RAPID FLUOROMETRIC ASSAY OF HIV-INDUCED CELL FUSION

HIV 诱导的细胞融合的快速荧光测定

• 批准号: 2067703
• 负责人: PAUL J MADDON
• 金额: $ 19.64万
• 依托单位: PROGENICS PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2067703
• 关键词: CD4 molecule; HIV envelope protein gp120; HIV envelope protein gp41; HIV infections; antiviral antibody; biomedical automation; cell fusion; human immunodeficiency virus 1; human tissue; immunofluorescence technique; membrane fusion; method development; microorganism immunology; monoclonal antibody; neutralizing antibody; virus infection mechanism; virus receptors

Cells that express gpl20/gp4l, the HIV-1 envelope glycoprotein, can fuse with cells expressing CD4, the HIV-1 receptor, to form multinucleated giant cells or syncytia. Such cell-to-cell fusion appears to be a mechanism for virus transmission as well as a primary cause of cell death in HIV-1 infection. In addition, cell fusion can serve as a model system of viral entry to study HIV-1 envelope-mediated membrane fusion, the initial phase in the predicative cycle of the virus. During Phase I period, the investigators successfully developed an HIV-1 envelope- mediated fusion assay using cells that stably express gp120/gp4l from a laboratory-adapted strain and human cells expressing CD4. They then demonstrated that fusion between these cells can be measured using a novel fluorescence-based assay. Their system has several major advantages over already existing assays. It is virus-free, reproducible, sensitive, and rapid. It is also suitable for quantitative and kinetic analyses, providing an accurate and objective measurement of membrane fusion. During the Phase II period, they will automate and optimize the fluorescence- based fusion assay in order to rapidly analyze multiple samples. In addition, they will develop an automated fluorescence fusion assay using a cell line stably expressing gp2l0/gp4l from a primary isolate of HIV-1. These assays will permit a detailed analysis of membrane fusion mediated by envelope glycoproteins from viruses that exhibit markedly different tropisms for human cells. They will then use these assays to identify novel, functionally relevant human monoclonal antibodies (huMAbs) that interact with authentic, oligomeric gpl20/gp4l. To date, huMAbs have been selected by their ability to bind peptides or recombinant gp120 in an ELISA format that severely restricts the identification of functionally relevant huMAbs.huMAbs will be generated from HIV-l infected individuals, including long-term survivors, and tested as single agents or in combination with CD4-based molecules. In particular, huMAbs from long- term survivors, which are not currently available, may possess unusual characteristics and provide a unique insight into a protective immune response. Finally, they will extensively characterize the structural and functional properties of the huMAbs in order to select those most suitable for development as immunotherapeutics in Phase III.

表达 gpl20/gp4l（HIV-1 包膜糖蛋白）的细胞可以融合 与表达 CD4（HIV-1 受体）的细胞形成多核 巨细胞或合胞体。 这种细胞与细胞的融合似乎是 病毒传播机制以及细胞死亡的主要原因 HIV-1 感染。 此外，细胞融合可以作为模型系统 病毒进入研究 HIV-1 包膜介导的膜融合， 病毒预测周期的初始阶段。 第一阶段期间 期间，研究人员成功研制出一种HIV-1包膜—— 使用稳定表达 gp120/gp4l 的细胞进行介导的融合测定 实验室适应的菌株和表达 CD4 的人类细胞。 他们然后 证明可以使用一种新的方法来测量这些细胞之间的融合 基于荧光的测定。他们的系统相比之下有几个主要优点 已经存在的测定。 它无病毒、可重复、灵敏且 迅速的。 它还适用于定量和动力学分析， 提供准确、客观的膜融合测量。期间 在第二阶段期间，他们将自动化并优化荧光- 基于融合测定，以便快速分析多个样品。 在 此外，他们将开发一种自动荧光融合测定法，使用 来自HIV-1初级分离株的稳定表达gp210/gp4l的细胞系。 这些测定将允许对膜融合介导的详细分析 由表现出明显不同的病毒的包膜糖蛋白 人类细胞的趋向性。然后他们将使用这些测定来识别 新型、功能相关的人单克隆抗体 (huMAb) 与真实的寡聚 gpl20/gp4l 相互作用。 迄今为止，huMAb 已 根据其结合肽或重组 gp120 的能力进行选择 ELISA 格式严重限制了功能鉴定 相关的 hMAbs.huMAbs 将从 HIV-1 感染个体中产生， 包括长期幸存者，并作为单一药物或在 与基于 CD4 的分子结合。 特别是，来自长期 目前无法获得的足月幸存者可能拥有不寻常的能力 特征并提供对保护性免疫的独特见解 回复。最后，他们将广泛描述结构和 HuMAb 的功能特性，以便选择最合适的 开发为 III 期免疫治疗药物。