Improvement Of Hepatitis B Virus DNA Vaccine By Plasmid Coexpressing Hepatitis B Surface Antigen And Granulocyte Macrophage-Colony Stimulating Factor

ObjectiveAdministration of plasmid DNA encoding foreign proteins has proven to be an effective means of generating humoral immune responses and cellular immune responses in a number of animal models. Immune responses induced by DNA vaccination have been shown to be protective against challenge with various infectious agents. However, there is still a need to increase the potency of DNA vaccines. granulocyte macrophage-colony stimulating factor(GM-CSF) is one of immune adjuvants. GM-CSF can activate neutrophils, macrophages, DCs, and other mononuclear cells, and also stimulates progenitor/stem cells to mature and migrate from the bone marrow to the peripheral circulation. In order to enhance hepatitis B DNA vaccines antivirus effect, we constructed plasmid coexpressing GM-CSF and HBsAg and investigated whether GM-CSF can improve humoral and cellular immune responses and enhance HBsAg DNA vaccine antivirus effect.Methods1. Construction of plasmids(1) pcDNA3.1-SThe HBsAg-encoding fragment was created by PCR amplification of DNA sequence encoding the HBsAg from pEcob6 using primers designed to generate HindIII and XholI restriction sites at the 5＇and 3＇ends of the amplified fragments respectively.Then the amplified HBsAg DNA was cloned into unique HindIII and XholI cloning sites of the pcDNA3.1(+). (2)pcDNA3.1-GM-CSFThe GM-CSF -encoding fragment was created by PCR amplification of DNA sequence encoding the GM-CSF from pCD-hGM-CSF using primers designed to generate HindIII and XholI restriction sites at the 5＇and 3＇ends of the amplified fragments respectively. Then the amplified GM-CSF DNA was cloned into unique HindIII and XholI cloning sites of the pcDNA3.1(+).(3)pcDNA3.1-S-GM-CSFHBsAg DNA fragment (without stop codon ) of N terminus was amplified by PCR using primers designed to generate HindIII and BamH I restriction sites at the 5'and 3' ends of the amplified fragments respectively.Then the amplified HBsAg DNA was cloned into unique Hind III and BamH I cloning sites of the plasmid pcDNA3.1 (pcDNA3.1-S). Then, GM-CSF DNA fragment (without start codon ) of C terminus was amplified by PCR using primers designed to generate BamH I and Xhol I restriction sites at the 5'and 3' ends of the amplified fragments respectively was fused to the C terminus of pcDNA3.1-S. (4)pcDNA3.1-GM-CSF-S GM-CSF DNA fragment (without stop codon ) of N terminus was amplified by PCR using primers designed to generate HindIII and BamH Irestriction sites at the 5'and 3' ends of the amplified fragments respectively.Then the amplified GM-CSF DNA was cloned into unique Hind III and BamH I cloning sites of the plasmid pcDNA3.1 (pcDNA3.1- GM-CSF). Then, S DNA fragment (without start codon ) of C terminus was amplified by PCR using primers designed to generate BamH I and Xhol I restriction sites at the 5'and 3' ends of the amplified fragments respectively was fused to the C terminus of pcDNA3.1- GM-CSF.2. In vitro expression HepG2 cells and COS-7 cells were transfected with plasmid vectors using cationic liposome respectively. 48 hours later, HBsAg in culture supernatants and cell lysates were assayed by ELISA analysis. GM-CSF expressed by cells transfected with plasmids were detected using immunocytochemistry. Supernatants and cell lysates separated by SDS/PAGE, Proteins were transferred to poly (vinylidene difluoride) membrane, blocked with 4% nonfat dried milk. Blots were probed with anti-HBsAg antibodies, Western blots were developed with horseradish peroxidase-conjugated secondary antibodies. 3. DNA immunizaton All BALB/c(H-2d) mice and HBV-transgenic mice were immunized i.m.at 6-8 weeks of age. To enhance cellular uptake of plasmid DNA, the quadriceps muscles of mice were injected at multiple sites with a total volume of 100ul of 0.25% bupivacaine four days before the injection of plasmids. the plasmids were injected into the same region at three different sits in a final volume of 100ul. These mice were separated into 8 groups with 5 animals each. These mice were immunized...