Development of a novel Hsp70-based DNA vaccine as a multifunctional antigen delivery system.

DNA vaccination is a simple and effective method to induce immune responses against a variety of tumors as well as infectious diseases. Vaccination with major histocompatibility complex (MHC) class I tumor peptide has been carried out to induce an antigen-specific and tumor-reactive cytotoxic T lymphocytes (CTLs) response in vivo. In this study, we describe a novel DNA vaccine based on heat shock protein 70 (Hsp70), which can chaperon antigenic peptides and initiate innate and adaptive immune responses, to induce a more effective immune response. Ovalbumin (OVA) MHC class I epitope peptide (OVA(257-264): SIINFEKL) was selected as a model antigen and polyhistidine was used to facilitate the cytosolic delivery of the antigen-Hsp70 after endocytic uptake. A novel plasmid DNA vector encoding polyhistidine, Hsp70 and OVA(257-264) (pHis-Hsp70-pep) was designed. When mice were immunized with pHis-Hsp70-pep by intradermal injection in combination with electroporation, strong antigen-specific CTL responses were generated. pHis-Hsp70-pep also showed a significant protective effect against tumor challenge with an OVA-expression EL4 tumor line. These results indicate that the Hsp70-based DNA vaccine is useful as a multifunctional antigen delivery system to induce the antigen-specific immune response.

Enhanced generation of cytotoxic T lymphocytes by increased cytosolic delivery of MHC class I epitope fused to mouse heat shock protein 70 via polyhistidine conjugation.

Heat shock protein 70 (Hsp70)-associated antigens in a soluble form have been shown to elicit strong antigen-specific cytotoxic T lymphocyte (CTL) responses following immunization without any adjuvants. In order to improve the potential of Hsp70, we genetically designed a novel Hsp70-based antigen delivery system, in which the model MHC class I epitope of ovalbumin (OVA) (SIINFEKL; OVA257-264) was fused to mouse Hsp70. To facilitate the cytosolic delivery of the peptide following Hsp receptor-mediated endocytosis, polyhistidine of 25 or 50 residues was further fused to the fusion protein. Each fusion protein was then expressed in E. coli and purified. When added to DC2.4 cells, a mouse dendritic cell line, the fusion protein containing polyhistidine of 25 residues was efficiently taken up by the cells and efficiently distributed to the cytosol. The fusion protein also exhibited a significantly improved efficacy of MHC class I-restricted presentation of antigen. Vaccination of mice with the polyhistidine fusion protein generated strong antigen-specific CTL responses and antitumor activity. These findings suggest that polyhistidine fusion is a useful strategy to increase the potential of Hsp-based vaccination.

Enhanced antigen-specific antibody production following polyplex-based DNA vaccination via the intradermal route in mice.

DNA vaccination is an attractive approach with various advantages over conventional vaccination. The present study was undertaken to examine whether polyplex-based DNA vaccination could be used to modulate immune responses by plasmid DNA (pDNA). Methylated bovine serum albumin (mBSA) was used as a model of a cationic macromolecular carrier of pDNA encoding obalbumin (OVA) and the effects of polyplex formation of pDNA with mBSA on the antigen-specific immune responses were examined. Anti-OVA IgG antibody production was significantly increased following intradermal immunization with the polyplex compared with naked pDNA, although the induction of cytotoxic T lymphocyte activity was lowered by polyplex formation. We also demonstrated that the disposition and gene expression of pDNA following intradermal injection could be manipulated by polyplex formation. Intriguingly, we also found that the migration of dendritic cells to the injected site could be induced by polyplex formation probably due to a high level of tumor necrosis factor alpha production from the keratinocytes treated with mBSA/pDNA complexeses. Thus, the present study has demonstrated that the immune responses could be biased towards a Th2-type response by polyplex-based DNA vaccination through manipulation of not only pDNA disposition but also dendritic cell migration.