Dendritic cells transduced with an adenovirus vector encoding Epstein-Barr virus 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 stimulating 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 donors were cultured with autologous DC infected with recombinant adenovirus vector AdEGFP, encoding an enhanced green fluorescent protein (EGFP), or AdLMP2B at a multiplicity of infection of 250. After 48 h, >95% of the DC were positive for EGFP expression as assessed by fluorescence-activated cell sorting analysis, indicating efficient gene transfer. AdLMP2-transduced DC were used to stimulate CD8(+) T cells. Responder CD8(+) T cells were tested for gamma interferon (IFN-gamma) release by enzyme-linked spot (ELISPOT) assay and cytotoxic activity. Prior to in vitro stimulation, the frequencies of T-cells directed against two HLA-A2-presented LMP2 peptides (LMP2 329-337 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 showed a significant (2-log) increase of the day 0 frequency of T cells reactive against the LMP2 329-337 peptide, from 0.003 to 0.3 (P < 0.001). Moreover, 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 genetically modified with an adenovirus encoding EBV antigens stimulate the generation of EBV-specific CD8(+) effector T cells in vitro, supporting the potential application of EBV-based adenovirus vector vaccination for the immunotherapy of the EBV-associated malignancies.
Ranieri
E
E
Departments of Surgery, Pittsburgh, Pennsylvania 15261, USA.
Herr
W
W
Gambotto
A
A
Olson
W
W
Rowe
D
D
Robbins
P D
PD
Kierstead
L S
LS
Watkins
S C
SC
Gesualdo
L
L
Storkus
W J
WJ
D000260:Adenoviruses, Human / Q000276:immunology*; D017951:Antigen Presentation / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000821:virology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000276:immunology*; D015789:HLA-A2 Antigen / Q000276:immunology; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000276:immunology*; D006801:Humans; D016130:Immunophenotyping; D010455:Peptides / Q000276:immunology; D014161:Transduction, Genetic; D019444:Vaccines, DNA / Q000235:genetics* / Q000276:immunology; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000276:immunology*; D014765:Viral Vaccines / Q000235:genetics* / Q000276:immunology
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D015789:HLA-A2 Antigen; D010455:Peptides; D019444:Vaccines, DNA; D014763:Viral Matrix Proteins; D014765:Viral Vaccines