Construction And Identification Of Schistosoma Japonicum Antigen Epitope PDDV Inducing High Level Endogenous IFN-γ

Schistosomiasis, a disease that affects more than 200 millionindividuals worldwide, is a major cause of morbidity in hepatic fibrosis. Atthe same time, the development of hepatic fibrosis and portal hypertensionare the principal causes of mortality in Schistosomiasis japonicum.In both murine and human studies, high level of Th2 cytokines andreduced Th1 cytokine have been associated with an increased risk of severehepatic fibrosis, and IL-10 is thought to play a critical immunoregulatoryrole. Evidence indicates that CD4~+Th2 cell is the key components whichaffects liver Pathology.IL-13 and IL-4 is the main cytokines leading to hepatic fibrosis,and IFN-γ,IL-12,IL-10 take important parts in protectionagainst fibrosis, among which IFN-γis a strong antifibrogenic cytokinecertificated by clinical experiments. In the current study, to exploreantifibrosis effect of Th1 immun response eliciting by SchistosomaJapanicum antigen, and the same time to provide an useful evidence forother fibrotic disease, we constructed several peptide-DNA dualvaccines(PDDV) with antigen epitopes from Schistosoma Japanicum,thenapplied the one or more which cliciting higher level of Th1 cytokineexpression to acute and chronic Schistosoma hepatic disease. The researchincludes the following two parts:Part one: We have identified four T cell epitopes from SchistosomaJapanicum, including P4,P6,P18 and P22 in previous study. At first, wedevised and synthesized the coding sequences of the epitopes, then weligated the epitopes gene with CpG ODN1826 or IL-12 into pCI-neo in thepresense of low melt agarose gel. Restrict endonuelease analysis and PCRindicated the genomic reconstruction is successful. Sequence analysisverified the results were consistent with devisition.The cationic epitope peptides were synthesized with 18 lysine residueswhich act as a bridge between antigenic peptide and encoding gene. Weprepared condensed DNA complexes by titrating cationic peptide into asolution of plasmid DNA at certain concemtration of NaCl and certaincharge ratio (the ratio of moles of lysine to nucleotide). By DNAPrecipitation Assays, Gel retardation Assay, DNaseⅠDigestion Assay andElectron Microscopy Assay,we discovered that at 150mM NaClconcemtration and a charge ratio of 4,. DNA could be protected fromdigestion by DNaseⅠand was completely condensed into homogeneousparticles with a diameter of about 20 nm that is already at the size-exclusion limit for the nuclear import.We prepared DNA peptide complexes with cationic antigen peptides andrecombinant plasmids pCI-neo-epitope-CpG1826 or PCI-neo-epitope-IL-12following the conditions refered above, resulting twelve sorts of PDDV andinjection at either side of the tail base.The groups of animal experiment1,[K]18P4-PCI-P4-IL-122,[K]18P6-PCI-P6-IL-123,[K]18P18-PCI-P18-IL-124,[K]18P22-PCI-P22-IL-125,Mixtrue PDDV of thefour kinds of above6,[K]18-PCI-neo-IL-127,[K]18P4-PCI-P4-CpG18268,[K]18P6-PCI-P6-CpG18269,[K]18P18-PCI-PI8-CpG182610,[K]18P22-PCI-P22-CpG182611,Mixtrue PDDV of the four kinds of above12,[K]18-PCI-neo-CpG182613,HBSThirteen groups of mouse were immunized three times with two-weekinterval. As controls, mice were immunized with HBS. Seven days after thefinish of immunizations, spleen cells were collected, re-stimulated in vitrowith epitope peptide. Lymphocytes proliferation and the level of IFN-γandIL-4 in the supematant of cell culture were tested.The levels of lymphocyte proliferation and IFN-γin No 8 group whichis immunized with [K]18P6-PCI-P6-CpG1826 PDDV is higher than controlafter stimulation with epitope peptide and SWAP, and the levels of IL-4 islower than the control, all of the above show significant difference. Itindicated that [K]18P6-PCI-P6-CpG1826 PDDV can strongly induced Th1type immune response.Significant lymphocyte proliferation occurs in the mice immunizedwith the mixed PDDV which made separately with four epitope peptides andIL-12 recombinant plasmids, meanwhile the levels of IFN-γand IL-4increased significantly, especially IL-4. But for the mice which immunized with the mixed PDDV which made with four epitope peptides and CpGODN 1826 recombinant plasmids, even though lymphocyte proliferationoccurs, the levels of IFN-γdecreased obviously, but the levels of IL-4increased significantly,it is suggested that the mixed PDDV can inducedtype 1 helper T (Th1) cell immune immune response and type 2 helper T(Th2) cell immune immune response, but the latter can affect strongly.In summary, for the four epitope peptides, some may be Th1 typeepitopes,others are Th2 type epitopes, the conclusion requires furtherexperiment to confirm because it come from once experimentIn conclusion, we construsted eight recombinant plasmids succesfully.By DNA Precipitation Assays, Gel retardation Assay, DNaseⅠDigestionAssay and Electron Microscopy Assay,we discovered that at 150mM NaClconcemtration and a charge ratio of 4,. DNA could be protected fromdigestion by DNaseⅠand was completely condensed into homogeneousPDDV particles with a diameter of about 20 nm. After immunizing,Lymphocytes proliferation and the IFN-γand IL-4 in the supematant of cellculture demonstrated that [K]18P6-PCI-P6-CpG1826 PDDV can stronglyinduced Th1 type immune response.