ADVANCES IN DNA VACCINES AGAINST EBOLA & DENGUE VIRUSES

本实验室和相关实验室的两项新进展为将该研究应用于埃博拉病毒和登革热病毒以及潜在的其他新兴病毒的DNA疫苗奠定了基础：(1)将溶酶体膜蛋白的靶向序列偶联到DNA疫苗编码的蛋白抗原上；我们已经证明，这可以显著增强对几种抗原的免疫反应，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，在免疫反应机制的辅助T细胞臂上加工和呈递抗原表位。(2)作为DNA-明胶纳米球凝聚体的非病毒系统，用于体内递送基因，作为将DNA疫苗递送到抗原呈递细胞的手段，并用于共同递送靶向配体和细胞因子，增强免疫系统反应。拟议研究的主要目标是用编码埃博拉病毒和登革热病毒蛋白质的DNA疫苗完善这些系统，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。本研究设计首先制备含有LAMP溶酶体膜蛋白信号序列和靶向序列的嵌合埃博拉病毒和登革热病毒蛋白抗原的DNA质粒。用这些质粒接种小鼠的免疫应答将与用编码野生型蛋白抗原的DNA接种小鼠的免疫应答进行比较。这些实验还将比较小鼠对接种裸DNA疫苗的反应与纳米球DNA疫苗的反应。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。其他实验将分析细胞因子共递送的影响，包括白细胞介素2和4、粒细胞-巨噬细胞集落刺激因子和γ干扰素。此外，对靶向溶酶体膜的抗原的免疫应答将与编码蛋白质抗原的DNA偶联到与抗原提呈细胞B7蛋白结合的CTLA4配体的效果以及对靶向抗原提呈细胞的纳米球DNA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.