The Study Of Recombinant Plasmid CpG-HBcAg Immune Effects On BALB/c Mouse And Human Dendritic Cells

Hepatitis B virus (HBV) causes a common infectious disease, and there are anestimated 350 million chronic HBV carriers worldwide. Patients with chronic hepatitisB are at high risk of developing liver cirrhosis, and this is associate with a higher rate ofmortality due to the development of hepatocellular carcinoma or noncarcinomatouscomplications of cirrhosis.Currently, the only therapy for chronic hepatitis that has a lasting beneficialeffect is systemic treatment with alpha interferon (IFN-a), but a sustained response isachieved in only one-third of patients with chronic hepatitis B. Nucleoside analoguessuch as lamivudine provide a therapeutic alternative leading to a rapid decrease inserum HBV DNA levels and to histopathological improvement of liver disease.However, cessation of treatment usually leads to a rapid relapse of disease,and longtermtreatment often results in the selection of resistant viral variants. These outcomesemphasize the need for novel therapeutic approaches. Although the pathogenesis ofchronic liver disease is not well understood, there is a consensus that liver damage isimmune mediated. Specific immunotherapeutic strategies have been proposed aspossible alternatives to the use of IFN or antiviral drugs to enhance or to broadenpatients with chronic hepatitis B. Unmethylated cytosine-guanine dinucleotides within the context of certainflanking sequences (CpG motifs), as originally identified in bacterial DNA, have diversestimulatory effects on the innate and adaptive immune systems. Several of these effectscontribute to the strong Th1-type adjuvant activity for antigen-specific responses. Forexample, CpG DNA triggers most (95%) B cells to proliferate, secrete immunoglobulin(Ig) and cytokines, and be protected from apoptosis, all of which contribute to astronger humoral response.CpG DNA also directly activates monocytes,macrophages,and dendritic cells to secrete various Th1 cytokines,which in turn inducesT and NK cells to secrete additional cytokines. Overall, CpG induces a strong Th1-likepattern of cytokine production dominated by inter- leukin-12 (IL-12) and IFN-g, withlittle secretion of Th2 cytokines, and these cytokines can provide additional T-cell helpfor both humoral and cell-mediated immune responses.CpG ODN have been shown tobe effective Th1-type vaccine adjuvants in animals with a variety of antigens. Forexample,mice immunized by i.m. injection of antigen with CpG ODN have strongcytotoxic T lymphocytes (CTL) and predominantly IgG2a antibodies, also indicative ofa Th1-type response. Since such Th1-type immune responses are thought to benecessary tbr clearance of HBV infection,it is possible, that CpG ODN with recombinantHBcAg may be an effective therapeutic vaccine for the treatment of patients chronicallyinfected with HBV.Therefore, in our study, we first constructed eukaryotic expression recombinantvectors for expressing HBcAg and CpG. Then,BALB/c Mouse immunized by i.m.injection of recombinant vectors pZeoSV2 (+) /CpG-HBcAg (ISS) .We explored theimmune effects of pZeoSV2(+)/CpG-HBcAg(ISS) action on BALB/c mice. Meantime,recombinant vector pEGFP-N1/ CpG-HBcAg(ISS) was used to transfect DCs.Then,we have investigated the alteration of the surthce molecules expression on theDCs after transfection and effective to its fucation. In additon,we have investigated theeffects and mechanisms of apoptosis HepG2 induced by DCs culture supernatant,whichwas the DCs after transfected recombinant plasmid pEGFP-N1/CpG-HBcAg(ISS) tohuman monocyte-derived dendritic cells. At last, we have investigated the expression of Toll-like receptors (TLR) 9 in peripheral blood mononuclear cells (PBMC) of patientswith chronic hepatitis B and C of different virus copies infection.Objective1. To construct the recombinant eukaryotic expression vectors pZeoSV2 (+) / CpG-HBcAg(ISS) and pEGFP-N1/CpG-HBcAg(ISS), which provided the basic materialfor the development of Hepatitis B virus remedial DNA vaccine.2. To explore the immune effects of recombinant plasmids pZeoSV2(+)/ CpG-HBcAg(ISS) action on BALB/c mice.3. To explore the alteration of the surface molecules expression on the DCs aftertransfection recombinant plasmid pEGFP-N1/CpG-HBcAg(ISS) to human monocytederiveddendritic cells and effective to its fucation.4. To explore the effects and mechanisms of apoptosis HepG2 induced by DCs culturesupernatant,which was the DCs after transfection recombinant plasmid pEGFP-N1/CpG-HBcAg(ISS) to human monocyte-derived dendritic cells.5. The aim of the present study was to investigate the expression of Toll-like receptors(TLR) 9 in peripheral blood mononuclear cells (PBMC) of patients with chronichepatitis B and C of different virus copies infection.Methods1. Three couples of primers were designed for PCR according to the known sequenceof HbcAg. The CpG fragments sensitive to human or murine and Non-CpG fragmentsare pulled into primers. The HBcAg gene was amplified by PCR from genome ofserumal DNA of chronic HBV patients, and PCR product was subcloned into eukaryoticexpression vector pZeoSV2 (+) and pEGFP-N1. The constructed pZeoSV2 (+) /CpG-HBcAg (ISS) and pEGFP-N1/CpG-HBcAg(ISS) were identified by restrictingenzyme digestion analysis, PCR amplifying, and DNA sequencing.2. Recombinant plasmid pZeoSV2 (+) /CpG-HBcAg (ISSb,c) was used to immuneBALB/c mice. The serum HBcAb, IFN-γ,IL-2,IL-12,IL-4 and IL-10 level weredetected by ELISA.3. Recombinant plasmid pEGFP-N1/CpG-HBcAg(ISSa,c) was constructed and used to transfect DCs.The expression of CD80 and CD86 on the transfected DCs were analyzedby flow cytometry. The IFN-γ,IL-12,IL-4 and IL-10 in the culture supematant of thetransfected DCs were detected by ABC-ELISA.4. Recombinant plasmid pEGFP-N1/CpG-HBcAg(ISSa,c) was constructed and used totransfect DCs. The alteration of apoptosis HepG2 were analyzed by flow cytometry.5. The serum viral load of HBV and HCV was detected in the patients and we analyzedthe correlation between HBV-DNA copies or HCV-RNA copies and the TLR9expression. The protein level of TLR9 was evaluated using flow cytometry. The studygroup was comprised of 90 patients (60 with chronic hepatitis B, 30 with chronichepatitis C) and 20 healthy controls.Results1. The size of amplified HBcAg gene was 530bp. Restriction enzyme digestion, PCRamplifying and DNA sequencing confirmed that pZeoSV2 (+) / CpG-HBcAg (ISS)and pEGFP-N1/ CpG-HBcAg(ISS) had been constructed successfully. Theharvestedfull- length sequence of HBcAg gene was identical with that registered in theGenBank.2. The pZeoSV2 (+) /CpG-HBcAg (ISSb,c) recombinant plasmids can produceHBcAb in BALB/c mice. The anti-HBc titers of pZeoSV2 (+) /CpG-HBcAg (ISSb)groups are obviously higher than those of pZeoSV2 (+) /CpG-HBcAg (ISSc) group(P＜0.01 ) . The result showed that high level of Th1 type cytokines including IL-2,IFN-γand IL-12 were induced in pZeoSV2 (+) /CpG-HBcAg (ISSb) recombinantplasmid groups,where the level of Th2 type cytokines such as IL-4 and IL-10 wereinhibited obviously.3. pEGFP-N1/CpG-HBcAg (ISSa)-transfected DCs expressed higher level of CD80and CD86 (P＜0.01) .The result showed that high level of Th1 type cytokinesincluding IFN-γand IL-12 were induced in pEGFP-N1/CpG-HBcAg(ISSa)recombinantplasmid groups (P＜0.01 ) ,where the level of Th2 type cytokines such as IL-4 and IL-10 were inhibited obviously (P＜0.05) .4..The culture supematant of pEGFP-N1/CpG-HBcAg (ISSa) group increasedapoptosis rate of HepG2 with training time [0h: ( 2.8±0.8 ) % , 6h:(7.6±1.0)%,12h:(9.2±1.1)%, 18h:(12.6±1.2)%,24h:(17.7±0.7)%, P＜0.05].5. Our results demonstrated that HBV or HCV infection led to a decreased expression ofTLR9 protein compared to the healthy group(P＜0.05). The TLR9 protein level isnegative correlation related to serum viral load of HBV and HCV(r=-0.632, r=-0.909, P＜0.01).Conclusions1. The pZeoSV2 (+) /CpG-HBcAg (ISS) has been successfully constructed, whichprovides the basic material for further study the function of CpG and the developmentof Hepatitis B virus remedial DNA vaccine.2. pZeoSV2 (+) /CpG-HBcAg (ISSb,c) has recombinant plasmid an obvious role onHBcAb Production in BALB/c mice. The recombinant plasmids include CpG-HBcAg(ISSb) gene sequence has induced the expression of Th1 type cytokineswherease inhibited the expression of Th2 type cytokines.3. The pEGFP-N1/CpG-HBcAg(ISSa) recombinant plasmid can improve the costimularymolecule on DCs and has induced the production of Th1 type cytokines(IFN-γand IL-12) wherease inhibited the expression of Th2 type cytokines (IL-4and IL-10) .4. The culture supematant of pEGFP-N1/CpG-HBcAg (ISSa) group could significantincrease apoptosis of HepG2.5. There is downregulation of TLR9 protein in PBMC of HBV-infected or HCV-infectedpatients.They are negative correlation related to serum viral load and have an importantrole in detecting viral replication of HBV and HCV.