Studies On The Responses Of PBMCs From HCV Infected Individuals To A Novel CpG ODNs

Hepatitis C Virus is a major public health problem which estimatedly infected 180,000,000 worldwide and 380,000 in China. Infection with HCV is usually subclinical and associated with mild symptoms. So most infection progressed into chronic liver disease, including recurring hepatitis, liver cirrhosis and hepatocellular carcinoma. The current therapy for chronic HCV infection is combination of alpha interferon (INFα) and ribavirin.Bacterial DNA with CpG motifs can efficiently stimulate various cells of the vertebrate immune system in a non-specific way. It has been reported that synthetic oligodeoxynucleotides that contain CpG motifs (CpG-ODNs) can stimulate B cell, dendritic cells (DCs) and activate macrophages and monocytes, and NK cells in vitro. In vivo, CpG-ODNs induce transient splenomegaly, increase antibody responses and promote T helper cell type 1 (Th1) immune responses. These observations make CpG-ODNs a interesting adjuvant for vaccines and a powerful potent drug for the treatment of cancer, allergies and infectious diseases. The experimental use of CpGODNs as natural adjuvants or cancer and allergy drugs has been extensively studied. However, the treatment of infectious diseases is a relatively new area in CpG-ODNs research. There is little information about immune responses of HCV infected individuals to CpG-ODNs. It is our intension to identify the immunostimulatory effects of CpG-ODNs to the PBMCs from HCV infectioned individuals and the potential application of CpG-ODNs in the treatment of viral hepatitis C. We selected a noval CpG-ODNs designed by our colleagues and three CpG-ODNs reported to carry on our study. We use the 3H-Thymidine incorporation assay for measurement of cell proliferation, the cell protection test for measurement of anti-virus substances producted by the PBMCs, the ELISA for measurement of cytokines IFNα,IFNγ,IL-6 and IL-12 induced by CpG-ODNs. In this research, we also compared the differential responses of PBMCs from individuals infected with HBV or HCV using the statistical methods (Cooperating with other colleagues, we obtained Data of HBV infected individuals). To our knowledge, this is the first study conducted to evaluate the immune stimulatory activities of CpG-ODNs PBMCs from patients infected with HBV or HCV.Materials and Meathods:1. CpG-ODNs: Based on their stimulatory effects on human PBMCs, CpG ODNs can be divided into 3 types: CpG-A ODNs (also known as"type D"), CpG-B ODNs (also known as"type K") and CpG-C ODNs. 2216, a published typical CpG-A ODNs, is a potent inducer of IFNαand IFNγfrom pDC and NK cells respectively whereas stimulates weak proliferation of B cells and differentiation of plasmocytoid dendritic cells (pDC). 2006, a well-studied CpG-B ODNs, is able to induce proliferation of B cells and maturation of DC, has little or no effects on pDC. C274, CpG-C ODNs share the above effects of both type A and type B CpG ODNs. BW001 is a novel CpG-C ODNs designed by our colleagues Bao Musheng[Bao Musheng, 2006]. All CpG-ODNs used in the experiment are synthesized by Shanghai Sangon Biological Engineering Thechnolgy and Service Co. Ltd.2. PBMCs islation and culture: Totally 65 HBV infected and 19 HCV infected individuals and 8 healthy donors are inclued in this study. All blood samples were obtained from the first hospital of Jilin university. All the donors included in this study have not received IFN or other antiviral therapy recently or ever. PBMCs of freshly drawn heparinized blood were prepared from buffy coats by Ficoll-Hypaque (GIBCO/BRL, Invitrogen) density gradient centrifugation and cultured in 10%FBS/IMDM in an incubator with 5% CO2 at 37℃.3. 3H-Thymidine incorporation assay: For in vitro proliferation assays, PBMCs were cultured with 10% FBS/IMDM with CpG-ODNs of a final concentration of 6μg/ml. 5×105 cells were incubated in 200μl of medium in 96 well U-bottom plates for 48 h. 0.5μCi of 3H-thymidine was added per well 16-18h before cell harvesting. Incorporation of 3H-thymidine was determined using aγcounter. CPM (count per minute) were recoreded to calculate the stimulation index.4. Protection effects of antiviral substances produced by CpG-ODNs stimulated PBMCs: We collected the supernants before addition of 3H-thymidine in the 3H-thymidine incorporation assay. The supernatants were then tested for their ability to inhibit the cytopathic effect (CPE) caused by VSV on Vero E6 cells. The Vero E6 cells were seeded into 96-well flat-bottomed plates (3×104 cells/100μl/well) and were directly incubated with the collected supernatants (10μl/well) for 16h. 10TCID50 (50% tissue culture infectious doses) of VSV was added for another incubation of 48-72h. The cytopathic effects caused by VSV were examined by crystal violet staining method. Anti-virus effects were expressed as OD values at 578nm, which are correlated to the anti-virus effects of the supernatants.5. ELISA of cytokines induced by CpG-ODNs: Cytokines in the collected supernatants were quantified by ELISA Kits according to the protocols provided by the ELISA kits (U-Theq.)6. Data analyses: All data obtained were analyzed by SPSS 15.0. The value of p<0.05 is considered to be significant.Results and Disscussion:1. Antiviral activity of BW001:We have identified in this study that BW001 can stimulate the proliferation and antiviral-substance production of PBMCs from HCV infected individuals (p<0.001).When acts on PBMCs from HCV infected individuals, BW001 exhibits the charateristics of type-C CpG ODNs. Whatsmore, BW001 can stimulate the production of antiviral cytokines including IFNαand IFNγby PBMCs from HCV infected individuals (p<0.01).2. The effects of BW001 and other CpG ODNs on the PBMCs from HCV infected individualsComparing to typical CpG ODNs (2216: type-A, 2006: type-B, C274: type-C), BW001 exhibits the characteristics of typical type-C CpG ODNs when acts on the PBMCs from HCV infected individuals. It can induce stronger proliferation of PBMCs than 2216 and stronger production of antiviral substances than 2006.3. The effects of CpG ODNs on the PBMCs from healthy, HBV infected and HCV infected individuals.3.1 The effects of BW001 on the PBMCs from healthy, HBV infected and HCV infected individuals.BW001 can significantly stimulate the proliferation and the production of antiviral substances by PBMCs from healthy donors, HBV infected individuals and HCV infected individuals. The effects of BW001 on the proliferation of PBMCs from HCV infected individuals are stronger than that of normal donors and HBV infeted individuals (p<0.01).The plasmal IFNαlevel of HCV infected and HBV infected individuals are higher than that of the normal donors (p<0.01). Interestingly, when cultured in absence of CpG ODNs, the PBMCs from normal donors produced more IFNαthan those from HCV or HBV infected individuals (p<0.01). Furthemore, BW001 was demonstrated capable of stimulating PBMCs from HCV infected individuals to produce less IFNαthan those produced from the PBMCs derived normal donors or HBV infeted individuals (p<0.01).The plasmal IFNγlevel of HCV infected individuals is lower than that of normal donors and HBV infected individuals (p<0.01). The production of IFNγby isolated PBMCs increased when cultured in 10%FBS/IMDM. The PBMCs from HCV infected individuals produced more IFNγthan the PBMCs from normal and HBV infected individuals (p<0.01). The effects of BW001 on the producion of IFNγby PBMCs from HCV infected individuals are stronger than that on the PBMCs from normal and HBV infected individuals (p<0.01).3.2 The effects of BW001 and 2216 on the PBMCs from healthy, HBV infected and HCV infected individuals.The effects of BW001 on the proliferation of PBMCs from HCV infected individuals are stronger than that of 2216. There are no differences between the effects of BW001 and 2216 on the producion of antiviral substances, IFNαand IFNγ.3.3 The effects of CpG ODNs on the production of IFNγby PBMCs from normal, HBV infected and HCV infected individuals.It was found that PBMCs from HCV infected individuals produced more IFNγthan those from HBV infected individuals when exposed to various CpG ODNs.3.4 The effects of CpG ODNs on the production of IL-6 by PBMCs from normal donors, HBV infected individuals and HCV infected individuals.CpG ODNs stimulate higher IL-6 producion by PBMCs from HBV infected individuals than PBMCs from HCV infected individuals.As we known, CpG ODNs can activate the cells of innate immune system, including dendritic cells, macrophages and natural killer (NK) cells, consequently stimulate the production of Th1 cytokines and chemokines and up-regulate the costimulatory molecules which facilitate the generation of adaptive immune responses. What's more, a phase Ia study with health volunteers shows that CpG ODNs are safe and well tolerated with a wide dose range. To identify the potential of CpG ODNs in clinical applications of treating hepatitis B and hepatitis C, we tend to investigate the responsiveness of PBMCs from HBV/HCV infected individuals in vitro.Together, we conclude that CpG ODNs could be used as a potential treatment for both hepatitis B and hepatitis C, preferentially for hepatitis C.