EBV Genome Research Articles

Direct Sequencing and Characterization of a Clinical Isolate of Epstein-Barr Virus from Nasopharyngeal Carcinoma Tissue by Using Next-Generation Sequencing Technology

Epstein-Barr virus (EBV)-encoded molecules have been detected in the tumor tissues of several cancers, including nasopharyngeal carcinoma (NPC), suggesting that EBV plays an important role in tumorigenesis. However, the nature of EBV with respect to genome width in vivo and whether EBV undergoes clonal expansion in the tumor tissues are still poorly understood. In this study, next-generation sequencing (NGS) was used to sequence DNA extracted directly from the tumor tissue of a patient with NPC. Apart from the human sequences, a clinically isolated EBV genome 164.7 kb in size was successfully assembled and named GD2 (GenBank accession number HQ020558). Sequence and phylogenetic analyses showed that GD2 was closely related to GD1, a previously assembled variant derived from a patient with NPC. GD2 contains the most prevalent EBV variants reported in Cantonese patients with NPC, suggesting that it might be the prevalent strain in this population. Furthermore, GD2 could be grouped into a single subtype according to common classification criteria and contains only 6 heterozygous point mutations, suggesting the monoclonal expansion of GD2 in NPC. This study represents the first genome-wide analysis of a clinical isolate of EBV directly extracted from NPC tissue. Our study reveals that NGS allows the characterization of genome-wide variations of EBV in clinical tumors and provides evidence of monoclonal expansion of EBV in vivo. The pipeline could also be applied to the study of other pathogen-related malignancies. With additional NGS studies of NPC, it might be possible to uncover the potential causative EBV variant involved in NPC.

The Members of an Epstein-Barr Virus MicroRNA Cluster Cooperate To Transform B Lymphocytes

Epstein-Barr virus (EBV) transforms B lymphocytes through the expression of the latent viral proteins EBNA and latent membrane protein (LMP). Recently, it has become apparent that microRNAs (miRNAs) also contribute to EBV's oncogenic properties; recombinant EBVs that lack the BHRF1 miRNA cluster display a reduced ability to transform B lymphocytes in vitro. Furthermore, infected cells evince a marked upregulation of the EBNA genes. Using recombinant viruses that lack only one member of the cluster, we now show that all three BHRF1 miRNAs contribute to B-cell transformation. Recombinants that lacked miR-BHRF1-2 or miR-BHRF1-3 displayed enhanced EBNA expression initiated at the Cp and Wp promoters. Interestingly, we find that the deletion of miR-BHRF1-2 reduced the expression level of miR-BHRF1-3 and possibly that of miR-BHRF1-1, demonstrating that the expression of one miRNA can potentiate the expression of other miRNAs located in the same cluster. Therefore, the phenotypic traits of the miR-BHRF1-2 null mutant could result partly from reduced miR-BHRF1-1 and miR-BHRF1-3 expression levels. Nevertheless, using an miR-BHRF1-1 and miR-BHRF1-3 double mutant, we could directly assess and confirm the contribution of miR-BHRF1-2 to B-cell transformation. Furthermore, we found that the potentiating effect of miR-BHRF1-2 on miR-BHRF1-3 synthesis can be reproduced with simple expression plasmids, provided that both miRNAs are processed from the same transcript. Therefore, this enhancing effect does not result from an idiosyncrasy of the EBV genome but rather reflects a general property of these miRNAs. This study highlights the advantages of arranging the BHRF1 miRNAs in clusters: it allows the synchronous and synergistic expression of genetic elements that cooperate to transform their target cells.

Epstein-Barr virus exploits intrinsic B-lymphocyte transcription programs to achieve immortal cell growth

Epstein-Barr virus nuclear antigen 2 (EBNA2) regulation of transcription through the cell transcription factor RBPJ is essential for resting B-lymphocyte (RBL) conversion to immortal lymphoblast cell lines (LCLs). ChIP-seq of EBNA2 and RBPJ sites in LCL DNA found EBNA2 at 5,151 and RBPJ at 10,529 sites. EBNA2 sites were enriched for RBPJ (78%), early B-cell factor (EBF, 39%), RUNX (43%), ETS (39%), NFκB (22%), and PU.1 (22%) motifs. These motif associations were confirmed by LCL RBPJ ChIP-seq finding 72% RBPJ occupancy and Encyclopedia Of DNA Elements LCL ChIP-seq finding EBF, NFκB RELA, and PU.1 at 54%, 31%, and 17% of EBNA2 sites. EBNA2 and RBPJ were predominantly at intergene and intron sites and only 14% at promoter sites. K-means clustering of EBNA2 site transcription factors identified RELA-ETS, EBF-RUNX, EBF, ETS, RBPJ, and repressive RUNX clusters, which ranked from highest to lowest in H3K4me1 signals and nucleosome depletion, indicative of active chromatin. Surprisingly, although quantitatively less, the same genome sites in RBLs exhibited similar high-level H3K4me1 signals and nucleosome depletion. The EBV genome also had an LMP1 promoter EBF site, which proved critical for EBNA2 activation. LCL HiC data mapped intergenic EBNA2 sites to EBNA2 up-regulated genes. FISH and chromatin conformation capture linked EBNA2/RBPJ enhancers 428 kb 5' of MYC to MYC. These data indicate that EBNA2 evolved to target RBL H3K4me1 modified, nucleosome-depleted, nonpromoter sites to drive B-lymphocyte proliferation in primary human infection. The primed RBL program likely supports antigen-induced proliferation.

Treatment of Epstein-Barr Virus-related Hemophagocytic Lymphohistiocytosis (EBV-HLH); Update 2010

The clinical features of Epstein-Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH) vary significantly, from mild to severe, at the time of the treatment decision. There are many reports of successful treatments, for example conservative treatment without etoposide, HLH-94/2004-type immunochemotherapy with etoposide, and hematopoietic stem cell transplantation. When considering the treatment of EBV-HLH, the most important factor is the finding that a survival benefit is obtained when etoposide-containing therapy is initiated within 4 weeks of diagnosis. This indicates that there may be a window for observation or conservative corticosteroid/cyclosporine A or intravenous immunoglobulin (IVIG) treatment; however, once the disease is defined as "high risk" and/or refractory to such therapy, prompt introduction of etoposide (ideally within 4 wk) is recommended. In deciding whether the disease is "high-risk," evaluation of clinical staging, EBV genome copy numbers in the serum, cellular EBV tropism, chromosome analysis, and screening for hereditary immuno-deficient diseases such as familial HLH, are required.

Inflammation and Epstein-Barr Virus Infection Are Common Features of Myasthenia Gravis Thymus: Possible Roles in Pathogenesis

The thymus plays a major role in myasthenia gravis (MG). Our recent finding of a persistent Epstein-Barr (EBV) virus infection in some MG thymuses, combined with data showing that the thymus is in a proinflammatory state in most patients, supports a viral contribution to the pathogenesis of MG. Aim of this study was to gain further evidence for intrathymic chronic inflammation and EBV infection in MG patients. Transcriptional profiling by low density array and real-time PCR showed overexpression of genes involved in inflammatory and immune response in MG thymuses. Real-time PCR for EBV genome, latent (EBER1, EBNA1, LMP1) and lytic (BZLF1) transcripts, and immunohistochemistry for LMP1 and BZLF1 proteins confirmed an active intrathymic EBV infection, further supporting the hypothesis that EBV might contribute to onset or perpetuation of the autoimmune response in MG. Altogether, our results support a role of inflammation and EBV infection as pathogenic features of MG thymus.

EBV Latent Membrane Protein 1 Is a Negative Regulator of TLR9

EBV infects most of the human population and is associated with a number of human diseases including cancers. Moreover, evasion of the immune system and chronic infection is an essential step for EBV-associated diseases. In this paper, we show that EBV can alter the regulation and expression of TLRs, the key effector molecules of the innate immune response. EBV infection of human primary B cells resulted in the inhibition of TLR9 functionality. Stimulation of TLR9 on primary B cells led to the production of IL-6, TNF-α, and IgG, which was inhibited in cells infected with EBV. The virus exerts its inhibitory function by decreasing TLR9 mRNA and protein levels. This event was observed at early time points after EBV infection of primary cells, as well as in an immortalized lymphoblastoid cell line. We determined that the EBV oncoprotein latent membrane protein 1 (LMP1) is a strong inhibitor of TLR9 transcription. Overexpression of LMP1 in B cells reduced TLR9 promoter activity, mRNA, and protein levels. LMP1 mutants altered in activating the NF-κB pathway prevented TLR9 promoter deregulation. Blocking the NF-κB pathway recovered TLR9 promoter activity. Mutating the NF-κB cis element on the TLR9 promoter restored luciferase transcription in the presence of LMP1. Finally, deletion of the LMP1 gene in the EBV genome abolished the ability of the virus to induce TLR9 downregulation. Our study describes a mechanism used by EBV to suppress the host immune response by deregulating the TLR9 transcript through LMP1-mediated NF-κB activation.

Epstein-Barr virus-encoded EBNA1 enhances RNA polymerase III-dependent EBER expression through induction of EBER-associated cellular transcription factors

BackgroundEpstein-Barr Virus (EBV)-encoded RNAs (EBERs) are non-polyadenylated RNA molecules transcribed from the EBV genome by RNA polymerase III (pol III). EBERs are the most abundant viral latent gene products, although the precise mechanisms by which EBV is able to achieve such high levels of EBER expression are not fully understood. Previously EBV has been demonstrated to induce transcription factors associated with EBER expression, including pol III transcription factors and ATF-2. We have recently demonstrated that EBV-encoded nuclear antigen-1 (EBNA1) induces cellular transcription factors, and given these findings, we investigated the role of EBNA1 in induction of EBER-associated transcription factors.ResultsOur data confirm that in epithelial cells EBNA1 can enhance cellular pol III transcription. Transient expression of EBNA1 in Ad/AH cells stably expressing the EBERs led to induction of both EBER1 and EBER2 and conversely, expression of a dominant negative EBNA1 led to reduced EBER expression in EBV-infected Ad/AH cells. EBNA1 can induce transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc. A variant chromatin precipitation procedure showed that EBNA1 is associated with the promoters of these genes but not with the promoters of pol III-transcribed genes, including the EBERs themselves. Using shRNA knock-down, we confirm the significance of both ATF-2 and c-Myc in EBER expression. Further, functional induction of a c-Myc fusion protein led to increased EBER expression, providing c-Myc binding sites upstream of EBER1 were intact. In vivo studies confirm elevated levels of the 102 kD subunit of TFIIIC in the tumour cells of EBV-positive nasopharyngeal carcinoma biopsies.ConclusionsOur findings reveal that EBNA1 is able to enhance EBER expression through induction of cellular transcription factors and add to the repertoire of EBNA1's transcription-regulatory properties.

Multicenter evaluation of prototype real‐time PCR assays for Epstein‐Barr virus and cytomegalovirus DNA in whole blood samples from transplant recipients

Quantitative PCR is becoming widespread for diagnosing and monitoring post-transplantation diseases associated with EBV and CMV. These assays need to be standardized to manage patients in different facilities. Five independent laboratories in Japan compared home-brew assays and a prototype assay system to establish a standard quantitative procedure for measuring EBV and CMV. Reference standards and a total of 816 (642 EBV and 174 CMV) whole blood samples from post-transplantation recipients were used for this multicenter evaluation. The prototype reference standard for EBV was compared to a panel of samples, with a theoretical expected value made using EBV-positive cells containing two virus genome copies per cell. The mean ratio of the reference standard at each site to the standard of the prototype assay was ≤ 4.15 for EBV among three different sites and ≤ 3.0 for CMV between two laboratories. The mean of the theoretical expected number of the EBV genome: prototype reference was close to 1.0. The correlation coefficients between the viral copy numbers determined using the prototype assay and those using each home-brew assay were high (EBV, 0.73-0.83, median = 0.78; CMV, 0.54-0.60, median = 0.57). The dynamics of the EBV and CMV loads in transplant recipients were similar between the assay types. There was an inter-laboratory difference among the quantification results, indicating that a unified protocol and kit are favorable for standardizing the quantification of EBV and CMV. Such standardization will help to standardize the diagnosis and monitoring of diseases associated with EBV and CMV.

Diagnosis of infectious mononucleosis caused by Epstein–Barr virus in infants

The diagnosis of infectious mononucleosis (IM) is usually on serologic tests. The responses of anti-Epstein-Barr virus (anti-EBV) antibodies are weak in infants. The authors encountered some IM infants in whom anti-EBV antibodies were undetectable during early stage, although EBV genome was found in their blood. The aim of the present study was therefore to clarify the frequency of anti-EBV-antibody negative IM cases. The EBV serostatus of 104 IM children diagnosed on Sumaya criteria was retrospectively studied. The EBV genome in peripheral blood mononuclear cells was measured. The anti-viral capsid antigen-IgM (anti-VCA-IgM)-positive rate in the acute phase was only 25% in infants but 80% in patients ≥ 4 years of age. Twenty percent of the infants were negative for all anti-EBV antibodies and required repeated serologic tests. For infants, the significant rise in anti-VCA-IgG was the most sensitive marker. Three seronegative infants with IM symptoms, with circulating EBV genome during acute phase, were eventually considered as having IM on anti-VCA-IgG seroconversion thereafter. To diagnose IM in infants the serologic test alone in the acute phase is not sensitive enough. It is proposed that the EBV genome be evaluated in peripheral blood mononuclear cells when infants presenting with IM symptoms are negative for anti-EBV antibodies during the acute phase.

Oral Presentations

Background: Previous work has shown high levels of anti-Epstein Barr Virus (EBV) antibodies in individuals with multiple sclerosis (MS).Objectives: To examine history of glandular fever, anti-EBV antibody profile and whole blood viral load at first clinical diagnosis of central nervous system demyelination (FCD), compared to community controls. Methods: The Australian Multicentre Study of Environment and Immune Function (Ausimmune Study) recruited FCD cases and unaffected controls matched on age (18–59 years), sex and study region, between 1 November 2003 and 31 December 2006. Data included history of glandular fever (yes/no), serum IgG antibody titres for EBNA complex, viral capsid antigen (VCA) and early antigen (EA), and whole blood quantitative EBV DNA viral load (measured using real-time polymerase chain reaction [PCR], primers and probe derived from BamH1 of the EBV genome and Namalwa cell line with 2 EBV copies per cell for the standard curve) expressed as EBV copies per ug of DNA. Conditional logistic regression was used to analyse case-control differences. Results: Past history of glandular fever (n=277 cases, n=538 controls) was associated with increased odds of being a FCD case (OR=1.87, 95% confidence interval [CI] 1.30–2.67). In a subsample (n=204 cases, n=210 controls), higher titres (log2) of anti-EBNA (OR=1.26, 95% CI 1.13–1.40), anti-VCA (OR=1.16, 95% CI 1.03– .32) and anti-EA-restricted (OR=1.16, 95% CI 1.04–1.29) but not anti-EAdiffuse (OR=1.06, 95% CI 0.93–1.21) antibodies, were associated with increased risk of being a FCD case. EBV load (n=215 cases, n=216 controls) was more likely to be positive in FCD cases than controls (56.3% vs. 49.5%), and was significantly correlated with anti-EBNA (p=0.02), anti-VCA (p<0.001) and anti-EA-restricted (p=0.001), but not anti-EAdiffuse (p=0.16) antibodies. Multivariate analyses are underway. Conclusions: History of glandular fever, or evidence of prior infection with EBV is associated with increased odds of being a FCD case, strengthening evidence that EBV infection is a risk factor for MS onset.

Epstein–Barr virus in bone marrow of rheumatoid arthritis patients predicts response to rituximab treatment

Objectives. Viruses may contribute to RA. This prompted us to monitor viral load and response to anti-CD20 therapy in RA patients.Methods. Blood and bone marrow from 35 RA patients were analysed for CMV, EBV, HSV-1, HSV-2, parvovirus B19 and polyomavirus using real-time PCR before and 3 months after rituximab (RTX) treatment and related to the levels of autoantibodies and B-cell depletion. Clinical response to RTX was defined as decrease in the 28-joint disease activity score (DAS-28) >1.3 at 6 months.Results. Before RTX treatment, EBV was identified in 15 out of 35 patients (EBV-positive group), of which 4 expressed parvovirus. Parvovirus was further detected in eight patients (parvo-positive group). Twelve patients were negative for the analysed viruses. Following RTX, EBV was cleared, whereas parvovirus was unaffected. Eighteen patients were responders, of which 12 were EBV positive. The decrease in the DAS-28 was significantly higher in EBV-positive group compared with parvo-positive group (P = 0.002) and virus-negative patients (P = 0.04). Most of EBV-negative patients that responded to RTX (75%) required retreatment within the following 11 months compared with only 8% of responding EBV-positive patients. A decrease of RF, Ig-producing cells and CD19+ B cells was observed following RTX but did not distinguish between viral infections. However, EBV-infected patients had significantly higher levels of Fas-expressing B cells at baseline as compared with EBV-negative groups.Conclusions. EBV and parvovirus genomes are frequently found in bone marrow of RA patients. The presence of EBV genome was associated with a better clinical response to RTX. Thus, presence of EBV genome may predict clinical response to RTX.

The enhanced transcriptional activity of the V-val subtype of Epstein-Barr virus nuclear antigen 1 in epithelial cell lines

The Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) is required for the replication and maintenance of the episomal EBV genome and for the transactivation of viral gene expression. EBNA1 has been classified into five subtypes, among which the V-val subtype was reported to be associated with nasopharyngeal carcinoma (NPC). Here we report a higher transcriptional activity of the V-val subtype of EBNA1 than for the prototype derived from B95.8 cells to transactivate FR-containing luciferase plasmid, which was mainly a consequence of the mutations in the carboxy-terminus of EBNA1. This interpretation was further supported by the finding that the variant form of EBNA1 has a higher binding affinity for the FR sequence than the prototype by electrophoretic mobility shift assays. The functional advantage of the V-val EBNA1 investigated in this study may contribute to the oncogenesis of NPC.

Hsp90 inhibitors block outgrowth of EBV-infected malignant cells in vitro and in vivo through an EBNA1-dependent mechanism

EBV causes infectious mononucleosis and is associated with certain malignancies. EBV nuclear antigen 1 (EBNA1) mediates EBV genome replication, partition, and transcription, and is essential for persistence of the viral genome in host cells. Here we demonstrate that Hsp90 inhibitors decrease EBNA1 expression and translation, and that this effect requires the Gly-Ala repeat domain of EBNA1. Hsp90 inhibitors induce the death of established, EBV-transformed lymphoblastoid cell lines at doses nontoxic to normal cells, and this effect is substantially reversed when lymphoblastoid cell lines are stably infected with a retrovirus expressing a functional EBNA1 mutant lacking the Gly-Ala repeats. Hsp90 inhibitors prevent EBV transformation of primary B cells, and strongly inhibit the growth of EBV-induced lymphoproliferative disease in SCID mice. These results suggest that Hsp90 inhibitors may be particularly effective for treating EBV-induced diseases requiring the continued presence of the viral genome.

The Latent Origin of Replication of Epstein-Barr Virus Directs Viral Genomes to Active Regions of the Nucleus

The Epstein-Barr virus efficiently infects human B cells. The EBV genome is maintained extrachromosomally and replicates synchronously with the host's chromosomes. The latent origin of replication (oriP) guarantees plasmid stability by mediating two basic functions: replication and segregation of the viral genome. While the segregation process of EBV genomes is well understood, little is known about its chromatin association and nuclear distribution during interphase. Here, we analyzed the nuclear localization of EBV genomes and the role of functional oriP domains FR and DS for basic functions such as the transformation of primary cells, their role in targeting EBV genomes to distinct nuclear regions, and their association with epigenetic domains. Fluorescence in situ hybridization visualized the localization of extrachromosomal EBV genomes in the regions adjacent to chromatin-dense territories called the perichromatin. Further, immunofluorescence experiments demonstrated a preference of the viral genome for histone 3 lysine 4-trimethylated (H3K4me3) and histone 3 lysine 9-acetylated (H3K9ac) nuclear regions. To determine the role of FR and DS for establishment and subnuclear localization of EBV genomes, we transformed primary human B lymphocytes with recombinant mini-EBV genomes containing different oriP mutants. The loss of DS results in a slightly increased association in H3K27me3 domains. This study demonstrates that EBV genomes or oriP-based extrachromosomal vector systems are integrated into the higher order nuclear organization. We found that viral genomes are not randomly distributed in the nucleus. FR but not DS is crucial for the localization of EBV in perichromatic regions that are enriched for H3K4me3 and H3K9ac, which are hallmarks of transcriptionally active regions.

Hypoxia-targeting by tirapazamine (TPZ) induces preferential growth inhibition of nasopharyngeal carcinoma cells with Chk1/2 activation

Hypoxia is commonly developed in solid tumors, which contributes to metastasis as well as radio- and chemo-resistance. Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic head and neck cancer prevalent in Southeast Asia with a high incidence rate of 15-30/100,000 persons/year (comparable to that of pancreatic cancer in the US). Previous clinical studies in NPC showed that hypoxia is detected in almost 100% of primary tumors and overexpression of hypoxia markers correlated with poor clinical outcome. Tirapazamine (TPZ) is a synthetic hypoxia-activated prodrug, which preferentially forms cytotoxic and DNA-damaging free radicals under hypoxia, thus selectively eradicate hypoxic cells. Here, we hypothesized that specific hypoxia-targeting by this clinical trial agent may be therapeutic for NPC. Our findings demonstrated that under hypoxia, TPZ was able to induce preferential growth inhibition of NPC cells, which was associated with marked cell cycle arrest at S-phase and PARP cleavage (a hallmark of apoptosis). Examination of S-phase checkpoint regulators revealed that Chk1 and Chk2 were selectively activated by TPZ in NPC cells under hypoxia. Hypoxia-selectivity of TPZ was also demonstrated by preferential downregulation of several important hypoxia-induced markers (HIF-1α, CA IX and VEGF) under hypoxia. Furthermore, we demonstrated that TPZ was equally effective and hypoxia-selective even in the presence of the EBV oncoprotein, LMP1 or the EBV genome. In summary, encouraging results from this proof-of-concept study implicate the therapeutic potential of hypoxia-targeting approaches for the treatment of NPC.

Four Cases of Chronic Active Epstein-Barr Virus Infection That Performed Reduced-Intensity Conditioning Bone Marrow Transplantation.

Abstract 4734Epstein-Barr virus (EBV) is B cell tropic virus. However, the EBV-infected T and NK cells play a critical role on the pathogenesis of chronic active EBV infection (CAEBV). The diagnostic criteria are fever, lymphadenopathy and hepatosplenomegaly with abnormal antibody titer to EBV and increased amount of the EBV genome in the blood. But the mechanism on how EBV infects T and NK cells is not fully known yet. Once leukemic transformation occur the prognosis of the disease becomes very poor. The evidence that hematopoietic stem cell transplantation (SCT) is effective on CAEBV has accumulated, however, controversy still remains on the necessity of pre-transplant chemotherapy, and what kind of conditioning regimen is suitable during SCT.In this article, we reported 2 cases of T cell type CAEBV and 2 cases of NK cell type CAEBV that have been treated by bone marrow transplantation (BMT) with reduced intensity regimen (RIC) without pre-transplant chemotherapy. RIC consists of Cyclophosphamide (CY, 100-120 mg/Kg), Fludarabine (Flu, 120-150 mg/m2) and low dose TBI (3-4 Gy).Case 1 is an 8-year-old boy with EBV infected CD4 positive T cells. He showed elevated serum enzyme, coronary artery aneurysm, generalized rash, and positive EBV DNA in cerebrospinal fluid. The EBV genome in peripheral mononuclear cells was 1.3+105 copies /μgDNA. Southern blot analysis with EBV terminal repeat probe revealed clonal proliferation of virus. Tetramer analysis did not detect EBV specific CTL. He received modified HLH 2004 protocol (Henter et al, 2006). Immediately after normalization of liver dysfunction, BMT were performed from unrelated donor. Now he is in complete remission.Case 2 is an 8-year-old boy with EBV infected CD4 positive T cells. He also showed liver dysfunction, lymphadenopathy and positive EBV DNA in cerebrospinal fluid. The EBV genome in peripheral mononuclear cells is 2.6+105 copies /μgDNA. EBV clonality was detected by Southern bolt analysis. Tetramer analysis of BRLF1, EBNA3A and EBNA3B peptides showed EBV specific CTL. BMT from HLA-matched sibling donor achieved complete chimeras. But the EBV genomes in his peripheral blood increased to 4.7 +103 copies /μgDNA after BMT. EBV positive CD4 T cells were present. At the moment, it is not clear whether the origin of EBV positive T cells is from donor or not.Case 3 is a 21-year-old female with mosquito allergy with EBV infected NK cells. Liver dysfunction and lymphadenopathy were occasionally complained. The EBV genome in her peripheral blood is 1.2+105 copies /μgDNA. EBV showed monoclonal amplification. BRLF1 specific CTL were detected by tetramer analysis. HLA one antigen mismatched BMT from unrelated donor induced complete remission after grade 3 skin GVHD.Case 4 is a 19-year-old boy with CAEBV secondary to EBV-hemophagocytic lymphohistiocytosis. EBV positive NK cells were present. EBV genome in his peripheral blood was 1.2+104 copies /μgDNA. EBV showed monoclonal amplification. EBV specific CTL were not detected. After BMT from HLA-matched unrelated donor, EBV DNA was rapidly eliminated from his blood. But he suffered from pure red cell aplasia caused by major Blood type incompatibility and now treated with Rituximab.Four cases of CAEBV were described. RIC was demonstrated to be effective for engraftment of donor bone marrow cells without any serious adverse effects except pure red cell aplasia. Addition of pre-transplant chemotherapy seemed to be non-essential for engraftment or remission induction. In case 2 EBV infected peripheral CD4 positive cells were demonstrated after BMT. Determining the origin of the EBV infected CD4 positive T cells may provide hints for understanding on how EBV infects T cells or how EBV infected Tcells survive after BMT. Disclosures:No relevant conflicts of interest to declare.

Epstein-Barr Virus-Associated Haemophagocytic Lymphohistiocytosis in Adults Characterised by High Viral Genome Load within Circulating Natural Killer Cells.

Abstract 3672Poster Board III-608 IntroductionEpstein Barr virus (EBV) is predominantly B lymphotrophic both in-vitro and in vivo, where in immunocompetent individuals the virus persists asymptomatically in the B lymphoid compartment under host T cell control. EBV's association with B cell malignancies, such as Hodgkin and Burkitt lymphoma, can be viewed as rare accidents of the virus' lifelong interaction with the B cell system. By contrast, EBV infection of NK and T cells is considered a rare event but is nonetheless strongly associated with a spectrum of rare lymphoproliferations: EBV-associated haemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV infection (CAEBV), aggressive NK leukaemia and NK/T lymphoma all characterised pathogenically by the presence of monoclonal EBV in the T and/or NK cells. The mechanism of viral entry and its contribution to lymphoproliferation in these cell lineages remains to be clearly defined. The majority of reported cases of EBV-HLH occur in the context of primary EBV infection in children or adolescents, some of whom have a defined inherited immune defect. Adult cases of EBV-HLH occur extremely rarely and appear to be more frequent in individuals of East Asian origin. Furthermore, the vast majority of analysed cases of EBV-HLH have identified CD8+ lymphocytes as the predominant virus-bearing cell. To-date, EBV infection of (CD3-CD56+) circulating NK cells has not been reported and the pattern of viral gene expression remains unclear. Methods/PatientsWe analysed peripheral blood from three consecutive cases of EBV-HLH, referred to our laboratory between 2007-2009, to identify the predominant virus-harbouring cell. All three cases occurred in adults (mean age 44yrs), with no history of inherited immunodeficiency, who presented with clinical and laboratory features consistent with a diagnosis of HLH; fever, hepatosplenomegaly, pancytopenia, markedly elevated serum ferritin and lactate dehydrogenase and EBV copy number of 105-106 per millilitre of whole blood. Haemophagocytosis was unequivocally present on tissue biopsy from two patients. Mononuclear cells were separated using the MoFlo™ cell sorter into pure populations. Patient 1 and 2: CD19+CD3 −CD56−, CD3+CD19−CD56−, and CD56+CD3−CD19−. Patient 3: CD19+CD3 –CD16−, CD3+CD19−CD16−, CD16+CD3−CD19− and CD3−CD19−CD16−. DNA was subsequently extracted from each population and assayed by quantitative PCR, expressed as genome copies per million cells. ResultsIn all three cases we found the predominant EBV load within the non-B, non-T lymphocyte populations; definitively shown to be the CD56+CD3− cell fraction in 2 cases and for case 3 within CD3−CD19−CD16− lymphocytes likely to represent CD56+CD16- NK cells (a minority population in normal peripheral blood). A representative figure is shown: [Display omitted] We then quantitatively examined latent and lytic viral gene transcripts by real-time PCR and, in contrast to previously published data, we found a tightly restricted pattern of EBV gene expression with extremely high levels of EBER (EBV-encoded RNA) transcripts present. Lymphocytes derived from tonsillar tissue and peripheral blood, from both healthy and immunosuppressed individuals, served as control samples and demonstrated the predominant EBV genome load in the CD19+ B-cells but not the T or NK fractions. ConclusionThis novel finding of high EBV genome copy numbers and a restricted pattern of viral gene expression, within circulating natural killer cells in the context of adult EBV-HLH, is both pathogenically intriguing and importantly, has relevance for the investigation of targeted therapies for this aggressive disease. Disclosures:No relevant conflicts of interest to declare.

Viral-load and B-lymphocyte monitoring of EBV reactivation after allogeneic hemopoietic SCT in children

EBV-associated post transplant lymphoproliferative disease (EBV-PTLD) is a life-threatening complication that may occur after hemopoietic SCT. We prospectively screened 80 children on a weekly basis using nested quantitative PCR to evaluate EBV genome copies. EBV viral load <1000 copies per 10(5) PBMC was observed in 63% of transplants, whereas it was between 1000 and 9999 copies per 10(5) PBMC in 13%, and between 10 000 and 19 999 in 10%, with no significant increase in percentage of CD20+ lymphocytes. Viral load reached > or = 20 000 copies per 10(5) PBMC in 14% of patients, and rituximab was administered to 75% of them. None of the patients except one developed a lymphoproliferative disease. Our study found that only 13% of unrelated donor HSCT recipients had a very high risk of EBV-PTLD defined as > or = 20 000 geq per 10(5) PBMC associated with an increase in CD20+ lymphocyte. We suggest that rituximab could be administered in the presence of very high levels of EBV-DNA viral load or in the presence of mid levels of EBV-DNA viral load associated with an increase in the percentage of CD20+ lymphocytes. Through this approach, we significantly reduced the number of patients treated with rituximab, and consequently the acute and chronic adverse events related to this treatment.

Elevation of clonal serum free light chains in patients with HIV-negative primary effusion lymphoma (PEL) and PEL-like lymphoma

Elevation of clonal serum free light chains in patients with HIV-negative primary effusion lymphoma (PEL) and PEL-like lymphoma

CD4+ T-cell clones recognizing human lymphoma-associated antigens: generation by in vitro stimulation with autologous Epstein-Barr virus–transformed B cells

AbstractEpstein-Barr virus (EBV)–specific T-cell preparations, generated by stimulating immune donor lymphocytes with the autologous virus-transformed B-lymphoblastoid cell line (LCL) in vitro, can be used to target EBV-positive malignancies. Although these preparations are enriched for EBV antigen–specific CD8+ T cells, most also contain a CD4+ T-cell population whose specificity is unknown. Here, we show that, although CD4+ T-cell clones derived from such cultures recognize HLA class II–matched LCLs but not mitogen-activated B lymphoblasts, many (1) do not map to any known EBV antigen, (2) can be raised from EBV-naive as well as EBV-immune persons, and (3) can recognize a broad range of human B lymphoma–derived cell lines irrespective of EBV genome status, providing those lines to express the relevant HLA class II–restricting allele. Importantly, such CD4+ clones not only produce IFNγ but are also cytotoxic and can control the outgrowth of HLA-matched lymphoma cells in cocultivation assays. We infer that such CD4+ T cells recognize cellular antigens that are preferentially up-regulated in EBV-transformed but not mitogen-activated B lymphoblasts and that are also expressed in a range of B-cell malignancies. Such antigens are therefore of potential value as targets for CD4+ T cell–based immunotherapy.