Establishment of real-time polymerase chain reaction-based assay for quantitation of Epstein-Barr virus DNA in healthy donors and in patients with EBV associated lymphoma.

Epstein-Barr virus (EBV) is associated with several malignancies including nasopharyngeal carcinoma and lymphoma in immunocompromised patients. Quantitative monitoring of EBV DNA in these patients has recently become essential for management of the disease. In the present study the authors developed a rapid and reliable real-time PCR to quantify the EBV DNA in peripheral blood mononuclear cell (PBMC) using hybridization probe technique. The real-time primers and probes in this real-time PCR system were designed based on EBNA-1 sequence. The newly-established real-time PCR demonstrated its high sensitivity (as few as 10 copies of EBV could be detected) and specificity. The intra- and inter-assay variations of the assay were shown to be within a 0.5-log10-difference range. A total of 2 EBV-seronegative, 14 EBV-seropositive healthy donors and 4 patients with PCNSL were enrolled into the study. Our results revealed the median of EBV-DNA in lymphoma patients (7886 copies/10(6) PBMC or 15,150 copies /microg DNA) was higher than that of healthy donors (<10 copies/l0(6) PBMC or <10 copies/microg DNA) with statistic significance (P < 0.01). Assessment of this assay in larger number of donors and patients will provide clinical cut-off values which are essential for monitoring and diagnosis of EBV-associated diseases.

Establishment of cytotoxic T lymphocytes specific for autologous Epstein-Barr virus in HIV-infected patients: the feasibility study of EBV-specific immunotherapy for patients with EBV-associated lymphoma.

Cytotoxic T lymphocytes specific for Epstein-Barr virus (EBV) have previously been successfully used in immunotherapy of Posttransplant lymphoproliferative disease (PTLD) and Hodgkin's disease. A similar strategy has never been employed in HIV/AIDS patients who also have high risk of developing EBV-associated lymphoma. A total of 5 HIV-infected patients were enrolled to evaluate their EBV-specific T cell responses by Interferon-gamma (IFNgamma) ELISpot assays. Most patients had detectable T cell responses, mainly directed at Epstein-Barr nuclear antigen (EBNA-3). The authors wanted to see whether it was possible to augment magnitude and spectrum of the EBV responses by stimulating patient PBMC with cells presenting autologous EBV antigens. The authors successfully established spontaneously EBV-transformed lymphoblastoid cell lines (EBVh-BCL) and used them for generation of EBV-specific CTL (EBV-CTL). The EBVh-CTL lines established in the present study were not only highly cytotoxic against the autologous virus but also able to secrete IFNgamma detected by ELISpot. The authors are now in the process of generating these lines in a large number and in a clinical grade for adoptive immunotherapy.