ABSTRACT
Objective:To develop an effective therapeutic double HBV DNA vaccine with two eukaryotic expressing motif,namely pS2.S and pIIF,a fusion ORF of hIL-2/hIFN-?.Methods:Two genes were amplified,which separately encoded HBV preS2.S middle envelope protein as a vaccine and human IL-2/IFN-? fusion protein as an adjuvant by PCR technique from plasmids pcDNA3.1/preS2.S and pcDNA3.1/hIL-2/IFN-?.Then the genes subcloned into eukaryotic vector pVAX1.The new plasmid was analysed by restriction endonuclease and DNA sequencing.The constructed plasmids were transfected into COS-7 cells in vitro with LipofectamineTM 2000.The expressing products in supernatants were quantified by ELISA.Space structure of fusion hIL-2/IFN-? expressed by the adjuvant plasmid was simulated by CE software.The double plasmid association were injected into BALB/c mice by in situ electroporation method,and specific humoral and cellular immune responses were examined.Results:The gene segments and inserting direction of pVAX1-S2.S(pS2.S)and pVAX1-IL-2IFN-?(pIIF)were both correct by genetic analysises.At 48 h after transfection,HBsAg and the cytokines of IL-2 and IFN-? were respectively 45.1 ng/ml,10.03 ng/ml and 11.5 ng/ml by ELISA.The activity domains of the two cycokines in fusion protein were entirely exposed on surface by CE software.Experiments were pefrormed in mice by injection of plasmid pS2.S.Protective antibodies of anti-HBs were produced in terms of dose-dependment pattern for the efficacy.Co-injection of plasmid pS2.S together with adjuvant pIIF resulted in more evident immune responses and dose of the plasmid pS2.S needed was diminished.There was strongly specific humoral and cell immunity by pS2.S and pIIF co-injection with EP technique.Conclusion:The double plasmids pS2.S and pIIF of anti-HBV are constructed succeedly and induce strongly specific immune activity in vitro and vivo.
ABSTRACT
Objective To investigate the effect of therapeutic dual-plasmid HBV DNA vaccine on specific humoral and cellular immunity in cancrivorous monkey (Macaca fascicularis). Methods The eukaryotic expression plasmids encoding human IL-2 and IFN-? fusion protein (pFP) were constructed to enhance the cellular immunity of therapeutic HBV DNA vaccine (pS2.S) encoding HBV envelope middle protein in the form of dual-plasmid (pS2.S+pFP). Thirty cancrivorous monkeys were randomly divided into 5 groups (6 each) with sex rotio of 1∶1, namely the dual-plasmid DNA vaccine group in high dosage (2000?g/kg), medium dosage (660?g/kg), low dosage (200?g/kg), and EP-mediated pFP (330?g/kg) or PBS administration groups as the controls. The monkeys were vaccinated repeatedly with the dual-plasmid HBV DNA vaccine, and then the immune responses including level of serum anti-HBs and number of IFN-? secreting T-cells induced by HBsAg were examined by means of enzyme-linked immunosobent assay (ELISA) and enzyme-linked immunosobent spot assay (ELISPOT) respectively. Results Ten weeks after immunization of HBV DNA vaccine, various levels of serum anti-HBs was detected in all the monkeys of three different dose groups. Sixteen weeks after administration of EP-mediated HBV DNA vaccine, the positive HBsAg-specific INF-? T cell responses was found in 3, 2 and 3 out of 3 monkeys, respectively in the high, medium and low losage groups, and HBsAg-specific INF-? T cell responses were positive in all the animals with the respective cell count of 30.0?13.5 SFCs/3?105 PBMCs, 30.7?26.3 SFCs/3?105 PBMCs and 17.7?6.4 SFCs/3?105 PBMCs in each corresponding group at the 29th week. However, HBsAg-specific INF-? T cell responses were negative in the pFP and PBS group at the same time. Conclusion Electroporation-mediated vaccination of the HBV DNA vaccine can effectively induce both humoral and cellular HBsAg-specific immune responses in cancrivorous monkeys.
ABSTRACT
To evaluate the safety of the therapeutic DNA vaccine against the hepatitis B virus. Plasmid DNA was detected by PCR in tissues of BALB/c mice immunized with the plasmids combined with electrotransfer in doses of 10 and 50?g. In long term toxicity study, HBV DNA vaccine was administered by repeated intramuscular injections combined with electrotransfer of pDNA to NIH mice in doses of 30, 60 and 120?g, twice a week for four consecutive weeks. Morbid manifestations, behaviors, hematology, blood chemistry, anti nuclear antibodies, and histopathology were analyzed. Results showed that plasmid DNA was detected primarily in the muscle at the site of injection, where it remained for up to 8 weeks. Eight repeated intramuscular injections of HBV DNA vaccine showed no adverse effects on behaviors, hematology, blood chemistry, and histopathology. No evidences of autoimmune mediated pathology and anti nuclear antibodies were observed in the mice. These results suggested that the therapeutic HBV DNA vaccine was safe and well tolerated.
ABSTRACT
To evaluate the immune response and safety of the therapeutic DNA vaccine against the hepatitis B virus in rhesus macaques immunized by intramuscular injection of therapeutic DNA vaccine combined with electroporation. HBV DNA vaccine was administered to rhesus macaques by repeated intramuscular injections combined with electroporation in doses of 0 2,1 and 2mg, monthly for three consecutive months. The anti HBs antibody level was used to evaluate the efficacy of induction of humoral immunity. After that, the vaccine was intramuscularly injected daily in the same dose for six days to evaluate its safety. It was found that immunization with the therapeutic DNA vaccine against hepatitis B virus by means of intramuscular injections combined with electroporation induced a strong and specific anti HBs humoral immune response in rhesus macaques.Nine repeated intramuscular injections of the vaccine did not show adverse effects on behaviors, hematology, blood chemistry, and histopathology. No evidences of autoimmune mediated pathology and anti nuclear antibodies were observed in the rhesus macaques. These results suggested that electroporation significantly improves the efficacy of the therapeutic HBV DNA vaccine in rhesus macaques.The vaccine is safe and well tolerated.