EVALUATION OF HIV GAG DNA VACCINE TO MAX T HELPER/CTL

描述：(改编自申请者摘要)我们的总体目标是 CD8+细胞毒性T淋巴细胞(CTL)和CD4+T细胞的作用机制 针对高度保守的内部病毒粒子可引发辅助反应 HIV-1 Gag.为了实现这一目标，一个新的发展是 构建能够在REV中表达Gag的DNA表达载体 和RRE独立和物种独立的时尚。使用这种新方法，我们 将系统地评估其表达和免疫应答， 不同亚细胞靶向形式的HIV-1 Gag最大限度诱导 小鼠抗GAG的CTL和T辅助细胞反应。 我们将落实两个具体目标：(一)。对能力的表征 不同HIV-1 Gag表达载体诱导CD8+CTL的实验研究HIV-1 Gag蛋白的表达 使我们能够在小鼠细胞中表达HIV-1 Gag分子的载体 已生成REV和RRE缺勤。衍生的GAG表达载体， 它们针对不同的细胞内和细胞外隔间 将构建和评估用于抗原处理和呈递的。 这些不同形式的恶作剧包括：(A)野生型恶作剧，它组装成 有效地从细胞中释放的颗粒；(B)失败的突变堵塞物 以质膜为靶标并留在细胞质中；(C)突变堵住 不能组装成颗粒；(D)快速降解的突变的插嘴 在细胞内，(E)分泌但非颗粒形式的GAG；(F)细胞表面 固定形式的恶作剧。(A)各种形式的HIV-11 Gag分子将被 在p815细胞中表达，并作为CTL靶细胞进行比较。(B)DNA载体 表达不同形式的HIV-1 Gag分子将进行CTL测试 对小鼠的诱导作用。(2)。不同HIV-1 Gag的功能特性研究 将优化T辅助应答的表达载体，特别是那些 可增强CTL反应。(A)不同形式的Hiva Gag DNA疫苗 还将评估上述对T辅助反应的诱导 DNA免疫后小鼠T细胞增殖和细胞因子的检测 体外GAG刺激后的反应。(B)CTLA-4-HIV-1 Gag融合 由单一DNA疫苗载体编码的分子将被评估。这 策略将允许针对抗原呈递的GAG分子进行特定靶向 MHC 11类演示的单元格。(C)利用DNA进行组合研究 从该特定目标识别的载体和从以下来源识别的DNA载体 具体目标1.

DESCRIPTION: (Adapted from Applicant's Abstract) Our overall goal is to determine the mechanisms by which CD8+ cytotoxic T lymphocyte (CTL) and CD4+ T helper responses can be elicited against highly conserved internal virion proteins of HIV-1 Gag. Toward this goal, a novel development is the construction of DNA expression vectors, which enable us to express Gag in a Rev and RRE independent and species independent fashion. Using this new method, we will systematically evaluate the expression of, and immune response to, different subcellularly targeted forms of HIV-1 Gag to maximize the induction of CTL and T helper responses against Gag in mice. We will carry out two specific aims: (1). Characterization of the ability of various HIV-1 Gag expression vectors to induce CD8+ CTL. HIV-1 Gag expression vectors which enable us to express HIV-1 Gag molecules in mouse cells in the absence of Rev and RRE have been generated. Derivative Gag expression vectors, which are targeted to different intracellular and extracellular compartments for antigen processing and presentation will be constructed and evaluated. These different forms of Gag include: (a) wild type Gag that assembles into particles which are released from cells efficiently; (b) mutant Gags that fail to target to plasma membrane and remain in the cytoplasm; (c) mutant Gags that fail to assemble into particles; (d) mutant Gags that are rapidly degraded intracellularly, (e) secreted but non-particle forms of Gag; (f) cell surface anchored forms of Gag. (A) Various forms of HIV-11 Gag molecules will be expressed in p815 cells and compared as CTL target cells. (B) DNA vectors expressing various forms of HIV-1 Gag molecules will be tested for CTL induction in mice. (2). Characterization of the ability of various HIV-1 Gag expression vectors that will optimize T helper response especially those that could enhance CTL response. (A) The different forms of HIVA Gag DNA vaccine described above will also be evaluated for induction of T helper responses after DNA immunization in mice by measuring T cell proliferative and cytokine responses after Gag stimulation in vitro. (B) A CTLA-4-HIV-1 Gag fusion molecule encoded by a single DNA vaccine vector will be evaluated. This strategy will allow specific targeting of Gag molecules to antigen presentation cells for MHC class 11 presentation. (C) Combination studies by using DNA vectors identified from this specific aim with DNA vectors identified from specific aim 1.