Vector development for gene-vaccination targeting dendritic cells

我们试图开发针对专业抗原提呈细胞(APC)的弓形虫病DNA疫苗，如朗格汉斯细胞(LC)和树突状细胞(DC)。由于初始T细胞只能在专业APC的刺激下被激活，因此LC/DC在诱导宿主产生有效的保护性细胞免疫特别是对抗细胞内寄生虫病方面起着至关重要的作用。DC以未成熟细胞的形式存在于非淋巴组织内，并在收到成熟信号后迁移至淋巴组织。因此，DC具有分化/成熟而不是增殖的特性。由于非增殖性细胞的特性，DNA转染树突状细胞一直非常困难。经过努力，我们决定使用编码细菌CpG基序的质粒载体。含有未甲基化的CpG基序的人工合成的寡脱氧核苷酸(ODN)，即ODN1466和ODN1555，编码在质粒载体中，能激发强烈的天然免疫反应(PNAS，93：2879,1996；J.169：5590,2002)。通过基因枪免疫，我们分析了弓形虫热休克蛋白70(T.G.HSP70)、T.G.HSP30(慢殖子特异性)和SAG1(速殖子特异性)基因对弓形虫病的免疫效果。与T.G.HSP30和SAG1基因相比，弓形虫感染急性期和慢性期T.G.HSP70基因免疫B6和BALB/c小鼠各器官的弓形虫载量下降最为显著。与肌肉或腹腔注射50μg弓形虫HSP70基因相比，单基因枪法接种2μg弓形虫诱导的弓形虫数量显著减少。T.G.HSP70基因的疫苗效力持续3个月以上。因此，我们利用基因枪成功地建立了针对LC/DC的DNA疫苗方法(Vaccine，2003)。

We have attempted to develop DNA vaccine against toxoplasmosis targeting the professional antigen presenting cells (APC) such as Langerhans cells (LC) and dendritic cells (DC). As naive T cells can only be activated by the stimulation of professional APC, roles of LC/DC are crucial in inducing effective protective cellular immunities in host especially against intracellular parasitic diseases. DC reside within non-lymphoid tissues as immature cells and migrate to lymphoid tissues after receiving a maturation signal. Thus, DC possess a property of differentiation /maturation but not proliferation. Because of the property of non-proliferative cells, DNA transfection to DC has been very difficult. After struggling, we have decided to use a plasmid vector encoding bacterial CpG motifs. The synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs, i.e. ODN1466 and ODN1555, encoded in the plasmid vector, stimulate a strong innate immune response (PNAS, 93 : 2879, 1996 ; J. Immunol. 169 : 5590, 2002).By using gene gun vaccination with this plasmid vector, we have analyzed the vaccine effects of Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) (a virulent tachyzoite-specific), T.g.HSP30 (a bradyzoite-specific) and SAG1 (a tachyzoite- specific) genes against toxoplasmosis. The vaccination with T.g.HSP70 gene induced the most prominent reduction in T. gondii loads in various organs of B6 and BALB/c mice at the acute and chronic phases of toxoplasmosis compared with T.g.HSP30 and SAG1 genes. A single gene gun vaccination with 2 μg of T.g.HSP70 gene induced a significant reduction in the number of T. gondii organisms compared with 50 μg of T.g.HSP70 gene vaccination by intramuscular or intraperitoneal injection. The vaccine effects of T.g.HSP70 gene persisted for more than 3 months. Thus, we have successfully established DNA vaccination method targeting LC/DC by using the gene gun (Vaccine, 2003 in press).