The haemagglutinin (HA) glycoprotein of influenza A virus is a major antigen that initiates humoral immunity against infection; however, the cellular immune response against HA is poorly understood. Furthermore, HA-derived cytotoxic T-lymphocyte (CTL) epitopes are relatively rare in comparison to other internal gene products. Here, CTL epitopes of the HA serotype H5 protein were screened. By using prediction, refolding and a T2 cell-binding assay, followed by immunization of HLA-A2.1/K transgenic mice, an HLA-A*0201-restricted decameric epitope, RI-10 (H5 HA205–214, RLYQNPTTYI), was shown to elicit a robust CTL epitope-specific response. In addition, RI-10 and its variant, KI-10 (KLYQNPTTYI), were also demonstrated to be able to induce a higher CTL epitope-specific response than the influenza A virus dominant CTL epitope GL-9 (GILGFVFTL) in peripheral blood mononuclear cells of HLA-A*0201-positive patients who had recovered from H5N1 virus infection. Furthermore, the crystal structures of RI-10–HLA-A*0201 and KI-10–HLA-A*0201 complexes were determined at 2.3 and 2.2 Å resolution, respectively, showing typical HLA-A*0201-restricted epitopes. The conformations of RI-10 and KI-10 in the antigen-presenting grooves in crystal structures of the two complexes show significant differences, despite their nearly identical sequences. These results provide implications for the discovery of diagnostic markers and the design of novel influenza vaccines.


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  1. Ahmed, R., Oldstone, M. B. & Palese, P.(2007). Protective immunity and susceptibility to infectious diseases: lessons from the 1918 influenza pandemic. Nat Immunol 8, 1188–1193.[CrossRef] [Google Scholar]
  2. Altman, J. D., Moss, P. A., Goulder, P. J., Barouch, D. H., McHeyzer-Williams, M. G., Bell, J. I., McMichael, A. J. & Davis, M. M.(1996). Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96.[CrossRef] [Google Scholar]
  3. Bender, B. S. & Small, P. A., Jr(1992). Influenza: pathogenesis and host defense. Semin Respir Infect 7, 38–45. [Google Scholar]
  4. Bennink, J. R., Yewdell, J. W., Smith, G. L., Moller, C. & Moss, B.(1984). Recombinant vaccinia virus primes and stimulates influenza haemagglutinin-specific cytotoxic T cells. Nature 311, 578–579.[CrossRef] [Google Scholar]
  5. Bertoletti, A., Costanzo, A., Chisari, F. V., Levrero, M., Artini, M., Sette, A., Penna, A., Giuberti, T., Fiaccadori, F. & other authors(1994). Cytotoxic T lymphocyte response to a wild type hepatitis B virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope. J Exp Med 180, 933–943.[CrossRef] [Google Scholar]
  6. Bouvier, M., Guo, H. C., Smith, K. J. & Wiley, D. C.(1998). Crystal structures of HLA-A*0201 complexed with antigenic peptides with either the amino- or carboxyl-terminal group substituted by a methyl group. Proteins 33, 97–106.[CrossRef] [Google Scholar]
  7. Braciale, T. J., Sweetser, M. T., Morrison, L. A., Kittlesen, D. J. & Braciale, V. L.(1989). Class I major histocompatibility complex-restricted cytolytic T lymphocytes recognize a limited number of sites on the influenza hemagglutinin. Proc Natl Acad Sci U S A 86, 277–281.[CrossRef] [Google Scholar]
  8. Bui, H. H., Peters, B., Assarsson, E., Mbawuike, I. & Sette, A.(2007). Ab and T cell epitopes of influenza A virus, knowledge and opportunities. Proc Natl Acad Sci U S A 104, 246–251.[CrossRef] [Google Scholar]
  9. Cao, W., Myers-Powell, B. A. & Braciale, T. J.(1996). The weak CD8+ CTL response to an influenza hemagglutinin epitope reflects limited T cell availability. J Immunol 157, 505–511. [Google Scholar]
  10. Chen, H., Smith, G. J., Zhang, S. Y., Qin, K., Wang, J., Li, K. S., Webster, R. G., Peiris, J. S. & Guan, Y.(2005). Avian flu: H5N1 virus outbreak in migratory waterfowl. Nature 436, 191–192.[CrossRef] [Google Scholar]
  11. Chen, H., Li, Y., Li, Z., Shi, J., Shinya, K., Deng, G., Qi, Q., Tian, G., Fan, S. & other authors(2006). Properties and dissemination of H5N1 viruses isolated during an influenza outbreak in migratory waterfowl in western China. J Virol 80, 5976–5983.[CrossRef] [Google Scholar]
  12. Chu, F., Lou, Z., Chen, Y. W., Liu, Y., Gao, B., Zong, L., Khan, A. H., Bell, J. I., Rao, Z. & other authors(2007). First glimpse of the peptide presentation by rhesus macaque MHC class I: crystal structures of Mamu-A*01 complexed with two immunogenic SIV epitopes and insights into CTL escape. J Immunol 178, 944–952.[CrossRef] [Google Scholar]
  13. Claas, E. C., Osterhaus, A. D., van Beek, R., De Jong, J. C., Rimmelzwaan, G. F., Senne, D. A., Krauss, S., Shortridge, K. F. & Webster, R. G.(1998). Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351, 472–477.[CrossRef] [Google Scholar]
  14. Doherty, P. C. & Kelso, A.(2008). Toward a broadly protective influenza vaccine. J Clin Invest 118, 3273–3275. [Google Scholar]
  15. Garboczi, D. N., Hung, D. T. & Wiley, D. C.(1992). HLA-A2–peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc Natl Acad Sci U S A 89, 3429–3433.[CrossRef] [Google Scholar]
  16. Gotch, F., McMichael, A., Smith, G. & Moss, B.(1987). Identification of viral molecules recognized by influenza-specific human cytotoxic T lymphocytes. J Exp Med 165, 408–416.[CrossRef] [Google Scholar]
  17. Gricks, C. S., Rawlings, E., Foroni, L., Madrigal, J. A. & Amlot, P. L.(2001). Somatically mutated regions of immunoglobulin on human B-cell lymphomas code for peptides that bind to autologous major histocompatibility complex class I, providing a potential target for cytotoxic T cells. Cancer Res 61, 5145–5152. [Google Scholar]
  18. Heiny, A. T., Miotto, O., Srinivasan, K. N., Khan, A. M., Zhang, G. L., Brusic, V., Tan, T. W. & August, J. T.(2007). Evolutionarily conserved protein sequences of influenza A viruses, avian and human, as vaccine targets. PLoS One 2, e1190[CrossRef] [Google Scholar]
  19. Kees, U. & Krammer, P. H.(1984). Most influenza A virus-specific memory cytotoxic T lymphocytes react with antigenic epitopes associated with internal virus determinants. J Exp Med 159, 365–377.[CrossRef] [Google Scholar]
  20. Khan, A. R., Baker, B. M., Ghosh, P., Biddison, W. E. & Wiley, D. C.(2000). The structure and stability of an HLA-A*0201/octameric tax peptide complex with an empty conserved peptide-N-terminal binding site. J Immunol 164, 6398–6405.[CrossRef] [Google Scholar]
  21. Kilpatrick, A. M., Chmura, A. A., Gibbons, D. W., Fleischer, R. C., Marra, P. P. & Daszak, P.(2006). Predicting the global spread of H5N1 avian influenza. Proc Natl Acad Sci U S A 103, 19368–19373.[CrossRef] [Google Scholar]
  22. Kreijtz, J. H., de Mutsert, G., van Baalen, C. A., Fouchier, R. A., Osterhaus, A. D. & Rimmelzwaan, G. F.(2008). Cross-recognition of avian H5N1 influenza virus by human cytotoxic T-lymphocyte populations directed to human influenza A virus. J Virol 82, 5161–5166.[CrossRef] [Google Scholar]
  23. Kuzushima, K., Hayashi, N., Kimura, H. & Tsurumi, T.(2001). Efficient identification of HLA-A*2402-restricted cytomegalovirus-specific CD8 (+) T-cell epitopes by a computer algorithm and an enzyme-linked immunospot assay. Blood 98, 1872–1881.[CrossRef] [Google Scholar]
  24. Lagging, L. M., Meyer, K., Hoft, D., Houghton, M., Belshe, R. B. & Ray, R.(1995). Immune responses to plasmid DNA encoding the hepatitis C virus core protein. J Virol 69, 5859–5863. [Google Scholar]
  25. Lee, J. K., Stewart-Jones, G., Dong, T., Harlos, K., Di Gleria, K., Dorrell, L., Douek, D. C., van der Merwe, P. A., Jones, E. Y. & other authors(2004). T cell cross-reactivity and conformational changes during TCR engagement. J Exp Med 200, 1455–1466.[CrossRef] [Google Scholar]
  26. Lee, L. Y., Ha Do, L. A., Simmons, C., de Jong, M. D., Chau, N. V., Schumacher, R., Peng, Y. C., McMichael, A. J., Farrar, J. J. & other authors(2008). Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals. J Clin Invest 118, 3478–3490. [Google Scholar]
  27. Li, H., Zhou, M., Han, J., Zhu, X., Dong, T., Gao, G. F. & Tien, P.(2005). Generation of murine CTL by a hepatitis B virus-specific peptide and evaluation of the adjuvant effect of heat shock protein glycoprotein 96 and its terminal fragments. J Immunol 174, 195–204.[CrossRef] [Google Scholar]
  28. Liu, J., Xiao, H., Lei, F., Zhu, Q., Qin, K., Zhang, X. W., Zhang, X. L., Zhao, D., Wang, G. & other authors(2005). Highly pathogenic H5N1 influenza virus infection in migratory birds. Science 309, 1206[CrossRef] [Google Scholar]
  29. Madden, D. R., Garboczi, D. N. & Wiley, D. C.(1993). The antigenic identity of peptide-MHC complexes: a comparison of the conformations of five viral peptides presented by HLA-A2. Cell 75, 693–708.[CrossRef] [Google Scholar]
  30. Normile, D.(2006). Avian influenza. Evidence points to migratory birds in H5N1 spread. Science 311, 1225[CrossRef] [Google Scholar]
  31. Olsen, B., Munster, V. J., Wallensten, A., Waldenstrom, J., Osterhaus, A. D. & Fouchier, R. A.(2006). Global patterns of influenza A virus in wild birds. Science 312, 384–388.[CrossRef] [Google Scholar]
  32. Passoni, L., Scardino, A., Bertazzoli, C., Gallo, B., Coluccia, A. M., Lemonnier, F. A., Kosmatopoulos, K. & Gambacorti-Passerini, C.(2002). ALK as a novel lymphoma-associated tumor antigen: identification of 2 HLA-A2.1-restricted CD8+ T-cell epitopes. Blood 99, 2100–2106.[CrossRef] [Google Scholar]
  33. Reid, S. W., McAdam, S., Smith, K. J., Klenerman, P., O'Callaghan, C. A., Harlos, K., Jakobsen, B. K., McMichael, A. J., Bell, J. I. & other authors(1996). Antagonist HIV-1 Gag peptides induce structural changes in HLA B8. J Exp Med 184, 2279–2286.[CrossRef] [Google Scholar]
  34. Rimmelzwaan, G. F., Fouchier, R. A. & Osterhaus, A. D.(2007). Influenza virus-specific cytotoxic T lymphocytes: a correlate of protection and a basis for vaccine development. Curr Opin Biotechnol 18, 529–536.[CrossRef] [Google Scholar]
  35. Salomon, R. & Webster, R. G.(2009). The influenza virus enigma. Cell 136, 402–410.[CrossRef] [Google Scholar]
  36. Sourdive, D. J., Murali-Krishna, K., Altman, J. D., Zajac, A. J., Whitmire, J. K., Pannetier, C., Kourilsky, P., Evavold, B., Sette, A. & other authors(1998). Conserved T cell receptor repertoire in primary and memory CD8 T cell responses to an acute viral infection. J Exp Med 188, 71–82.[CrossRef] [Google Scholar]
  37. Stevens, J., Blixt, O., Tumpey, T. M., Taubenberger, J. K., Paulson, J. C. & Wilson, I. A.(2006). Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312, 404–410.[CrossRef] [Google Scholar]
  38. Stewart-Jones, G. B., McMichael, A. J., Bell, J. I., Stuart, D. I. & Jones, E. Y.(2003). A structural basis for immunodominant human T cell receptor recognition. Nat Immunol 4, 657–663. [Google Scholar]
  39. Thomas, P. G., Keating, R., Hulse-Post, D. J. & Doherty, P. C.(2006). Cell-mediated protection in influenza infection. Emerg Infect Dis 12, 48–54.[CrossRef] [Google Scholar]
  40. Townsend, A. R. & Skehel, J. J.(1984). The influenza A virus nucleoprotein gene controls the induction of both subtype specific and cross-reactive cytotoxic T cells. J Exp Med 160, 552–563.[CrossRef] [Google Scholar]
  41. Townsend, A. R., Bastin, J., Gould, K. & Brownlee, G. G.(1986). Cytotoxic T lymphocytes recognize influenza haemagglutinin that lacks a signal sequence. Nature 324, 575–577.[CrossRef] [Google Scholar]
  42. Uebel, S. & Tampe, R.(1999). Specificity of the proteasome and the TAP transporter. Curr Opin Immunol 11, 203–208.[CrossRef] [Google Scholar]
  43. Uebel, S., Kraas, W., Kienle, S., Wiesmuller, K. H., Jung, G. & Tampe, R.(1997). Recognition principle of the TAP transporter disclosed by combinatorial peptide libraries. Proc Natl Acad Sci U S A 94, 8976–8981.[CrossRef] [Google Scholar]
  44. Vitiello, A., Marchesini, D., Furze, J., Sherman, L. A. & Chesnut, R. W.(1991). Analysis of the HLA-restricted influenza-specific cytotoxic T lymphocyte response in transgenic mice carrying a chimeric human-mouse class I major histocompatibility complex. J Exp Med 173, 1007–1015.[CrossRef] [Google Scholar]
  45. Wabuke-Bunoti, M. A., Taku, A., Fan, D. P., Kent, S. & Webster, R. G.(1984). Cytolytic T lymphocyte and antibody responses to synthetic peptides of influenza virus hemagglutinin. J Immunol 133, 2194–2201. [Google Scholar]
  46. Wahl, A., Schafer, F., Bardet, W., Buchli, R., Air, G. M. & Hildebrand, W. H.(2009). HLA class I molecules consistently present internal influenza epitopes. Proc Natl Acad Sci U S A 106, 540–545.[CrossRef] [Google Scholar]
  47. Wang, G., Zhan, D., Li, L., Lei, F., Liu, B., Liu, D., Xiao, H., Feng, Y., Li, J. & other authors(2008). H5N1 avian influenza re-emergence of Lake Qinghai: phylogenetic and antigenic analyses of the newly isolated viruses and roles of migratory birds in virus circulation. J Gen Virol 89, 697–702.[CrossRef] [Google Scholar]
  48. WHO(2009). Cumulative number of confirmed human cases of avian influenza A/(H5N1) reported to WHO. http://www.who.int/csr/disease/avian_influenza/country/cases_table_2009_04_23/en/index.html
  49. Wilson, I. A. & Cox, N. J.(1990). Structural basis of immune recognition of influenza virus hemagglutinin. Annu Rev Immunol 8, 737–771.[CrossRef] [Google Scholar]
  50. Yap, K. L., Ada, G. L. & McKenzie, I. F.(1978). Transfer of specific cytotoxic T lymphocytes protects mice inoculated with influenza virus. Nature 273, 238–239.[CrossRef] [Google Scholar]
  51. Zhou, J. Y., Shen, H. G., Chen, H. X., Tong, G. Z., Liao, M., Yang, H. C. & Liu, J. X.(2006a). Characterization of a highly pathogenic H5N1 influenza virus derived from bar-headed geese in China. J Gen Virol 87, 1823–1833.[CrossRef] [Google Scholar]
  52. Zhou, M., Xu, D., Li, X., Li, H., Shan, M., Tang, J., Wang, M., Wang, F. S., Zhu, X. & other authors(2006b). Screening and identification of severe acute respiratory syndrome-associated coronavirus-specific CTL epitopes. J Immunol 177, 2138–2145.[CrossRef] [Google Scholar]
  53. Zwart, P. H., Afonine, P. V., Grosse-Kunstleve, R. W., Hung, L. W., Ioerger, T. R., McCoy, A. J., McKee, E., Moriarty, N. W., Read, R. J. & other authors(2008). Automated structure solution with the PHENIX suite. Methods Mol Biol 426, 419–435. [Google Scholar]

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