Preparation Of HLA-Specific CML Polyepitope Vaccine In Chinese People And Valuation Its Immune Activity

BackgroundChronic myeloid leukemia (CML) is a clonal disease of the hematopoietic stem cells and is characterized by Philadelphia (Ph) chromosome or the BCR-ABL fusion gene. BCR-ABL fusion gene was also expressed in some acute lymphoblastic leukemia (ALL) patients. Despite the great success of the tyrosine kinase inhibitors (TKIs), CML remains incurable, probably due to treatment resistance of leukemia stem cells, which are responsible for rapid disease relapse after discontinuation of therapy and the existence of minimal residual disease (MRD). Thus, how to treat CML patients with minimal residual disease (MRD) is an urgent and important issue for clinical hematology. Nowadays, increasing effective immunotherapies involving vaccination or adoptive cellular immunotherapy are used to treat CML. Peptides which derived from amino acid sequences crossing the b3a2breakpoint in p210was demonstrated to elicit class I restricted cytotoxic T cells proliferation in vitro. WT1, a transcription factor, was expressed at low levels by immature CD34+progenitor cells, whereas over-expressed in hematopoietic malignancies including acute and chronic myeloid leukemia. WT1peptide vaccines were showed to elicit and boost an immune response to WT1and could be used as treatment for patients with CML who have residual disease. To date, some studies have focused on HLA-restricted BCR-ABL or WT1epitope vaccines and made great progress. However, combined BCR-ABL and WT1epitopes and targeted on more Chinese-restricted HLA alleles and broader antigens have not been tried. In this study, we designed a novel chimeric polyepitope vaccine that used recombinant lentivirus transduced DC vaccines carrying target BCR-ABL and WT1genes, and evaluated its immunological effects in vitro.ObjectivePrediction and decision of HLA-specific CML polyepitope and found parallel nucleotide sequences. Then cascaded a single nucleotide sequence and ligated into lentiviral vector. Transfected DCs, prepared HLA-specific CML polyepitope vaccine and valuation its immune activity in vitro. This research provided a new strategy to treat CML.Materials and Methods1. Polyepitope selection:Consulted related SCI literature about CML epitope,and selected CTL epitopes and B cell epitopes in different BCR-ABL protein antigens which can induce cellullar immunologic response and humoral immunoresponse and stably bind class I and class II MHC molecules. Though SYFPEITHI combined with MAPPP algorisms, the selected epitopes were predicted for the specific value of peptide and proteasomal cleavage site. Furthermore, the nucleotide sequences of these epitope were cascaded a single nucleotide sequence.2. Preparation of HLA-Specific CML polyepitope vaccine:This nucleotide sequence was ligated into lentiviral vector. PBMCs were collected from health donors, and DCs were prepared successfully. The DC vaccine was constructed using lentiviral vector transduced DCs. To confirm target gene mRNA expression, PT-PCR analysis were conducted. And fluorescence was observed in DC cells and HEK293cells after transduced by recombinant lentivirus.3. Evaluation of the immune activity of this polyepitope vaccine:CD3+T cells were separated from PBMC by magnetic bead and identified T cells purity by Flow cytometry. T lymphocytes were stimulated with DC vaccine and then co-cultured in vitro with peripheral blood mononuclear cells (PBMCs) from CML or ALL patients, respectively. The cytotoxicity of proliferous cytotoxic T lymphocytes (CTLs) was determined by the LDH assay. The IFN-y production of CTLs was detected using ELISPOT assay.Results1. Consulted SCI literature about CML epitope which have been published in PubMed since1995, the study selected50CTL epitopes and B cell epitopes in different BCR-ABL protein antigens.2. The polyepitope DNA sequence has been synthesized successfully, and the recombinant lentiviral have been successfully packaged. After tranfected DC cells, the target gene were verified at the level of mRNA. And fluorescence was observed in DC cells and HEK293cells after transduced by recombinant lentivirus.3.LDH assay confirmed that the cytotoxic activity about experimental group apparently higher than control group. Furthermore, the in vivo stimulation of CTLs with this DC vaccine were produced high level of IFN-y.Conclusion1. We have successfully prepared HLA-specific CML polyepitope vaccine.2. We evaluated its immune activity in vitro. And provided a new way about immunotherapy of CML and eliminate MRD.