Using the model system of the murine immune response to the vesicular stomatitis virus (VSV) G protein, studies on the regulation and specificity of the immune response to viruses will be performed. These studies will provide critical insights into cellular immunology. Fine structural analysis of the thymus derived (T cell ) lymphocyte clones and hybridomas elicited in immunized mice and the epitopes they recognize will be undertaken. Both CD4+ and CD8+ G-reactive T cells will be studied; the MHC restriction of the epitopes recognition will be defined. The sequences recognized by the T cells will be characterized by a variety of methods including: infections vs. killed virus wild type vs. defective interfering virus, purified G protein micelles or isolated trimers of truncated soluble Gs, serotype (NJ vs Indiana) restriction, field isolates, vaccinia-G vectors, transfected antigen presenting cells (APCs), proteolytic peptides of G, lacZ fusion proteins, synthetic oligopeptides. Cytokines secreted upon stimulation will be characterized by bioassay, ELISA, or in situ hybridization. The clones/hybridomas will be characterized for utilization of T cell receptor (TCR) variable region genes, and anti-idiotypic (anti-ID) reagents will be generated and used in studies of immunization and perturbation of the hybridomas will be studied both in vitro and in vivo. The influence of the hosts genotype on the epitopes recognized and the IDs utilized will be examined. Whether T cell or B cell immunity, or a combination of both is critical for immune protection will be studied. The influence of loss of principal restricting elements on the hosts' response will be investigated. The techniques used in the pursuit range from basic cellular immunologic culture and assays of T cells, to molecular cloning and transfections, in situ hybridization, biochemical isolation of fragments and fusion proteins, cell biology viral infection of living hosts, and viral propagation in vitro.
