ENHANCEMENT OF DNA AND MINIGENE VACCINE IMMUNOGENICITY

DNA疫苗提供了许多优于传统方法的优点， 免疫，但这种新方法诱导的免疫水平往往 相当低。在几项研究中，针对病毒攻击的保护 已经赋予了DNA免疫，但诱导免疫的水平， 通过标准的体外方法低或不可检测。该提案的重点是 通过优化几种DNA疫苗的免疫原性， 标准（目标1-3），并通过特异性增强免疫原性， 免疫调节分子的施用（目的4）。具体目标 是： L.评估不同的DNA运载工具。DNA递送效率 将在与阳离子脂质复合的“裸”递送时进行评估。或 包封在脂质体中。评估将采用两种标记基因， 免疫原性病毒序列，并且体外和体内实验将 进行。 2.评估不同的接种途径。DNA将由 几种途径，并测定其表达和免疫原性。 此外，该实验室还可以使用“基因枪”， 经皮DNA递送。将比较这一过程的效率 简单的接种。 3.评估不同的促销员。为了配合目标1和2，我将尝试 为了优化DNA表达和免疫原性， 转录启动子将雇用三名推销员;一名给予 一般表达，一个直接转录到肌肉，另一个直接转录到肌肉。 直接转录至单核细胞/巨噬细胞。 4.通过共表达重组质粒提高现有DNA疫苗的免疫原性 免疫调节分子。将尝试增强免疫原性 通过免疫原性DNA序列和免疫调节剂的共接种， 分子。将使用两类免疫调节剂：粘附分子， 在T细胞的紧密贴壁和T细胞之间的信号传导中很重要， 抗原呈递细胞：和细胞因子，其可增强T细胞 应答 5.评价DNA疫苗在产生MHC中的相对效率 I类限制性免疫应答，与MHC类 非限制性反应。就其性质而言，DNA疫苗可能有利于抗原 通过MHC I类途径呈递，而不利于通过MHC II类;这可能导致偏向于CTL应答（而不利于 抗体应答）。将进行实验来解决这个问题 关心 这些实验将使用LCMV模型系统进行。 我们已经广泛地描述了对这种药剂的免疫反应。和 已经鉴定了当用作疫苗时赋予免疫力的序列。 在这个系统中，DNA免疫提供保护，尽管水平较低 比传统的疫苗接种更有效。因此，我们理想地 来优化这种DNA疫苗诱导的抗病毒保护性免疫。

DNA vaccines offer many advantages over more traditional approaches to immunization, but the levels of immunity induced by this new approach tend to be rather low. In several studies, protection against viral challenge has been conferred by DNA immunization, but levels of induced immunity were low or undetectable by standard in vitro methods. This proposal focuses on improving the immunogenicity of DNA vaccination, by optimizing several criteria (aims 1-3), and by specifically enhancing immunogenicity by co- administration of immunomodulatory molecules (aim 4). The specific aims are: l. Evaluate different delivery vehicles for DNA. Efficiency of DNA delivery will be assessed when delivered "naked', complexed with cationic lipid. or enclosed in liposomes. The evaluations will employ both marker genes, and immunogenic viral sequences, and both in vitro and in vivo experiments will be undertaken. 2. Evaluate different routes of inoculation. DNA will be administered by several routes, and its expression and immunogenicity determined. Additionally, the laboratory has access to a "gene gun", which allows transdermal DNA delivery. The efficiency of this process will be compared to simple inoculations. 3. Evaluate different promoters. In concert with aims 1 & 2, I will attempt to optimize DNA expression and immunogenicity by using different transcriptional promoters. Three promoters will be employed; one to give general expression, one to direct transcription to muscle, and one to direct transcription to monocyte/macrophages. 4.Improve the immunogenicity of current DNA vaccines by co-expression of immunomodulatory molecules. Enhancement of immunogenicity will be attempted by co-inoculation of immunogenic DNA sequences and immunomodulatory molecules. Two classes of immunomodulator will be used; adhesion molecules, important in the close apposition of, and signaling between, T cells and antigen presenting cells: and cytokines, which may enhance T cell responses. 5.Evaluate the relative efficiency of DNA vaccines in generation of MHC class I-restricted immune responses, compared to generation of MHC class Il-restricted responses. By their nature, DNA vaccines may favor antigen presentation by the MHC class I path way, and disfavor presentation by MHC class II; this may lead to skewing in favor of CTL responses (and against antibody responses). Experiments will be carried out to address this concern. These experiments will be carried out using the LCMV model system. We have extensively characterized the immune response to this agent. and have identified sequences which confer immunity when employed as vaccines. DNA immunization in this system confers protection, though at a level lower than that conferred by more traditional vaccination. Thus we are ideally placed to optimize this DNA vaccine-induced antiviral protective immunity.