Three T-cell epitopes within the C-terminal 265 amino acids of the matrix protein pp65 of human cytomegalovirus recognized by human lymphocytes

Abstract

Although a T-cell response in human cytomegalovirus (HCMV)-immune individuals exists against the most abundantly expressed protein pp65 of the virus matrix, less is known about the determinants that evoke this response. The aim of the study was to identify regions within HCMV pp65 (ppUL83) that contain sequences for the cellular immune response by the use of three recombinant overlapping beta-galactosidase pp65 fusion proteins (C74, C35, and C47), covering the C-terminal 265 amino acids of the entire pp65 sequence. Two T-cell epitope determinants were recognized by human lymphocytes of healthy, HCMV-seropositive, human leukocyte antigen (HLA)-typed individuals. One T-cell determinant (amino acids [aa] 303-388) was localized in the mid-region of the entire pp65 sequence and a second T-cell determinant (aa 477-561) within the C-terminal region. By fine mapping with synthetic hexadecamer peptides three T-cell epitopes were identified within these two regions: P10-I (aa 361-376) in the mid-region, P3-II (aa 485-499), and P6-II (aa 509-524) in the C-terminal region. Inhibition studies with monoclonal antibodies to HLA class I or class II revealed a class II restricted response to peptides P10-I or P6-II, respectively. P10-I responders shared the HLA-DR11 allele and P6-II responders the -DR3 allele. Therefore, these T-cell epitopes of HCMV pp65 might be presented in association with particular HLA class II alleles.

Purification of Ag-Specific T Lymphocytes After Direct Peripheral Blood Mononuclear Cell Stimulation Followed by CD25 Selection. I. Application to CD4+ or CD8+ Cytomegalovirus Phosphoprotein pp65 Epitope Determination

“…Finally, the new clinical possibilities offered by the strategy presented can be rapidly tested because its relies on methods and reagents, namely immunomagnetic sorting, canarypox vector, and anti-CD25 mAbs, that have already been validated for clinical applications. b Epitope that has also been detected by others (26). c Epitope that has also been detected by others (27).…”

Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines

“…Two previously described CMV epitope specificities [21,22], restricted through HLA-DRB1*01 and DRB1*13, were confirmed. Our DRB5-restricted T cell clone recognized amino acids 489-507, already described as a presumably DR11-restricted epitope [21,22]. This sequence, which also contains the HLA class I (HLA-A2) epitope NLVPMVATV, can therefore be presented by at least two different class II molecules.…”

The Immunodominant CD8 T Cell Response to the Human Cytomegalovirus Tegument Phosphoprotein pp65495–503 Epitope Critically Depends on CD4 T Cell Help in Vaccinated HLA-A*0201 Transgenic Mice

“…CD4 T cell help is thus important to induce e6-specific (but not e3-and e8-specific) CD8 T cell responses by monospecific (peptidebased) and multispecific (DNA-based) vaccines. The pp65 Ag encodes several HLA-DR-binding motifs (44)(45)(46) that could bind to HLA-DR1 molecules in A2-DR1 mice and trigger CD4 T cell help (58). It is thus difficult to identify CD4 T cell specificities that contribute to the induction of the immunodominant, e6-specific CD8 T cell response in pp65-immune mice.…”

“…The immunodominant, HLA-A*0201-restricted pp65 [495][496][497][498][499][500][501][502][503] epitope is positioned in the pp65 Ag within an overlapping (nested) CD4 T cell helper domain (44)(45)(46). We generated DNA-based vaccines encoding wild-type pp65 or mutant pp65 Ags, in which the pp65 495-503 epitope is deleted and/or cloned into a different position within the pp65 protein, and compared CD8 T cell frequencies with eight HLA-A*0201-restricted pp65 epitopes (e1-e8).…”