The increased utility of the rhesus macaque animal model in both HIV vaccine development and pathogenesis studies necessitates the development of reference MHC typing laboratories for this species. In this proposal the applicants plan to develop two reference laboratories for MHC typing of the rhesus macaque. Both of these laboratories will be directed by individuals who have had more than ten years of experience in analyzing the MHC of the rhesus macaque. One of these laboratories will be at the WRPRC under the direction of Dr. David Watkins and the other will be located at the Biomedical Primate Research Center (the Netherlands) under the direction of Dr. Ronald Bontrop. The applicants plan to offer services to both the North American and European scientific community for MHC typing of rhesus macaques. Initially, this will include PCR-SSP tests for alleles encoding MHC class I molecules that bind peptides derived from SIV and SHIV. The applicants will also offer PCR-SSP typing for MHC class II alleles that similarly encode MHC class II molecules that bind peptides from SIV or SHIV. The investigators propose to develop additional molecular techniques for analysis of the macaque MHC class I and class II alleles. These will include reference strand conformational analysis (RSCA) and direct sequencing of MHC class II alleles in the rhesus macaque. The investigators plan to utilize the resources of a company (PFI) that develops and markets these technologies for the analysis of the human MHC alleles. Thus, the applicants have proposed a subcontract to PFI to provide PCR-SSP plates and to develop RSCA for the rhesus macaque. Additionally, the applicants will offer training for individuals that wish to set up MHC typing in their own laboratories. Finally, the applicants will have a panel of well- characterized, MHC-defined cell lines that will be invaluable for the analysis of the MHC in the rhesus macaque. These will include a panel of rhesus macaque B-lymphoblastoid cell lines (BLCL) for which the investigators know all of the expressed MHC class I and class II molecules. These will be useful in mapping both cytotoxic T cell (CTL) and helper T cell (HTL) responses and epitopes in the macaque. The applicants will also have a panel of transfectants expressing rhesus macaque MHC class I and II alleles as well as a panel of extracted DNA samples from animals of known MHC type. These will be useful in standardizing MHC typing in several different laboratories. The principals in this proposal have extensive experience in the analysis of both rhesus and human MHC. Dr. Watkins has been studying nonhuman primate (NHP) MHC class I and II alleles for the last 15 years, and is also an ASHI- certified clinical laboratory Director at the UW Hospital and Clinics HLA Typing Laboratory. Dr. Bontrop has been involved in MHC typing in both humans and rhesus macaques for the last 15 years, and is Director of the Dutch Primate Research Center. PFI has been supplying kits and reagents for the analysis of MHC molecules in humans for the last 15 years.
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