Quantitative as well as qualitative parameters of T cell stimulation in vivo are critical for vaccine efficacy. one of the most important factors in T cell activation is the antigen presenting cell. Different types of antigen presenting cells (APCs) vary widely in their ability to process and present antigens. In addition, APCs differ widely in their expression of costimulatory molecules of T cell activation. These costimulatory molecules are presented either by cell membrane proteins such as B7 or secreted cytokines such as interleukin 1. Strategies of vaccine design should therefore consider the targeting of antigens to different classes of APCs as well as potentially modifying the antigen-presenting cells to enhance their T cell stimulatory activity. The most potnt class of APC is the dendritic cell. These cells are distinct from macrophages and appear to possess much greater T cell stimulatory capacity in in vitro assays. Recently, GM-CSF has been identified as an extremely important cytokine in both maintaining dendritic cell function as well as inducing differentiation of dendritic cell precursors into the high potency APC state. Based on these concepts, this project seeks to analyze the efficacy of a recombinant chimeric vaccine aimed at simultaneously inducing dendritic cell differentiation and targeting antigen to the endosomal compartment of these same dendritic cells. The concept of the chimeric vaccine is to link GM-CSF to the antigen via a linker sequence that does not interfere with the structural integrity or receptor binding capacity of either component of the molecule. A recombinant molecule linking GM-CSF to the HPV oncogene product E7 has been synthesized and demonstrated to preserve GM-CSF functional activity as well as maintain multiple E7 antibody epitopes. in principle, such a chimeric molecule could stimulate the differentiation of dendritic cells via the GM-CSF component while concentrating the antigen into those same dendritic cells as a consequence of receptor mediated endocytosis subsequent to binding to the GM-CSF receptor. We plan to test the ability of this recombinant chimeric vaccine to induce both helper and cytolytic T cell responses as well as antibody responses to E7. If evidence of enhanced responses is gained, this approach will be extended to a second antigen system, influenza hemagglutinin. Ultimately, if this recombinant chimeric vaccine is shown to enhance T cell and/or antibody responses, this strategy will be tested for its ability to protect against lethal infection with influenza as well as immunize against challenge with tumors expressing HPV E7.
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