Research & Development Of Novel Vaccines For Whilms Tumor

Objective to develop novel candidate vaccines for Willms 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 Willms Tumor,HLA-A*0201,HLA-A*2402,constructinging three DNA vaccines,we produce the peptide,then immunized the Balb/c mice.We select the HLA-A*2402 antigen epitope as our research subject matter after the results of MTS and FCM were detected.The DNA fragments encoding the WT1 antigens HLA-A*2402,and the DNA fragments encoding the couple antigens HLA-A*2402-HBc were cloned and inserted into the eukaryotic expression vector pcDNA3.1(+) in sense orientation and baculovirus expression pFactbac respectively, and transfected into COS-7 cells and Sf9 cell respectively, and the expression of the target proteins in the cells were identified by immunocytochemistry and Westen blot. At last Balb/c mice were immunized separately with the novel vaccine(VLP as the vector )and DNA vaccines(as control), and the immune response was assayed by ELISA,FCM,MTS and so on. And the immune protection was carried 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 were better than HLA-A*0201 in stimulating lymphocyte cell..So we select the HLA-A*2402 peptide to study.(3) Construct a DNA vaccines including the antigen epitope HLA-A*2402. The cloned DNA fragment was consistent with the corresponding sequences reported in Genbank.(4) Construct a couple vaccine including the antigen epitope HLA-A*2402 separately which use VLP as vector. The cloned DNA fragment was consistent with the corresponding sequences reported in Genbank.The result of WB certificated that the couple proteins can express in baculovirus expression system.(5) Immunized Balb/c mice with the couple vaccine and theDNA vaccine as control.All 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-dominated immune 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 vaccine. Immunized Balb/c mice with the couple vaccine could produce functional immune response, which was better than that elicited by the DNA vaccines.(6) All the results indicated that the antigen epitopes HLA-A*2402 and was important to immunotherapy for Willms Tumor.And the new vaccine which use VLP as vector is one of good ways for tumor immunotherapy.Conclusions The prepared couple vaccine could elicited cellular 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.We need tumor animal model to study further.