RAPID FLUOROMETRIC ASSAY OF HIV-INDUCED CELL FUSION

表达HIV-1包膜糖蛋白gp 120/gp 41的细胞可以融合 与表达HIV-1受体CD 4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一种 病毒传播的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 研究HIV-1病毒介导的膜融合， 病毒预测周期的初始阶段。 在阶段I期间 在此期间，研究人员成功地开发了一种HIV-1信封- 使用稳定表达来自人的gp 120/gp 41的细胞的介导的融合测定 实验室适应菌株和表达CD 4的人细胞。 然后他们 证明了这些细胞之间的融合可以使用一种新的 基于荧光的测定。他们的系统有几个主要的优势， 现有的化验。 它是无病毒的，可重复的，敏感的， 迅速。 它也适用于定量和动力学分析， 提供了膜融合的准确和客观的测量。期间 在第二阶段，他们将自动化和优化荧光- 基于融合分析，以快速分析多个样品。 在 此外，他们还将开发一种自动荧光融合检测方法， 一种稳定表达来自HIV-1原代分离株的gp 210/gp 41的细胞系。 这些试验将允许详细分析膜融合介导的 通过病毒的包膜糖蛋白， 人类细胞的趋向性然后他们将使用这些分析来识别 本发明涉及一种新型的功能相关的人单克隆抗体（huMAb）， 与真实的寡聚gp 120/gp 4l相互作用。 到目前为止，huMAb已经 通过它们结合肽或重组gp 120的能力来选择， ELISA形式严重限制了功能性 相关的huMAb. huMAb将从HIV-1感染的个体产生， 包括长期存活者，并作为单一药剂或 与基于CD 4的分子组合。 特别是，来自长- 长期幸存者，目前还没有，可能拥有不寻常的， 特性，并提供了一个独特的见解，保护性免疫 反应最后，他们将广泛表征结构和 huMAb的功能特性，以选择那些最合适的 作为第三阶段的免疫治疗药物。

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.