Epstein–Barr virus vector-mediated gene transfer into human B cells: potential for antitumor vaccination

Abstract

The efficient gene transfer of immunostimulatory cytokines into autologous tumor cells or the transfer of tumor-associated antigens into professional antigen-presenting cells is a prerequisite for many immunotherapeutic approaches. In particular with B cells, the efficiency of gene uptake is one of the limiting factors in cell-based vaccine strategies, since normal and malignant human B cells are commonly refractory to transducing gene vectors. Due to its natural tropism for human B cells, Epstein-Barr virus (EBV), a human herpes virus, might be an option, which we wanted to explore. EBV efficiently infects human B cells and establishes a latent infection, while the viral genome is maintained extrachromosomally. Although these characteristics are attractive, EBV is an oncogenic virus. Here, we present a novel EBV-derived vector, which lacks three EBV genes including two viral oncogenes and an essential lytic gene, and encodes granulocyte-macrophage colony-stimulating factor (GM-CSF) as a cytokine of therapeutic interest. We could show that EBV vectors efficiently transduce different B-cell lines, primary resting B cells, and tumor cells of B-cell lineage. Vectorderived GM-CSF was expressed in sufficient amounts to support the maturation of dendritic cells and their presentation of model antigens to cognate T-cell clones in autologous settings and an allogeneic, HLA-matched assay. We conclude that the EBV vector system might offer an option for ex vivo manipulation of B cells and gene therapy of B-cell lymphomas. Gene Therapy (2006) 13, 150-162.

The Lytic Phase of Epstein-Barr Virus Requires a Viral Genome with 5-Methylcytosine Residues in CpG Sites

“…Another example is safe viral vectors for human B cells, which are particularly recalcitrant to the transduction of therapeutic genes (17). Based on the highly specific B-cell tropism of EBV, such gene vectors could be engineered in sufficient quantities for the immune therapy of B-cell lymphomas or leukemias (22).…”

Oncolytic viruses in cancer therapy

“…In this vector the Tag protein responsible for cellular transformation has been deleted, and although the recombinant SV40 vectors still integrate, tumorigenesis has not been observed [86]. Moreover, despite that the Epstein-Barr virus is associated with a number of different types of cancers and is therefore classified as a type 1 carcinogen, it has recently been developed into an expression vector for non-lytic tumor vaccination against B cell lymphomas [87]. The vector has been rendered safe (non-oncogenic and replication-deficient) by deleting two oncogenic genes and one gene critical for the lytic cycle of the virus.…”

Genetic design of an optimized packaging cell line for gene vectors transducing human B cells

“…22,28,37 Briefly, 3 Â 10 5 Raji cells were incubated at 371C in 24-well cluster plates with different dilutions of the gene vector stocks to be analyzed. The absolute number of GFP + cells was determined by UV microscopy in a defined fraction of the total Raji cell population 3 days after infection.…”

“…Supernatants were collected and used to infect Raji cells according to our standard quantification method. 22,28,37 With the p1933/GFP_amp gene vector, we obtained approximately 1. 19,20 To analyze the tropism of gene vectors generated with the 293-VII+ packaging cell line, we transduced primary resting B cells prepared from adenoids or peripheral blood, activated B blasts 39 and B cells from patients with chronic lymphocytic leukemia.…”