A recombinant adenovirus expressing an Epstein-Barr virus (EBV) target antigen can selectively reactivate rare components of EBV cytotoxic T-lymphocyte memory in vitro

Abstract

While the bulk of a virus-induced cytotoxic T-lymphocyte (CTL) response may focus on a few immunodominant viral antigens, in certain tumor virus systems the detectability of clones recognizing other, subdominantantigens can assume particular importance. By using the human CTL response to Epstein-Barr virus (EBV) as a model system, here we show that even rare components of virus-specific memory can be selectively reactivated in vitro when the relevant target antigen is expressed in autologous stimulator cells from a recombinant adenovirus (RAd) vector. We generated a replication-deficient adenovirus, RAd-E3C, which in skin fibroblast cultures expressed the EBV nuclear antigen EBNA3C at a 10-to 100-fold-higher level than that naturally present in EBV-transformed lymphoblastoid cell lines (LCLs). Initial experiments with a donor whose polyclonal CTL response to LCL stimulation contained a strong EBNA3C-specific component showed that these CTLs could be efficiently reactivated by in vitro stimulation either with RAd-E3C-infected fibroblasts or with RAd-E3C-infected peripheral blood mononuclear cells. Then we studied donors whose responses to LCL stimulation contained little if any detectable EBNA3C reactivity but were dominated by clones recognizing other EBV target antigens; in vitro stimulation with RAd-E3C-infected peripheral blood mononuclear cells selectively reactivated EBNA3C-specific CTL clones from these individuals, with the epitope specificities of responses subsequently identified at the peptide level. This RAd-based approach could be applied more generally to screen for human CTL responses against any candidate target antigen expressed by tumor cells.

Selective amino acid substitutions of a subdominant Epstein-Barr virus LMP2-derived epitope increase HLA/peptide complex stability and immunogenicity: implications for immunotherapy of Epstein-Barr virus-associated malignancies

“…In this investigation we first evaluated CLG-specific responses in a group of HLA-A*0201-positive EBVseropositive individuals. Our results demonstrate that all donors have CLG-specific CTL precursors that can be reactivated by specific peptide stimulations, a finding which further favors the use of the CLG epitope as target of EBV-specific immunotherapies and the employment of specific stimulations to amplify a desired CTL population [6][7][8].…”

“…EBV induces long-lasting CTL memory, which is believed to play an important role in controlling EBV-carrying cells in the infected host. Thus, EBV-specific CTL precursors can be reactivated in a relatively large number from the peripheral blood of EBV-seropositive individuals by in vitro stimulation with autologous virus-infected B cells [5], or by selective stimulations [6][7][8]. Recent studies have shown that, in each individual, such CTL responses are a composite of several virus-induced reactivities [9][10][11][12].…”

The generation of LMP2a-specific cytotoxic T lymphocytes for the treatment of patients with Epstein-Barr virus-positive Hodgkin disease

“…EBV, a widespread lymphotrophic herpes virus, induces long‐lasting CTL memory which is believed to play an important role in controlling EBV‐harboring cells in the infected host. Therefore, EBV‐specific CTL precursors can be reactivated from the peripheral blood of EBV‐seropositive individuals by in vitro stimulation with autologous LCL 18 or by selective restimulation 19, 20. In each individual, these CTL responses are a mixture of several virus‐induced reactivities, each reactivity being directed against one or more of the viral proteins presented in the context of particular HLA class I restriction determinants 8, 9, 21.…”

Immediate-Early Transactivator Rta of Epstein-Barr Virus (EBV) Shows Multiple Epitopes Recognized by EBV-Specific Cytotoxic T Lymphocytes

“…a Obtained by subtracting the lysis of target cells pulsed with the control peptide HIV-env from the lysis of target cells pulsed with peptide Rta [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] . The E/T ratio was 10:1. and target cells.…”

“…Eight CTL lines recognizing the seven different epitopes were examined in this way; six lines showed reactivity against the vaccinia virus-infected cells, whereas two (B-3 and E-1, specific for peptides Rta-67 and Rta-50, respectively) obviously were not able to kill vaccinia virus-infected cells. Inappropriate processing of vaccinia virus antigens has been described (2,45); in such cases, CTL reactivities against a certain protein or epitope can be demonstrated by use of target cells transiently transfected with an expression vector for the protein in question or by application of other, e.g., adenovirus-based, expression systems (32,42).…”