, Volume 59, Issue 9, pp 693–703

Molecular typing of major histocompatibility complex class I alleles in the Indian rhesus macaque which restrict SIV CD8+ T cell epitopes

  • Masahiko Kaizu
  • Gretta J. Borchardt
  • Chrystal E. Glidden
  • Debra L. Fisk
  • John T. Loffredo
  • David I. Watkins
  • William M. Rehrauer
Original Paper


The utility of the rhesus macaque as an animal model in both HIV vaccine development and pathogenesis studies necessitates the development of accurate and efficient major histocompatibility complex (MHC) genotyping technologies. In this paper, we describe the development and application of allele-specific polymerase chain reaction (PCR) amplification for the simultaneous detection of eight MHC class I alleles from the rhesus macaque (Macaca mulatta) of Indian descent. These alleles were selected, as they have been implicated in the restriction of CD8+ T cell epitopes of simian immunodeficiency virus (SIV). Molecular typing of Mamu-A*01, Mamu-A*02, Mamu-A*08, Mamu-A*11, Mamu-B*01, Mamu-B*03, Mamu-B*04, and Mamu-B*17 was conducted in a high throughput fashion using genomic DNA. Our amplification strategy included a conserved internal control target to minimize false negative results and can be completed in less than 5 h. We have genotyped over 4,000 animals to establish allele frequencies from colonies all over the western hemisphere. The ability to identify MHC-defined rhesus macaques will greatly enhance investigation of the immune responses, which are responsible for the control of viral replication. Furthermore, application of this technically simple and accurate typing method should facilitate selection, utilization, and breeding of rhesus macaques for AIDS virus pathogenesis and vaccine studies.


Rhesus macaque SIV MHC Genotyping 

Supplementary material


  1. Allen TM, Sidney J, del Guercio MF, Glickman RL, Lensmeyer GL, Wiebe DA, DeMars R, Pauza CD, Johnson RP, Sette A, Watkins DI (1998) Characterization of the peptide binding motif of a rhesus MHC class I molecule (Mamu-A*01) that binds an immunodominant CTL epitope from simian immunodeficiency virus. J Immunol 160:6062–6071PubMedGoogle Scholar
  2. Allen TM, O’Connor DH, Jing P, Dzuris JL, Mothe BR, Vogel TU, Dunphy E, Liebl ME, Emerson C, Wilson N, Kunstman KJ, Wang X, Allison DB, Hughes AL, Desrosiers RC, Altman JD, Wolinsky SM, Sette A, Watkins DI (2000) Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia. Nature 407:386–390PubMedCrossRefGoogle Scholar
  3. Allen TM, Mothe BR, Sidney J, Jing P, Dzuris JL, Liebl ME, Vogel TU, O’Connor DH, Wang X, Wussow MC, Thomson JA, Altman JD, Watkins DI, Sette A (2001) CD8(+) lymphocytes from simian immunodeficiency virus-infected rhesus macaques recognize 14 different epitopes bound by the major histocompatibility complex class I molecule mamu-A*01: implications for vaccine design and testing. J Virol 75:738–749PubMedCrossRefGoogle Scholar
  4. Bontrop RE (2006) Comparative genetics of MHC polymorphisms in different primate species: duplications and deletions. Hum Immunol 67:388–397PubMedCrossRefGoogle Scholar
  5. Bontrop RE, Watkins DI (2005) MHC polymorphism: AIDS susceptibility in non-human primates. Trends Immunol 26:227–233PubMedCrossRefGoogle Scholar
  6. Boyson JE, Shufflebotham C, Cadavid LF, Urvater JA, Knapp LA, Hughes AL, Watkins DI (1996) The MHC class I genes of the rhesus monkey. Different evolutionary histories of MHC class I and II genes in primates. J Immunol 156:4656–4665PubMedGoogle Scholar
  7. Brody JR, Kern SE (2004) Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. Biotechniques 36:214–216PubMedGoogle Scholar
  8. Burton DR, Desrosiers RC, Doms RW, Koff WC, Kwong PD, Moore JP, Nabel GJ, Sodroski J, Wilson IA, Wyatt RT (2004) HIV vaccine design and the neutralizing antibody problem. Nat Immunol 5:233–236PubMedCrossRefGoogle Scholar
  9. Carrington M, Nelson GW, Martin MP, Kissner T, Vlahov D, Goedert JJ, Kaslow R, Buchbinder S, Hoots K, O’Brien SJ (1999) HLA and HIV-1: heterozygote advantage and B*35-Cw*04 disadvantage. Science 283:1748–1752PubMedCrossRefGoogle Scholar
  10. Carrington M, O’Brien SJ (2003) The influence of HLA genotype on AIDS. Annu Rev Med 54:535–551PubMedCrossRefGoogle Scholar
  11. Daza-Vamenta R, Glusman G, Rowen L, Guthrie B, Geraghty DE (2004) Genetic divergence of the rhesus macaque major histocompatibility complex. Genome Res 14:1501–1515PubMedCrossRefGoogle Scholar
  12. Dzuris JL, Sidney J, Appella E, Chesnut RW, Watkins DI, Sette A (2000) Conserved MHC class I peptide binding motif between humans and rhesus macaques. J Immunol 164:283–291PubMedGoogle Scholar
  13. Egan MA, Kuroda MJ, Voss G, Schmitz JE, Charini WA, Lord CI, Forman MA, Letvin NL (1999) Use of major histocompatibility complex class I/peptide/beta2M tetramers to quantitate CD8(+) cytotoxic T lymphocytes specific for dominant and nondominant viral epitopes in simian-human immunodeficiency virus-infected rhesus monkeys. J Virol 73:5466–5472PubMedGoogle Scholar
  14. Evans DT, Knapp LA, Jing P, Mitchen JL, Dykhuizen M, Montefiori DC, Pauza CD, Watkins DI (1999a) Rapid and slow progressors differ by a single MHC class I haplotype in a family of MHC-defined rhesus macaques infected with SIV. Immunol Lett 66:53–59PubMedCrossRefGoogle Scholar
  15. Evans DT, O’Connor DH, Jing P, Dzuris JL, Sidney J, da Silva J, Allen TM, Horton H, Venham JE, Rudersdorf RA, Vogel T, Pauza CD, Bontrop RE, DeMars R, Sette A, Hughes AL, Watkins DI (1999b) Virus-specific cytotoxic T-lymphocyte responses select for amino-acid variation in simian immunodeficiency virus Env and Nef. Nat Med 5:1270–1276PubMedCrossRefGoogle Scholar
  16. Evans DT, Jing P, Allen TM, O’Connor DH, Horton H, Venham JE, Piekarczyk M, Dzuris J, Dykhuzen M, Mitchen J, Rudersdorf RA, Pauza CD, Sette A, Bontrop RE, DeMars R, Watkins DI (2000) Definition of five new simian immunodeficiency virus cytotoxic T-lymphocyte epitopes and their restricting major histocompatibility complex class I molecules: evidence for an influence on disease progression. J Virol 74:7400–7410PubMedCrossRefGoogle Scholar
  17. Furchner M, Erickson AL, Allen T, Watkins DI, Sette A, Johnson PR, Walker CM (1999) The simian immunodeficiency virus envelope glycoprotein contains two epitope presented by the Mamu-A*01 class I molecule. J Virol 73:8035–8039PubMedGoogle Scholar
  18. Gilbert PB, Peterson ML, Follmann D, Hudgens MG, Francis DP, Gurwith M, Heyward WL, Jobes DV, Popovic V, Self SG, Sinangil F, Burke D, Berman PW (2005) Correlation between immunologic responses to a recombinant glycoprotein 120 vaccine and incidence of HIV-1 infection in a phase 3 HIV-1 preventive vaccine trial. J Infect Dis 191:666–677PubMedCrossRefGoogle Scholar
  19. Hirsch VM, Lifson JD (2000) Simian immunodeficiency virus infection of monkeys as a model system for the study of AIDS pathogenesis, treatment, and prevention. Adv Pharmacol 49:437–477PubMedCrossRefGoogle Scholar
  20. Horton H, Rehrauer W, Meek EC, Shultz MA, Piekarczyk MS, Jing P, Carter DK, Steffen SR, Calore B, Urvater JA, Vogel TU, Wilson NA, Watkins DI (2001) A common rhesus macaque MHC class I molecule which binds a cytotoxic T-lymphocyte epitope in Nef of simian immunodeficiency virus. Immunogenetics 53:423–426PubMedCrossRefGoogle Scholar
  21. Kaslow RA, Carrington M, Apple R, Park L, Munoz A, Saah AJ, Goedert JJ, Winkler C, O’Brien SJ, Rinaldo C, Detels R, Blattner W, Phair J, Erlich H, Mann DL (1996) Influence of combinations of human major histocompatibility complex genes on the course of HIV-1 infection. Nat Med 2:405–411PubMedCrossRefGoogle Scholar
  22. Kelley J, Walter L, Trowsdale J (2005) Comparative genomics of major histocompatibility complexes Immunogenetics 56:683–695PubMedCrossRefGoogle Scholar
  23. Kiepiela P, Leslie AJ, Honeyborne I, Ramduth D, Thobakgale C, Chetty S, Rathnavalu P, Moore C, Pfafferott KJ, Hilton L, Zimbwa P, Moore S, Allen T, Brander C, Addo MM, Altfeld M, James I, Mallal S, Bunce M, Barber LD, Szinger J, Day C, Klenerman P, Mullins J, Korber B, Coovadia HM, Walker BD, Goulder PJ (2004) Dominant influence of HLA-B in mediating the potential co-evolution of HIV and HLA. Nature 432:769–775PubMedCrossRefGoogle Scholar
  24. Knapp LA, Cadavid LF, Eberle ME, Knechtle SJ, Bontrop RE, Watkins DI (1997a) Identification of new mamu-DRB alleles using DGGE and direct sequencing. Immunogenetics 45:171–179PubMedCrossRefGoogle Scholar
  25. Knapp LA, Lehmann E, Piekarczyk MS, Urvater JA, Watkins DI (1997b) A high frequency of Mamu-A*01 in the rhesus macaque detected by polymerase chain reaction with sequence-specific primers and direct sequencing. Tissue Antigens 50:657–661PubMedGoogle Scholar
  26. Koff WC, Johnson PR, Watkins DI, Burton DR, Lifson JD, Hasenkrug KJ, McDermott AB, Schultz A, Zamb TJ, Boyle R, Desrosiers RC (2006) HIV vaccine design: insights from live attenuated SIV vaccines. Nat Immunol 7:19–23PubMedCrossRefGoogle Scholar
  27. Lobashevsky AL, Thomas JM (2000) Six Mamu-A locus alleles defined by polymerase chain reaction sequence specific primer method. Hum Immunol. 61:1013–1020PubMedCrossRefGoogle Scholar
  28. Loffredo JT, Sidney J, Wojewoda C, Dodds E, Reynolds MR, Napoe G, Mothe BR, O’Connor DH, Wilson NA, Watkins DI, Sette A (2004) Identification of seventeen new simian immunodeficiency virus-derived CD8+ T cell epitopes restricted by the high frequency molecule, Mamu-A*02, and potential escape from CTL recognition. J Immunol 173:5064–5076PubMedGoogle Scholar
  29. Loffredo JT, Sidney J, Piaskowski S, Szymanski A, Furlott J, Rudersdorf R, Reed J, Peters B, Hickman-Miller HD, Bardet W, Rehrauer WM, O’Connor DH, Wilson NA, Hildebrand WH, Sette A, Watkins DI (2005) The high frequency Indian rhesus macaque MHC class I molecule, Mamu-B*01, does not appear to be involved in CD8+ T lymphocyte responses to SIVmac239. J Immunol 175:5986–5997PubMedGoogle Scholar
  30. McNeil AJ, Yap PL, Gore SM, Brettle RP, McColl M, Wyld R, Davidson S, Weightman R, Richardson AM, Robertson JR (1996) Association of HLA types A1-B8-DR3 and B27 with rapid and slow progression of HIV disease. QJM 89:177–185PubMedGoogle Scholar
  31. Migueles SA, Sabbaghian MS, Shupert WL, Bettinotti MP, Marincola FM, Martino L, Hallahan CW, Selig SM, Schwartz D, Sullivan J, Connors M (2000) HLA B*5701 is highly associated with restriction of virus replication in a subgroup of HIV-infected long term nonprogressors. Proc Natl Acad Sci USA 97:2709–2714PubMedCrossRefGoogle Scholar
  32. Miller MD, Yamamoto H, Hughes AL, Watkins DI, Letvin NL (1991) Definition of an epitope and MHC class I molecule recognized by gag-specific cytotoxic T lymphocytes in SIVmac-infected rhesus monkeys. J Immunol 147:320–329PubMedGoogle Scholar
  33. Mothe BR, Sidney J, Dzuris JL, Liebl ME, Fuenger S, Watkins DI, Sette A (2002) Characterization of the peptide-binding specificity of Mamu-B*17 and identification of Mamu-B*17-restricted epitopes derived from simian immunodeficiency virus proteins. J Immunol 169:210–219PubMedGoogle Scholar
  34. Mothe BR, Weinfurter J, Wang C, Rehrauer W, Wilson N, Allen TM, Allison DB, Watkins DI (2003) Expression of the major histocompatibility complex class I molecule Mamu-A*01 is associated with control of simian immunodeficiency virus SIVmac239 replication. J Virol 77:2736–2740PubMedCrossRefGoogle Scholar
  35. Muhl T, Krawczak M, Ten Haaft P, Hunsmann G, Sauermann U (2002) MHC class I alleles influence set-point viral load and survival time in simian immunodeficiency virus-infected rhesus monkeys. J Immunol 169:3438–3446PubMedGoogle Scholar
  36. Nathanson N, Hirsch VM, Mathieson BJ (1999) The role of nonhuman primates in the development of an AIDS vaccine. AIDS 13(Suppl A):S113–S120PubMedGoogle Scholar
  37. Newberg MH, Kuroda MJ, Charini WA, Miura A, Lord CI, Schmitz JE, Gorgone DA, Lifton MA, Kuus-Reichel K, Letvin NL (2002) A simian immunodeficiency virus nef peptide is a dominant cytotoxic T lymphocyte epitope in Indian-origin rhesus monkeys expressing the common MHC class I allele mamu-A*02. Virology 301:365–373PubMedCrossRefGoogle Scholar
  38. O’Connor DH, Mothe BR, Weinfurter JT, Fuenger S, Rehrauer WM, Jing P, Rudersdorf RR, Liebl ME, Krebs K, Vasquez J, Dodds E, Loffredo J, Martin S, McDermott AB, Allen TM, Wang C, Doxiadis GG, Montefiori DC, Hughes A, Burton DR, Allison DB, Wolinsky SM, Bontrop R, Picker LJ, Watkins DI (2003) Major histocompatibility complex class I alleles associated with slow simian immunodeficiency virus disease progression bind epitopes recognized by dominant acute-phase cytotoxic-T-lymphocyte responses. J Virol 77:9029–9040PubMedCrossRefGoogle Scholar
  39. Olerup O, Zetterquist H (1991) HLA-DRB1*01 subtyping by allele-specific PCR amplification: a sensitive, specific and rapid technique. Tissue Antigens 37:197–204PubMedGoogle Scholar
  40. Olerup O, Zetterquist H (1992) HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching cadaveric transplantation. Tissue Antigens 39:225–235PubMedCrossRefGoogle Scholar
  41. Otting N, Heijmans CM, Noort RC, de Groot NG, Doxiadis GG, van Rood JJ, Watkins DI, Bontrop RE (2005) Unparalleled complexity of the MHC class I region in rhesus macaques. Proc Natl Acad Sci USA 102:1626–1631PubMedCrossRefGoogle Scholar
  42. Robinson J, Waller MJ, Fail SC, Marsh SG (2006) The IMGT/HLA and IPD databases. Hum Mutat 27:1192–1199PubMedCrossRefGoogle Scholar
  43. Robinson S, Charini WA, Newberg MH, Kuroda MJ, Lord CI, Letvin NL (2001) A commonly recognized simian immunodeficiency virus Nef epitope presented to cytotoxic T lymphocytes of Indian-origin rhesus monkeys by the prevalent major histocompatibility complex class I allele Mamu-A*02. J Virol 75:10179–10186PubMedCrossRefGoogle Scholar
  44. Schramm RD, Paprocki AM, Watkins D (2002) Birth of MHC-defined rhesus monkeys produced by assisted reproductive technology. Vaccine 20:603–607Google Scholar
  45. Sette A, Sidney J, Bui HH, del Guercio MF, Alexander J, Loffredo J, Watkins DI, Mothe BR (2005) Characterization of the peptide-binding specificity of Mamu-A*11 results in the identification of SIV-derived epitopes and interspecies cross-reactivity. Immunogenetics 57:53–68PubMedCrossRefGoogle Scholar
  46. Shiina T, Ota M, Shimizu S, Katsuyama Y, Hashimoto N, Takasu M, Anzai T, Kulski JK, Kikkawa E, Naruse T, Kimura N, Yanagiya K, Watanabe A, Hosomichi K, Kohara S, Iwamoto C, Umehara Y, Meyer A, Wanner V, Sano K, Macquin C, Ikeo K, Tokunaga K, Gojobori T, Inoko H, Bahram S (2006) Rapid evolution of major histocompatibility complex class I genes in primates generates new disease alleles in humans via hitchhiking diversity. Genetics 173:1555–1570PubMedCrossRefGoogle Scholar
  47. Sibal LR, Samson KJ (2001) Nonhuman primates: a critical role in current disease research. ILAR J 42:74–84PubMedGoogle Scholar
  48. Su J, Luscher MA, Xiong Y, Rustam T, Amara RR, Rakasz E, Robinson HL, MacDonald KS (2005) Novel simian immunodeficiency virus CTL epitopes restricted by MHC class I molecule Mamu-B*01 are highly conserved for long term in DNA/MVA-vaccinated, SHIV-challenged rhesus macaques. Int Immunol 17:637–648PubMedCrossRefGoogle Scholar
  49. Tanaka-Takahashi Y, Yasunami M, Naruse T, Hinohara K, Matano T, Mori K, Miyazawa M, Honda M, Yasutomi Y, Nagai Y, Kimura A (2007) Reference strand-mediated conformation analysis-based typing of multiple alleles in the rhesus macaque MHC class I Mamu-A and Mamu-B loci. Electrophoresis 28:918–924PubMedCrossRefGoogle Scholar
  50. Vogel TU, Friedrich TC, O’Connor DH, Rehrauer W, Dodds EJ, Hickman H, Hildebrand W, Sidney J, Sette A, Hughes A, Horton H, Vielhuber K, Rudersdorf R, De Souza IP, Reynolds MR, Allen TM, Wilson N, Watkins DI (2002) Escape in one of two cytotoxic T-lymphocyte epitopes bound by a high-frequency major histocompatibility complex class I molecule, Mamu-A*02: a paradigm for virus evolution and persistence? J Virol 76:11623–11636PubMedCrossRefGoogle Scholar
  51. Voss G, Letvin NL (1996) Definition of human immunodeficiency virus type 1 gp120 and gp41 cytotoxic T-lymphocyte epitopes and their restricting major histocompatibility complex class I alleles in simian-human immunodeficiency virus-infected rhesus monkeys. J Virol 70:7335–7340PubMedGoogle Scholar
  52. Watanabe N, McAdam SN, Boyson JE, Piekarczyk MS, Yasutomi Y, Watkins DI, Letvin NL (1994) A simian immunodeficiency virus envelope V3 cytotoxic T-lymphocyte epitope in rhesus monkeys and its restricting major histocompatibility complex class I molecule Mamu-A*02. J Virol 68:6690–6696PubMedGoogle Scholar
  53. Watkins DI, Kannagi M, Stone ME, Letvin NL (1988) Major histocompatibility complex class I molecules of nonhuman primates. Eur J Immunol 18:1425–1432PubMedCrossRefGoogle Scholar
  54. Welsh K, Bunce M (1999) Molecular typing for the MHC with PCR-SSP. Rev Immunogenet 1:157–176PubMedGoogle Scholar
  55. Wojcechowskyj JA, Yant LJ, Wiseman RW, O’Connor SL, O’Connor DH (2007) Control of simian immunodeficiency virus SIVmac239 is not predicted by inheritance of Mamu-B*17-containing haplotypes. J Virol 81:406–410PubMedCrossRefGoogle Scholar
  56. Yant LJ, Friedrich TC, Johnson RC, May GE, Maness NJ, Enz AM, Lifson JD, O’Connor DH, Carrington M, Watkins DI (2006) The high-frequency major histocompatibility complex class I allele Mamu-B*17 is associated with control of simian immunodeficiency virus SIVmac239 replication. J Virol 80:5074–5077 (Erratum in: J Virol 2006 80:6720)PubMedCrossRefGoogle Scholar
  57. Yasutomi Y, McAdam SN, Boyson JE, Piekarczyk MS, Watkins DI, Letvin NL (1995) A MHC class I B locus allele-restricted simian immunodeficiency virus envelope CTL epitope in rhesus monkeys. J Immunol 154:2516–2522PubMedGoogle Scholar
  58. Zhang ZQ, Fu TM, Casimiro DR, Davies ME, Liang X, Schleif WA, Handt L, Tussey L, Chen M, Tang A, Wilson KA, Trigona WL, Freed DC, Tan CY, Horton M, Emini EA, Shiver JW (2002) Mamu-A*01 allele-mediated attenuation of disease progression in simian-human immunodeficiency virus infection. J Virol 76:12845–12854PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Masahiko Kaizu
    • 1
    • 2
  • Gretta J. Borchardt
    • 1
  • Chrystal E. Glidden
    • 1
  • Debra L. Fisk
    • 1
  • John T. Loffredo
    • 1
  • David I. Watkins
    • 1
    • 2
  • William M. Rehrauer
    • 2
    • 3
  1. 1.Wisconsin National Primate Research CenterUniversity of WisconsinMadisonUSA
  2. 2.Department of Pathology and Laboratory MedicineUniversity of WisconsinMadisonUSA
  3. 3.University of Wisconsin Hospital and ClinicsMadisonUSA

Personalised recommendations