The Study On Anti-HBs Fab-IFNα Effect In HBV Infection

Infection with hepatitis B virus (HBV) can cause transient and chronic liver in?am- mation. A long-term chronic HBV infection can lead to liver cirrhosis and development of hepatocellular carcinoma. Although HBV infection can be prevented through vaccination, interferon-γ(IFNγ) and nucleosidic inhibitors such as lamivudine have been approved for the treatment of HBV. Low response rates to therapy, severe side-effects, high cost and indefinite outcomes of long-term therapy are the disadvantages of their therapy. HBV has still remained as one of the most significant viral pathogens.Interferon-α(IFNα), an immunomodulatory antiviral drug, that inhibits HBV-DNA replication, is frequently used drugs in CHB. IFNαis effective only in less than 30% of the chronic carriers, and about 50% of IFNα-treated patients experience recurrence after cessation of treatment.Because the recovery on IFNαis limited in CHB, Anti-HBs Fab, a fully human monoclonal antibody against a surface antigen of hepatitis B virus (HBsAg), was generated and its immunological and biochemical characteristics were studied. The recombination of anti HBs Fab and IFNαmaybe has a synergistic activity for HBV-DNA and HBV antigen clearance.Part 1 Construction and expression of the fusion protein consisting of IFNαand anti-HBs Fab in prokaryoticAnti-HBs Fab have been shown its immunological and biochemical characteristics for against a surface antigen of hepatitis B virus. The purpose of this section is to construct and express the fusion protein consisting of IFNαand anti-HBs Fab. Using pBAD-IFNαplasmid as template, the IFNαwere amplified with corresponding endonuclease sites and artificial linker at 5',3'termini by polymerase chain reaction (PCR) techniques. IFNαPCR products were digested with XbaI for gel purification. The vector pBAD-HBs Fab was digested with XbaI. Then two productions were ligation with T4 ligase at 16℃30min. Following bacterial transformation, the recombinant plasmid was identified with restriction enzyme digestion and PCR.Single clones were picked from the resulting transformation, grown in 100ml of LB broth with ampicillin (100μg/L), and induced with arabinose. Fusion protein was purified by Ni2+ charged His tag affinity chromatography. After purification of the fusion protein, Human anti-HBs Fab-IFNαwere concentrated and separated in a 12% SDS-PAGE. The proteins were transferred to nitrocellulose, block, incubated with HRP-conjugated goat anti-human IgG Fab.The molecular weight of the purified protein was about 47.5Kd and 25Kd. We supposed that the band about 47.5Kd fusion protein consistingλfragment and IFNα, another band about 25Kd containing heave chain Fd fragment. The fusion protein showed well bioactivity with western blot and Dot-blot, and HBsAg affinity which is similar to anti-HBs Fab fragment. Theλ-IFNαactivity was 4.2×103~4.85×104IU/ml.The new constructed expression of the fusion protein with both HBsAg affinity andλ-IFNαactivity make it possible for immunobiotherapy drug targeted to Hepatitis B virus.Part 2 Construction and expression of the fusion protein consisting of anti-HBsλfragment and IFNαin PichiaAs a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli. It is faster, easier, and less expensive to use than other eukaryotic expression systems and generally gives higher expression levels. Pichia pastoris is methylotrophic yeast, capable of metabolizing methanol as its sole carbon source. Heterologous expression in Pichia pastoris can be secreted. Secretion requires the presence of a signal sequence on the expressed protein to target it to the secretory pathway. Pichia may have an advantage in the glycosylation of secreted proteins because it may not hyperglycosylate.After cloned and sequenced gene of interest into the pPICZαC behind the AOX1 promoter, isolated plasmid DNA by miniprep for restriction analysis and sequencing. The plasmid DNA was linearized with BstX I, then was transformed into X33 using electro- poration methods. Transformants are plated on YPDS plates containing 100μg/ml Zeocin to isolate Zeocin-resistant clones for small-scale expression.λ-IFNαwas purified using BrCN-sepherose 4B from the culture media, and purifica- tion of recombination protein was analyzed by Western blot. The molecular weight of the purified protein was about 50Kd, which was higher than its expression in E.coli. Because of Pichia had many of the advantages of higher eukaryotic expression such as protein processing, protein folding, and posttranslational modification. The antigenicity and binding activity to HBsAg of fusion protein were dectected with ELISA and the bioactivity of IFNαwas detected with WISH cells. This anti HBsλ-IFNαantibody had the HBsAg affinity similar to that of anti-HBs Fab fragment, and exhibited high affinity which estimated by dot blot where the HBsAg concentration was 3μg/ml. Theλ-IFNαactivity was 7.8×104~5.1×105IU/ml, which was higher than prokaryotic expression.Part 3 The study on anti-HBsλ-IFNαeffect in HBV infectionCurrent in vitro models for hepatitis B virus (HBV) are based on human hepato- blastoma cell lines transfected with HBV genome. HepG2.2.15 was established from the hepatoblastoma cell line HepG2. HepG2.2.15 exhibited prolonged expression of HBV, as was demonstrated by secreted levels of HBsAg, HBeAg, and HBV DNA in the culture medium of the growing cells. The quantity of HBV-DNA ranged between 1.2×105~3.7×105 copies/ml. The concentration of HBsAg was between 16.2~20.5 COI (normal<1.0 COI), HBeAg was between 230~320Ncu/L (normal<30 Ncu/L), which were evaluated with chemiluminescence immunoassay method. HepG2.2.15 can serve as an important tool for further exploration of HBV host–pathogen interaction, and for assessing new antiviral agents.λ-IFNαused in this study is a recombination protein contained humanλantibody fragment and IFNα, which was raised against the surface antigen of hepatitis B virus. To assess our hypothesis that the HBV DNA detected in the cell culture medium originated from viral particles. Cytotoxicity of fusion protein was determined by mitochondrial toxicity testing (MTT) of cells. The inhibitory activities ofλ-IFNαwas further examined at different conditions. Different concentrations ofλ-IFNαand IFNαin DMEM were added to the wells with the monolayer growth of HepG2.2.15 cells and the cells and supernatant were collected at day 3, 6 and 9. The concentrations of HBsAg and HBeAg in the culture supernatant were determined by ELISA respectively, and the extracellular HBV DNA was measured by quantitative PCR with HBV specific primers.Flow cytometry was used to determine the Fas expression of HepG 2.2.15 and which was treated withλ-IFNα. Resistance of HepG 2.2.15 to Fas-mediated apoptosis and the effect ofλ-IFNαon the apoptosis were studied using anti-Fas agonistic monoclonal antibody CHl1. Over a 9-day period in culture, groupλ-IFNαwith different concentration produced and secreted HBsAg and HBeAg and HBV- DNA at increasing concentrations at any time. The cytotoxicity ofλ-IFNαhad no statistical differences which treated under the same conditions (p>0.05).The inhibitory rate ofλ-IFNαon HBV- DNA and HBsAg and HBeAg showed no statistical differences between the concentration of 2 and 10 IU/ml (P>0.05). At the concentration of 20,50,100 IU/ml, the inhibitory rate ofλ-IFNαon HBV-DNA and HBsAg and HBeAg showed higher statistical differences(P<0.05). A significantly higher HBsAg and HBeAg and HBV- DNA inhibitory rate was byλ-IFNαcultures at every assayed conditions (p<0.05). The results proved that targetingλ-IFNαcan enhance the anti-HBV effect of IFNα.λ-IFNαmight take advantage of interaction both receptors of IFNαand HBsAg byλcarried. IFNαcould be easier to get to the targeting cells. The result makes it porsible to carry out further studies on targeted therapy.Fas expression of HepG2.2.15 was low and increased after the cells were treated withλ-IFNα. The HepG2.2.15 could resist Fas-mediated apoptosis. CH11 could not induce HepG2.2.15 cells to apoptosis. But the apoptosis induced by CH11 increased when the cells up-regulated the Fas expression after treatment withλ-IFNα.Increasing evidence demonstrates that humoral immunity is important for protection from HBV infection. In a word, this part of experiment confirmed thatλ-IFNαwas able to neutralize HBsAg in vitro.λ-IFNαis able to up-regulate the Fas expression and subsequently promotes the CHll-mediated apoptosis. Anti HBsλ-IFNαin vitro models of HBV infection have shown some promise, the efficacy of neutralizing antibodies in vivo requires evaluation in animal experiment.