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-A*10) is important for the control of SIV infection by CD8+ T cells 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 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 induction of SIV-specific CD8+ T cell responses detected by Mane-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.
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Acknowledgements
This study was supported by the Australian NHMRC award 510488 and US NIH awards HHSN266200400088C/N01A140088, P51RR000167 and P40RR019995. We thank Sheilajen Alcantara, Thakshila Amarasena, Leanne Smith, Julie Karl and the Non-Human Primate Nomenclature Committee for expert assistance. The authors declare they have no conflict of interest.
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Supplemental Fig. 1
Summary of MHC class I genotypes for 50 Mane-A1*084-positive pig-tailed macaques determined by pyrosequencing. This table gives the number of sequence reads detected for each class I transcript in each animal examined by pyrosequencing with the 568-bp cDNA-PCR amplicon. Where multiple transcripts are noted, the class I genotype is ambiguous due to sequence identity within the region sequenced. Sequence reads that are ambiguous for more than four transcript variants are given as the allele lineage followed by “g” to indicate group. Limited pedigree information was available for a subset of these animals including the four half sibs illustrated in Fig. 1 and three pairs of full sibs as indicated at the top of this table. The various colours in this table highlight combinations of transcripts that are inferred to represent haplotypes based on these known pedigree relationships as well as more distant ancestral relationships. It should be noted that MID6-tagged 568-bp amplicons for four independent RNA samples (highlighted in yellow) yielded an average of nearly tenfold fewer sequence reads for evaluation compared to the other 13 MIDs used in this study. Since the performance of the MID6-tagged primers during the primary cDNA-PCR reactions was indistinguishable from the other primers, this appears to reflect undesirable secondary interactions that arise during the emulsion PCR and/or sequencing-by-synthesis phases of pyrosequencing. (PDF 512 kb)
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Fernandez, C.S., Reece, J.C., Saepuloh, U. et al. Screening and confirmatory testing of MHC class I alleles in pig-tailed macaques. Immunogenetics 63, 511–521 (2011). https://doi.org/10.1007/s00251-011-0529-5
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DOI: https://doi.org/10.1007/s00251-011-0529-5