Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia: implications for vaccine development.

Abstract

Summal~There is considerable interest in designing an effective vaccine to the ubiquitous Epstein-Barr virus (EBV). An important role for EBV-specific cytotoxic T lymphocytes (CTLs) in eliminating virus-infected cells is well established. Limited studies using a small number of immune donors have defined target epitopes within the latent antigens of EBV. The present study provides an extensive analysis of the distribution of class I-restricted CTL epitopes within EBV-encoded proteins. Using recombinant vaccinia encoding individual EBV latent antigens (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, LP, and LMP 1), we have successfully localized target epitopes recognized by CTL clones from a panel of 14 EBV-immune donors. Of the 20 CTL epitopes localized, five were defined at the peptide level. Although CTL clones specific for nine epitopes recognized both type I and type 2 transformants, a significant number of epitopes (7/16 epitopes for which EBV type specificity was determined) were detected only on type 1 EBV transformants. Vaccinia recombinants encoding EBNA 3A and EBNA 3C were recognized more frequently than any other vaccinia recombinants used in this study, while no CTL epitopes were localized in EBNA 1. Surprisingly, epitope specificity for a large number of EBV-specific CTL clones could not be localized, although vaccinia recombinants used in this study encoded most of the latent antigens of EBV. These results suggest that any EBV vaccine based on CTL epitopes designed to provide widespread protection will need to include not only latent antigen sequences but also other regions of the genome. The apparent inability of human CTLs to recognize EBNA I as a target antigen, often the only latent antigen expressed in Burkitt's lymphoma and nasopharyngeal carcinoma, suggests that EBV-specific CTL control of these tumors will not be feasible unless the downregulation of latent antigens can be reversed.

EBV-Induced Human CD8+ NKT Cells Synergise CD4+ NKT Cells Suppressing EBV-Associated Tumours Upon Induction of Th1-Bias

“…Human fetal thymic cells (containing 1  10 7 thymocytes, thymocytes: thymic epithelial cells and other stromal cells  1 : 1; immature and mature NKT cells were positively depleted) (17,18) were transplanted into thymi of anaesthetised SCID mice. The chimaeras were then intrathymically challenged with EBV (10 7 pfu) (14,21). The challenge was repeated once after 6 days.…”

Assessment and characterization of the cytolytic T lymphocyte response against Epstein–Barr virus in patients with non-Hodgkin’s lymphoma after autologous peripheral blood stem cell transplantation

“…Although blocking experiments were not undertaken in all patients investigated, the data obtained showed the same characteristics for the EBV-specific CTL response as reported for healthy individuals. [13][14][15] It can be substantially blocked by antibodies directed against CD3 or CD8, while anti-CD4 or anti-MHC class II show no effect, and the main effector cells are CD8-positive CTLs. MHC class I restriction is further underlined by experiments using EBNA-derived nonapeptides presented by autologous PHA blasts, which demonstrated that the EBV-directed CTLs recognize only the peptide known to be associated with HLA-B8 (therefore matching the patients HLA-haplotype), whereas another, HLA-A11-restricted EBV-peptide did not lead to a significant lysis.…”

“…11,12 This surveillance is based mainly on CD8-positive HLA-restricted memory T cells found in the peripheral blood of seropositive individuals which can be reactivated in vitro by challenge with autologous EBVinfected B cells. [13][14][15][16][17] In immunosuppressed individuals, reactivation of this latent EBV infection can lead to the outgrowth of EBV-transformed B lymphocytes with unregulated polyclonal or monoclonal lymphoproliferation, of which the latter has a rapidly progressive and uniformly fatal course. 18,19 For this reason, the EBV-directed CTL response has been carefully investigated after allogeneic BMT, where up to 30% of the recipients of allografts from mismatched or matched unrelated donors may develop EBV lymphoma.…”

Creation of immune ‘stealth’ genes for gene therapy through fusion with the Gly-Ala repeat of EBNA-1

“…45 The observation that the GAr does not inhibit priming of CTL is important and consistent with reports describing the presence of EBNA-1-specific CTL in EBV-seropositive individuals. [31][32][33][34][35][36] As antigen presentation by professional APC, most likely DC, is crucial to the initiation of virusspecific CTL responses, the presence of EBNA-1-specific CTL in EBV-positive donors suggests that antigen processing for MHC class I by specialized APC is not hampered by the GAr. In case EBV-specific CTL are induced through the direct route following EBV infection of DC, these observations would indicate that antigen presentation in DC differs intrinsically from presentation by 'nonprofessional' APC.…”

“…Although EBNA-1-specific CTL have been described in infectious mononucleosis patients and healthy carriers, they cannot recognize EBV-infected cells. [31][32][33][34][35][36] The failure to recognize endogenously expressed EBNA-1 has been attributed to the glycine-alanine repeat (GAr) domain in the EBNA-1 sequence that protects EBNA-1 from proteasomal degradation and subsequent presentation in the context of MHC class I. 37,38 This successful immune-evasion strategy points to the unique opportunity to hide cells expressing transgenes from CTLmediated target-cell destruction by incorporation of the GAr sequence into the transgene.…”