JC virus (JCV) is a human polyoma virus and is the etiologic agent of progressive multifocal leukoencephalopathy (PML), a central demyelinating disease. PML is seen in immunocompromised individuals, suggesting a role for immune mechanisms in controlling disease progression. Approximately 70% of the population exhibit serum antibodies to JCV proteins. Most of the studies to date have foused on determination of humoral immune responses to JCV. The goal of this research project is to investigate the cell-mediated immune response to JCV in normal healthy individuals as a prelude to understanding its failure in PML. The CD4+ and CD8+ T cell response to the JCV large tumor (T) antigen (Ag) will be examined. T cell cultures will be initiated in vitro with autologous antigen-presenting cells (APCs) that have been """"""""fed"""""""" purified T Ag. Virus-infected cells will be used for subsequent T cell stimulations. Two viral systems will be utilized: a recombinant vaccinia virus system and an EBV-expression vector system. T cell function will be analyzed by measuring cell proliferation and/or cytokine release after stimulation with JCV T Ag-expressing cell lines. In addition, CD8+ T cell lytic activity will be determined by cytotoxicity assays. Progress has been made in purifying JCV T antigen, developing the EBV-expression system, and generating vaccinia virus recombinants. The coding region for JCV T ag has been ligated into the pTrcHis vector. This vector will encode for a fusion protein made of the protein of interest containing 6 histidine residues at the amino-terminus. Presence of the 6 histidines allows for protein purification over a nickel-coated column. JCV T ag has been successfully produced in bacterial cells. The conditions for optimal purification are being determined. The EBV-expression vector has been genetically engineered to express JCV T Ag. In addition. EBV-transformed cell lines have been generated and will be used to express the JCV T Ag from the EBV-expression vector. A recombinant vaccinia virus containing JCV T ag sequences has been generated and plaque purified. The expression of JCV T ag in recombinant vaccinia-JCV T ag virus infected cells is being determined. Once these systems are fully defined, CD4+ and CD8+ T lymphocyte cultures will be generated and examined for responsiveness to JCV T Ag.
