Donor lymphocytes have potential as a treatment for adenovirus (Ad) disease in haematopoietic stem cell transplant (SCT) recipients, but better understanding of Ad-specific T-cell responses is required. Most healthy adults exhibit memory T-cell responses to hexon, a capsid protein synthesized late after infection. However, since the Ad E3-19k downregulates major histocompatibility complex (MHC) class I molecules, cytotoxic T cells (CTLs) targeted to early viral proteins may be more effective in eliminating Ad-infected cells . Here we show that Ad-specific CTLs recognize the early region 2 proteins DNA polymerase (Pol) and DNA-binding protein (DBP). Firstly, memory Ad-specific CD8 T cells were amplified from healthy donors by stimulation with Ad-infected dendritic cells and found to exhibit MHC-restricted cytotoxicity to targets expressing Pol and DBP. Secondly, gamma interferon responses to HLA A2-binding motif peptides from Pol and DBP were directly detected in peripheral blood mononuclear cells (PBMCs) from a recently infected normal donor. Peptide-specific CTLs generated to Pol and DBP epitopes were confirmed to exhibit HLA A2-restricted killing of targets expressing Pol or DBP. Lastly, Pol-epitope-specific T cells were detected at similar or higher frequencies than hexon and DBP in three of three SCT recipients recovering from invasive Ad disease. Pol epitopes were well conserved among different Ad serotypes. Therefore, Pol is a promising target for immunotherapy of Ad disease.


Article metrics loading...

Loading full text...

Full text loading...



  1. Bennett, E. M., Bennink, J. R., Yewdell, J. W. & Brodsky, F. M.(1999). Cutting edge: adenovirus E19 has two mechanisms for affecting class I MHC expression. J Immunol 162, 5049–5052. [Google Scholar]
  2. Bennink, J. R., Yewdell, J. W. & Gerhard, W.(1982). A viral polymerase involved in recognition of influenza virus-infected cells by a cytotoxic T-cell clone. Nature 296, 75–76.[CrossRef] [Google Scholar]
  3. Bordigoni, P., Carret, A. S., Venard, V., Witz, F. & Le Faou, A.(2001). Treatment of adenovirus infections in patients undergoing allogeneic hematopoietic stem cell transplantation. Clin Infect Dis 32, 1290–1297.[CrossRef] [Google Scholar]
  4. Bunde, T., Kirchner, A., Hoffmeister, B., Habedank, D., Hetzer, R., Cherepnev, G., Proesch, S., Reinke, P., Volk, H. D. & other authors(2005). Protection from cytomegalovirus after transplantation is correlated with immediate early 1-specific CD8 T cells. J Exp Med 201, 1031–1036.[CrossRef] [Google Scholar]
  5. Burgert, H. G. & Kvist, S.(1985). An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens. Cell 41, 987–997.[CrossRef] [Google Scholar]
  6. Catalina, M. D., Sullivan, J. L., Bak, K. R. & Luzuriaga, K.(2001). Differential evolution and stability of epitope-specific CD8+ T cell responses in EBV infection. J Immunol 167, 4450–4457.[CrossRef] [Google Scholar]
  7. Chakrabarti, S., Collingham, K. E., Fegan, C. D., Pillay, D. & Milligan, D. W.(2000). Adenovirus infections following haematopoietic cell transplantation: is there a role for adoptive immunotherapy? Bone Marrow Transplant 26, 305–307.[CrossRef] [Google Scholar]
  8. Chakrabarti, S., Mautner, V., Osman, H., Collingham, K. E., Fegan, C. D., Klapper, P. E., Moss, P. A. & Milligan, D. W.(2002). Adenovirus infections following allogeneic stem cell transplantation: incidence and outcome in relation to graft manipulation, immunosuppression, and immune recovery. Blood 100, 1619–1627.[CrossRef] [Google Scholar]
  9. Cleghon, V., Piderit, A., Brough, D. E. & Klessig, D. F.(1993). Phosphorylation of the adenovirus DNA-binding protein and epitope mapping of monoclonal antibodies against it. Virology 197, 564–575.[CrossRef] [Google Scholar]
  10. Einsele, H., Roosnek, E., Rufer, N., Sinzger, C., Riegler, S., Loffler, J., Grigoleit, U., Moris, A., Rammensee, H. G. & other authors(2002). Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy. Blood 99, 3916–3922.[CrossRef] [Google Scholar]
  11. Feuchtinger, T., Matthes-Martin, S., Richard, C., Lion, T., Fuhrer, M., Hamprecht, K., Handgretinger, R., Peters, C., Schuster, F. & other authors(2006). Safe adoptive transfer of virus-specific T-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation. Br J Haematol 134, 64–76.[CrossRef] [Google Scholar]
  12. Flomenberg, P., Babbitt, J., Drobyski, W. R., Ash, R. C., Carrigan, D. R., Sedmak, G. V., McAuliffe, T., Camitta, B., Horowitz, M. M. & Bunin, N.(1994). Increasing incidence of adenovirus disease in bone marrow transplant recipients. J Infect Dis 169, 775–781.[CrossRef] [Google Scholar]
  13. Flomenberg, P., Piaskowski, V., Truitt, R. L. & Casper, J. T.(1995). Characterization of human proliferative T cell responses to adenovirus. J Infect Dis 171, 1090–1096.[CrossRef] [Google Scholar]
  14. Flomenberg, P., Piaskowski, V., Truitt, R. L. & Casper, J. T.(1996). Human adenovirus-specific CD8+ T-cell responses are not inhibited by E3-19K in the presence of gamma interferon. J Virol 70, 6314–6322. [Google Scholar]
  15. Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B.(1986). Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 83, 8122–8126.[CrossRef] [Google Scholar]
  16. Green, M. & Wold, W. S. M.(1979). Human adenoviruses: growth, purification, and transfection assay. Methods Enzymol 58, 425–435. [Google Scholar]
  17. Haveman, L. M., Bierings, M., Legger, E., Klein, M. R., de Jager, W., Otten, H. G., Albani, S., Kuis, W., Sette, A. & Prakken, B. J.(2006). Novel pan-DR-binding T cell epitopes of adenovirus induce pro-inflammatory cytokines and chemokines in healthy donors. Int Immunol 18, 1521–1529.[CrossRef] [Google Scholar]
  18. Heemskerk, B., Lankester, A. C., van Vreeswijk, T., Beersma, M. F., Claas, E. C., Veltrop-Duits, L. A., Kroes, A. C., Vossen, J. M., Schilham, M. W. & van Tol, M. J.(2005). Immune reconstitution and clearance of human adenovirus viremia in pediatric stem-cell recipients. J Infect Dis 191, 520–530.[CrossRef] [Google Scholar]
  19. Heemskerk, B., van Vreeswijk, T., Veltrop-Duits, L. A., Sombroek, C. C., Franken, K., Verhoosel, R. M., Hiemstra, P. S., van Leeuwen, D., Ressing, M. E. & other authors(2006). Adenovirus-specific CD4+ T cell clones recognizing endogenous antigen inhibit viral replication in vitro through cognate interaction. J Immunol 177, 8851–8859.[CrossRef] [Google Scholar]
  20. Horton, T. M., Ranheim, T. S., Aquino, L., Kusher, D. I., Saha, S. K., Ware, C. F., Wold, W. S. & Gooding, L. R.(1991). Adenovirus E3 14.7K protein functions in the absence of other adenovirus proteins to protect transfected cells from tumor necrosis factor cytolysis. J Virol 65, 2629–2639. [Google Scholar]
  21. Hromas, R., Cornetta, K., Srour, E., Blanke, C. & Broun, E. R.(1994). Donor leukocyte infusion as therapy of life-threatening adenoviral infections after T-cell-depleted bone marrow transplantation. Blood 84, 1689–1690. [Google Scholar]
  22. Ison, M. G.(2006). Adenovirus infections in transplant recipients. Clin Infect Dis 43, 331–339.[CrossRef] [Google Scholar]
  23. Izzedine, H., Launay-Vacher, V. & Deray, G.(2005). Antiviral drug-induced nephrotoxicity. Am J Kidney Dis 45, 804–817.[CrossRef] [Google Scholar]
  24. Jooss, K., Ertl, H. C. & Wilson, J. M.(1998). Cytotoxic T-lymphocyte target proteins and their major histocompatibility complex class I restriction in response to adenovirus vectors delivered to mouse liver. J Virol 72, 2945–2954. [Google Scholar]
  25. Joung, I. & Engler, J. A.(1992). Mutations in two cysteine-histidine-rich clusters in adenovirus type 2 DNA polymerase affect DNA binding. J Virol 66, 5788–5796. [Google Scholar]
  26. Kast, W. M., Offringa, R., Peters, P. J., Voordouw, A. C., Meloen, R. H., van der Eb, A. J. & Melief, C. J.(1989). Eradication of adenovirus E1-induced tumors by E1A-specific cytotoxic T lymphocytes. Cell 59, 603–614.[CrossRef] [Google Scholar]
  27. Kastenmuller, W., Gasteiger, G., Gronau, J., Baier, R., Ljapoci, R., Busch, D. & Drexler, I.(2007). Cross-competition of CD8+ T cells shapes the immunodominance hierarchy during boost vaccination. J Exp Med 204, 2187–2198.[CrossRef] [Google Scholar]
  28. Kitchingman, G. R.(1985). Sequence of the DNA-binding protein of a human subgroup E adenovirus (type 4): comparisons with subgroup A (type 12), subgroup B (type 7), and subgroup C (type 5). Virology 146, 90–101.[CrossRef] [Google Scholar]
  29. Knopf, C. W.(1998). Evolution of viral DNA-dependent DNA polymerases. Virus Genes 16, 47–58.[CrossRef] [Google Scholar]
  30. Kring, S. C. & Spindler, K. R.(1996). Lack of effect of mouse adenovirus type 1 infection on cell surface expression of major histocompatibility complex class I antigens. J Virol 70, 5495–5502. [Google Scholar]
  31. La Rosa, A. M., Champlin, R. E., Mirza, N., Gajewski, J., Giralt, S., Rolston, K. V., Raad, I., Jacobson, K., Kontoyiannis, D. & other authors(2001). Adenovirus infections in adult recipients of blood and marrow transplants. Clin Infect Dis 32, 871–876.[CrossRef] [Google Scholar]
  32. Leen, A. M., Sili, U., Vanin, E. F., Jewell, A. M., Xie, W., Vignali, D., Piedra, P. A., Brenner, M. K. & Rooney, C. M.(2004). Conserved CTL epitopes on the adenovirus hexon protein expand subgroup cross-reactive and subgroup-specific CD8+ T cells. Blood 104, 2432–2440.[CrossRef] [Google Scholar]
  33. Leen, A. M., Myers, G. D., Sili, U., Huls, M. H., Weiss, H., Leung, K. S., Carrum, G., Krance, R. A., Chang, C. C. & other authors(2006). Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals. Nat Med 12, 1160–1166.[CrossRef] [Google Scholar]
  34. Lichtenstein, D. L., Toth, K., Doronin, K., Tollefson, A. E. & Wold, W. S.(2004). Functions and mechanisms of action of the adenovirus E3 proteins. Int Rev Immunol 23, 75–111.[CrossRef] [Google Scholar]
  35. Liu, H., Naismith, J. H. & Hay, R. T.(2000). Identification of conserved residues contributing to the activities of adenovirus DNA polymerase. J Virol 74, 11681–11689.[CrossRef] [Google Scholar]
  36. Ljungman, P., Ribaud, P., Eyrich, M., Matthes-Martin, S., Einsele, H., Bleakley, M., Machaczka, M., Bierings, M., Bosi, A. & other authors(2003). Cidofovir for adenovirus infections after allogeneic hematopoietic stem cell transplantation: a survey by the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 31, 481–486.[CrossRef] [Google Scholar]
  37. McKelvey, T., Tang, A., Bett, A. J., Casimiro, D. R. & Chastain, M.(2004). T-cell response to adenovirus hexon and DNA-binding protein in mice. Gene Ther 11, 791–796.[CrossRef] [Google Scholar]
  38. Mizukoshi, E., Sidney, J., Livingston, B., Ghany, M., Hoofnagle, J. H., Sette, A. & Rehermann, B.(2004). Cellular immune responses to the hepatitis B virus polymerase. J Immunol 173, 5863–5871.[CrossRef] [Google Scholar]
  39. Muller, W. J., Levin, M. J., Shin, Y. K., Robinson, C., Quinones, R., Malcolm, J., Hild, E., Gao, D. & Giller, R.(2005). Clinical and in vitro evaluation of cidofovir for treatment of adenovirus infection in pediatric hematopoietic stem cell transplant recipients. Clin Infect Dis 41, 1812–1816.[CrossRef] [Google Scholar]
  40. Neofytos, D., Ojha, A., Mookerjee, B., Wagner, J., Filicko, J., Ferber, A., Dessain, S., Grosso, D., Brunner, J. & other authors(2007). Treatment of adenovirus disease in stem cell transplant recipients with cidofovir. Biol Blood Marrow Transplant 13, 74–81. [Google Scholar]
  41. Olive, M., Eisenlohr, L. C. & Flomenberg, P.(2001). Quantitative analysis of adenovirus-specific CD4+ T cell responses from healthy adults. Viral Immunol 14, 403–413.[CrossRef] [Google Scholar]
  42. Olive, M., Eisenlohr, L., Flomenberg, N., Hsu, S. & Flomenberg, P.(2002). The adenovirus capsid protein hexon contains a highly conserved human CD4+ T-cell epitope. Hum Gene Ther 13, 1167–1178.[CrossRef] [Google Scholar]
  43. Papadopoulos, E. B., Ladanyi, M., Emanuel, D., Mackinnon, S., Boulad, F., Carabasi, M. H., Castro-Malaspina, H., Childs, B. H., Gillio, A. P. & Small, T. N.(1994). Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation. N Engl J Med 330, 1185–1191.[CrossRef] [Google Scholar]
  44. Parker, K. C., Bednarek, M. A. & Coligan, J. E.(1994). Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J Immunol 152, 163–175. [Google Scholar]
  45. Rammensee, H. G., Friede, T. & Stevanović, S.(1995). MHC ligands and peptide motifs: first listing. Immunogenetics 41, 178–228.[CrossRef] [Google Scholar]
  46. Rooney, C. M., Smith, C. A., Ng, C. Y., Loftin, S. K., Sixbey, J. W., Gan, Y., Srivastava, D. K., Bowman, L. C., Krance, R. A. & other authors(1998). Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 92, 1549–1555. [Google Scholar]
  47. Routes, J. M., Bellgrau, D., McGrory, W. J., Bautista, D. S., Graham, F. L. & Cook, J. L.(1991). Anti-adenovirus type 5 cytotoxic T lymphocytes: immunodominant epitopes are encoded by the E1A gene. J Virol 65, 1450–1457. [Google Scholar]
  48. Shisler, J., Yang, C., Walter, B., Ware, C. F. & Gooding, L. R.(1997). The adenovirus E3–10.4K/14.5K complex mediates loss of cell surface Fas (CD95) and resistance to Fas-induced apoptosis. J Virol 71, 8299–8306. [Google Scholar]
  49. Smith, C. A., Woodruff, L. S., Rooney, C. & Kitchingman, G. R.(1998). Extensive cross-reactivity of adenovirus-specific cytotoxic T cells. Hum Gene Ther 9, 1419–1427.[CrossRef] [Google Scholar]
  50. Sparer, T. E., Wynn, S. G., Clark, D. J., Kaplan, J. M., Cardoza, L. M., Wadsworth, S. C., Smith, A. E. & Gooding, L. R.(1997). Generation of cytotoxic T lymphocytes against immunorecessive epitopes after multiple immunizations with adenovirus vectors is dependent on haplotype. J Virol 71, 2277–2284. [Google Scholar]
  51. Tang, J., Olive, M., Champagne, K., Flomenberg, N., Eisenlohr, L., Hsu, S. & Flomenberg, P.(2004). Adenovirus hexon T-cell epitope is recognized by most adults and is restricted by HLA DP4, the most common class II allele. Gene Ther 11, 1408–1415.[CrossRef] [Google Scholar]
  52. Tang, J., Olive, M., Pulmanausahakul, R., Schnell, M., Flomenberg, N., Eisenlohr, L. & Flomenberg, P.(2006). Human CD8+ cytotoxic T cell responses to adenovirus capsid proteins. Virology 350, 312–322.[CrossRef] [Google Scholar]
  53. Tsomides, T. J., Aldovini, A., Johnson, R. P., Walker, B. D., Young, R. A. & Eisen, H. N.(1994). Naturally processed viral peptides recognized by cytotoxic T lymphocytes on cells chronically infected by human immunodeficiency virus type 1. J Exp Med 180, 1283–1293.[CrossRef] [Google Scholar]
  54. Vos, H. L., van der Lee, F. M., Reemst, A. M., van Loon, A. E. & Sussenbach, J. S.(1988). The genes encoding the DNA binding protein and the 23K protease of adenovirus types 40 and 41. Virology 163, 1–10.[CrossRef] [Google Scholar]
  55. Walter, E. A., Greenberg, P. D., Gilbert, M. J., Finch, R. J., Watanabe, K. S., Thomas, E. D. & Riddell, S. R.(1995). Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N Engl J Med 333, 1038–1044.[CrossRef] [Google Scholar]
  56. Wysocka, M., Eisenlohr, L. C., Otvos, L., Jr, Horowitz, D., Yewdell, J. W., Bennink, J. R. & Hackett, C. J.(1994). Identification of overlapping class I and class II H-2d restricted T cell determinants of influenza virus N1 neuraminidase that require infectious virus for presentation. Virology 201, 86–94.[CrossRef] [Google Scholar]
  57. Yang, S. Y., Milford, E., Hammerling, U. & Dupont, B.(1989). Description of the reference panel of B-lymphoblastoid cell lines for factors of the HLA system: the B-cell line panel designed for the Tenth International Histocompatibility Workshop. In Immunobiology of HLA, vol. I: Histocompatibility Testing 1987, pp. 11–19. Edited by B. Dupont. New York: Springer-Verlag.
  58. Yusuf, U., Hale, G. A., Carr, J., Gu, Z., Benaim, E., Woodard, P., Kasow, K. A., Horwitz, E. M., Leung, W. & other authors(2006). Cidofovir for the treatment of adenoviral infection in pediatric hematopoietic stem cell transplant patients. Transplantation 81, 1398–1404.[CrossRef] [Google Scholar]

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error