This project will explore a novel vaccine strategy designed to enhance the induction of virus-specific CD8+ cytolytic T lymphocytes (CTL). The strategy involves the use of viral vectors or other delivery systems to introduce into host cells viral genes modified to contain sequences that will target the relevant viral protein for rapid degradation in the cytoplasm. Because cytoplasmic degradation of viral proteins provides the substrate for the class I antigen processing pathway, we hypothesize that these targeting strategies will facilitate the induction of CD8+ CTL responses specific for the relevant viral protein. We will directly test the hypotheses that such targeting strategies will result in increased loading of class I MHC molecules with viral peptides and enhanced induction of CTL responses. Several independent approaches will be taken to defining transferable sequence elements that will target viral proteins for rapid degradation in the cytoplasm of human cells. Modified viral proteins designed to undergo ubiquitin (Ub)-dependent degradation by the N-end rule pathway or by the related second codon rule pathway will be expressed in human cell lines. Kinetics of degradation will be measured biochemically, and the Ub dependence of degradation will be evaluated using cell lines with temperature sensitive defects in the E1 Ub activating enzyme. Constructs designed to undergo rapid cytoplasmic degradation by other two other mechanisms will also be analyzed. Based on preliminary studies of processing of viral envelope proteins for class I-restricted recognition by a TAP 1/2-dependent pathway, it is proposed that insertion that an """"""""out of context"""""""" hydrophobic domain in a cytoplasmic protein will trigger extremely rapid degradation, resulting in efficient generation of peptides that can be presented with class I molecules. in addition, the C-terminal degradation-targeting domain of the short lived RAG-2 protein will be used to force the rapid degradation of test viral proteins. For each degradation targeting strategy, quantitative analysis of naturally processed peptides will be carried out to determine whether targeting results in increased loading of class I MHC molecules with the relevant viral peptides. In addition and IL-2-independent clonal expansion of antigen-specific CTL will be compared. Five different approaches will be taken to measuring the immunogenicity of cells expressing degradation- targeted viral proteins. These include in vitro studies of antigen- specific proliferative and cytolytic responses of human T cell clones, and studies of secondary in vitro CTL responses using responding cells from infected human donors. In addition, in vivo studies in the mouse model will be used to evaluate the generation of CTL responses from naive T cell populations and to evaluate this vaccine strategy in a influenza virus immunization/challenge model. Together, these studies would provide a foundation for the development of optimized CTL-based vaccine strategies.
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