ADVANCES IN DNA VACCINES AGAINST EBOLA & DENGUE VIRUSES

这项研究和相关实验室的两个新进展奠定了这项研究应用于针对埃博拉和登革热病毒以及潜在的其他新兴病毒的DNA疫苗的基础：(1)溶酶体膜蛋白的靶向序列与DNA疫苗编码的蛋白质抗原的偶联；我们已经证明这显著增强了对几种抗原的免疫应答，可能是通过将抗原引导到主要的组织相容性II类(MHC II)途径，以处理抗原表位并将其呈递到免疫反应机制的辅助T细胞臂。(2)一种作为DNA-明胶纳米球凝聚体的体内递送基因的非病毒系统，作为将DNA疫苗递送到抗原提呈细胞以及用于共递送靶向配体和增强免疫系统反应的细胞因子的手段。拟议研究的主要目标是用编码埃博拉和登革病毒蛋白的DNA疫苗来完善这些系统，并证明这些方法可能应用于其他新出现的病毒的可行性。这项研究将与维护埃博拉和登革热病毒挑战动物模型系统的其他实验室合作进行。研究设计是，首先，制备编码含有LAMP溶酶体膜蛋白信号和靶向序列的埃博拉和登革病毒嵌合蛋白抗原的DNA质粒构建体。将用这些质粒免疫的小鼠的免疫反应与用编码野生型蛋白抗原的DNA免疫的小鼠进行比较。这些实验还将比较小鼠对裸露DNA疫苗和纳米球DNA疫苗的反应。这些实验将包括DNA剂量、给药途径、疫苗接种动力学和免疫反应持续时间的研究。其他实验将分析共同传递细胞因子的影响，包括白细胞介素2和4，粒细胞-巨噬细胞集落刺激因子，和伽马干扰素。此外，针对溶酶体膜的抗原的免疫应答将与编码与抗原提呈细胞的B7蛋白结合的CTLA4配体的蛋白质抗原的DNA的效果以及对纳米球DNA针对抗原提呈细胞的应答进行比较。

Two novel advances of this and associated laboratories underlie the application of this research to DNA vaccines against Ebola and dengue viruses, and potentially other emerging viruses: (1) The coupling of the targeting sequence of lysosomal membrane proteins to the protein antigen encoded by DNA vaccines; we have shown this to enhance markedly the immune response to several antigens, presumably by directing the antigen to the major histocompatability class II (MHC II) pathway for processing and presentation of antigenic epitopes to the helper T cell arm of the immune response mechanism. (2) A non-viral system for the in vivo delivery of genes as a DNA-gelatin nanosphere coacervate as a means to deliver the DNA vaccine to antigen presenting cells and for the co-delivery of targeting ligands and cytokines that augment the immune system response. The principle goals of the proposed research are to perfect these systems with DNA vaccines encoding Ebola and dengue virus proteins and to prove the feasibility of these approaches for possible application to other emerging viruses. This research will be conducted in collaboration with other laboratories that maintain animal model systems for Ebola and dengue virus challenge. The research design is, first, to prepare DNA plasmid constructs encoding chimeric Ebola and dengue virus protein antigens containing the signal and targeting sequences of the LAMP lysosomal membrane protein. The immune response of mice vaccinated with these plasmids will be compared to that of mice vaccinated with DNA encoding the wild type protein antigens. These experiments will also compare the response of mice to vaccination with naked DNA as compared to nanosphere DNA. The experiments will include studies of DNA dose, route of administration, kinetics of vaccination, and duration of the immune response. Additional experiments will analyze the effect of co-delivery of cytokines, including interleukins 2 and 4, granulocyte-macrophage colony stimulating factor, and gamma interferon. Additionally, the immune response to of antigens targeted to the lysosomal membrane will be compared to the effect of DNA encoding the protein antigens coupled to the CTLA4 ligand that binds to the B7 protein of antigen presenting cells, and to the response to nanosphere DNA directed to antigen presenting cells.