| Since more and more tumor antigens were identified, the therapeutic cancer vaccine based on tumor antigens had showed an attractive outlook. Cervical cancer is one of most common cancer in women. Human papillomavirus(HPV) oncogenic protein E7 is expressed in most HPV-contains cervical cancers and is important in the induction and maintenance of cellular transformation. And it is attractive targer for specific immunotherapy agaist cervical carcinoma. Therefore, we designed and synthesized the therapeutic vaccine against cervical cancer on base of HPV oncogenic protein E7 and studied its immunological effect. The antigen specific CD8+ Cytolytic T lymphocytes(CTLs) are thought to play a key role in tumor immunotherapy. Therefore, the stimulation of specific CTL represents one major goal in the design of vaccines for tumor immunotherapy. CTLs recognize immunogenic peptides derived from cellular proteins presented at the cell surface in the content of MHC class â… molecules. As the result of the specific interaction between the T cell receptor(TCR) and MHC class â… /peptide complexs, CTLs are able to kill target cells expressing tumor-associated antigen(TAA). Identification of tumor antigens and CTL epitopes provides more opportunities for the development of synthetic peptide vaccines. Peptide-based approaches offer several potential advantages in terms of purity, lot-to-lot consistency, cost of production, and a high specificity in eliciting immune response. This technology has proven an extremely useful strategy to avoid potentially harmful immune responses. Unfortunately, immunization with antigenic peptides alone is not sufficient to elicit an efficient CTL response, either because of their inherent lack of immunogenicity, or because of their rapid degradation, or because of poor uptake by APCs. It is important for peptide-based vaccine development to improve the immunogenicity of peptides. In the present study, according to the modern immunological theories and the methods of molecular design, we designed the therapeutic vaccine against human cervical cancer. All peptides were synthesized on an ABI 431A instrument using standard F-moc technology. These crude peptides were purified by reversed-phase high pressure liquid chromatography(RP-HPLC). All peptides were checked for homogeneity by analytical RP-HPLC and for identity by electrospray mass spectrometer. The results showed that the purity was >80 % for all peptides and that they had the expected molecular mass. To investigate the capacity of peptides to mobilize a human CTL repertoire, specific CTLs were generated in vitro using healthy HLA-A2.1+ donor PBMCs plused with peptides. The result showed that CTLs induced by peptide containing Th epitope, CTL epitope and lipid part elicited the strongest cytotoxic activity. CTLs induced with peptides lysed T2 cells pulsed with CTL epitope, but did not lyse T2 cells alone. To analyze the ability of specific CTLs to lyse endogenously HPV E7-expression tumor cell, the HPV E7 positive, HLA-A2-expressing cervical cancer cell line Caski was used as target cell in a standard 51Cr-release assay. The result showed CTLs were able to efficiently lyse Caski tumor cells, which showed that the tumor antigen E7 could be naturally processed and presented. To assess their in vivo immunogenicity, HLA-A2/Kb transgenic(Tg) mice were immunized with the peptides. Our study showed peptidecontaining Th and CTL epitopes could induce CTL response but CTL epitope could not do so in Tg mouse. The result showed the necessity for the help effect of Th cells in the induction of CTL. Our study also showed the peptide containing Th, CTL and lipid part had already induced the strongest CTL response, which showed that linkage of Th, CTL epitope and lipid part could improve the immunogenicity of CTL epitope. Peptide binding affinity to HLA-A2 molecules was analyzed by a binding assay on TAP-deficient human T2 cell line. Our observations revealed that peptide containing Th, CTL and lipid part had a marked increase in the stability and persistence...
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