Screening and confirmatory testing of MHC class I alleles in pig-tailed macaques
Author: Caroline S. Fernandez, Jeanette C. Reece, Uus Saepuloh, Robert De Rose, Diah Iskandiati, David H. O’Connor, Roger W. Wiesman, Stephen J. Kent Abstract
Pig-tailed macaques (Macaca nemestrina) are a commonly studied primate model of human AIDS. The Mane-A1*084:01 MHC class I allele (previously named Mane-A1*10) is important for the control of SIV infection by CD8+ T cells in this model. Validated methods to detect this allele in large in this model. Validated methods to detect this allele in large numbers of macaques are lacking. We studied this MHC allele using sequence-specific PCRs in 217 pig-tailed macaques and identified 75 (35%) positive animals. We then performed massively parallel pyrosequencing with a universal 568-bp MHC class I cDNA-PCR amplicon for 50 of these 75 macaques. All 50 animals expressed Mane-A1 *084:01 or closely related variants of the Mane-A1 *084 lineage. Mane-A1 *084:01 transcripts accounted for an average of 20.9% of all class I sequences identified per animal. SIV infection of a subset of these macaques resulted in the introduction of SIV-specific CD8+ T cell responses detected by Man-A1*084:01 tetramers. An average of 19 distinct class I transcripts were identified per animal by pyrosequencing. This analysis revealed 89 new Mane class I sequences as well as 32 previously described sequences that were extended with the longer amplicons employed in the current study. In addition, multiple Mane class I haplotypes that had been inferred previously based on shared transcript profiles between unrelated animals were confirmed for a subset of animals where pedigree information was available. We conclude that sequence specific PCR is useful to screen pig-tailed macaques for Mane-A1*084:01, although pyrosequencing permits a much broader identification of the repertoire of MHC class I sequences and haplotypes expressed by individual animals.
