Characterization of binding specificities of bovine leucocyte class I molecules: impacts for rational epitope discovery

Abstract

The binding of peptides to classical major histocompatibility complex (MHC) class I proteins is the single most selective step in antigen presentation. However, the peptide-binding specificity of cattle MHC (bovine leucocyte antigen, BoLA) class I (BoLA-I) molecules remains poorly characterized. Here, we demonstrate how a combination of high-throughput assays using positional scanning combinatorial peptide libraries, peptide dissociation, and peptide-binding affinity binding measurements can be combined with bioinformatics to effectively characterize the functionality of BoLA-I molecules. Using this strategy, we characterized eight BoLA-I molecules, and found the peptide specificity to resemble that of human MHC-I molecules with primary anchors most often at P2 and P9, and occasional auxiliary P1/P3/P5/P6 anchors. We analyzed nine reported CTL epitopes from Theileria parva, and in eight cases, stable and high affinity binding was confirmed. A set of peptides were tested for binding affinity to the eight BoLA proteins and used to refine the predictors of peptide–MHC binding NetMHC and NetMHCpan. The inclusion of BoLA-specific peptide-binding data led to a significant improvement in prediction accuracy for reported T. parva CTL epitopes. For reported CTL epitopes with weak or no predicted binding, these refined prediction methods suggested presence of nested minimal epitopes with high-predicted binding affinity. The enhanced affinity of the alternative peptides was in all cases confirmed experimentally. This study demonstrates how biochemical high-throughput assays combined with immunoinformatics can be used to characterize the peptide-binding motifs of BoLA-I molecules, boosting performance of MHC peptide-binding prediction methods, and empowering rational epitope discovery in cattle.

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References

  1. Birch J, Murphy L, MacHugh ND, Ellis SA (2006) Generation and maintenance of diversity in the cattle MHC class I region. Immunogenetics 58:670–679. doi:10.1007/s00251-006-0137-y

    CAS  PubMed  Article  Google Scholar 

  2. Codner GF, Stear MJ, Reeve R, Matthews L, Ellis SA (2012) Selective forces shaping diversity in the class I region of the major histocompatibility complex in dairy cattle. Anim Genet 43:239–249. doi:10.1111/j.1365-2052.2011.02239.x

    CAS  PubMed  Article  Google Scholar 

  3. De Groot AS, Nene V, Hegde NR, Srikumaran S, Rayner J, Martin W (2003) T cell epitope identification for bovine vaccines: an epitope mapping method for BoLA A-11. Int J Parasitol 33:641–653

    PubMed  Article  Google Scholar 

  4. Ferre H et al (2003) Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding. Protein Sci 12:551–559

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  5. Ferre H, Ruffet E, Nielsen LL, Nissen MH, Hobley TJ, Thomas OR, Buus S (2005) A novel system for continuous protein refolding and on-line capture by expanded bed adsorption. Protein Sci 14:2141–2153. doi:10.1110/ps.051396105

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  6. Follin E, Karlsson M, Lundegaard C, Nielsen M, Wallin S, Paulsson K, Westerdahl H (2013) In silico peptide-binding predictions of passerine MHC class I reveal similarities across distantly related species, suggesting convergence on the level of protein function. Immunogenetics 65:299–311

    CAS  PubMed  Article  Google Scholar 

  7. Gaddum RM, Willis AC, Ellis SA (1996) Peptide motifs from three cattle MHC (BoLA) class I antigens. Immunogenetics 43:238–239

    CAS  PubMed  Google Scholar 

  8. Graham SP et al (2006) Theileria parva candidate vaccine antigens recognized by immune bovine cytotoxic T lymphocytes. Proc Natl Acad Sci U S A 103:3286–3291. doi:10.1073/pnas.0511273103

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  9. Graham SP et al (2008) Characterization of the fine specificity of bovine CD8 T-cell responses to defined antigens from the protozoan parasite Theileria parva. Infect Immun 76:685–694

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  10. Harndahl M, Justesen S, Lamberth K, Roder G, Nielsen M, Buus S (2009) Peptide binding to HLA class I molecules: homogenous, high-throughput screening, and affinity assays. J Biomol Screen 14:173–180

    CAS  PubMed  Article  Google Scholar 

  11. Harndahl M, Rasmussen M, Roder G, Buus S (2011) Real-time, high-throughput measurements of peptide-MHC-I dissociation using a scintillation proximity assay. J Immunol Methods 374:5–12

    CAS  PubMed  Article  Google Scholar 

  12. Harndahl M, Rasmussen M, Roder G, Dalgaard Pedersen I, Sorensen M, Nielsen M, Buus S (2012) Peptide-MHC class I stability is a better predictor than peptide affinity of CTL immunogenicity. Eur J Immunol 42:1405–1416. doi:10.1002/eji.201141774

    CAS  PubMed  Article  Google Scholar 

  13. Hegde NR, Ellis SA, Gaddum RM, Tregaskes CA, Sarath G, Srikumaran S (1995) Peptide motif of the cattle MHC class I antigen BoLA-A11. Immunogenetics 42:302–303

    CAS  PubMed  Article  Google Scholar 

  14. Hegde NR, Deshpande MS, Godson DL, Babiuk LA, Srikumaran S (1999) Bovine lymphocyte antigen-A11-specific peptide motif as a means to identify cytotoxic T-lymphocyte epitopes of bovine herpesvirus 1. Viral Immunol 12:149–161

    CAS  PubMed  Article  Google Scholar 

  15. Holmes EC, Roberts AF, Staines KA, Ellis SA (2003) Evolution of major histocompatibility complex class I genes in Cetartiodactyls. Immunogenetics 55:193–202. doi:10.1007/s00251-003-0560-2

    CAS  PubMed  Article  Google Scholar 

  16. Hoof I et al (2009) NetMHCpan, a method for MHC class I binding prediction beyond humans. Immunogenetics 61:1–13

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  17. Hunter WM, Greenwood FC (1962) Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature 194:495–496

    CAS  PubMed  Article  Google Scholar 

  18. Joosten I, Teale AJ, van der Poel A, Hensen EJ (1992) Biochemical evidence of the expression of two major histocompatibility complex class I genes on bovine peripheral blood mononuclear cells. Anim Genet 23:113–123

    CAS  PubMed  Article  Google Scholar 

  19. Leisner C et al (2008) One-pot, mix-and-read peptide-MHC tetramers. PLoS One 3:e1678

    PubMed Central  PubMed  Article  Google Scholar 

  20. Li X et al (2011) Two distinct conformations of a rinderpest virus epitope presented by bovine major histocompatibility complex class I N*01801: a host strategy to present featured peptides. J Virol 85:6038–6048

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  21. Lundegaard C, Lamberth K, Harndahl M, Buus S, Lund O, Nielsen M (2008) NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11. Nucleic Acids Res

  22. Macdonald IK et al (2010) MHC class I bound to an immunodominant Theileria parva epitope demonstrates unconventional presentation to T cell receptors. PLoS Pathog 6:e1001149. doi:10.1371/journal.ppat.1001149

    PubMed Central  PubMed  Article  Google Scholar 

  23. MacHugh ND et al (2011) Extensive polymorphism and evidence of immune selection in a highly dominant antigen recognized by bovine CD8 T cells specific for Theileria annulata. Infect Immun 79:2059–2069

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  24. McKeever DJ, Taracha EL, Innes EL, MacHugh ND, Awino E, Goddeeris BM, Morrison WI (1994) Adoptive transfer of immunity to Theileria parva in the CD8+ fraction of responding efferent lymph. Proc Natl Acad Sci U S A 91:1959–1963

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  25. McMahon RM, Friis L, Siebold C, Friese MA, Fugger L, Jones EY (2011) Structure of HLA-A*0301 in complex with a peptide of proteolipid protein: insights into the role of HLA-A alleles in susceptibility to multiple sclerosis Acta crystallographica Section D. Biol Crystallogr 67:447–454. doi:10.1107/S0907444911007888

    CAS  Article  Google Scholar 

  26. Nene V et al (2012) Designing bovine T cell vaccines via reverse immunology. Ticks Tick-Borne Dis 3:188–192. doi:10.1016/j.ttbdis.2011.12.001

    PubMed  Article  Google Scholar 

  27. Nielsen M et al (2003) Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci 12:1007–1017

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  28. Nielsen M et al (2007) NetMHCpan, a method for quantitative predictions of peptide binding to any HLA-A and -B locus protein of known sequence. PLoS One 2:e796

    PubMed Central  PubMed  Article  Google Scholar 

  29. Ostergaard Pedersen L et al (2001) Efficient assembly of recombinant major histocompatibility complex class I molecules with preformed disulfide bonds. Eur J Immunol 31:2986–2996. doi:10.1002/1521-4141(2001010)31:10<2986::AID-IMMU2986>3.0.CO;2-R

    CAS  PubMed  Article  Google Scholar 

  30. Parker KC et al (1992) Sequence motifs important for peptide binding to the human MHC class I molecule, HLA-A2. J Immunol 149:3580–3587

    CAS  PubMed  Google Scholar 

  31. Pedersen LE et al (2011) Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities. Immunogenetics 63:821–834. doi:10.1007/s00251-011-0555-3

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  32. Robinson J, Halliwell JA, McWilliam H, Lopez R, Marsh SG (2013) IPD—the immuno polymorphism database. Nucleic Acids Res 41:D1234–D1240

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  33. Shields MJ, Ribaudo RK (1998) Mapping of the monoclonal antibody W6/32: sensitivity to the amino terminus of beta2-microglobulin. Tissue Antigens 51:567–570

    CAS  PubMed  Article  Google Scholar 

  34. Sidney J, Peters B, Frahm N, Brander C, Sette A (2008) HLA class I supertypes: a revised and updated classification. BMC Immunol 9:1

    PubMed Central  PubMed  Article  Google Scholar 

  35. Sinnathamby G, Seth S, Nayak R, Shaila MS (2004) Cytotoxic T cell epitope in cattle from the attachment glycoproteins of rinderpest and peste des petits ruminants viruses. Viral Immunol 17:401–410. doi:10.1089/0882824041857094

    CAS  PubMed  Article  Google Scholar 

  36. Stryhn A, Pedersen LO, Romme T, Holm CB, Holm A, Buus S (1996) Peptide binding specificity of major histocompatibility complex class I resolved into an array of apparently independent subspecificities: quantitation by peptide libraries and improved prediction of binding. Eur J Immunol 26:1911–1918

    CAS  PubMed  Article  Google Scholar 

  37. Svitek N et al (2014) Use of “one-pot, mix-and-read” peptide-MHC class I tetramers and predictive algorithms to improve detection of cytotoxic T lymphocyte responses in cattle. Vet Res 45:50

    PubMed Central  PubMed  Article  Google Scholar 

  38. Thomsen MC, Nielsen M (2012) Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion. Nucleic Acids Res 40:W281–W287

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  39. Yewdell JW, Bennink JR (1999) Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu Rev Immunol 17:51–88

    CAS  PubMed  Article  Google Scholar 

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Acknowledgments

This research was funded through an award (No. 0965346) from the BREAD program of the National Science Foundation (USA); MN is a researcher at the Argentinean national research council (CONICET).

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No competing interests

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Correspondence to Morten Nielsen.

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Hansen, A.M., Rasmussen, M., Svitek, N. et al. Characterization of binding specificities of bovine leucocyte class I molecules: impacts for rational epitope discovery. Immunogenetics 66, 705–718 (2014). https://doi.org/10.1007/s00251-014-0802-5

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Keywords

  • Bovine leucocyte antigen
  • BoLA
  • Rational epitope discovery
  • CTL epitopes
  • Immunoinformatics