Research On DNA Vaccine Against Hantavirus

ObjectiveHemorrhagic fever with renal syndrome( HFRS ) is a severe, high mordent disease,which is caused by Hantavirus , a kind of virus belonging to bunyaviri-dae . Because of the unknown mechanism , different clinical presents and the severe condition , there are no special drugs to cure the disease. Those clearly indicate the need for effective vaccines. Nucleic vaccine beings the new way to prepare the Hantavirus vaccine. It has the good quality which is compared with the traditional vaccine , such as the low cost , long time immunity. Hantavirus G2 glycoprotein has two sites to neutralize the antigens, which has the more cross reaction of different type than G1. Those characteristics provide the theory foundation as the hopeful candidate immunogens. So we constructed the eukary-otic expression vector based on the G2 gene, observed the expressing in the eu-karyote and its immunity in the mice. Because of the poor immunogenicity of DNA vaccine we used the CpG - DNA as adjuvant to immoprove its immunogenicity. There is the dispute to the safety of the DNA vaccine,so we study the possible integration of the vaccine to mice gene besides its immunity, so those provide the basic to the later study.MethodsWe designed two pair of primers based on the Hantavirus 76-118 all gene sequence ,one pair was led in two digestive sites of EcoR I /Xba I at the two ends. The other was introduced a immunity stimulating sequence GAGATT , which is sutible to the mice , and two digestive sites HindIII/Xho I. We obtained two gene segments , which one contained the immunity stimulating seg-uence,but the other didn' t. By amplificating the gene segment through the PCR, we connected the two productions to T vector pMD18 - T and constructed the vector pMD18 -T/G2 and pMD18 - T/G2(ISS) 0 After identificating ,we digested pMD18 -T/G2 ,pMD18 -T/G2(ISS)and pcDNA3. 1 + with two endo-nucleases, and connect the two gene segment with pcDNA3. 1 + and constructed the eukaryotic expression vector pcDINA3. 1 +/G2 and pcDNA3. 1 +/G2(ISS) , the two were transfected into Vero - E6 cells and we detected their expression in the eukaryotic. After extraction of the plasmid ,we adjusted the plasmid concentration to lmg/ml and used BALB/c male mice, aged 6 to 8 week , were randomly divided into 6 groups, receivingPBS, pcDNA3.1 + , pcDNA3. 1 +/G2, pcDNA3. 1 + /G2(ISS) ,pcDNA3.1 +/G2 and CpG - ODN by injection three times , two weeks apart. After the first immunity we collected the serum and the culter supernatant of spleen cells at day 15 ,30,45 ,60,to detect the the level of IL -4 IFN - -y and antibody of G2 glycoprotein by ELISA;T cells proliferation by MTT;the changes of subset CD4 + N CD8 + by FACS, to know the immunity effect of the vaccine by the results, futher more we can know wether the CpG -DNA has the immunity modulation . At day 90 , the mice receiving PBS and pcDNA3. 1 +/G2 were killed to get their lungs and spleens to extract the DNA. Using original primer , we observed wether the recombinant pcDNA3. 1 +/G2 can integret into the host DNA by PCR with the extracted gene templet .ResultsThe recombinants of pMD18 - T /G2 ,PMD18 - T /G2( ISS ) were verified by PCR of clone and endonucleases digestion, and the recombinants also be verified by endonucleases digestion. The green fluorescence was seen in the kyto-plasm after transfection of the two eukaryotic expression vector into Vero — E6, which showed that G2 glycoprotein was expressed in the eukaryotic cell and had the antigenicity . The results of immunized to mice show that the levels of IL - 4 , IFN - Y, antibody , the CD4+ ^ CD8 + T cell percentages and the condition of spleen cell proliferation are higher after immnity of pcDN A3. 1 + /G2 and pcD-NA3. 1 +/G2( ISS) and CpG - ODN three times later. But all of those in thegroup immunized with pcDNA3. 1 + were significantly lower than that of the group immunized with pcDNA3. 1 +/G2 (ISS) or group immunized with pcD-NA3. 1 + /G2and CpG - ODN , the results of group immunized with pcDNA3. 1 +/G2 were higher than group of pcDNA3. 1 + , otherwise the group injected with PBS had the lower results of all than every group. At day 15 ,30 after first immunizing, all indexers didn' t go up , but at day 45 they were at the highest level and at day 60 decreased at some level. Those of group immunized with pcDNA3. 1 +/G2( ISS) increased at the same level compared with pcDNA3.1 + and CpG - ODN . Especially , the group of immunized with pcDNA3. 1 + had markedly increased the production of IFN - 7 and the percentage of CD8 + cells . In the test of gene integrating there was no integrating between recombinant pcDNA3. 1 +/G2 and the host chromosome.ConclusionG2 glycoprotein mainly induces humoral immunity namely Th2 mediated immunity. In our experiment we constructed the eukaryotic expression vector pcDNA3. 1 +/G2 based on the G2 glycoprotein gene. In the following test we found that the mice can product the antibody after immunized with the recombi-nentors, so the gene of G2 glycoprotein could be considered as a candidate DNA vaccine. At the same time G2 glycoprotein has the low immunogenicity ,we use CpG - ODN as adjuvant to immunize the mice with the gene vaccines that can elevate the level of humoral immunity. So CpG - ODN can be regarded as a promising adjuvant for the DNA vaccine of Hantavirus. And we also found that the DNA vaccine of G2 glycoprotein can not integrted with host DNA , so that provide the foundation for the study on the DNA vaccine about Hantavirus.