Research Project
3096905
This is the first competitive renewal application of thus multidisciplinary program project to define the molecular and cellular basis of immune recognition of human cytomegalovirus (HCMV), and to determine the role of specific immune responses in host defense during permissive and latent HCMV infections. The project includes an Administrative Core to provide overall organizational direction and a Scientific Core comprised of three subsections: (i) a Cell Culture/Immunology Core for virus and cell culture production and flow cytometry services; (ii) a Biochemistry Core for protein sequencing and peptide synthesis; and (iii) a Monoclonal Antibody Core for generation of anti-HCMV, HLA-DR/DQ, and TcR mcAbs. In addition, there are five interrelated scientific proposals. The first project will determine the extent of expression of two HCMV glyco-protein gene families, HXLF and HQLF; characterize the association among products of these genes to form disulfide-linked complexes; and analyze the enhancer regulatory region which potentially influences the temporal and directional transcription of these two sets of glycoprotein genes. A second project will isolate and characterize HCMV glycoproteins using biochemical and molecular methods; and identify their antigenic domains by reactivity of mcAbs with synthetic peptides and functional domains using in vitro binding and plaque reduction assays. The third project will define the major HCMV proteins recognized by helper T cells (Th) and cytotoxic T cells (Tc), identify the immunodominant T cell epitopes on these proteins and characterize the molecular mechanisms involved in Th-B cell and Th-Tc interactions. The fourth project will determine if the immune deficiency in congenital HCMV patients is due to lack of clonal selection of HCMV-Th or antigen-specific T suppressor cells (Ts), and will determine the possible role of defective cell-mediated immunity in persistence of replicating virus. The final project will characterize the molecular basis for MHC-restricted recognition of HCMV by Th using synthetic HCMV peptides and transfectants expressing HLA class II alpha beta genes with or without site-directed mutations; and analyze the HCMV-T cell receptor repertoire by studying Ti alpha beta gene rearrangements in HCMV-Th and Th precursor frequencies using anti-Ti mcAbs. We anticipate that further understanding of structure-function relationships involved in the biology of HCMV infection and protective immune responses to this virus will eventually contribute to the development of improved approaches to prevention and treatment of HCMV-related disease.
