RAPID FLUOROMETRIC ASSAY OF HIV-INDUCED CELL FUSION

表达HIV-1包膜糖蛋白gpl20/gp4l的细胞可以融合 与表达HIV-1受体CD4的细胞形成多核 巨细胞或合胞体。这种细胞间的融合似乎是一种 病毒传播的机制以及细胞死亡的主要原因 在HIV-1感染中。此外，细胞融合可以作为一个模型系统。 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。在第一阶段期间 在此期间，调查人员成功地开发出一种HIV-1信封- 利用稳定表达gp120/gp4l的细胞进行介导性融合试验 实验室适应的菌株和表达CD4的人类细胞。然后他们 证明了这些细胞之间的融合可以用一种新的 基于荧光的分析。他们的系统有几个主要的优点 已有的化验结果。它是无病毒的，可复制，敏感， 速度很快。它还适用于定量和动力学分析， 为膜融合提供准确、客观的测量。在.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……里面 此外，他们将开发一种自动荧光融合分析方法，使用 稳定表达HIV-1原代分离株gp210/gp41的细胞系。 这些检测将允许对膜融合介导的详细分析。 通过来自病毒的包膜糖蛋白表现出明显不同的 人类细胞的向向性。然后，他们将使用这些化验来确定 新型的、功能相关的人类单抗(HuMAbs) 与真实的寡聚体gp20/gp4l相互作用。到目前为止，HuMAb已经 根据它们结合多肽或重组gp120的能力而选择 严重限制功能鉴定的EL ISA格式 将从L感染者身上产生相关的huMAbs.huMcAb， 包括长期幸存者，并作为单一药物或在 与以CD4为基础的分子结合。特别是，来自Long-Rong的HuMAb 目前无法获得的定期幸存者可能拥有不同寻常的 特征，并提供了对保护性免疫的独特见解 回应。最后，他们将广泛地描述结构和 为了选择那些最合适的HuMAb的功能性质 作为第三阶段免疫疗法的发展。

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.