HIV Molecular Biology and DNA Vaccine Approaches Against

HIV 分子生物学和 DNA 疫苗方法

• 批准号: 6758418
• 负责人: George N. Pavlakis
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6758418
• 关键词: AIDS; AIDS vaccines; Primates; cell growth regulation; gene expression; gene induction /repression; genetic manipulation; genetic regulation; genetic transcription; host organism interaction; human immunodeficiency virus; intracellular transport; laboratory mouse; life cycle; messenger RNA; molecular biology; pathologic process; point mutation; protein protein interaction; protein structure function; species difference; vaccine development; vector vaccine; virus infection mechanism; virus protein

This project aims to understand the role and mechanism of function of HIV regulatory and accessory proteins in the virus life cycle and disease development; to dissect the mechanisms of nucleocytoplasmic trafficking of macromolecules; and to examine the mechanism of Vpr function as transcription activator and as cell cycle modulator. We study the function of several HIV-1 proteins, including Tat, Rev, and Vpr. We showed that the defect of Rev in rodent cells is caused by delayed import of Rev in the nucleus. The factors responsible for this effect are under investigation and may lead to new types of interventions targeted against the essential Rev factor. A new inhibitor of CRM1-directed nuclear export has been identified and characterized. This inhibitor will be useful for studies of Rev and many cellular proteins exported through the CRM1 pathway. We identified a new RNA element (named RTE) from the mouse genome that is able to replace the Rev and RRE of HIV-1, using a mutated HIV-1 DNA proviral clone as a new type of molecular trap. The factors binding to this element are under investigation. This result showed the presence of additional RNA elements in the genome that are able to direct nuclear export and may define a distinct pathway for nucleocytoplasmic transport. The role of Vpr on virus life cycle is complex and needs to be examined in more detail. Although several cellular proteins were shown to interact with Vpr, the mechanisms of its function are not fully elucidated. We have detected direct binding of Vpr to additional cellular proteins and have studied the effect of this binding on the cell cycle and on transactivator effects of Vpr. We have shown that Vpr interacts directly with the glucocorticoid and other nuclear receptors, and is found in complexes with these factors during active transcription. We also found that Vpr binds directly to p300, an important coactivator of many cellular promoters. This binding leads to increased activation of such promoters, and explains some of the transactivation effects of Vpr. The understanding of the regulatory mechanisms of HIV gene expression has been applied for the development of improved DNA vaccination approaches. We have developed a general method to increase the expression of unstable mRNAs by introducing multiple point mutations in their coding regions. This results in efficient transport, and increased stability and translation of several mRNAs. Based on our results with gag and env sequences of HIV, we developed efficient expression vectors for DNA-based immunization. We showed that better antigen expression results in increased immunogenicity. This led to DNA vaccines that are efficiently expressed and achieve the production of antibodies, CTL, and helper responses in mouse. In contrast, primates do not achieve an equally effective immune response, indicating differences among the species. We developed additional DNA vaccine vectors producing modified antigens in order to elicit more potent immune responses in primates. We tested several such vectors expressing either secreted or intracellularly degraded antigens and showed that some combinations appear to boost immunogenicity. Results in mice were used to design monkey vaccination experiments. We work on further optimization of the vaccine vectors to improve the methodology of DNA vaccination, aiming at efficient vaccination using combinations of vectors against multiple HIV antigens.

该项目旨在了解HIV调节和附属蛋白在病毒生命周期和疾病发育中的作用和机制。剖析大分子的核质流量的机制；并检查VPR功能作为转录激活剂和细胞周期调节剂的机制。 我们研究了几种HIV-1蛋白的功能，包括TAT，REV和VPR。我们表明，啮齿动物细胞中的Rev缺陷是由核中Rev进口的延迟引起的。负责这种效果的因素正在研究中，可能导致针对基本转速因素的新型干预措施。 已经确定并表征了CRM1定向核出口的新抑制剂。该抑制剂将对通过CRM1途径导出的REV和许多细胞蛋白的研究有用。 我们使用突变的HIV-1 DNA病毒前克隆作为一种新型的分子陷阱，从小鼠基因组中鉴定出一种新的RNA元素（称为RTE），该元件能够替代HIV-1的RER和RRE。与该元素结合的因素正在研究中。该结果表明，基因组中存在其他RNA元素，能够引导核输出，并可能定义了核总质转运的独特途径。 VPR在病毒生命周期中的作用很复杂，需要更详细地检查。尽管显示了几种细胞蛋白与VPR相互作用，但其功能的机制尚未完全阐明。我们已经检测到VPR与其他细胞蛋白的直接结合，并研究了这种结合对细胞周期和VPR的反式激活效应的影响。我们已经表明，VPR与糖皮质激素和其他核受体直接相互作用，并且在活性转录过程中与这些因素的复合物中发现。我们还发现，VPR直接与P300结合，P300是许多细胞启动子的重要共激活因子。这种结合导致这种启动子的激活增加，并解释了VPR的一些反式激活效应。 对HIV基因表达的调节机制的理解已应用于改进的DNA疫苗接种方法的发展。我们已经开发了一种通用方法，可以通过在其编码区域引入多点突变来增加不稳定的mRNA的表达。这会导致有效的运输，并增加了几个mRNA的稳定性和翻译。基于我们使用HIV的GAG和ENV序列的结果，我们开发了有效的基于DNA免疫的表达向量。我们表明，更好的抗原表达会导致免疫原性增加。这导致了有效表达的DNA疫苗，并实现了小鼠中抗体，CTL和辅助反应的产生。相反，灵长类动物没有达到同等有效的免疫反应，表明该物种之间的差异。我们开发了产生改良的抗原的其他DNA疫苗载体，以便在灵长类动物中引起更有效的免疫反应。我们测试了表达分泌或细胞内降解抗原的几个这样的载体，并表明某些组合似乎增强了免疫原性。小鼠的结果用于设计猴子疫苗接种实验。我们致力于进一步优化疫苗向量，以改善DNA疫苗接种的方法，旨在使用对多种HIV抗原的载体组合进行有效疫苗接种。