title: Identification of major histocompatibility complex class II-restricted antigens and epitopes of the Epstein-Barr virus by a novel bacterial expression cloning approach.
abstract: 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.
authors.affiliation: Children's Hospital, Technical University Munich, Kölner Platz 1, D-80804 Munich, Germany.
authors.forename: Slavoljub
authors.initials: S
authors.lastname: Milosevic
authors.forename: Uta
authors.initials: U
authors.lastname: Behrends
authors.forename: Dinesh
authors.initials: D
authors.lastname: Adhikary
authors.forename: Josef
authors.initials: J
authors.lastname: Mautner
mesh_terms: D017951:Antigen Presentation; D000956:Antigens, Viral / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000276:immunology*; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology / Q000821:virology
chemical_list: D000956:Antigens, Viral; D004274:DNA, Recombinant; D000939:Epitopes; D000949:Histocompatibility Antigens Class II; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase