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Journal of Virology, November 2006, p. 10357-10364, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.01193-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of Major Histocompatibility Complex Class II-Restricted Antigens and Epitopes of the Epstein-Barr Virus by a Novel Bacterial Expression Cloning Approach

Slavoljub Milosevic, Uta Behrends, Dinesh Adhikary, and Josef Mautner*

Clinical Cooperation Group, Technical University Munich, Children's Hospital, Hematology-Oncology, Kölner Platz 1, 80804 Munich, Germany, and Institute for Clinical and Molecular Biology, GSF-National Research Center for Environment and Health, Marchioninistr. 25, 81377 Munich, Germany

Received 8 June 2006/ Accepted 7 August 2006

Epstein-Barr virus (EBV)-specific T cells have been successfully used to treat or prevent EBV-positive lymphoproliferative disease in hematopoietic stem cell transplant recipients, but the antigens recognized by the infused CD4+ T cells have remained unknown. Here, we describe a simple procedure that permits the identification of viral T-helper (TH)-cell antigens and epitopes. This direct antigen identification method is based on the random expression of viral polypeptides fused to chloramphenicol acetyltransferase (CAT) in bacteria, which are subsequently fed to major histocompatibility complex class II+ antigen-presenting cells and probed with antigen-specific T cells. The fusion of antigenic fragments to CAT offers several advantages. First, chloramphenicol treatment allows the selection of bacteria expressing antigen-CAT fusion proteins in frame, which greatly reduces the number of colonies to be screened. Second, antigenic fragments fused to CAT are expressed at high levels, even when derived from proteins that are toxic to bacteria. Third, the uniformly high expression level of antigen-CAT fusion proteins permits the establishment of large and representative pool sizes. Finally, antigen identification does not require knowledge of the restriction element and often leads directly to the identification of the T-cell epitope. Using this approach, the BALF4 and BNRF1 proteins were identified as targets of the EBV-specific T-helper-cell response, demonstrating that lytic cycle antigens are a relevant component of the EBV-specific TH-cell response.

* Corresponding author. Mailing address: Children's Hospital, Technical University Munich, Kölner Platz 1, D-80804 Munich, Germany. Phone: 49-89-7099518. Fax: 49-89-7099500. E-mail: mautner{at}gsf.de.

Journal of Virology, November 2006, p. 10357-10364, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.01193-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Adhikary, D., Behrends, U., Feederle, R., Delecluse, H.-J., Mautner, J. (2008). Standardized and Highly Efficient Expansion of Epstein-Barr Virus-Specific CD4+ T Cells by Using Virus-Like Particles. J. Virol. 82: 3903-3911 [Abstract] [Full Text]  


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