| The cytotoxic T lymphocytes (CTLs) play a crucial role in anti-tumor immunity. The elicitation of strong CTL responses capable of mediating tumor regression in vivo has become the main aim of the immuotherpies for tumor. Peptide-based vaccines and naked DNA have been considered of the most rapidly evolving technologies for tumor vaccination due to their safety and feasibility. The major drawback Peptide-based vaccine is their weak inherent immunogenicity. Immunazition with peptide or recombinant proteins generally fails to elicit CTLs, and even in some case the induction of tolerance was observed. Although naked DNA vaccines in some cases may be effective in inducing immunity, however, the immune response that they elicit is often too weak or too late in developing to provide protective immunity.Previous studies demonstrated the differences between the dynamics for the presentations of these two forms of antigens. Antigenic peptides could be presented directly and quickly, however, for a short period of time due to their short half-life. Whereas, antigens in the form of genes could be expressed for a long time, but have a time lag owing to the process of gene expression. In addition, the dynamics of antigen presentation is thought a critical parameter in activating CTL response. Thus, the functional complementation of antigen forms of peptide and genes may be reasonably proposed and the collocation of antigenic peptide with gene encoding the same antigen might have a synergistic effect on elicitation of anti-tumor immunity.In the present study based on the previous research on "mimovirus" in our institute, we designed a peptide-DNA dual vaccine, which combines the peptide and DNA vaccines on the base of the probable functional complementation of antigen forms of peptide and genes. The cationic polylysine has been widely described as a self-assembly nonviral vector for gene therapy, which can condense plasmid DNA into small particles through electrostatic interactions between the positively charged lysine residues and the negatively charged DNA. As a test system, we chose a CTL epitope from the well-defined tumor Ag, P815A, as atarget and synthesized a bifunctional linear cationic peptide, which comprises a P815A CTL epitope and a DNA-binding moiety consisting of polylysine residues.In the present study, cationic antigenic peptide was synthesized by standard Fmoc, purified by middle-performance liquid chromatography and the purity was confirmed by reversed-phase high-performance liquid chromatography and the molecule weight by mass spectrometry. The gene for GM-CSF was amplified by RT-PCR and the plasmid encoding P815A and GM-CSF was constructed by standard recombinant technology. The condition of peptide-DNA vaccine preparation was determined by precipitation assay, gel retardation assay, DNase I protection assay and electronic microscopy. Further, the expressions of P815A and GM-CSF mediated by peptide-DNA vaccine particles were accessed by cell transfection and Western blotting. Finally, the efficacy of inducing antigen specific CTL response and eliciting anti-tumor immunity were accessed by 51Cr release assay and protection assay in P815 model.Here, we report that the cationic peptide was successfully synthesized and purified and the bicistronic plasmid encoding GM-CSF and P815A was constructed correctly. As bias of the results from precipitation, gel retardation, DNase I protection and electron microscopy assays, all vaccines used for immunization of animals were prepared by titrating cationic peptide into a solution of DNA with charge ratio of 2 at 87.5 mM NaCl concentration. The efficient expression of P815A and GM-CSF genes in cells mediated by peptide-DNA dual vaccine was conformed by cell transfection and Western blotting. The peptide-DNA dual vaccine elicited P815A specific CTL response, which was assayed in 51Cr release. Moreover, the peptide-DNA dual vaccine effectively protected DBA/2 mice from the fatal P815 tumor challenge and cure tumor-bearing DBA/2 mice with fatal P815 tumor,... |
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