cells. Cell clones were selected by the adding of both blasticidin and G418, then serial detection were done. RESULTS: After 36 cell clones were picked and expanded. Three cell clones were defined as the best. Quantity of their HBV DNA were 4.1 x 10(6), 3.6 x 10(6) and 1.2 x 10(6) copies/ml, respectively. Enveloped recombinant, but not wild type HBV were confirmed in the culture medium. CONCLUSIONS: The stable cell lines can realize large preparation of recombinant HBV virions. This will contribute to the use of HBV vector for gene therapy and HBV susceptible cell lines screening.

[Study on anti-HBV effects of genetically engineered replication-defective hepatitis B virus expressing dominant negative mutants of core protein].

BACKGROUND: To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular expression of dominant negative mutants of core protein. METHODS: Two kinds of full length mutant HBV genome, which express Core-partial P and Core-S fusion protein, were transfected into HepG 2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA and intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. RESULTS: The mean inhibitory rates of HBsAg were 2.74+/-3.83%, 40.08+/-2.05% (P less than 0.01) and 52.94+/-1.93% (P less than 0.01) in group 2.2.15-pMEP4, 2.2.15-CP and 2.2.15-CS, respectively. The mean inhibitory rates of HBeAg were 4.46+/-4.25%, 52.86+/-1.32% (P less than 0.01) and 41.60+/-1.65% (P less than 0.01), respectively. The inhibitory rates of HBc related HBV DNA were 15.3%, 82.0% and 67.2%, respectively. Recombinant HBV virion was detectable in the culture medium of only group 2.2.15-CP. CONCLUSION: Dominant negative mutants of core protein can efficiently suppress wt-HBV replication and the expressions of HBV antigens. With the help of wild-type HBV, the recombinant HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified.

[Development of hygromycin-resistant packaging cell line for hepatitis B virus-derived vectors].

OBJECTIVE: To cooperate with the study of HBV vector, hygromycin-resistant packaging cell line was developed that allows encapsidation of plasmids into HBV particles. METHODS: Free of packaging signal, HBV genome was inserted into plasmid pMEP4, which expresses the HBV structural proteins including core, pol and preS/S proteins. HepG2 cell lines were employed to transfect with the construct. Hygromycin selection was done at a concentration of 150 micrograms/ml in the culture medium. The hygromycin-resistant clones with the best expressions of HBsAg and HBcAg were theoretically considered as packaging cell line and propagated under the same conditions. It was infected with recombinant retrovirus vector and hen selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR. RESULTS: Hygromycin-resistant HBV packaging cell line was generated, which harbored an HBV mutant whose packaging signal had been deleted. Expressions of HBsAg and HBcAg were detectable. Infected with recombinant retrovirus pRV-CP, the hygromycin-resistant packaging cell line was found to secrete mutant HBV particles and no wild-type HBV was detectable in the culture medium. CONCLUSION: After the packaging signal was deleted and transfected into HepG2 cell lines, the partial HBV genome lost its ability to form wild-type HBV, but conserves cis-action providing structural proteins for the packaging of the replication-defective HBV.