Dendritic cells transduced with an adenovirus vector encoding Epstein-Barrvirus latent membrane protein 2B: A new modality for vaccination

Epstein-Barr virus (EBV) is a herpesvirus commonly associated with several malignancies, particularly in immunocompromised hosts. As a strategy for st imulating immunity against EBV for the treatment of EBV-associated tumors, we have genetically engineered dendritic cells (DC) to express EBV antigens , such as latent membrane protein 2B (LMP2B), using recombinant adenovirus vectors. CD8(+) T lymphocytes from HLA-A2.1(+), EBV-seropositive healthy do nors were cultured with autologous DC infected with recombinant adenovirus vector AdEGFP, encoding an enhanced green fluorescent protein (EGFP), or Ad LMP2B at a multiplicity of infection of 250. After 48 h, >95% of the DC wer e positive far EGFP expression as assessed by fluorescence-activated cell s orting analysis, indicating efficient gene transfer. AdLMP2-transduced DC w ere used to stimulate CD8(+) T cells. Responder CD8(+) T cells were tested for gamma interferon (IFN-gamma) release by enzyme-linked spot (ELISPOT) as say and cytotoxic activity. Prior to in vitro stimulation, the frequencies of T-cells directed against two HLA-A2-presented LMP2 peptides (LMP2 329-33 7 and LMP2 426-434) were very low as assessed by IFN-gamma spot formation ( T-cell frequency, <0.003%)). IFN-gamma ELISPOT assays performed at day 14 s howed a significant (2-log) increase of the day 0 frequency of T cells reac tive against the LMP2 329-337 peptide, from 0.003 to 0.3 (P < 0.001). Moreo ver, specific cytolytic activity was observed against the autologous EBV B- lymphoblastoid cell lines after 21 days of stimulation of T-cell responders with AdLMP2-transduced DC (P < 0.01), In summary, autologous mature DC gen etically modified with an adenovirus encoding EBV antigens stimulate the ge neration of EBV-specific CD8(+) effector T cells in vitro, supporting the p otential application of EBV-based adenovirus vector vaccination for the imm unotherapy of the EBV-associated malignancies.