Abstract
The incidence of Varicella Zoster Virus (VZV) reactivation after T cell depleted allogeneic stem cell transplantation (TCD alloSCT) is 40% the first year after transplantation. CD8 T cells are important in herpes virus reactivations. To analyze the role of CD8 T cells in VZV reactivation, we set out to develop multimeric MHC peptide complexes to be able to identify VZV specific CD8 T cells. Predictive algorithms were used to generate potential immunogenic peptides from Immediate Early (IE) proteins 4, 62 and 63 of VZV that bind in HLA-A2. 127 nonamer peptides were synthesized and tested by a high throughput peptide binding assay (REVEAL5™ assay). Of the peptides that were capable of sufficient binding to the MHC molecules, ProVE™ Pentamers were synthesized. 61 Pentamers were produced and used to screen for HLA-A2 restricted VZV specific T cells in peripheral blood derived from patients with clinical VZV reactivation after TCD alloSCT. In 6 out of 18 HLA-A2 positive patients after VZV reactivation, at a median of 43 days after onset of reactivation, T cells stained positive with the multimeric MHC complex that binds the ALW peptide of the IE62 protein of VZV (IE62-ALW-A2). The percentage of IE62-ALW-A2 positive T cells detected in these 6 patients ranged from 0.02% – 0.13% of CD8 T cells, with a median of 0.04% of CD8 T cells. To confirm the specificity of the Pentamer positive T cells, the IE62-ALW-A2 Pentamer positive T cells were sorted from one patient single cell per well, and expanded. The T cell clones exerted cytolytic activity against HLA-A2 positive EBV-lymphoblastoid cell lines loaded with the IE62-ALW-A2 peptide, whereas unloaded target cells were not lysed. HLA-A2 positive COS cells transfected with the IE62 gene and thus endogenously expressing the IE62 protein were recognized by IE62-ALW-A2 positive T cells as demonstrated by IFNg production. In addition, peripheral blood lymphocytes were stimulated in vitro with the IE62-ALW-A2 peptide in combination with IL-2 and IL-15, and we demonstrated with this procedure that IE62-ALW-A2 positive CD8 T cells were present at a low frequency in another 3 of the 12 IE62-ALW-A2 negative patients. To study whether the immune response against the IE62-ALW-A2 epitope correlated with the course of the clinical infection, we studied the percentage of IE62-ALW-A2 positive T cell during a VZV reactivation in one patient. Six days prior to reactivation, only 0.03% of the CD8 T cells were IE62-ALW-A2 positive. At 42 days after onset of the reactivation, 0.23% of the CD8 T cells were Pentamer positive. After the reactivation resolved, IE-62-ALW-A2 specific CD8 T cells declined to 0.09% at day 49 and 0.03% at day 145 after reactivation. In conclusion, we identified by using a high throughput peptide binding assay and Pentamer production, a new immune dominant epitope of VZV that is recognized by HLA-A2 restricted CD8+ T cells. We demonstrate that IE62-ALW-A2 specific T cells can recognize and lyse target cells that endogenously express the IE62 protein of VZV. In addition, we demonstrate that IE62-ALW-A2 specific T cells expand during the VZV specific immune response. This dominant epitope of VZV in combination with multimeric complexes can be a useful tool to study the immune response to VZV infection and possibly to VZV vaccination.
Identification of wild-type and mutant p53 peptides binding to HLA-A2 assessed by a peptide loading-deficient cell line assay and a novel major histocompatibility complex class I peptide binding assay