title: Identification of an atypical CD8 T cell epitope encoded by murine cytomegalovirus ORF-M54 gaining dominance after deletion of the immunodominant antiviral CD8 T cell specificities.
abstract: Control of murine cytomegalovirus (mCMV) infection is mediated primarily by CD8 T cells, with four specificities dominating in BALB/c mice. Functional deletion of the respective immunodominant epitopes (IDEs) in mutant virus Δ4IDE revealed a still efficient control of infection. In a murine model of hematopoietic cell transplantation and infection with Δ4IDE, an mCMV-specific open reading frame (ORF) library screening assay indicated a strong CD8 T cell reactivity against the ORF-M54 product, the highly conserved and essential mCMV homolog of human CMV DNA polymerase UL54, which is a known inducer of in vivo protection against mCMV by DNA immunization. Applying bioinformatic algorithms for CD8 T cell epitope prediction, the top-scoring peptides were used to stimulate ex vivo-isolated CD8 T cells and to generate cytolytic T cell lines; yet, this approach failed to identify M54 epitope(s). As an alternative, a peptide library consisting of 549 10-mers with an offset of two amino acids (aa), covering the complete aa-sequence of the M54 protein, was synthesized and used for the stimulation. A region of 12 aa proved to encompass an epitope. An 'alanine walk' over this antigenic 12-mer and all possible 11-, 10- and 9-mers derived thereof revealed aa-residues critical for antigenicity, and terminal truncations identified the H-2D(d) presented 8-mer M5483-90 as the optimal epitope. An increased frequency of the corresponding CD8 T cells in the absence of the 4 IDEs indicated immunodomination by the IDE-specific CD8 T cells as a mechanism by which the generation of M54-specific CD8 T cells is inhibited after infection with wild-type mCMV.
authors: Institute for Virology and Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg-University Mainz, Obere Zahlbacher Strasse 67, Hochhaus am Augustusplatz, 55131, Mainz, Germany, r.holtappels@uni-mainz.de.
authors: Rafaela
authors: R
authors: Holtappels
authors: Niels A W
authors: NA
authors: Lemmermann
authors: Doris
authors: D
authors: Thomas
authors: Angélique
authors: A
authors: Renzaho
authors: Matthias J
authors: MJ
authors: Reddehase
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D019295:Computational Biology; D003602:Cytotoxicity, Immunologic; D018604:Epitope Mapping; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000276:immunology*; D005260:Female; D016679:Genome, Viral; D006566:Herpesviridae Infections / Q000276:immunology* / Q000821:virology; D064532:Histocompatibility Antigen H-2D / Q000276:immunology; D016056:Immunodominant Epitopes / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D018146:Muromegalovirus / Q000235:genetics / Q000276:immunology*; D009154:Mutation; D016366:Open Reading Frames / Q000235:genetics / Q000276:immunology*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000276:immunology
chemical_list: D000956:Antigens, Viral; D018984:Epitopes, T-Lymphocyte; D064532:Histocompatibility Antigen H-2D; D016056:Immunodominant Epitopes; D019151:Peptide Library; D010455:Peptides