USE OF ACCESSORY GENE MUTANTS FOR THE DEVELOPMENT OF ATTENUATED HIV VACCINES

使用辅助基因突变体开发减毒 HIV 疫苗

• 批准号: 5200718
• 负责人: K PEDEN
• 金额: --
• 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/5200718
• 关键词: AIDS vaccines; attenuated microorganism; cell growth regulation; gene mutation; host organism interaction; human immunodeficiency virus 1; human immunodeficiency virus 2; macrophage; mutant; protein structure function; tissue /cell culture; transposon /insertion element; virus protein; virus replication

The long-term goals of this project are: 1) to evaluate the feasibility of generating live attenuated virus vaccines of HIV-1 and HIV-2 that are rendered non-pathogenic by mutation of accessory genes, either individually or in combination; and 2) to explore the possibility that the accessory gene proteins can be targets for anti-viral therapy. Recent studies using the SIV system have shown that viruses defective in the production of Nef are non-pathogenic in monkeys and confer protection to those monkeys from subsequent challenge with wild-type SIV. Mutations in the other accessory genes, vif, vpr and vpu of HIV-1 and vif, vpr and vpx of HIV-2, have yet to be explored in animal model systems. As prerequisites to both the development of candidate live attenuated virus vaccines and the development of anti-HIV drugs directed against the accessory gene products, we have been engaged on studies to determine the role of these proteins in the life cycle of HIV-1 and HIV-2 in vitro, since a knowledge of how they function is critical to both goals. Our earlier work had demonstrated the critical role of HIV-1 Vif to virus replication in primary T cells (peripheral blood mononuclear cells, PBMC), although this protein is not obligatory for HIV-1 replication in most CD4-positive cell lines. We have gone on to show that Vif also plays a critical role for HIV-1 replication in primary monocyte-derived macrophages (MDM). In the case of Nef, there has been a controversy as to whether this protein positively or negatively influences virus growth. We have shown that whether or not Nef has a measurable effect on virus replication depends on the particular virus-host system used. While Nef mutants of several HIV-1 strains all replicate slightly less well than wild type in PBMC and, in the case of a macrophage-tropic HIV-1, in MDM, there can be either no effect or dramatic reductions in virus replication when Nef mutants of HIV-1 and HIV-2 are assayed in CD4-positive cell lines. For Vpr, we have shown that there is little consequence of mutating this gene in HIV-1 and HIV-2 for the replication in CD4-positive cell lines or PBMC, although this protein appears to be a positive factor for growth in MDM. We have commenced studies to investigate the molecular mechanism of Nef, Vif and Vpr. Results so far have indicated that expression of these proteins in several mammalian cells results in the cessation of cell growth (cytostasis), and we are currently investigating the cellular pathways that bring this about. As part of our goal to develop attenuated HIV vaccine candidates, we have modified an HIV-1 genome to allow the insertion of different genes into the nef open reading frame. Using this virus, we plan to investigate the consequences of ectopic expression of the vpr and vif genes on virus replication and whether different HIV-1 and HIV-2 alleles of these genes and nef can complement the appropriate Vif, Vpr or Nef mutants of HIV-1.

本项目的长期目标是：1）评估可行性 生产HIV-1和HIV-2的减毒活病毒疫苗， 通过辅助基因突变而使其非致病性， 单独或组合;以及2）探讨 辅助基因蛋白可以作为抗病毒治疗的靶点。 最近使用SIV系统的研究表明， Nef产生在猴子中是非致病性的，并提供保护 对那些猴子进行后续的野生型SIV攻击。 突变 HIV-1的vif、vpr和vpu以及vif、vpr和vpu的其它辅助基因中， vpx的HIV-2，尚未在动物模型系统中探索。 作为候选减毒活疫苗开发的先决条件， 病毒疫苗和针对艾滋病毒的抗艾滋病毒药物的开发 辅助基因产物，我们一直从事研究，以确定 这些蛋白质在体外HIV-1和HIV-2生命周期中的作用， 因为了解它们如何运作对这两个目标都至关重要。 我们 早期的工作已经证明了HIV-1 Vif对病毒的关键作用， 在原代T细胞（外周血单核细胞， PBMC），尽管这种蛋白质对于HIV-1在PBMC中的复制不是强制性的。 大多数CD 4阳性细胞系。 我们已经继续表明， 在原发性单核细胞来源的HIV-1复制中起关键作用。 巨噬细胞（MDM）。 在Nef的情况下，有一个争议， 这种蛋白质对病毒生长的影响是积极的还是消极的。 我们已经证明Nef是否对病毒有可测量的影响， 复制取决于所使用的特定病毒宿主系统。 当Nef 几种HIV-1病毒株的突变体的复制能力都略低于 PBMC中的野生型，以及在嗜巨噬细胞的HIV-1的情况下，在MDM中， 病毒复制可能没有效果或急剧减少， 当在CD 4阳性细胞中测定HIV-1和HIV-2的Nef突变体时， 线 对于Vpr，我们已经证明， 使HIV-1和HIV-2中的该基因突变，以便在CD 4阳性细胞中复制， 细胞系或PBMC，尽管这种蛋白质似乎是一种阳性因子， 在MDM中成长。 我们已经开始研究Nef的分子机制， Vif和Vpr。 迄今为止的结果表明，这些表达 一些哺乳动物细胞中的蛋白质会导致细胞 生长（细胞停滞），我们目前正在研究细胞 这些途径带来了这一切。 作为我们开发减毒HIV候选疫苗目标的一部分，我们 修改了HIV-1基因组，允许将不同的基因插入 NEF开放式阅读框架。 利用这种病毒，我们计划调查 vpr和vif基因异位表达对病毒的影响 复制以及这些基因的不同HIV-1和HIV-2等位基因 nef可以补充HIV-1的适当Vif、Vpr或Nef突变体。