Identification of HLA-A2 binding peptides from cytomegalovirus and its recognition by cytotoxic T lymphocytes..

The human pathogen CMV, is a major cause of mortality in the case of immunocompromised recipients of allogeneic bone marrow transplants. The CD8⁺ class I restricted response to CMV plays a crucial role in the control of CMV infection in asymptomatic immunocompetent hosts; however, the viral antigen recognised by CD8⁺ CTLs are not well characterised. The lower matrix 65 kD phosphoprotein is a prime candidate for production of CMV antigenic peptides and it been has shown that it can act as target for CTL's. We have used an in vitro assay to investigate potential viral antigens recognised by HLA-A2 restricted CTLs. Synthetic peptides were designed using the published pp65 protein sequence to contain the consensus binding motif for HLA-A2. These peptides were used in a standard T2 binding assay. T2 cells (HLA - A2, B5) were incubated overnight in the presence of the synthetic peptides. The positive control HLA-A2-binding influenza matrix peptide, AE41, resulted in a 3 fold increase in cell surface HLA-A2 expression. Incubation with the designed CMV pp65 peptides resulted in varying degrees of HLA-A2 expression. In particular, peptide AE45 showed a two-fold increase in expression. The aim of our project is to define CMV specific epitopes recognised by cytotoxic T cells (CTL). Using the T2 binding assay we have identified certain CMV pp65 synthetic peptides that bind specifically to the HLA-A2 molecule. We are now in the process of analysing the recognition of such pp65 peptides by CTL's especific for the pp65 protein. Further definition of CMV specific peptide epitopes presented by particular Class I molecules will allow studies of the CTL response to CMV in infected patients with defined HLA haplotypes.