Chronic Active Epstein-Barr Virus Research Articles

Immune Modulation by Epstein–Barr Virus Lytic Cycle: Relevance and Implication in Oncogenesis

EBV infects more than 90% of people globally, causing lifelong infection. The phases of the EBV life cycle encompass primary infection, latency, and subsequent reactivation or lytic phase. The primary infection usually happens without noticeable symptoms, commonly in early life stages. If it manifests after childhood, it could culminate in infectious mononucleosis. Regarding potential late consequences, EBV is associated with multiple sclerosis, rheumatoid arthritis, chronic active EBV infection, lymphomas, and carcinomas. Previous reports that the lytic phase plays a negligible or merely secondary role in the oncogenesis of EBV-related tumors are steadily losing credibility. The right mechanisms through which the lytic cycle contributes to carcinogenesis are still unclear, but it is now recognized that lytic genes are expressed to some degree in different cancer-type cells, implicating their role here. The lytic infection is a persistent aspect of virus activity, continuously stimulating the immune system. EBV shows different strategies to modulate and avoid the immune system, which is thought to be a key factor in its ability to cause cancer. So, the principal goal of our review is to explore the EBV’s lytic phase contribution to oncogenesis.

CD274 structural variants for guiding treatment with PD-1 blockade in a patient with relapsed/refractory chronic active EBV transformed to NK lymphoma

CD274 structural variants for guiding treatment with PD-1 blockade in a patient with relapsed/refractory chronic active EBV transformed to NK lymphoma

Research progress on systemic chronic active Epstein-Barr virus disease

Research progress on systemic chronic active Epstein-Barr virus disease

Recurrent oral and nasopharyngeal ulcers, facial erythema, vesicles and scars in an elderly female.

A 71-year-old woman presented with a 30-year history of recurrent fever, fatigue, oral and nasopharyngeal ulcers, facial oedematous erythema and vesicles that ulcerated and healed by forming scars.

Highly sensitive detection of Epstein-Barr virus-infected cells by EBER flow FISH.

When Epstein-Barr virus (EBV) infection is suspected, identification of infected cells is important to understand the pathogenesis, determinine the treatment strategy, and predict the prognosis. We used the PrimeFlow™ RNA Assay Kit with a probe to detect EBV-encoded small RNAs (EBERs) and multiple surface markers, to identify EBV-infected cells by flow cytometry. We analyzed a total of 24 patients [11 with chronic active EBV disease (CAEBV), 3 with hydroa vacciniforme lymphoproliferative disorder, 2 with X-linked lymphoproliferative disease type 1 (XLP1), 2 with EBV-associated hemophagocytic lymphohistiocytosis, and 6 with posttransplant lymphoproliferative disorder (PTLD)]. We compared infected cells using conventional quantitative PCR methods and confirmed that infected cell types were identical in most patients. Patients with CAEBV had widespread infection in T and NK cells, but a small amount of B cells were also infected, and infection in patients with XLP1 and PTLD was not limited to B cells. EBV-associated diseases are believed to be complex pathologies caused by EBV infecting a variety of cells other than B cells. We also demonstrated that infected cells were positive for HLA-DR in patients with CAEBV. EBER flow FISH can identify EBV-infected cells with high sensitivity and is useful for elucidating the pathogenesis.

Lymphocytes in Patients with Chronic Active Epstein-Barr Virus Disease Exhibited Elevated PD-1/PD-L1 Expression and a Prevailing Th2 Immune Response.

Chronic active Epstein-Barr virus disease (CAEBV) is a proliferative disease of EBV+ T or natural killer (NK) cells with an unclear pathogenesis. This study aimed to examine the frequency and exhaustion levels of lymphocyte subsets in patients with CAEBV to further investigate the pathogenesis. Using flow cytometry, we detected the frequency, expression levels of programmed cell death 1 (PD-1) and programmed death ligand 1 (PD-L1), and EBV infection status of peripheral T subsets and NK cells in patients with CAEBV and healthy individuals. 24 patients and 15 healthy individuals were enrolled in this study. Patients showed notably higher expression levels of PD-1 and PD-L1 in peripheral T subsets and NK cells compared to healthy individuals (P < 0.05). EBV+ lymphocytes exhibited significantly higher PD-L1 expression levels than EBV- lymphocytes. Additionally, the frequency of effector memory T (Tem) cells was significantly increased in patients, and the PD-L1 expression level was positively correlated with the EBV load. Besides, helper T cell 2 (Th2) immune bias, also favoring EBV amplification, was found in patients, including increased Th2 cell frequency, enhanced response capacity, and elevated serum levels of associated cytokines. The distribution and PD-1 expression levels of peripheral T subsets returned to normal in patients who responded to PD-1 blockade therapy. The up-regulation of the PD-1/PD-L1 pathway of peripheral T and NK cells and Th2 immune predominance jointly promoted EBV replication and the development of CAEBV. PD-1 blockade therapy reduced the PD-1 expression level of lymphocytes and helped normalize the distribution of the T subsets.

Single-Cell Transcriptomic Analysis of Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis.

Epstein-Barr virus (EBV) infection can lead to infectious mononucleosis (EBV-IM) and, more rarely, EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), which is characterized by a life-threatening hyperinflammatory cytokine storm with immune dysregulation. Interferon-gamma (IFNγ) has been identified as a critical mediator for primary HLH; however, the detailed role of IFNγ and other cytokines in EBV-HLH is not fully understood. In this study, we used single-cell RNA sequencing to characterize the immune landscape of EBV-HLH and compared it with EBV-IM. Three pediatric patients with EBV-HLH with different backgrounds,one with X-linked lymphoproliferative syndrome type 1 (XLP1), two with chronic active EBV disease (CAEBV), and two patients with EBV-IM were enrolled. The TUBA1B + STMN1 + CD8 + T cell cluster, a responsive proliferating cluster with rich mRNA detection, was explicitly observed in EBV-IM, and the upregulation of SH2D1A-the gene responsible for XLP1-was localized in this cluster. This proliferative cluster was scarcely observed in EBV-HLH cases. In EBV-HLH cases with CAEBV, upregulation of LAG3 was observed in EBV-infected cells, which may be associated with an impaired response by CD8 + T cells. Additionally, genes involved in type I interferon (IFN) signaling were commonly upregulated in each cell fraction of EBV-HLH, and activation of type II IFN signaling was observed in CD4 + T cells, natural killer cells, and monocytes but not in CD8 + T cells in EBV-HLH. In conclusion, impaired responsive proliferation of CD8 + T cells and upregulation of type I IFN signaling were commonly observed in EBV-HLH cases, regardless of the patients' background, indicating the key features of EBV-HLH.

Infectious Diseases and Basal Ganglia Calcifications: A Cross-Sectional Study in Patients with Fahr's Disease and Systematic Review.

Background: It is unclear whether patients with basal ganglia calcifications (BGC) should undergo infectious disease testing as part of their diagnostic work-up. We investigated the occurrence of possibly associated infections in patients with BGC diagnosed with Fahr's disease or syndrome and consecutively performed a systematic review of published infectious diseases associated with BGC. Methods: In a cross-sectional study, we evaluated infections in non-immunocompromised patients aged ≥ 18 years with BGC in the Netherlands, who were diagnosed with Fahr's disease or syndrome after an extensive multidisciplinary diagnostic work-up. Pathogens that were assessed included the following: Brucella sp., cytomegalovirus, human herpesvirus type 6/8, human immunodeficiency virus (HIV), Mycobacterium tuberculosis, rubella virus, and Toxoplasma gondii. Next, a systematic review was performed using MEDLINE and Embase (2002-2023). Results: The cross-sectional study included 54 patients (median age 65 years). We did not observe any possible related infections to the BGC in this population. Prior infection with Toxoplasma gondii occurred in 28%, and in 94%, IgG rubella antibodies were present. The positive tests were considered to be incidental findings by the multidisciplinary team since these infections are only associated with BGC when congenitally contracted and all patients presented with adult-onset symptoms. The systematic search yielded 47 articles, including 24 narrative reviews/textbooks and 23 original studies (11 case series, 6 cross-sectional and 4 cohort studies, and 2 systematic reviews). Most studies reported congenital infections associated with BGC (cytomegalovirus, HIV, rubella virus, Zika virus). Only two studies reported acquired pathogens (chronic active Epstein-Barr virus and Mycobacterium tuberculosis). The quality of evidence was low. Conclusions: In our cross-sectional study and systematic review, we found no convincing evidence that acquired infections are causing BGC in adults. Therefore, we argue against routine testing for infections in non-immunocompromised adults with BGC in Western countries.

End-stage ADPKD with a low-frequency PKD1 mosaic variant accelerated by chemoradiotherapy

Autosomal dominant polycystic kidney disease (ADPKD) is commonly caused by PKD1, and mosaic PKD1 variants result in milder phenotypes. We present the case of a 32 year-old male with chronic active Epstein–Barr virus who underwent bone marrow transplantation with chemoradiotherapy at age 9. Despite a low-frequency mosaic splicing PKD1 variant, he developed severe renal cysts and end-stage renal disease in his 30 s. This case highlights how environmental factors may contribute to the genetic predisposition to ADPKD.

Novel plasma microRNA expression features in diagnostic use for Epstein-Barr virus-associated febrile diseases

BackgroundEpstein-Barr virus (EBV) is widely infected in humans and causes various diseases. Among them, microRNAs of EBV play a key role in the progression of EBV-associated febrile diseases. There're few specific indicators for rapid differential diagnosis of various febrile diseases associated with EBV, and the lack of more reliable screening methods with high diagnostic utility has led to spaces for improvement in the accurate diagnosis and efficient treatment of relevant patients, making EBV infection a complicated clinical problem. With recent advances in plasma microRNA testing, the apparent presence of EBV microRNAs in plasma can help screen for EBV infection. The gene networks targeted by these microRNAs can also indicate potential biomarkers of EBV-associated febrile diseases. This study aimed to identify some novel miRNAs as potential biomarkers for early diagnosis of respectively EBV-associated febrile diseases. Materials and methodsA total of 110 participants were recruited for this task. First, we performed high-throughput sequencing and preliminary PCR validation of differentially expressed miRNAs in 15 participants with EBV-associated fever (divided into 5 groups, common EBV carriers, infectious mononucleosis (IM) and chronic active EBV infection (CAEBV), EBV-associated Hemophagocytic Lymphohistiocytosis (EBV-HLH), and 3 healthy individuals). After a comprehensive analysis, 10 miRNAs with abnormal expression were screened, and then qRT-PCR was performed in the rest of 95 participants to detect the validation of miRNAs expression in plasma samples. Thereafter, we further investigated their potential for clinical application in EBV-related febrile diseases by using a combination of Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis, and Protein-protein interaction network analysis. ResultsThrough identification and detailed analysis of the obtained data, we found significant differences in the expression of Hsa-miR-320d, EBV-miR-BART22, and EBV-miR-BART2-3p in blood samples from patients with different EBV-related febrile diseases. We found that the expression levels of Hsa-miR-320d, EBV-miR-BART22, and EBV-miR-BART2-3p in plasma are indicative of determining different disease types of EBV-related febrile diseases, while EBV-miR-BART22 and EBV-miR-BART2-3p may be potential therapeutic targets. ConclusionThe expression levels of Hsa-miR-320d, EBV-miR-BART22, and EBV-miR-BART2-3p suggest that they may be used as transcriptional features for early differential diagnosis of EBV-related febrile diseases, and EBV-miR-BART22 and EBV-miR-BART2-3p may be potential therapeutic targets.

Ultra-low doses of microARNs restore a strong immune response in patients with Chronic Active Epstein-Barr Virus infection (CAEBV)

Chronic active Epstein-Barr Virus infection (CAEBV) is an abnormal response of the immune system to EBV infection, characterized by the absence of anti-EBNA (EBV Nuclear Antigens) IgG and associated with chronic fatigue and several immune related diseases. We present here an innovative therapeutic approach of CAEBV based on the use of ultra-low doses of non-coding RNAs (ncRNAs) involved in the host-virus interaction. We performed a retrospective study on 26 patients treated with our remedy. All the patients expect one developed positive EBNA IgG within eighteen months of starting treatment. Among the 25 cases that seroconverted, 75% report a significant improvement of their symptoms, especial of chronic fatigue. This study gives the proof of the efficiency of ultra-low doses of ncRNAs in restoring a strong immune response against EBV. More generally, this therapeutic approach can potentially be used in almost all pathologies.

Differentiating Between Epstein-Barr Virus-positive Lymphoid Neoplasm Relapse and Post-transplant Lymphoproliferative Disorder After Sex-mismatched Hematopoietic Stem Cell Transplantation.

After allogeneic hematopoietic stem cell transplantation (HSCT), accurate differentiation between donor-derived post-transplant lymphoproliferative disorder (PTLD) and relapse of recipient-derived lymphoproliferative disorder (LPD) is crucial for determining treatment. Conventional diagnostic approaches for PTLD include histopathological examination, flow cytometry, and chimerism analysis of bulk tumor tissue. However, these methods are inconclusive in cases in which the primary disease is an Epstein-Barr virus (EBV)-positive LPD and is of the same lineage as that of the post-HSCT LPD tumor cells. Particularly, in cases where the number of tumor cells in the tissue is low, it is difficult to determine the origin of tumor cells. In this study, we developed a new method to simultaneously detect signals using sex chromosome fluorescence in situ hybridization, immunofluorescence staining, and EBV-encoded small RNA in situ hybridization on a single section of formalin-fixed paraffin-embedded histopathological specimen. The utility of the method was validated using specimens from 6 cases of EBV-positive LPD after sex-mismatched HSCT that were previously difficult to diagnose, including Hodgkin lymphoma-like PTLD that developed after HSCT for Hodgkin lymphoma and recurrence of chronic active EBV infection. This method successfully preserved the histologic structure after staining and allowed accurate determination of tumor cell origin and lineage at the single-cell level, providing a definitive diagnosis in all cases. This method provides a powerful tool for the diagnosis of LPDs after sex-mismatched HSCT.

Clinical significance of anti-Epstein-Barr virus antibodies in systemic chronic active Epstein-Barr virus disease.

Systemic chronic active Epstein-Barr virus disease (sCAEBV) is a rare and fatal neoplasm, involving clonally proliferating Epstein-Barr virus (EBV)-infected T cells or natural killer cells. Patients with sCAEBV have abnormal titers of anti-EBV antibodies in their peripheral blood, but their significance is unknown. We retrospectively investigated titers and their relationship with the clinical features of sCAEBV using the data collected by the Japanese nationwide survey. Eighty-four patients with sCAEBV were analyzed. The anti-EBV nuclear antigen (EBNA) antibody, targeting EBNA-expressing EBV-positive cells, was found in 87.5% of children (<15 years old), 73.7% of adolescents and young adults (15-39 years old), and 100% of adults (≥40 years old). Anti-EBNA antibody titers were significantly lower and anti-VCA-IgG antibody titers significantly higher in patients with sCAEBV than those in healthy controls (p < 0.0001). Patients with high anti-VCA-IgG and anti-early antigen-IgG antibody (antibodies against the viral particles) levels had significantly better 3-year overall survival rates than those with low titers, suggesting that patients with sCAEBV have a reduced immune response to EBV-infected cells.

Cytokine Storm Syndromes Associated with Epstein-Barr Virus.

Epstein-Barr virus (EBV) is a ubiquitous and predominantly B cell tropic virus. One of the most common viruses to infect humans, EBV, is best known as the causative agent of infectious mononucleosis (IM). Although most people experience asymptomatic infection, EBV is a potent immune stimulus and as such it elicits robust proliferation and activation of the B-lymphocytes it infects as well as the immune cells that respond to infection. In certain individuals, such as those with inherited or acquired defects affecting the immune system, failure to properly control EBV leads to the accumulation of EBV-infected B cells and EBV-reactive immune cells, which together contribute to the development of often life-threatening cytokine storm syndromes (CSS). Here, we review the normal immune response to EBV and discuss several CSS associated with EBV, such as chronic active EBV infection, hemophagocytic lymphohistiocytosis, and post-transplant lymphoproliferative disorder. Given the critical role for cytokines in driving inflammation and contributing to disease pathogenesis, we also discuss how targeting specific cytokines provides a rational and potentially less toxic treatment for EBV-driven CSS.

Haploidentical Transplantation with Post-Transplant Cyclophosphamide Versus Single Cord Blood Transplantation for Adults with Relapsed/Refractory Malignant Lymphoma

Introduction Although the treatment of malignant lymphoma has diversified with the advent of chimeric antigen receptor T-cell therapy and other novel agents, allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a curative treatment for some patients with relapsed/refractory malignant lymphoma (R/R ML). R/R ML eligible for allo-HSCT often requires urgent transplantation, making human leukocyte antigen (HLA)-haploidentical transplantation with post-transplant cyclophosphamide (PTCY-haplo) or cord blood transplantation (CBT) a promising option when HLA-matched donors are unavailable. PTCY-haplo has recently been reported to achieve similar or better clinical outcomes than CBT for other hematological malignancies, though data regarding R/R ML are insufficient. Therefore, this study compares PTCY-haplo and CBT for patients with R/R ML using the Transplant Registry Unified Management Program 2 (TRUMP2) in Japan. Methods This study included adult patients with R/R ML registered with TRUMP2 who underwent PTCY-haplo and CBT for their first allo-HSCT between January 2013 and December 2021. In this study, R/R ML included mature B-, T-, and NK-cell neoplasms, as defined in the World Health Organization's 2017 classification. Patients undergoing allo-HSCT for Hodgkin's lymphoma, aggressive NK-cell leukemia, chronic active Epstein-Barr virus (EBV) infection, systemic EBV-positive T-cell lymphoma of childhood, or adult T-cell leukemia/lymphoma were excluded. The primary endpoint was 3-year overall survival (OS). A Cox proportional hazard regression model for OS was used to estimate hazard ratios (HR) with 95% confidence intervals (CI) in the multivariate analysis. The following factors were included in the multivariate model: donor type, age, sex, performance status, Hematopoietic Cell Transplantation-specific Comorbidity Index score, disease status at allo-HSCT, conditioning regimen, cytomegalovirus seropositivity, and disease linage. The secondary endpoints were 3-year progression-free survival (PFS), 3-year graft versus host disease (GVHD)/relapse-free survival (GRFS), 3-year cumulative incidence of relapse and non-relapse mortality (NRM), 28-day cumulative incidence of neutrophil engraftment, 100-day cumulative incidence of platelet engraftment, 100-day cumulative incidence of grade II-IV and grade III-IV acute GVHD, and 3-year cumulative incidence of any and extensive chronic GVHD. Results A total of 1093 patients, including 223 who underwent PTCY-haplo and 870 who underwent CBT, were included in the study. The median age was 54 years (interquartile range, 44-61 years), and 616 (56%) patients had B-cell lymphoma. The PTCY-haplo group included older patients, more patients who were positive for cytomegalovirus antibodies in recipients or donors, and more patients who received a reduced-intensity conditioning regimen. The 3-year OS was 49% (95% CI: 41-56%) in the PTCY-haplo group and 42% (95% CI: 38-45%) in the CBT group (P=0.090). The adjusted HR was 0.88 (95% CI: 0.70-1.09, P=0.20). The PTCY-haplo group had comparable 3-year PFS, 3-year GRFS, 3-year relapse rates, and 3-year NRM as the CBT group. The PTCY-haplo group had higher neutrophil engraftment at 28 days (92.8% [95%CI: 88.5-95.6%] vs 81.6% [95%CI: 78.9-84.0%], P&amp;lt;0.001) and platelet engraftment at 100 days (77.6% [95%CI: 71.5-82.5%] vs 71.0% [95%CI: 67.9-73.9%], P&amp;lt;0.001); similar grade II-IV acute GVHD at 100 days and 3-year cumulative incidences of chronic GVHD and extensive chronic GVHD; and lower grade III-IV acute GVHD at 100 days (7.17% [95%CI: 4.27-11.1%] vs 13.6% [95%CI 11.5-16.0], P=0.01). Conclusion In this population, PTCY-haplo and CBT result in similar 3-year OS after allo-HSCT. Faster engraftment and less severe acute GVHD were noted in the PTCY-haplo group.

Caebv Disease Originates from Infected Hematopoietic Stem Cells

Background Chronic active Epstein-Barr Virus (EBV) disease (CAEBV) is lethal syndrome due to persistent EBV infection. When diagnosed as CAEBV, EBV infection was observed in multiple hematopoietic lineages, but the etiology of CAEBV is still elusive. Methods Bone marrow and peripheral cells derived from five CAEBV patients, one EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) patient, and two healthy controls were analyzed. Multiple assays were applied to identify and characterize EBV-infected cells, including quantitative PCR (qPCR), PrimeFlow and single-cell RNA-sequencing (scRNA-seq). Based on scRNA-seq data, alterations in gene expression of particular cell types were analyzed between CAEBV patients and controls, and between infected and uninfected cells. One CAEBV patient was treated with allogeneic hematopoietic stem cell transplantation (HSCT), and the samples derived from this patient were analyzed again six month after HSCT. Results EBV infected the full spectrum of the hematopoietic system including both lymphoid and myeloid lineages, so as hematopoietic stem cells (HSCs) in the CAEBV patients. EBV-infected HSCs exhibited a higher differentiation rate towards downstream lineages, and the EBV infection had an impact on both the innate and adaptive immunity, resulting in inflammatory symptoms. EBV infected cells were thoroughly removed from the hematopoietic system after HSCT. Conclusions Token together,multiple lines of evidence presented in this study suggest that CAEBV disease originates from the infected hematopoietic stem cells. Figure 1. EBV-infected HSCs were detected in CAEBV patients with multiple assays.

Chronic active Epstein-Barr virus infection mimicking tuberculosis - A diagnostic enigma.

Chronic active EBV infection is a rare disorder prone for misdiagnosis. They present with a wide range of symptoms from indolent to aggressive clinical course. Clinico-pathological correlation with confirmation by ancillary techniques is inevitable to diagnose this disease. We present a case of a 29-year-old male with fever, weight loss, and lymphadenopathy for 6 months. Lymph node biopsy showed occasional granuloma with preserved architecture. Suspected to have tuberculosis, he received antitubercular treatment (ATT) with no response for 3 months. Subsequently, additional workup showed many EBV-positive cells in sinusoids with high serum EBV titer, confirming the difficult diagnosis of CAEBV. The present case highlights the difficulty in the diagnosis of this entity and also emphasizes the necessity to recognize this disorder in countries endemic for tuberculosis, as it is no longer bound by ethnicity and geographical boundaries.

Combining BCL-XL Inhibition with Brentuximab Vedotin to Overcome Chemoresistance in EBV-Related T/NK Lymphoma

Introduction Epstein Barr Virus (EBV) related T and NK malignancies, such as extra-nodal NK/T lymphoma (ENKTL) or chronic active EBV (CAEBV), are intrinsically chemoresistant. Conventionally, this is overcome in the clinic by using high-intensity treatment protocols. However, this makes therapy difficult to deliver, especially in patients with co-morbidities, and there are few options for those with relapsed or refractory disease. Intrinsic resistance to apoptosis, driven primarily by the anti-apoptotic BH3 protein BCL-XL, appears to play a significant part in treatment resistance (Sejic, Blood Advances 2020). However, inhibition of anti-apoptotic pathways alone cannot kill all malignant cells in vitro and is not curative in animal models. Identification of drugs that function synergistically with BCL-XL inhibition is critical to unlocking the chemoresistance. We investigated the combination of BH3 inhibition with the promotion of apoptosis using the antibody-drug conjugate (ADC) Brentuximab Vedotin (BV). BV delivers an MMAE payload to CD30-positive cells triggering apoptotic cell death through cell cycle arrest and DNA damage. We identified synergism between BV and a BCL-XL BH3 mimetic that dramatically increased cell death at concentrations that should limit on-target toxicity. Methods A diverse panel of EBV+ T/NK lymphoma cell lines including SNK1, MECO4, SNK6, SNT8, SNK10, SNT15 and SNT16 were evaluated for sensitivity to MMAE alone and with specific BH3 family inhibitors including A1331852, inhibiting BCL-XL, venetoclax inhibiting and BCL-2 and the MCL-1 inhibitor S63845 (Generon). Drug sensitivity was assessed by staining with PI/Annexin (eBioscience) and then analysing on an Accuri C6 flow cytometer. Cell surface expression of CD30 was assessed by flow cytometry (eBiosience). We then tested the effect of BV in combination with A1331852 and assessed for the effect on viability using CellTitre Glo (Promega). Results Single-agent MMAE was toxic to all cell lines tested with an LD50 ranging from 0.6-33 nM and a maximal killing effect between 10%-45%.PI/Annexin staining suggested the mode of cell killing was apoptotic. As a single agent, A1331852 showed efficacy across all cell lines, apart from MEC04 which exhibited reduced susceptibility. In combination with MMAE, A1331852 enhanced both the sensitivity and maximal killing effect seen. Using a 1:50 fixed dose ratio then analysis in CompuSyn the effect of both agents was synergistic with a fraction affect (0.5) between 0.158 and 0.177 in the cell lines tested (Figure1) . Single-agent inhibition of BCL-2 or MCL-1 showed only limited efficacy and no synergy with MMAE. High levels of CD30 expression was evident on all cell lines making ENKTL a viable target for BV. While BV showed efficacy as a single agent, cell viability at a clinically achievable 100 ug/ml dose was between 30% and 78% after 72h. However, in combination with A1331852 at a low 4 nM dose maximal viability was between 1% and 25%, figure 2 shows results for the SNT8 cell line. In most resistant lines, SNK6 and MECO4, sensitivity to BV could be increased using doses of A1331852 up to 500 nM, leading to 5-6% viability at 72h. Based on murine PK data this should still be clinically achievable. Discussion Combining targeted delivery of MMAE via BV with the BCL-XL inhibitor A1331852 is highly toxic to a panel of CAEBV and ENKTL-derived cell lines. These cells are intrinsically resistant to apoptosis but are sensitive to BCL-XL inhibition. The use of BCL-XL inhibitors has been limited by on-target thrombocytopenia and the combination of BH3 mimetics with chemotherapy can cause marked haematological toxicity. Therefore, using a targeted agent such BV in combination with BCL-XL inhibition has the potential to enhance efficacy without triggering excessive toxicity. Additionally, the synergistic nature of the combination allows the use of low-dose A1331852. We are working to better understand the cell line-specific differences in resistance to combination therapy, which does not appear to relate to either baseline BCL-XL protein levels, CD30 expression or the rate of cell division. These data show the potential of BV with BCL-XL inhibition to be an effective and tolerable treatment for ENKTL. We are now planning a detailed preclinical study to better understand the efficacy and safety of this combination in vivo and progress towards a future clinical trial.

Establishment of Epstein-Barr Virus (EBV) Latent Gene-Expressing T-Cell Lines with an Expression Vector Harboring EBV Nuclear Antigen 1.

Chronic active Epstein-Barr virus (EBV) infection (CAEBV) is characterized by chronic or recurrent infectious mononucleosis-like symptoms and is associated with EBV-associated T/natural killer (NK)-cell lymphoproliferative disorders, which frequently lead to the development of life-threatening complications, such as virus-associated hemophagocytic syndrome and EBV-positive apparent leukemia/lymphoma mainly in T- and NK-cell lineages. In order to clarify the EBV genes responsible for the diseases, we introduced the plasmid coding sequences of EBV-encoded small RNAs (EBERs) and/or latent membrane protein (LMP) 1 into human T-lymphocyte virus-I-negative human T-cell lines using a gene expression vector harboring EBV nuclear antigen 1, established the G418-resistant transformants of five T-cell lines, and quantitatively examined the expression of EBERs and LMP1 using real-time reverse transcriptase-polymerase chain reaction. The expression levels of EBERs in T-cell transformants with EBER DNA paralleled those in EBV-positive human T- and NK-cell lines, SNTK cells. The expression of LMP1 mRNA varied in SNTK cells and in human T-cell transformants, and the expression of LMP1 mRNA in T-cell lines expressing both EBERs and LMP1 was much lower than that in the same cell line expressing LMP1 mRNA alone. The currently employed gene expression system and currently obtained transformants may be useful for the analyses of the pathophysiology of CAEBV and EBV-positive T/NK-cell lymphoproliferative disorders.

Chronic active Epstein-Barr virus disease originates from infected hematopoietic stem cells

AbstractChronic active Epstein-Barr virus (EBV) disease (CAEBV) is a lethal syndrome because of persistent EBV infection. When diagnosed as CAEBV, EBV infection was observed in multiple hematopoietic lineages, but the etiology of CAEBV is still elusive. Bone marrow and peripheral cells derived from 5 patients with CAEBV, 1 patient with EBV-associated hemophagocytic lymphohistiocytosis, and 2 healthy controls were analyzed. Multiple assays were applied to identify and characterize EBV-infected cells, including quantitative polymerase chain reaction, PrimeFlow, and single-cell RNA-sequencing (scRNA-seq). Based on scRNA-seq data, alterations in gene expression of particular cell types were analyzed between patients with CAEBV and controls, and between infected and uninfected cells. One patient with CAEBV was treated with allogeneic hematopoietic stem cell transplantation (HSCT), and the samples derived from this patient were analyzed again 6 months after HSCT. EBV infected the full spectrum of the hematopoietic system including both lymphoid and myeloid lineages, as well as the hematopoietic stem cells (HSCs) of the patients with CAEBV. EBV-infected HSCs exhibited a higher differentiation rate toward downstream lineages, and the EBV infection had an impact on both the innate and adaptive immunity, resulting in inflammatory symptoms. EBV-infected cells were thoroughly removed from the hematopoietic system after HSCT. Taken together, multiple lines of evidence presented in this study suggest that CAEBV disease originates from the infected HSCs, which might potentially lead to innovative therapy strategies for CAEBV.