Research&Development Of Novel Vaccines For Wilms' Tumor

Objective: To develop novel candidate vaccines for Wilms'tumor in order to lay a foundation for effective treatment and substitute for the radiotherapy and chemotherapy to this tumor.Methods: First we must prepare the necessary subject,including the antibody and the tumor cell line.Then to select 3 antigen epitope from the WT1 gene of Wilrns'tumor,HLA-A*0201,HLA-A*2402 and modified HLA-A*2402(HLA-A*2402x) ,using peptide synthesis tech,we produce the peptide,then immunized the Balb/c mice.We select the HLA-A*2402 and HLA-A*2402x antigen epitope as our research subject matter after the results of MTS and FCM were detected. The DNA fragments encoding the WT1 antigens (including, HLA-A*2402 and HLA-A*2402x),and the DNA fragments encoding the couple antigens HLA-A*2402-HBc and HLA-A*2402x-HBc were cloned and inserted into the eukaryotic expression vector pcDNA3.1(+) in sense orientation and prokaryotic expression vector pET32a(+) respectively, and transfected into COS-7 cells and cell respectively, and the expression of the target proteins in the cells were identified by immunocytochemistry, IFT(immunofluorescence technic) and Westen blot. At last Balb/c mice were immunized separately with the novel vaccine(VLP )and DNA vaccines(as control), and the immune response was assayed by ELISA, FCM, MTT and so on. And the immune protection wascarried out initial evaluation using the tumor cell line. Results:(1) We have prepared the qualified subject matter.using primary culture tech we established the tumor cell line.Expressing the WT1protein and purified,then immunizaed the rabbits for the antibody.(2) The result of MTS and FCM primarily certificated that the peptides HLA-A* 2402 and HLA-A*2402x were better than HLA-A*0201 in stimulating lymphocyte cell.But there is no different between the peptides HLA-A*2402 and HLA-A*2402x (p>0.05) .So we select the two peptides to study.(3) Construct two DNA vaccines including the antigen epitope HLA-A*2402 and HLA-A*2402x separately. All the cloned DNA fragments were consistent with the corresponding sequences reported in Genbank.(4) Construct two couple vaccines including the antigen epitope HLA-A*2402 and HLA-A*2402x separately which use VLP as vector. All the cloned DNA fragments were consistent with the corresponding sequences reported in Genbank.The result of WB and electron microscope certificated that the couple proteins can express in system.(5) Immunized Balb/c mice with the couple vaccines and theDNA vaccines as control.AU the vaccines can produce both effective specific humoral and cellular immune responses,especially the couple vaccines.The level of IgG2a-specific antibody was higher than that of IgG1-specific antibody,which indicated that a typical T-helper 1-dominatedimmune response was induced. T lymphocyte proliferation assay showed that the ConA-induced proliferation of spleenic T lymphocytes in the immunized mice was increased; ELISPOT assay showed that the number of the specific IFN-γ-producing CD8~+ T cells was much more than that of the control group; Flow cytometry analysis showed that the ratio of CD4~+/CD8~+ was lower than that of the control group. The results indicated that the specific CD8~+ T cell response was elicited by the couple vaccines. Immunized Balb/c mice with the couple vaccines could produce functional immune response, which was better than that elicited by the DNA vaccines.Conclusions: All the results indicated that the antigen epitopes HLA-A*2402 and HLA-A*2402x were important to immunotherapy for Wilms'tumor.And the new vaccines which use VLP as vector is one of good ways for Wilms'tumor immunotherapy.The prepared couple vaccines could elicited humoral immune response,and it is better than the DNA vaccines. The preliminary date in the present study might pave the way for the further study on developing effective immunotherapy methods to the infection of brucellosis based on vaccine strategy.