Combination of Epstein-Barr virus nuclear antigen 1, 3 and lytic antigen BZLF1 peptide pools allows fast and efficient stimulation of Epstein-Barr virus-specific T cells for adoptive immunotherapy.

Background: Epstein-Barr virus (EBV) infection is a major cause of morbidity following hematopoietic stem cell transplantation. EBV-infected B cells may not respond to rituximab treatment and may lead to a life-threatening post-transplantation lymphoproliferative disorder. Adoptive cellular immunotherapy using EBV-lymphoblastoid cell lines (LCL) as stimulating antigen has proved effective in restoring specific immunity. However, EBV presents several immunodominant antigens, and developing a swift and effective clinical-grade immunotherapy relies on the definition of a Good Manufacturing Practices (GMP) universal stimulating antigen.

Methods: Peripheral blood mononuclear cells (PBMCs) from six donors with a cellular immune response against EBV were immunoselected after stimulation with a new EBV antigen associated with an EBNA3 peptide pool.

Results: After immunoselection, a mean of 0.53 ± 0.25 × 10⁶ cells was recovered consisting of a mean of 24.77 ± 18.01% CD4⁺-secreting interferon (IFN)-γ and 51.42 ± 26.92% CD8⁺-secreting IFN-γ. The T memory stem cell sub-population was identified. EBV-specific T cells were expanded in vitro, and their ability to secrete IFN-γ and to proliferate after re-stimulation with EBV antigen was confirmed. A specific lysis was observed against autologous target cells pulsed with EBV peptide pools (57.6 ± 11.5%) and against autologous EBV-LCL (18.3 ± 7.3%). A mean decrease of 94.7 ± 3.3% in alloreactivity against third-party donor mononuclear cells with EBV-specific T cells was observed compared with PBMCs before selection.

Conclusions: Our results show that a combination of peptide pools including EBNA3 is needed to generate EBV-specific T cells with good specific cytotoxicity and devoid of alloreactivity, but as yet GMP grade is not fully achieved.