This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Background & Aims: Identify and characterize the complete Identification and characterization of the CD4 epitopes are important not only for the development of effective vaccine for AIDS virus infection but also help understand other diseases pathogenesis. Despite the enormous progress on basic immunology and epitope mapping strategies, the general property of a functional T cell epitope is not yet fully understood. Consequently, accurate screening strategy to identify the complete epitope repertoire is not available either. Moreover, understanding the property of functional T cell epitopes is crucial to understand cellular immunity, mechanism of viral evasion and will provide a rationale approach for improving AIDS vaccine design. In the studies described in this application, we propose to elucidate and identify the properties and characteristics of the functional CD4 T cell epitopes. More importantly, to develop a simple and accurate system to screen the complete antigen-specific CD4 epitope repertoire restricted to a specific MHC class II molecules in the rhesus monkey model. If the CD4 epitope-screening method is successfully developed, not only the scientific community that use the monkey model will be beneficiated but also could be directly apply in the human system. Our hypothesis is that the major characteristic of a functional T cell epitope is to have high binding affinity to the MHC class II proteins enough to bind as a long peptide fragment during the antigen processing steps. The hypothesis is based on data generated using our novel peptide binding affinity assay using the TCR tetramer for the CTL epitopes. The analysis of five well characterized SIV specific CTL epitopes restricted to the Mamu-A*01 molecule with consistent characteristics and common properties indicated that the TCR tetramer technology could be easily apply for the identification of the CD4 T cell epitopes. Finally, since the MHC class II peptide holding groove is wider compared to the MHC class I molecule, we also hypothesize that the number of available CD4 epitopes should be higher than the CTL epitopes.
Specific Aims :1. Construct a TCR tetramer specific for the p46/Mamu-DR*W201 complex. 2. CD4 epitope repertoire for the whole SIV protein restricted to the rhesus monkey MHC class II molecule Mamu-DR*W201 using overlapped pool peptides and the newly constructed TCR tetramer.3. Identify the complete repertoire of functional CD4+ T cell population specific for the whole SIV virus from vaccinated monkeys. Thus, conforming the utility of the TCR tetramer for the screening of functional CD4 eptopes. Results: Unfortunately, due to the delay in recruiting personnel with the necessary skills to perform the proposed study, the entire project was considerably delayed. Eileen Deharo, a Medical Research Specialist was originally assigned to this project, however, she moved back to the Division of Microbiology soon after the grant was awarded. A new postdoctoral fellow with the necessary background in molecular biology will join the project from April of 2008.
Aim 1. This study is currently in progress. In order to select the appropriate TCR ab sequences for the construction of the TCR tetramer, reassembling of a and b chains from an oligoclonal antigen-specific T cell population into a functional antigen-specific TCR molecule will be necessary. p46/Mamu-DR*W201 tetramer-binding T cells from two SHIV infected rhesus macaque monkey were isolated by a FACSAria cell sorter (Becton Dickinson, San Jose, CA). RNA was isolated from the sorted cells and cDNA was made for the amplification of the whole repertoire of the TCR a and b chains from these cells. All possible TCR ab combinations will be cotransfected into Schneider (S2) cells by calcium phosphate precipitation. Cell populations with positive staining will be considered to have a functional TCR alpha/beta pairing. The positive pair of alpha and beta chains will be selected to generate the soluble p46 TCR tetramer.
Aims 2 and 3. These studies will start upon completion of the SA1. A new reagent constructed from the SA1 is a crucial component for the completion of these studies.
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