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.
