HBV-positive Cells Research Articles

Targeting hepatitis B virus antigens to dendritic cells by heat shock protein to improve DNA vaccine potency

To investigate a novel DNA vaccination based upon expression of the HBV e antigen fused to a heat shock protein (HSP) as a strategy to enhance DNA vaccine potency. A pCMV-HBeAg-HSP DNA vaccine and a control DNA vaccine were generated. Mice were immunized with these different construct. Immune responses were measured 2 wk after a second immunization by a T cell response assay, CTL cytotoxicity assay, and an antibody assay in C57BL/6 and BALB/c mice. CT26-HBeAg tumor cell challenge test in vivo was performed in BALB/c mice to monitor anti-tumor immune responses. In the mice immunized with pCMV-HBe-HSP DNA, superior CTL activity to target HBV-positive target cells was observed in comparison with mice immunized with pCMV-HBeAg (44% +/- 5% vs 30% +/- 6% in E:T > 50:1, P < 0.05). ELISPOT assays showed a stronger T-cell response from mice immunized with pCMV-HBe-HSP than that from pCMV-HBeAg immunized animals when stimulated either with MHC class I or class II epitopes derived from HBeAg (74% +/- 9% vs 31% +/- 6%, P < 0.01). ELISA assays revealed an enhanced HBeAg antibody response from mice immunized with pCMV-HBe-HSP than from those immunized with pCMV-HBeAg. The lowest tumor incidence and the slowest tumor growth were observed in mice immunized with pCMV-HBe-HSP when challenged with CT26-HBeAg. The results of this study demonstrate a broad enhancement of antigen-specific CD4+ helper, CD8+ cytotoxic T-cell, and B-cell responses by a novel DNA vaccination strategy. They also proved a stronger antigen-specific immune memory, which may be superior to currently described HBV DNA vaccination strategies for the treatment of chronic HBV infection.

Nucleoporin 88 expression in hepatitis B and C virus-related liver diseases

To investigate the expression of nucleoporin 88 (Nup88) in hepatitis B virus (HBV) and C virus (HCV)-related liver diseases. We generated a new monoclonal Nup88 antibody to investigate the Nup88 protein expression by immunohistochemistry (IHC) in 294 paraffin-embedded liver specimens comprising all stages of hepatocellular carcinogenesis. In addition, in cell culture experiments HBV-positive (HepG2.2.15 and HB611) and HBV-negative (HepG2) hepatoma cell lines were tested for the Nup88 expression by Western-immunoblotting to test data obtained by IHC. Specific Nup88 expression was found in chronic HCV hepatitis and unspecific chronic hepatitis, whereas no or very weak Nup88 expression was detected in normal liver. The Nup88 expression was markedly reduced or missing in mild chronic HBV infection and inversely correlated with HBcAg expression. Irrespective of the HBV- or HCV-status, increasing Nup88 expression was observed in cirrhosis and dysplastic nodules, and Nup88 was highly expressed in hepatocellular carcinomas. The intensity of Nup88 expression significantly increased during carcinogenesis (P<0.0001) and correlated with dedifferentiation (P<0.0001). Interestingly, Nup88 protein expression was significantly downregulated in HBV-positive HepG2.2.15 (P<0.002) and HB611 (P<0.001) cell lines as compared to HBV-negative HepG2 cells. Based on our immunohistochemical data, HBV and HCV are unlikely to influence the expression of Nup88 in cirrhotic and neoplastic liver tissue, but point to an interaction of HBV with the nuclear pore in chronic hepatitis. The expression of Nup88 in nonneoplastic liver tissue might reflect enhanced metabolic activity of the liver tissue. Our data strongly indicate a dichotomous role for Nup88 in non-neoplastic and neoplastic conditions of the liver.

Hepatitis B virus enhances transduction of human hepatocytes by SV40-based vectors

Background/Aims: Chronic HBV infection, a world-wide epidemic, can lead to chronic hepatitis and eventually to cirrhosis and hepatocellular carcinoma. The liver poses obstacles for many available gene-transfer vectors. SV40-based vectors can transduce human hepatic and hematopoietic cells. We studied the effect of HBV on the transduction – efficiency of human hepatic cells by SV40 – based vectors. Methods: A SV40-vector carrying the luciferase gene, and wild-type SV40, were used to assess transduction efficiency of human HBV-positive and HBV-negative hepatic cells. Transduction efficiency was measured as luciferase activity or by T-antigen staining. To evaluate whether differences in transduction efficiency are due to cell recognition and/or nuclear transport, MHC-I receptors were measured by FACS analysis and SV40-DNA was extracted from the nuclei of transduced cells and quantified. Results: Two HBV-positive cell-lines, HepG2.2.2.15 and FLC4-A10II, were transduced significantly more efficiently than their parental HBV-negative cell-lines. Transient transfection of HuH-7 cells with the HBV genome also increased transduction efficiency. The level of MHC-I, the cellular receptor for SV40, was comparable in all the cell-lines studied. However, soon after infection with SV40, the nuclei of HepG2.2.2.15 contained >6-fold more SV40-DNA than HepG2. Conclusions: HBV increases transduction by SV40-vectors. This is due to enhanced vector entry and/or transport into the nucleus. SV40-vectors appear to have a potential for gene therapy for the treatment of HBV infections.

Nup88 expression in hepatocellular carcinogenesis indicates interaction of hepatitis B virus with the nuclear pore

Levels of atonal homolog 8 (ATOH8) are reduced in 48% of hepatitis B virus-associated hepatocellular carcinoma cells (HCCs). ATOH8 downregulation is associated with loss of tumor differentiation, indicating an effect mediated by cancer stem cells. We investigated the effects of loss of ATOH8 in human hepatocellular carcinoma (HCC) cells and cell lines.HCC and adjacent nontumor tissues were collected, from 2001 through 2012, from 242 patients undergoing hepatectomy at Sun Yat-Sen University Cancer Center in China; 83% of HCCs were associated with hepatitis B virus (HBV) infection. CD133+ cells were isolated from tumor tissues by flow cytometry. Experiments were performed in HBV-positive and HBV-negative HCC cell lines, the immortalized liver cell line LO2, and 8 other HCC cell lines. ATOH8 was expressed from lentiviral vectors in PLC8024 and Huh7 cells; levels were knocked down with small interfering RNAs in QSG7701 cells. Cells carrying empty vectors were used as controls. Gene regulation by ATOH8 was assessed in mobility shift and luciferase reporter assays. Cells were analyzed in proliferation, foci formation, and colony formation assays. The tumorigenic and chemo-resistant potential of cells were investigated by assessing growth of xenograft tumors in immunocompromised mice. Metastatic features of cells were assessed in Matrigel invasion assays and wound healing analyses.Levels of ATOH8 mRNA were reduced by more than 4-fold, compared to nontumor tissues, in 118 of 242 HCC samples (48.8%). Patients with tumor reductions in ATOH8 had significantly shorter times of disease-free survival (mean, 41.4 months) than patients with normal tissue levels (mean, 52.6 months). ATOH8 expression was reduced in HepG2, Huh7, PLC8024 and CRL8064 HCC cells, as well as CD133+ cells isolated from human HCC samples. Transgenic expression of ATOH8 in HCC cell lines significantly reduced proliferation and foci colony formation, as well as their invasive and migratory abilities. Transgenic expression of ATOH8 reduced the ability of HBV-positive PLC8024 cells to form tumors in mice, compared to control cells. Cells with ATOH8 knockdown formed xenograft tumors more rapidly, in more mice, than control cells. ATOH8 repressed transcription of stem-cell associated genes including OCT4, NANOG, and CD133. Knockdown of ATOH8 in CD133-negative QSG7701 cells caused them to express CD133; acquire self-renewal, differentiation, chemo-resistance properties; form more xenograft tumors in mice; and generate induced pluripotent stem cells (based on staining for alkaline phosphatase and their ability to form embryoid bodies and teratomas). Alternatively, expression of ATOH8 in PLC8024 and Huh7 cells significantly reduced the numbers of cells expressing CD133, and increased the chemo-sensitivity of Huh7 cells to 5-fluorouracil (5-FU) and cisplatin, in vitro and in mice.ATOH8 appears to be a tumor suppressor that induces stem-cell features and chemoresistance in HCC cells. Strategies to restore its levels and activities might be developed to treat patients with liver cancer.

Use of digoxigenin-labelled probes for the quantitation of HBV-DNA in antiviral drug evaluation

The use of digoxigenin-labelled probes was studied for quantitation of HBV-DNA during antiviral drug evaluation. Digoxigenin (dig)-labelled probes were generated either via incorporation of dig-dUTP in a polymerase chain reaction (PCR) or a random priming reaction. Using the PCR-labelled probe (delineating a 523 bp fragment in the core gene of the HBV) as little as 1 pg of immobilized HBV-DNA could be detected following an 8 h exposure of the hybridized membrane. A close correlation ( r=0.95) was found between the amount of HBV-DNA (range 2.5–200 pg) and the signal generated by the probe hybridized to its target DNA. By using a probe that was labelled with digoxigenin via random priming, the minimal quantity of immobilized HBV plasmid DNA that could be detected following an 8 h exposure was 4 pg, whereas a 32P-labelled probe, generated in parallel by random priming, allowed the detection of 16 pg of HBV plasmid DNA following a 4-day exposure. The PCR-generated digoxigenin-labelled probe proved to be useful for antiviral drug evaluation, i.e. to detect HBV-DNA in total cellular DNA from HBV-positive hepatoma cells (HepG2.2.15) that had either been treated with reference antiviral agents or left untreated. The 50% effective concentrations (EC 50) that were calculated for inhibition of HBV-DNA production by lamivudine (3TC), penciclovir (PCV), lobucavir (LBV), adefovir (PMEA) and tenofovir (PMPA) were comparable to those reported in the literature. The use of digoxigenin-labelled probes thus appears to be a simple, convenient, rapid, reliable and non-radioactive method for use for anti-HBV screening. In addition, and in contrast to 32P-labelled probes, digoxigenin-labelled probes can be stored for >1 year without loss of specific activity, which makes these probes particularly attractive for large-scale antiviral drug evaluation purposes.

HBV viral load within subpopulations of peripheral blood mononuclear cells in HBV infection using limiting dilution PCR

Extrahepatic viral load in peripheral blood mononuclear cells (PBMCs) of patients with hepatitis B virus (HBV) is still under debate. In this study, HBV infection rates and viral titers were examined within all PBMC subpopulations using limiting dilution-PCR (LD-PCR). PBMCs of patients with acute or chronic hepatitis B were separated by magnetic beads in monocytes, B-cells, CD4+ T-cells, CD8+ T-cells, and NK cells. Using two-round nested PCR, HBV-DNA sequences were detected in all patients examined within each PBMC subpopulation. The frequencies of HBV-positive cells and viral loads were calculated by Poisson analysis of HBV PCR results from serial dilutions of cells and cell lysates. Highest infection rates were found in monocytes and B-cells followed by CD8+ T-cells, NK cells, and CD4+ T-cells. Concerning all subsets, frequencies of HBV-positive cells were 50- to 500-fold higher in chronic than in acute hepatitis B. Viral loads were mostly estimated at about one HBV genome per HBV-positive cell. Moreover, slightly elevated HBV titers were seen in B-cells, CD4+ T-cells, and NK cells in both acute and chronic hepatitis B. It was demonstrated that beside a generally more latent HBV infection in PBMCs, elevated HBV titers point to replication or selective viral uptake within particular PBMC subsets. Therefore, the data suggest that HBV-infected PBMCs may participate in persistence of HBV.

Preparation of virus-free pyridoxylated hemoglobin from the blood of HBV or HTLV-I healthy carriers.

One of our major aims was to find ways to utilize outdated or virus-contaminated blood. Pyridoxylated hemoglobin (PLP-Hb), a possible substitute for red cells as an artificial oxygen carrier was prepared from outdated human blood. By conjugation with polyethylene glycol (PEG), the biological half life was increased about 3 folds at 82% blood replacement in rats without significant side effects in vivo or in vitro. We next tried to prepare virus-free PEG-PLP-Hb from HBV or HTLV-I positive blood. A considerable amount of HBV (Dane particles) could be removed from HBV-positive red cells by washing and during the preparation of PEG-PLP-Hb. When the hemoglobin preparations containing Dane particles were filtered through a porous cellulose filter, BMM-30 (30 nm pore size, Asahi Chemical Industry Co., Ltd., Osaka, Japan), HBV-DNA in the filtered fractions became less than 0.33% of the initial amount. More than 96% of blood leukocytes could be removed with a leukocyte removal filter, Sepacell R-500 (Asahi Medical Co., Ltd., Tokyo, Japan). The leukocytes collected from filtrated fractions of HTLV-I positive blood did not survive beyond 3 days. Since transmission of HTLV-I occurs by cell to cell contact and is rare in cell-Free condition, it is unlikely that the PLP-Hb prepared from HTLV-I positive blood which is deprived of leukocytes transmits HTLV-I infection.