Experimental Study On HCV Therapeutic DNA Vaccine

HCV infection is a global public health problem, now one hundred andseventy million people have infected it, which occupy approximately 3% ofthe world population. There are above forty million infectors in our country,and anti-HCV positive people approximately occupy 3.2%, furthermore, thenumber has the tendency to raise. HCV infection is the major cause of heap-titis C. Persistent HCV infection is a high risk of progressing to chronic hep-atitis,liver cirrhosis and hepatocellular carcinoma (HCC), usually morethan a decade after initial infection. Thus, the development of proper treatm-ent for HCV infection has been important.Up to now, no effective medicaltreatment is available for the majority of HCV infected patients, while newinfections are continuously emerging from transfuse, needle sharing, closecontact of HCV infected patient and other unidentified sources. Thus to con-trol the spread of HCV by developing new anti-HCV methods, such as ther-apeutic vaccines becomes an urgent task, especially in developing countriesincluding China, where there is a large infected population.In this study, we used HBc as the immuno-carrier to construct theexpression vector pcDNA-HBc-T1T2 ,and investigated its immunogenicity.First, got the plasmid pTrc-HBc at the basis of pTrc-HBV by usinggene engineering technique,and PCR to add NheI and SpeI site into theplasmid pTrc-HBc,then got the plasmid pTrc-HBcNheI SpeI .Inserted theHBcNheISpeI gene into the eukaryon expression plasmid pcDNA3.1 toconstructe the plasmid pcDNA3.1-HBcNheISpeI, In order to study thefeasibility of using HBcAg as the immuno-carrier for vaccine manufac-ture,the report genes GFP and RFP which came from plasmid pcDNA3-GFP and pcDNA3-RFP were inserted into plasmid pcDNA3.1-HBcNheISpeI,then,the reconstructed plasmids pcDNA3.1-HBcNheI-RFP and pcDNA3.1-HBcSpeI-GFP were transfected into the Hela cells with the company ofimmune liposomes Lipofectamine 2000,observing the expression of GFPand RFP under the confocal microscopy or inverted phase contrastmicroscope.Fused two synthetic T epitope antigen genes of HCV(1445-1453aa and 35-44aa)into the NheI or SpeI site of the plasmid pcDNA3.1-HBcNheISpeI DNA to got the plasmis pcDNA3.1-HBc-T1T2.The positiveplasmids pcDNA3.1-HBc-T1T2 were extracted and identified by restrictionenzyme,PCR and sequencing.The plasmids pcDNA3.1–HBc-T1T2 andpcDNA3.1-HBcNheISpeIwere transformed and expressed in E.coli JM109,discontinuous density gradient centrifugation to get the protein HBcAgand HBcAg-T1T2,SDS-PAGE to detect their purity,ultraviolet spectro-photometer to measure their concentration and with the HBeAg ELISAdetecting Kit to detect their immunogenicity.Then with them as thestimulant to stimulate the Lymphocytes from the blood of acute HCVinfector ,and used these lymphocytes as the effector cells,used the CM-Dillabelled and HCV infected Hela cells as the target cells to detect the activityof cytotoxic T lymphocyte(CTL).Last,immunized the plasmids pcDNA-HBc-T1T2 in animals and with the expression plasmid pcDNA-HBcNheISpeIas control.The tumor regression trial in mice was evaluated at appropriatetime;With ConA,HBcAg and HBcAg-T1T2 as the stimulant to stimulate thespleen cells from the normal and immunized mice ,then measured the levelof IL-2 in the culture medium of these cells with ABC-ELISAmethod.HBcAb titer was evaluated by ELISA;IL-12 and IL-10 in serumwere detected by ABC-ELISA;IL-5 in serum were detected by FACS;Nonspecific T lymphocytes proliferation response of spleenocytes wasrespectively examined by MTT assay;The cytokine INF-γ and IL-4 in spleencells,T cell subset of blood and spleen were all detected by FACS.The result showed that the identification of the expression plasmidspcDNA-HBc-T1T2 was agreed with expected,and the GFP and RFP in theplasmids pcDNA3-HBcSpeI-GFP and pcDNA3-HBcNheI-RFP were success-fully expressed;Tumor regression trial showed that the tumor from thepcDNA-HBc-T1T2 group was one only,different from the pcDNA3.1-HBcNheISpeI group(4 mice)and control group(24 mice);The detection of CTLactivity showed that the quantity of Hela cells infected by HCV inHBcAg-T1T2 stimulated group was almost killed(78.331±7.446),differentobviously from HBcAg(26.534±4.255)and control (7.425±0.119)group;Nonspecific lymphocytes proliferation response was induced stronger inexperimental group.FACS also showed that the ratio of CD8+ T cell in theexperimental group was higher than the control.The cytokine INF-γ inspleen cells and the IL-12 in serum from pcDNA-HBc-T1T2 immunizedgroup were higher than the control group.But different from above,theHBcAb in the serum from experimental group were almost similar with thecontrol group,the IL-4 in spleen cells and the IL-10 and IL-5 in serum ofpcDNA-HBc-T1T2 immunized group were lower.The range of IL-2 increasewas the greatest in the culture medium from spleen cells stimulated byHBcAg-T1T2.In conclusion, HBcAg can be used as the immuno-carrier of vaccine,the plasmid pcDNA-HBc-T1T2 could induce stronger cellular immune res-ponses, and it might be able to serve as a therapeutic vaccines candidatespecific for HCV.