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Long-Term Immunity against Actual Poxviral HLA Ligands as Identified by Differential Stable Isotope Labeling¹

Verena S. Meyer^*, Wolfgang Kastenmuller, Georg Gasteiger^,, Mirita Franz-Wachtel, Tobias Lamkemeyer, Hans-Georg Rammensee^*, Stefan Stevanovic^*,, Dagmar Sigurdardottir^2,* and Ingo Drexler^,

^* Department of Immunology, Institute for Cell Biology, University of Tubingen, Tubingen, Germany; Institute of Virology, Technical University Munich and Helmholtz Center Munich, Munich, Germany; Antigen-Specific Immunotherapy Clinical Cooperation Group, Helmholtz Center Munich, Munich, Germany; and Proteome Center Tubingen, Institute for Cell Biology, University of Tubingen, Tubingen, Germany

Viral peptides are presented by HLA class I on infected cells to activate CD8⁺ T cells. Several immunogenic peptides have been identified indirectly by epitope prediction and screening of T cell responses to poxviral vectors, including modified vaccinia virus Ankara (MVA) currently being tested as recombinant or smallpox vaccines. However, for the development of optimal vaccination and immunomonitoring strategies, it is essential to characterize the actual viral HLA ligand repertoire of infected cells. We used an innovative approach to identify naturally processed MVA HLA ligands by differential HPLC-coupled mass spectrometry. We describe 12 viral peptides presented by HLA-A*0201 and 3 by HLA-B*0702. All HLA-A*0201 ligands participated in the memory response of MVA-immune donors, and several were immunogenic in Dryvax vaccinees. Eight epitopes were novel. Viral HLA ligand presentation and viral protein abundance did not correlate. All ligands were expressed early during the viral life cycle, and a pool of three of these mediated stronger protection against a lethal challenge in mice as compared with late epitopes. This highlights the reliability of the comparative mass spectrometry-based technique to identify relevant viral CD8⁺ T cell epitopes for optimizing the monitoring of protective immune responses and the development of effective peptide-based vaccines.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ V.S.M. is supported by the Stiftung der deutschen Wirtschaft-Studienstiftung Klaus Muhrmann. This study is funded in part by European Union Project ALLOSTEM to H.-G.R. and Deutsche Forschungsgemeinschaft Project SFB456 TP B7 to I.D. The Proteome Center Tubingen is supported by the Ministerium fur Wissenschaft und Kunst, Landesregierung Baden-Wurttemberg.

² Address correspondence and reprint requests to Dr. Dagmar Sigurdardottir, Department of Immunology, Institute for Cell Biology, University of Tubingen, Auf der Morgenstelle 15, D-72076 Tubingen, Germany. E-mail address: dagmar.sigurdardottir{at}uni-tuebingen.de

³ Abbreviations used in this paper: VARV, variola virus; 2D, two-dimensional; B-LCL, B lymphoblastoid cell line; D₄, deuterated; LC-MS, nanoHPLC-coupled mass spectrometry analysis; LC-MS/MS, nanoHPLC-coupled tandem mass spectrometry analysis; MPXV, monkeypox virus; MS, mass spectrometry; MVA, modified vaccinia virus Ankara; m/z, mass to charge ratio of peptide ion; NIC, nicotinic acid; p.b., postboost; pI, isoelectric point; VACV, vaccinia virus; WR, Western Reserve; ORF, open reading frame.