Balb / C Mice And Hbv Transgenic Mouse Experimental Study, Different Gene Vaccine Immune Responses

Aims: To develop a HCV combined HBV DNA-based therapeutic vaccine and tostudy the immune responses of HBV-based immunization with plasmid by appendingimmune stimulatory DNA sequence (ISS) to HBV core gene or by coinjecting CpG-oligodeoxynucleotides in Balb/c mice and in HBV transgenic mice.Methods:1. The HBV core gene and HCV core cDNA were inserted into the eukaryoticexpression vector with two multiple cloning sites mammalian expression vector underthe CMV promoter and RSC promoter respectively, named pRSC-HBV/HCV. Theexpression of SP2/0 cells transfected with pRSC-HBV/HCV was assessed by Westernblot and immunofluorescence studies. The Balb/c mice were immunized by multiplesites intramuscular injection with pRSC-HBV/HCV. The anti-HBc Ab and anti-HCV Abwere detected by ELISA. CTL activity of splenocytes against SP2/0 expressing HBcAgand HCV nucleocasid cells was determined in standard LDH release method. The tumorsize and animal survival were evaluated as an in vivo index of CTL activity generated byimmunization with the plasmid DNA construct after inoculated with SP2/0 cellsexpressing both the HBcAg and HCV core protein.2. The HBV core gene or HBV core gene appended ISS, obtained by ploymerasechain reaction, were inserted into the eukaryotic expression vector under the CMVpromoter respectively, named V-HBc and V-HBc/CpG. CpG-ODN and non CpG-ODNwere synthesized and phosphorothioate-modified. The Balb/c mice were immunizedby multiple sites intramuscular injection:â‘ Group1, V-HBc+ non CpG ODN; â‘¡GrouP2, V-HBc/CpG; â‘¢Group3, V-HBc+CpG ODN; â‘£Group4, V-HBc. The anti-HBc Ab was measured by ELISA. CTL acivity of splenocytes against pRSC-HBV/HCVtransfected SP2/0 cells was determined in standard LDH release method. The tumor sizeand animal survival were evaluated as an in vivo index of CTL activity generated byimmunization with the plasmid DNA construct after inoculated with SP2/0 cellsexpressing HBcAg.3. The HBV transgenic mice were immunized by multiple sites intramuscularinjection with V-HBs and CpG ODN or V-HBs alone or V-1012 controls.The anti-HBsAb and HBsAg in serum from immunized mice was measured by ELISA. Theexpression of HBsAg in liver tissue was detected by inununohistochemistry method (SP).The histological activity index was calculated with Knodell's method and theultrasttuctAfe ofhepatocytes was observed by electfomicroscopy.Rcsults:1. The HBcAg and HCV nucleocaPsid expressed simultaneously in SP2/0 cellstransfected with pRSC-HBVMCV Both anti-HBc and ani-HCV core Ab was detectedin all inununized mice with pRSC-HBVMCV. The specific CTL activity of splenocytesagainst SP2/0 cells expressing both HBV and HCV nucleocaPsid were 8l% in miceimmunized with pRSC-HBV/HCV, Which is significantly higher than that in inununizedmice with pRSC controls. The boor size after inoculated with SP2/0 cells expressingHBcAg and HCV nucleocaPsid in mice immunized with pRSC-HBVnsCV wereremarkably reduced compared with mice injected with pRSC.2. The ani-HBc Ab could be detected in every mice inununized with V-HBC+CpGODN, V-HBC/CpG, V-HBC+ Non CpG ODN as well as V-HBc contrOl grouPs. Thespecific CTL activity of splenocytes apainst SP2/0 cells expressing HBcAg were l8% inmice immunized with V-HBC+CpG ODN and 60% in mice injected with V-HBC/CpG,Which is significantly higher than that in immunized mice with V-HBC+ Non CpG ODNas well as V-HBc control grouPs. The tUmor size after inoculated with SP2/0 cellsexpressing HBcAg in mice inUnwhzed with V-HBC+CpG ODN and in mice immunizedWth V-HBC/CpG were remarkably reduced comPared with mice injected with V-HBC+Non CpG ODN as we1l as V-HBc cothel grouPs.3. The ani-HBs Ab in seru!n could be detected and HBsAg in serum and livertissues could not be measured in 2 out of 6 HBV transgenic mice iInmunized With V-HBs+CpG ODN. There were not significanly changes in the level of anti-HBs Ab andHBsAg in serum from HBV transgenic mice inununized with V...