Development of molecular and cellular-targeting DNA vaccine against Mycobacterium tuberculosis and analysis of its mechanism

趋化因子与特定的细胞表面受体结合，这些受体在配体结合后被内化。趋化因子受体在多种免疫细胞上有不同的表达。前哨抗原提呈细胞(APC)，如未成熟的树突状细胞(DC)和巨噬细胞，表达趋化因子受体，如CCR5。我们将趋化因子的受体结合和内化应用于结核分枝杆菌疫苗接种。首先，我们通过DC和巨噬细胞的共聚焦显微镜分析检测了MIP-1CCR5Ligand-α融合蛋白的结合和内化，发现融合蛋白结合到这些细胞的表面并内化到细胞内，并与CCR5共定位。为了构建DNA疫苗，我们利用结核分枝杆菌的主要分泌蛋白MPT51，因为我们证明了MPT51能够诱导T细胞介导的免疫反应和对结核分枝杆菌攻击的保护性免疫。用短链14个氨基酸的…连接mip-1a和mpt51基因。将其克隆到哺乳动物表达载体pCI中。用基因枪系统将pCI-MPT51或pCI-MIP-1α-MPT51(融合)或pCI-MPT51+pCI-MIP1α(混合)免疫BALB/c小鼠，每周免疫3次。末次免疫3d后，采用MHC-四聚体实验检测MPT51特异性CD8~+T细胞。融合DNA疫苗免疫小鼠的CD8+T细胞数明显高于单独免疫MPT51基因疫苗的小鼠。与pCI-MPT51或pCI-MPT51+pCI-γα免疫小鼠相比，融合DNA疫苗免疫的小鼠脾细胞中抗原特异性的干扰素-MIP1mRNA量增加。在这三种DNA免疫模式中，融合DNA疫苗免疫的小鼠产生了最高水平的MPT51特异性干扰素-γ蛋白。此外，这些DNA疫苗免疫C57/BL6小鼠的脾细胞被MPT51的CDC T细胞表位多肽刺激。C5 7/BL6融合基因疫苗免疫C5 7/BL6小鼠产生的抗原特异性干扰素-γ基因和蛋白水平高于pCI-MPT5 1免疫小鼠。提示DNA疫苗载体编码的mip-1a抗原融合蛋白能有效地内化到抗原提呈细胞中，诱导更高水平的抗原特异性T细胞应答

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 … More 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