Multiplex MHC Typing of Rhesus Macaques

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DESCRIPTION (provided by applicant): The main goal of this small research Grant (RO3) application is to develop a new research technology that will allow the rapid identification of Major Histocompatibility Complex class I (MHC-I) alleles present in the genome of rhesus macaques (Macacca mulatta). MHC-I genes encode proteins central to the adaptive immune response, signaling infection by binding pathogenic peptides and presenting them on the cell surface to cytotoxic T cells. In addition, class I molecules are critical to the innate immune response, providing both inhibitory and stimulating signals to NK cells. Rhesus macaques are relevant preclinical models for human diseases and transplantation, and experimentally infected rhesus monkeys serve as an indispensable animal model to assess the pathogenesis, to validate therapy approaches and to develop vaccination strategies against AIDS. As in humans, the disease course in macaques is variable, and certain MHC-I molecules appear to be associated with better control SIV replication. Current molecular methods for genotyping rhesus monkeys use PCR techniques with individual sets of sequence-specific primers for a handful of Mamu-A and -B alleles. In order to develop a DNA assay that can simultaneously detect a large number of Mamu-A and -B alleles, we will combine the multiplexing capacity of the Luminex 100 platform with the high specificity of sequence-specific DNA probes (SSP) that contain minor groove binders (MGB). MGB allow the design of short probes with increase sensitivity to differentiate single nucleotide polymorphism (SNP), whereas the Luminex microspheres permit the simultaneous use of up to 100 different probes. We have designed primers that amplify the polymorphic exons 2 and 3 of all the known Mamu-A and -B alleles. We will probe these DNA fragments with Luminex microspheres coated with allele-specific probes. The rapid identification of these Mamu-A and -B alleles in Rhesus macaques will have significant consequences in understanding the role of MHC-I genes in transplantation, vaccine development, and infectious diseases studies that use this important animal model.