| Project/Area Number |
18590417
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bacteriology (including Mycology)
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| Research Institution | Hamamatsu University School of Medicine |
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Principal Investigator |
UCHIJIMA Masato Hamamatsu University School of Medicine, 医学部, Assistant Professor (20252174)
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| Co-Investigator(Kenkyū-buntansha) |
KOIDE Yukio Hamamatsu University School of Medicine, 医学部, Professor (30126809)
NAGATA Toshi Hamamatsu University School of Medicine, 医学部, Professor (90275024)
CERVANTES Jorge Hamamatsu University School of Medicine, 医学部, Post doctral Fellow (60377752)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,690,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2006: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | microbiology / infectious disease / immunology / vaccine / chemokine / mycobacterium / 細菌 |
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| Research Abstract |
Chemokines bind to specific cell-surface receptors which are internalized after binding of ligands. Chemokine receptors are differentially expressed on a variety of immune cells. Sentinel antigen-presenting cells (APCs), such as immature dendritic cells (DCs) and macrophages, express chemokine receptors such CCR5. We applied the receptor binding and internalization of chemokine to vaccination against Mycobacterium tuberculosis. First, we examined binding and internalization of MIP-1α (CCR5 ligand) -GFP fusion protein by confocal microscopy analysis using DCs and macrophage cells- GFP fusion protein was found to bind to surface of these cells and to be internalized into cells and colocalized with CCR5. To construct a DNA vaccine, we used MPT51, a major secreted protein of M tuberculosis, since we demonstrated that the MPT51 could induce T-cell-mediated immune responses and protective immunity upon challenge with M tuberculosis. MIP-la and MPT51 genes were ligated via short 14-amino acid
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spacer sequence and cloned into mammalian expression vector pCI. BALB/c mice were immunized three times biweekly with pCI-MPT51 or pCI- MIP-1α -MPT51 (fusion) or pCI-MPT51 +pCI-MIP1α (mixture) using Helios gene gun system. After three days form last immunization, MHC-tetramer assay was performed to measure MPT51-specific CD8^+ T cells. Numbers of the antigen specific CD8^+ T cells were higher in mice immunized with the fusion DNA vaccine than those of mice immunized with DNA vaccine encoding MPT51 alone. Amounts of antigen-specific IFN-γ mRNA increased in spleen cells from fusion DNA vaccine-immunized mice in compared with those of pCI-MPT51- or pCI-MPT51 +pCI-MIP1α -immunized mice. Mice immunized with the fusion DNA vaccine produced highest level of MPT51-specific IFN-γ protein among these three DNA vaccination patterns. Furthermore, spleen cells from C57/BL6 mice immunized with these DNA vaccines were stimulated by CDC T-cell epitope peptide of MPT51. C57/BL6 mice immunized with fusion DNA vaccine produced higher level of antigen-specific IFN-γ mRNA and protein than those of mice immunized with pCI-MPT51.These results suggest that MIP-la-antigen fusion proteins encoded by DNA vaccine vector are efficiently internalized into antigen presenting cells and induce higher level of antigen specific T cell responses Less
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