The Molecular Desige Of Mimovirus And Study On Its Effect Of Inducing Specific CTL Responses In Vivo

CD8+ cytotoxic T lymphocytes (CTLs) are now recognized as important mediators of immunity against tumors and intracellular pathogens including HIV. How to efficiently induce antigen-specific CTL responses in viva has become a major target in the development of moern vaccine strategies. Recent vaccination studies indicated that the key to elicit CTL responses is to efficiently delivery antigens into the MHC class I processing pathway of professional antigen-presenting cells (pAPCs), especially dendritic cells (DCs), which can thereby display high numbers of the MHC class I-peptide complexes in a rich costimulatory context to prime the specific nae CD8+ T cells. Till now, several particulate antigen delivery systems have been proved to be efficient, such as immune-stimulating complexes (ISCOMs), liposomes, virus-like particles (VLPs), microspheres and phage-displayed particles. Moreover, many studies suggested that coadministration of antigen in combination with cytokines or other immunoregulatory molecules, either in the form of recombinant proteins or plasmids, can always enhance immunity. Among many cytokin, murine IL- 12 has been shown to be potent as adjuvant to promote the induction of CTL responses in viva. Synthetic peptides corresponding to epitopes recognized by CTL have been investigated as the basis for safe, effective vaccines. However, it has been proved quite difficult for synthetic peptide vaccines to induce CTL response in viva. Here, rationally combining the epitope-based peptide vaccine and the cationic-peptide DNA delivery system of gene therapy, we firstly designed a completely novel CTL vaccine imovirus, which is a kind of virus-size particulate antigen delivery system. The cationic peptides represent a sort of synthetic DNA delivery system that is gaining prominence in gene therapy. These peptides (eg. oligolysine, [K] ~) can bind with plasmid DNA through electrostatic interactions between the positively charged lysine residues and the negatively charged phosphate backbone of the DNA, forming highly condensed particles that allow internalization by mammalian cells and protection of the packaged DNA from the effects of nucleases. In the present study, we designed and synthesized a cationic peptide composed of 18 lysines and a CTL epitope corresponding to residues 28-39 of HBsAg (H~Ld restricted). This cationic peptide can bind with the plasmid containing murine IL- 12 gene, then spontaneously forming particle at an appropriate charge ratio. With a size equivalent to a virus, the particle contains a molecule of DNA literally ackaged by presumably several thousands of antigenic peptides, and may simulate the mechanisms of cell entry and DNA delivery used by viruses into professional APCs. Therefore, we designate this kind of particle as mimovirus, which might also mimic the immunogenicity of a virus in viva to some extent. The formation of mimovirus was identified by DNA retardation assay and transmission electron microscopy analysis using a negative stain technique. And its effect of IL- 12 gene transfer was investigated on P815 cell lines In vitro with ELISA, showing that mimovirus formed at the peptide/DNA charge ratio of 4.0 might be more effective for gene delivery and expression. After single immunization of mice with mimovirus subcutaneously at the tail base, its effect of inducing specific CTL responses in viva was measured by standard 51Cr release assay. The mice immunized with the mimovirus were shown to mount an effective, specific anti-HB sAg 28-3...