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: 2568927
• 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/2568927
• 关键词: AIDS vaccines; attenuated microorganism; early diagnosis; gene mutation; helper T lymphocyte; host organism interaction; human immunodeficiency virus 1; human immunodeficiency virus 2; live vaccine; macrophage; monocyte; 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. 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?

该项目的长期目标是：1）评估可行性 生产 HIV-1 和 HIV-2 减毒活病毒疫苗 通过辅助基因突变而变得非致病性，或者 单独或组合； 2）探索可能性 辅助基因蛋白可以作为抗病毒治疗的靶点。 作为 开发候选减毒活病毒的先决条件 疫苗和针对艾滋病毒的药物的开发 辅助基因产品，我们一直致力于研究以确定 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于实现这两个目标都至关重要。 我们的 早期的工作已经证明了 HIV-1 Vif 对病毒的关键作用 原代 T 细胞（外周血单核细胞、 PBMC），我们继续证明 Vif 也发挥着关键作用 用于 HIV-1 在原代单核细胞衍生巨噬细胞 (MDM) 中的复制。 在 以 Nef 为例，我们已经证明 Nef 是否具有可测量的 对病毒复制的影响取决于特定的病毒宿主系统 用过的。 虽然几种 HIV-1 毒株的 Nef 突变体都略有复制 在 PBMC 和 MDM 中不如野生型，可能没有 当 Nef 突变体时，病毒复制的效果或显着减少 在 CD4 阳性细胞系中检测 HIV-1 和 HIV-2。 作为我们的一部分 为了开发减毒的艾滋病毒候选疫苗，我们修改了 HIV-1基因组允许将不同基因插入nef开放 阅读框架。 使用这种病毒，我们计划调查其后果 vpr 和 vif 基因异位表达对病毒复制的影响 这些基因和 nef 的不同 HIV-1 和 HIV-2 等位基因是否可以 补充适当的 HIV-1 Vif、Vpr 或 Nef 突变体。 作为一部分 我们确保疫苗和其他生物制品安全的计划 产品（参见项目 BK 03017），我们一直在描述敏感的 基于 PCR 的 RT (PBRT) 检测可检测是否存在低水平的 逆转录病毒污染。 该 PBRT 检测能够检测 1 到 10 个病毒体。 我们修改了测定方法，以便能够 去除背景信号并将其调整为与三个一起使用 不同的RNA模板。 因为 PBRT 检测可以检测到少量 逆转录病毒，我们正在应用它来解决艾滋病毒的某些问题 生物学。 例如，病毒可以从未刺激的PBMC中产生吗？ 来自未刺激的纯化 CD4 阳性 T 细胞，并来自淘析 单核细胞分化为巨噬细胞之前？ 能否检测到病毒 感染后早期？