ENHANCEMENT OF DNA AND MINIGENE VACCINE IMMUNOGENICITY

与更传统的方法相比，DNA疫苗提供了许多优势 免疫，但这种新方法诱导的免疫水平往往 是相当低的。在几项研究中，针对病毒挑战的保护 是由DNA免疫授予的，但诱导免疫水平是 低或不能用标准的体外方法检测到。这项提案的重点是 通过优化几种方法提高DNA疫苗的免疫原性 标准(目标1-3)，并通过联合- 免疫调节分子的给药(目标4)。具体目标 包括： L。评估不同的DNA递送工具。DNA传递的效率 将在“裸露”、与阳离子脂肪复合时进行评估。或 包裹在脂质体中。评估将使用这两个标记基因，以及 免疫原性病毒序列，体外和体内实验都将 要承担的责任。 2.评估不同的接种途径。DNA将由以下人员管理 几种途径，并对其表达和免疫原性进行测定。 此外，该实验室还拥有一把“基因枪”，这使得 经皮注射DNA。我们将比较这一过程的效率。 到简单的接种。 3.评估不同的推动者。与目标1和目标2一致，我将尝试 优化DNA表达和免疫原性的方法 转录启动子。将雇用三名推销员；一名将 一般表达，一个将转录定向到肌肉，一个定向到肌肉 直接转录到单核/巨噬细胞。 4.通过共表达提高现有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联合接种 分子。将使用两种免疫调节剂：黏附分子， 在T细胞和T细胞之间的紧密结合和信号传递中很重要 抗原提呈细胞：以及可能增强T细胞的细胞因子 回应。 5.评估DNA疫苗在MHC产生中的相对效率 与MHC类的生成相比，I类限制性免疫反应 IL-限制性反应。从本质上讲，DNA疫苗可能偏爱抗原。 由MHC I类路径方式表示，不赞成由MHC表示 第二类；这可能导致偏向于CTL反应(和反对 抗体反应)。将进行试验以解决这一问题 担忧。 这些实验将使用LCMV模型系统进行。 我们已经对这种制剂的免疫反应进行了广泛的表征。和 已经确定了被用作疫苗时具有免疫力的序列。 在这个系统中，DNA免疫提供了保护，尽管水平较低 而不是通过更传统的疫苗接种。因此，我们的理想状态是 以优化这种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.