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

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 由于辅助基因突变而变得非致病的 单独或结合在一起；以及2)探索 辅助基因蛋白可以作为抗病毒治疗的靶点。AS 两种候选减毒活病毒研制的先决条件 针对艾滋病的疫苗和抗HIV药物的开发 辅助基因产物，我们一直在进行研究，以确定 这些蛋白在HIV-1和HIV-2体外生命周期中的作用 因为对它们如何运作的了解对这两个目标都至关重要。我们的 早期的研究已经证明了HIV-1 Vif对病毒的关键作用 在初级T细胞(外周血单核细胞， PBMC)，我们进一步表明，VIF也发挥着关键作用 用于在原代单核细胞来源的巨噬细胞(MDM)中复制HIV-1。在……里面 在Nef的案例中，我们已经证明了Nef是否有一个可测量的 对病毒复制的影响取决于特定的病毒宿主系统 使用。而几个HIV-1毒株的Nef突变体都有轻微的复制 在PBMC和MDM中不如野生型好，可以没有 Nef突变体对病毒复制的影响或显著减少 HIV-1和HIV-2在CD4阳性细胞系中进行检测。作为我们的一部分 为了开发减毒HIV候选疫苗，我们已经修改了一种 HIV-1基因组允许将不同的基因插入nef开放 阅读框。利用这种病毒，我们计划调查其后果 Vpr和vif基因的异位表达对病毒复制和 这些基因和nef的不同HIV-1和HIV-2等位基因是否可以 补充HIV-1的适当Vif、Vpr或Nef突变体。作为以下内容的一部分 我们的计划是确保疫苗和其他生物疫苗的安全 产品(见BK 03017项目)，我们一直在表征一种敏感的 以聚合酶链式反应为基础的RT(PBRT)检测低水平的 逆转录病毒污染。这种PBRT检测方法能够检测到 在1到10个病毒粒子之间。我们已经修改了化验方法，以便能够 删除背景信号，并已将其调整为与三个 不同的RNA模板。因为PBRT检测可以检测到少量的 对于逆转录病毒，我们正在应用它来解决艾滋病毒中的某些问题 生物学。例如，病毒是否可以从非刺激的PBMC中产生， 从未经刺激的提纯的CD4阳性T细胞中提取，并从淋洗液中提取 单核细胞分化为巨噬细胞之前？能检测到病毒吗？ 感染后早期？

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. 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), and 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, 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 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. 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. As part of our program to ensure the safety of vaccines and other biological products (see project BK 03017), we have been characterizing a sensitive PCR-based RT (PBRT) assay to detect the presence of low levels of retrovirus contamination. This PBRT assay has the capacity to detect between 1 and 10 virions. We have modified the assay to be able to remove background signals and have adapted it for use with three different RNA templates. Because the PBRT assay can detect low amounts of retroviruses, we are applying it to address certain questions in HIV biology. For example, can virus be produced from non-stimulated PBMC, from non-stimulated purifed CD4-positive T cells, and fom elutriated monocytes before differentiation to macrophages? Can virus be detected early after infection?