Hot Spots Of Hepatitis B Virus Core Mutations And The Cytotoxic T Lymphocytes Response In Vitro

Taking Chinese common HBV/C mutants G87, L97 in mutation clustering region to analyze the HBV core mutation specific cytotoxic T-cell response in patients during chronic viral hepatitis. Target cells that express HBV-encoded core gene mutations antigens in the context of the appropriate HLA restriction element must be available for each subject studied. Since HBV is not infectious for human cells in vitro, so target cells must be produced by DNA-mediated transfer into cultured human primary cells or cell lines. For this purpose, we have developed a panel of Epstein-Barr virus-based episomal expression vectors containing HBV core gene mutants V60, G87, L97 under the transcriptional control of the simian virus 40 early promoter. Transfection of Epstein-Barr virus-immortalized B-cell lines with mutation recombinant consistently leads to stable expression of the HBV nucleocapsid protein. To assess the suitability of this system for the identification of HBV specific CTL in chronic hepatits B patients, a panel of EBO-HBV transfectants of defined HLA haplotype was used to monitor the HBV-specific CTL response in 9 patients with chronic active hepatitis and 5 asymptomatic carriers. Transfectants that stably express the HBV nucleocapsid (core) antigen were found to serve as excellent targets for the detection of HLA class I -restricted CTL that recognize endogenously synthesized HBV core antigen in these patients; they were also successfully used to stimulate the specific expansion of these CTL in vitro.Partâ… Establishment of immortalized B lymphoblastoid cell lines in patients with chronic hepatitis B virusEpstein-Barr virus (EBV) is a lymphotropic herpesvirus. It will latently infect any resting primary B cell in vitro, irrespective of its phenotype. As a consequence, the resting B cell becomes activated into a latently infected lymphoblast that will proliferate indefinitely. Infection by EBV in vitro easily transforms resting B cells from human peripheral blood into actively proliferating lymphoblastoid cell lines (LCLs). LCLs are used widely for various purposes: In etiological studies of tumorigenesis by EBV; As sources of DNA and cells to study various genetic disorders; and for studies of immunology and cellular biology.Lymphocyte separation and EBV stock preparationForty-six patients with chronic hepatitis B infection were selected for this study. For each selected patient, 30ml peripheral blood was collected in Vacutainer tubes containing heparin. PBMC were separated on Ficoll-Histopaque density gradient, washed three times in RPMI 1640, and either used immediately or cryopreserved in media contaning 90%FCS, 10%dimethyl sulfoxide by storage in the vapour phase of liquid nitrogen.Prepare virus stock from an EBV-transformed B95-8 marmoset cell line. 1×10⁷ cells were grown in 10ml RPMI 1640 medium, supplemented with 10% fetal bovine serum. These cultures were maintained for 12-14 days. After centrifugation for 3 min at 850g, the supernatant was filtered through a 0.22-μm membrane and stored in the vapour phase of liquid nitrogen until use. Immortalization of lymphocytesInitiation of immortalization was achieved by mixing the cells(1×10⁶ cells) in 1 ml RPMI-1640 complete medium, 1ml of active EBV supernatant and 2μg/ml CyA in 24 wells tissue culture flask. Each week, half of the supernatant was replaced by fresh medium containing 1μg/ml CyA and 2.Sμg/ml CpG. Immortalized B lymphoblastoid cell lines were established after about 3-4 weeks. Morphological characteristic of immortalized B-lymphocytes are normally visible by microscope. The LCL biologic properties expressed the CD19 and CD23 surface molecules were determined by flow cytometry. The cells aggregate of proliferative lymphoblast cells indicatie a successful transformation. The markers CD19 and CD23 are expressed strongly on LCLs membrane. CD19 and CD23 expressed on B cell blasts activated by EBV infected. EBV-immortalized B cell lines can be grown to large cell numbers in a relatively short time. These cellular models subsequently allow immune research for understanding the pathomechanism of chronic hepatitis B infection.Partâ…¡Stable expression of the EBO vector carrying HBV C gene mutants in immortalized human B-cell linesSince HBV is not infectious in vitro, Cytolytic target cells that express HBV gene products in the context of a large assortment of defined HLA molecules must be generated by alternative high-efficiency gene transfer methods. Herein we use the production and characterization of a panel of noninfectious Epstein-Barr virus (EBV)-based expression vectors specifically intended to generate stimulator/target cells for this purpose. Use the site-directed mutagenesis technique, HBV p3.8â…¡plasmids (adr subtype) which contans 1.2 copies of HBV genome were introduced to produce the C gene mutations at points V60, G87 and L97 and inserted into EBO-plpp vector. The recombinant plasmids contain HBV mutant were transfected into EBV-immortalized B cell lines.Construction of HBV p3.8â…¡mutants and EBO-HBV recombinantsPoint mutations were made in using the QuikChangc site-directed mutagenesis kit. Briefly, both of the mutagenic oligonucleotide primers for use were designed individually according to mutation of V60, G87 and L97 in HBV core gene. P3.8â…¡plasmid preparation. Denature the plasmid and anneal the oligonucleatide primer containing the desired mutation. Using the nonstrand-displacing action of Pfu Turbo DNA polymerase extend and incorporate the mutagenic primers resulting in nicked circular strands. Digest the methylated, nonmutated parental DNA template with Dpnâ… restriction enzyme. Transform the circular, nicked dsDNA into XL1-Blue supercompetent cells. After transformation, the XL1-Blue supercompetent cells repair the nicks in the mutated plasmid. Mutated Plasmids HBV-p3.8â…¡V60, G87, L97 were inserted the EBO-plpp vector after a double digestion with Sacâ… and Kpnâ… . Four strains of recombinant EBO vector carrying wild type and mutant HBV genomes were obtained.Cell culture and plasmid transfectionPBMCs from chronic HBV-infected patients whose HLA-A2 haplotypes had been determined by FCS were immortalized by infection with EBV as described at partâ… . Plasmid transfections were done by Lipofectamine^TM 2000 according to the operating manual. Briefly, plate 1×10⁷ cells in 2ml of growth medium without antibiotics in a 6-well format. Dilute 10μg DNA in 500μl of cultural medium without serum then mix gently. Mix Lipofectamine^TM 2000 gently before use, then dilute the 20μl in 500μl of cultural medium. Incubate for 5 minutes at room temperature. After 5 minute incubation, combine the diluted DNA with diluted Lipofectamine^TM 2000. Mix gently and incubate for 20 minutes at room temperature. Add the lml of complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth. Incubate cells at 37℃in a CO₂ incubator. Selection was applied after 24h with 250ug of hygromycin per ml.Determine the expected mutants and protein analysis of nucleocapsid construts in LCLsThe desired mutations identification in HBV p3.8â…¡were performed followed by cloning and sequencing analysis. The insertion of HBV core gene mutants in the recombinant EBO-HBV plasmid was confirmed by PCR as well as enzyme digestion with Sacâ… and Kpnâ… .By MEIA and Western blot analysis, the expressions of HBVantigens were detected in cell culture medium and cell lysate of transfected LCLs by each of four strains of EBO plasmids. EBO-HBV-transfected cells produce HBc/eAg and HBsAg which is readily detectable in the corresponding cell lysates and culture supernatants. The cell lysate from EBO-HBV transfectants displays an immunoreactive protein of 21 kDa.Partâ…¢Hot spots of hepatitis B virus core mutations and the cytotoxic T lymphocytes response in vitroThe cytotoxic T-cell response in chronic hepatitis B virus (HBV) infection has been described as weak and mono- or oligospecific in comparison to the more robust virus-specific T-cell response present in resolved infection. However, chronic hepatitis B is a heterogeneous disease with markedly variable levels of virus replication and liver disease activity. The HBcAg could be an immunologic target. Codon 48-60 and 84-101 substitution are prevalent in CAH patients with negative HBeAg. To analyze the role of C gene mutation in HBV-specific CTL response during chronic viral hepatitis, we have developed a panel of EBV-based episomal expression vectors containing wild HBV antigen and C gene mutants then transfeeted into the LCLs. These LCLs serve as a stimulator and a target to monitor the HBV-specific CTL response in patients with chronic viral hepatitis B.Generation of HBV-specific CTLPBMC were produced as previously described by partâ… . Cells(5×10⁵/ml) were plated in the presence of irradiated (3, 000 R) autogenous EBV-transformed B cell lines(5×10⁴/ml) and cultured for 4 weeks, rIL-2(20U/ml) and stimulated cells were complemented every four days of culture and the cytotoxic assays were performed after 2 weeks and 4 weeks.Cytotoxicity AssaysCytolytic activity was determined in the CytoTox 96(?) Non-Radioactive Cytotoxicity assay using round-bottom 96-well plates containing 10,000 targets/well. Stimulated PBMCs from patients were tested at E/T ratios of 50:1 on day 14 and 28. The percent target cell lysis was calculated from the formula: % Cytotoxicity =Experimental-Effector Spontaneous-Target Spontaneous/Target Maximum- Target Spontaneous×100.We now report the existence of CTLs able to lyse target cells that express endogenously synthesized HBV nucleocapsid antigen mutants (G87, L97) in the peripheral blood of patients with chronic active hepatitis B. The CTL response is HLA-A2 restricted, mediated by CD8-positive T cells. Equivalent lysis of target cells that express each of these proteins suggests that their intracellular trafficking pathways may intersect. The current report provides definitive evidence that HLA classâ… -restricted, CD8-positive CTLs that recognize endogenously synthesized HBV nucleocapsid antigen are induced during chronic HBV infection in humans and establishes a strategy that should permit a detailed analysis of the role played by HBV-specific CTLs in the immunopathogenesis of viral hepatitis. We demonstrate that chronically infected patiems with HBV C gene mutations who experience a spontaneous develop a CTL response. The results suggest that specific immunotherapeutic enhancement of the CTL response to HBV should be possible in chronically infected patients, and that it could lead to viral clearance in these individuals with resolution of chronic liver disease.