Treatment Of Epstein-Barr Virus Research Articles

Longitudinal tracing of on-treatment plasma Epstein-Barr virus DNA as a biomarker for real-time dynamic risk monitoring in patients with nasopharyngeal carcinoma: The EP-SEASON study.

136 Background: Plasma Epstein-Barr virus (EBV) DNA is a biomarker for nasopharyngeal carcinoma (NPC). However, whether longitudinal on-treatment EBV DNA tracing would inform real-time recurrence risk in NPC remains to be elucidated. To address this issue, we conducted EP-SEASON (NCT03855020), a prospective cohort study of longitudinal on-treatment EBV DNA in EBV-positive NPC patients. Methods: Non-metastatic NPC with detectable pretreatment EBV DNA that received standard sequential chemo-radiotherapy were recruited. Serial EBV DNA in blood specimens were collected across pretreatment, 3-week after each induction chemotherapy (IC), every-week during radiotherapy (RT), within 1-week and 1-3 months upon RT completion. EBV DNA extracted from plasma was quantified by a real-time quantitative polymerase chain reaction assay targeting the BamHI-W region of the EBV genome. Longitudinal changing patterns of EBV DNA and their clinical relevance were analyzed. Results: Between May 2019 and April 2021, a total of 1000 patients underwent per-protocol EBV DNA assessments and treatment; 248 and 752 patients received concurrent chemoradiotherapy (CCRT, subcochortno-IC) and IC plus CCRT (subcochortIC), respectively. Longitudinal changes and clearance rates of EBV DNA displayed unique patterns during IC and RT phases. Throughout IC and RT phases, the rate of EBV DNA clearance decreased with repeated IC cycles; interestingly, the rate re-rose upon the introduction of RT, and then dropped to low levels (<10%/week) after the 4th week of RT (post-RT4w). Patients in subcochortno-IC and subcochortIC also exhibited differential clearance patterns during RT, with the rate of clearance being higher in subcochortno-IC before the 2nd week of RT (post-RT2w). Notably, despite the prognostic significance of EBV DNA at each timepoint ( Pall < 0.05), longitudinal EBV DNA displayed a linkage changing map with dynamic recurrence risks: 3-year disease-free survival (DFS) rate steadily dropped with delayed EBV DNA clearance during IC; it re-rose at the 2nd RT week upon EBV DNA clearance, followed by a sharp decrease at the 4th RT week in subcochortIC. In contrast, 3-year DFS rate continuously decreased with delayed EBV DNA clearance in subcochortno-IC. Finally, we identified different phenotypes of whole-course circulating tumor DNA changing dynamics that showed distinct prognosis ( P < 0.05). Conclusions: Longitudinal on-treatment EBV DNA tracing could forecast real-time risks and facilitate risk-adapted decision-making in NPC.

Performance of the cobas EBV and cobas BKV assays: multi-site comparison of standardized quantitation.

Guidelines recommend monitoring of Epstein-Barr virus (EBV) and BK virus (BKV) in solid organ and hematopoietic stem cell transplant patients. The majority of quantitative DNA testing for EBV and BKV employs unstandardized individual laboratory-developed testing solutions (LDTs), with implications for accuracy, reproducibility, and comparability between laboratories. The performance of the cobas EBV and cobas BKV assays was assessed across five laboratories, using the World Health Organization International Standards (WHO IS) for EBV and BKV, and the National Institute of Standards and Technology Quantitative Standard for BKV, and results were compared with the LDTs in use at the time. Methods were also compared using locally sourced clinical specimens. Variation was high when laboratories reported EBV or BKV DNA values using LDTs, where quantitative values were observed to differ by up to 1.5 log10 unit/mL between sites. Conversely, results from the cobas EBV and cobas BKV assays were accurate and reproducible across sites and on different testing days. Adjustment of LDTs using the international standards led to closer alignment between the assays; however, day-to-day reproducibility of LDTs remained high. In addition, BKV continued to show bias, indicating challenges with the commutability of the BKV International Standard. The cobas EBV and cobas BKV assays are automated, aligned to the WHO IS, and have the potential to reduce the variability in viral load testing introduced by differences in LDTs. Standardization of reporting values may eventually allow different centers to compare data to allow clinical decision thresholds to be established supporting improvements in patient management.IMPORTANCEThe application of center-specific cut-offs for clinical decisions and the variability of LDTs often hinder interpretation; thus, the findings reported here support the need for standardization in the field of post-transplant monitoring of EBV and BKV to improve patient management. Alongside the choice of assay, it is also important to consider which standard to use when deciding upon a testing methodology. This is a call to action for standardization, as treatment for EBV and BKV is driven by viral load test results, and the more accurate and comparable the test results are across institutions, the more informed and better the treatment decisions can be.

Epstein Barr Virus Positive Hodgkin's Lymphoma in a Patient with NEMO Deficiency

IntroductionHypomorphic mutations in the NF-kappaB essential modulator (NEMO) can manifest in diverse ways, but are best characterized by early susceptibility to viral, pyogenic and mycobacterial infections, ectodermal dysplasia and absent pneumococcal responses. We describe a case of EBV+ Hodgkin's lymphoma in a 9-year-old patient with NEMO on antimicrobial prophylaxis and immunoglobulin replacement therapy (IgRT). Case reportOur patient was diagnosed with NEMO deficiency at 2 years with initial presentation of recurrent pneumonia, recurrent skin abscesses and severe eczema herpeticum. His disease was moderately controlled with IgRT, and prophylaxis with trimethoprim/sulfamethoxazole, azithromycin and acylovir prophylaxis, with primary complications of continued recurrent pneumonia and finger clubbing. At 9 years of age however, he developed acute back pain and weakness, along with night sweats. MRI revealed thoracic spinal cord compression at T5 secondary to a possible neoplastic soft tissue mass. A laminectomy revealed Epstein Barr virus (EBV) positive Hodgkin lymphoma, stage 4 high risk due to PET positivity with uptake at large cervical chain nodal conglomerates bilaterally, mediastinal mass, thoracic paraspinal mass, periportal lymph nodes, splenic, and right medial femoral neck. He also had low level EBV viremia (EBV viral load 7,515 IU/mL). He was treated according to AHOD1331 with ABVE-PC (doxorubicin/bleomycin/vincristine/etoposide/prednisone), and also received rituximab and valganciclovir for treatment of EBV. He is now in remission after 4 months of treatment, and continues on IgRT, pneumocystis jiroveci pneumonia, mycobacterium avium complex, and herpes simplex virus prophylaxis. ConclusionPatients with certain inborn errors of immunity have a higher incidence of malignancy due to impaired immune function. Despite their viral susceptibility, EBV-induced lymphomas are not common in NEMO deficiency. NEMO patients may need further enhanced screening for development of malignancy, including regular surveillance for chronic, cancer-causing infections such as EBV and HPV.

Epstein Barr virus nuclear antigens among University Students in Port Harcourt, Nigeria

Several diseases are associated with Epstein Barr virus (EBV) and they can be life threatening especially in immunocompromised individuals. Moreover, there is no well-established EBV prevention and control strategies in Nigeria. Therefore, this study was carried out to assess the prevalence of Epstein Barr Nuclear Antigen (EBNA) IgM antibody among university students in Port Harcourt, Nigeria. A hospital based cross-sectional survey was adopted to randomly analyze 91 students attending lectures in University of Port Harcourt, Rivers State, Nigeria. Enzyme Linked Immunosorbent Assay (ELISA) was used to analyze EBNA IgM antibody in the samples obtained. Chi-square analysis was used to determine the association of the infection with socio-demographic factors. Of the 91 subjects, 3(3.2%) were seropositive for EBNA IgM antibody while 88(96.7%) were observed to be seronegative for EBNA IgM antibody. Sera EBNA IgM positivity was highest in age group 20-25 years (4.7%), single (3.2%), male (3.8%), students (3.9), sexually inactive (4.0%), oral sex (2.4%), non-Anal sex (3.4%), never using condoms (5.0%), dry kissing (11.1%), Non-smokers (4.0%), no history of blood transfusion (3.3%), no history of tissue transplant (3.2%), no history of surgery (3.2%). This study confirms the presence of Epstein Barr virus primary infection among university students in Port Harcourt, and an onward risk of Infectious mononucleosis. This comes with the responsibility of establishing surveillance programs for detection, treatment and control of EBV in Port Harcourt and Nigeria at large.

Exploring the Impact of Humoral Immunogenicity with Tabelecleucel for the Treatment of EBV+ PTLD Following HCT and SOT

Background: Tabelecleucel is a non-genetically modified, allogeneic Epstein-Barr virus (EBV)-T-cell immunotherapy which targets and eliminates EBV-expressing cells in an HLA-restricted manner to treat patients (pts) with EBV+ post-transplant lymphoproliferative disease (PTLD) following hematopoietic stem cell transplantation (HCT) or solid organ transplantation (SOT). The theoretical risk of invoking a humoral or cell-mediated immune response against tabelecleucel is limited to the partially mismatched HLA allele. Tabelecleucel has proven to be safe and effective, with no confirmed evidence for clinical manifestation of immunogenicity in association with tabelecleucel. However, the theoretical consequences of eliciting an immune reaction against the mismatched HLA range from transient appearance of anti-HLA antibodies without any manifestation of clinically significant effects to the development of severe life-threatening conditions, which may include the potential for organ rejection. Therefore, evaluating the potential risk of immunogenicity against an allogeneic cell therapy like tabelecleucel is an important step in assessing the safety profile of the therapeutic agent. In this study we will assess the potential impact of humoral immunogenicity against tabelecleucel in pts with EBV+ PTLD after failure of rituximab or rituximab and chemotherapy enrolled in the ALLELE (NCT03394365) clinical study. Methods: Humoral immunogenicity was assessed through two methods of anti-HLA antibody detection that evaluated a total of 28 pts on study. The primary approach evaluated 9 pts by a pan anti-HLA detection method that provides a positive or negative result. In this approach evaluable pts must have a negative result in pre-treatment measurement and have at least one post-treatment measurement. Results are coupled with the pt's clinical safety profile to assess treatment related immunogenicity. The secondary approach evaluated 24 pts in a single-antigen bead method that provides not just a positive or negative result but also defines the specificity and strength of the positive result. Cross referencing the allele specificity of the Class I or Class II anti-HLA antibodies with the HLA type of the pt, the transplant and the infusion product aids in elucidating the source of the anti-HLA antibodies in these pts. Results: Two of the nine pts tested using the pan anti-HLA antibody detection approach had detection of anti-HLA antibodies in their pre-treatment sample. The remaining 7 evaluable pts had a median of 1 post-treatment measurement (min, max: 1, 3) and the median time post-treatment evaluated was 113 days (min, max: 40, 179). Six out of the 7 evaluable pts did not develop any anti-HLA antibodies post-treatment. One pt in the SOT cohort did develop anti-HLA antibodies 40 days after initial dosing on cycle 2 day 1. No treatment-emergent serious adverse events were reported for this pt, and only one adverse event of grade 1 fever was assessed as possibly related per investigator. The pt received additional doses of tabelecleucel yet did not manifest any subsequent events of fever or any signs of organ rejection. The second, single-antigen bead-based approach evaluated 24 pts. Four had anti-HLA antibodies present prior to tabelecleucel infusion. None of the pts evaluated had any incidence of de novo anti-HLA antibodies emerging post-treatment. For the 4 pts with pre-existing anti-HLA antibodies, evaluation of their safety profile and comparison to the total HLA immune profile of the pt is currently underway and will be reported at the time of presentation. Conclusions: To date, there has been no confirmed evidence for clinical manifestation of immunogenicity in association with tabelecleucel. Only 1 pt (tested using the pan anti-HLA method) out of 28 pts (tested using either method) had de novo emergence of anti-HLA antibodies post-infusion. This pt did not experience any severe events potentially reflecting immunogenicity such as hypersensitivity or SOT rejection. Taken together, these results reaffirm the low potential risk of developing humoral immunogenicity following tabelecleucel administration in HCT or SOT recipients with EBV+ PTLD after failure of rituximab or rituximab and chemotherapy.

Epstein-Barr virus-associated posttransplant lymphoproliferative disorders: new insights in pathogenesis, classification and treatment.

Posttransplant lymphoproliferative disorder (PTLD) is a serious complication following transplantation from an allogeneic donor. Epstein-Barr Virus (EBV) is involved in a substantial number of cases. In this review, we aim to summarize recent knowledge on pathogenesis, classification and treatment of EBV + PTLD. New insights in the complex oncogenic properties of EBV antigens noncoding Ribonucleic acids (RNAs), especially EBV MicroRNA (miRNAs), have increased our knowledge of the pathogenesis of EBV + PTLD. In addition the potential influence of EBV on the tumor microenvironment is becoming clearer, paving the way for new types of immunotherapy. Currently PTLD is classified according to the World Health Organization classification together with other lymphoproliferative disorders, based on the specific immunosuppression. However, a new framework integrating all types of lymphoproliferative disorders in all different settings of immune deficiency and dysregulation is needed. Although treatment of EBV + and EBV - PTLD was largely similar in the past, EBV-directed therapies are currently increasingly used. The use of EBV-directed therapies and new agents, based on better understanding of pathogenesis and classification of PTLD, will change the treatment landscape of EBV + PTLD in the next era.

Epstein Barr Virus: Development of Vaccines and Immune Cell Therapy for EBV-Associated Diseases.

Epstein-Barr virus (EBV) is the first human tumor virus discovered and is strongly implicated in the etiology of multiple lymphoid and epithelial cancers. Each year EBV associated cancers account for over 200,000 new cases of cancer and cause 150,000 deaths world-wide. EBV is also the primary cause of infectious mononucleosis, and up to 70% of adolescents and young adults in developed countries suffer from infectious mononucleosis. In addition, EBV has been shown to play a critical role in the pathogenesis of multiple sclerosis. An EBV prophylactic vaccine that induces neutralizing antibodies holds great promise for prevention of EBV associated diseases. EBV envelope proteins including gH/gL, gB and gp350 play key roles in EBV entry and infection of target cells, and neutralizing antibodies elicited by each of these proteins have shown to prevent EBV infection of target cells and markedly decrease EBV titers in the peripheral blood of humanized mice challenged with lethal dose EBV. Recent studies demonstrated that immunization with the combination of gH/gL, gB and/or gp350 induced markedly increased synergistic EBV neutralizing activity compared to immunization with individual proteins. As previous clinical trials focused on gp350 alone were partially successful, the inclusion of gH/gL and gB in a vaccine formulation with gp350 represents a promising approach of EBV prophylactic vaccine development. Therapeutic EBV vaccines have also been tested clinically with encouraging results. Immunization with various vaccine platforms expressing the EBV latent proteins EBNA1, LMP1, and/or LMP2 promoted specific CD4+ and CD8+ cytotoxic responses with anti-tumor activity. The addition of EBV envelope proteins gH/gL, gB and gp350 has the potential to increase the efficacy of a therapeutic EBV vaccine. The immune system plays a critical role in the control of tumors, and immune cell therapy has emerged as a promising treatment of cancers. Adoptive T-cell therapy has been successfully used in the prevention and treatment of post-transplant lymphoproliferative disorder. Chimeric antigen receptor T cell therapy and T cell receptor engineered T cell therapy targeting EBV latent proteins LMP1, LMP2 and/or EBNA1 have been in development, with the goal to increase the specificity and efficacy of treatment of EBV associated cancers.

Epstein-Barr DNA in advanced pediatric nasopharyngeal cancer

Background Studies suggest that the most common type of nasopharyngeal carcinoma (NPC) is WHO-3, which is strongly associated with Epstein-Barr virus (EBV).
 Objective To assess NPC patient characteristics in a national general referral hospital in Indonesia, with regards to EBV DNA load and treatment response.
 Methods Twenty-three pediatric patients diagnosed with NPC were included in the study. Data collected were history, physical examination, tissue biopsy, CT scan, staging and EBV DNA load from nasopharyngeal (NP) brushing as well as blood specimens. The NP brushing, blood specimens and CT scan evaluations were done two months post-treatment.
 Results Pediatric patients with symptoms such as blood tinged secretion, lymph node enlargement, and nasal congestion were more likely to have higher EBV DNA loads in their NP brushings (P<0.05) (including T3 and higher). Despite significant reduction of EBV DNA load in NP brushing post-treatment, it was not associated with treatment response, as evaluated by CT scan.
 Conclusion Higher DNA load from NP brushings is associated with a higher tumor stage. Larger sample size and follow-up data are needed to assess the usefulness of EBV DNA load assessment in pediatric patients.

Incidence and Risk Factors of Epstein-Barr Virus Viremia in Patients Undergoing Haploidential Transplantation with Post Translant Cyclophosphamide

INTRODUCTIONHaploidentical stem cell transplants are increasingly performed for a wide variety of malignant and non-malignant hematological conditions. Epstein - Barr virus (EBV) viremia and subsequent development of Post-Transplant Lymphoproliferative Disorder (PTLD) are potential adverse sequelae of the transplantation process and associated immunosuppression. Traditionally, haploidentical transplants have been associated with increased risk of EBV reactivation and subsequent PTLD. This was largely due to graft versus host disease (GVHD) prophylaxis protocols using intense immunosuppression with either Ex-vivo T-cell depletion or in-vivo T- cell depletion with anti-thymocyte globulin (ATG) and Alemtuzumab. However, most haploidentical transplants in recent years have utilized post-transplant cyclophosphamide (PTCy) for GVHD prophylaxis. Rituximab has been used for preemptive treatment of EBV viremia/PTLD, but there is scarce data on its use in this setting, as well as the risk factors and outcomes of EBV viremia associated with haploidentical transplants with PTCy. RESULTSA total of 63 patients with hematological malignancies received haploidentical transplant at our institution from 2013 from 2016. Patient characteristics are summarized in Table 1.All patients received EBV monitoring using plasma viral copies once weekly starting 20 days post-transplant. EBV monitoring continued for 6 months after immunosuppression ended or when CD4 count >200. Among these 63 patients, 18 (28.5%) became EBV DNA positive upon follow-up. Per institutional protocol, EBV titer was confirmed by repeat testing when viral load was 1000 copies/mL and treatment with Rituximab was initiated if repeat titer was >1000 copies/ml.The median time to detection of EBV viremia from the time of transplant was 99 days (range: 20-277). The median peak level for the EBV reactivation cohort was 7650 viral copies /ml. The median time from detection of viremia to peak viremia was 1.5 days (range 0-253).8 of the 18 (44.4%) patients that developed EBV viremia required Rituximab due to rising or persistent EBV viremia. The median EBV level at initiation of therapy was 31,000 copies/ml for the 8 patients that received Rituximab treatment. EBV seronegativity was achieved in 6 patients (75%) with treatment at a median of 13.5 days (range 3-26). The median number of Rituximab cycles administered was 1.5 (range: 1-16). None of the patients in either cohort developed PTLD or other EBV associated diseases.A univariate analysis was performed comparing EBV reactivation cohort and no EBV reactivation cohort to look for risk factors predisposing to EBV reactivation (Table 1) There were no statistically significant differences seen in the variables studied other than patients not in complete remission at the time of transplant were statistically more likely to develop EBV viremia. 9 out of 18 patients (50%) that developed EBV viremia were not in complete remission at the time of transplant, whereas 11 out of the 45 patients (24.44 %) that did not develop EBV viremia were in an incomplete remission at the time of transplant (p value: 0.05) (Table 1).With a median follow up of469 days, there were a total of 22 deaths among the 45 (48.9 %) patients that had no viremia, and 8 deaths amongst the 18 (44.4 %) patients that had viremia (p value: 0.75). There was no statistically significant difference in graft versus host disease (GVHD) incidence (p value: 0.79) or overall survival (555 days for patients with EBV reactivation versus not reached for patients without EBV reactivation, p value: 0.45) between these two groups (p value: 0.79) CONCLUSIONSIn this retrospective study assessing risk factors and outcomes of EBV viremia in patients following haploidentical transplants, we observed a 28.5 percent incidence of EBV viremia. Rituximab was effective in preemptive treatment of EBV viremia. However, no cases of PTLD were observed, even in those patients with sustained elevation in EBV titers. It is unclear how to manage asymptomatic elevations in viral titers after allogeneic transplantation in patients who are otherwise at low risk for development of PTLD. Previous data suggests that even high levels of EBV viremia lacks specificity for prediction of PTLD in post-transplant setting. Given these results, the utility of routine monitoring of EBV viral load after haploidentical transplant should be questioned in those who have received PTCy. [Display omitted] DisclosuresLin:Jazz Pharmaceuticals: Consultancy. Ganguly:Amgen: Other: Advisory Board; Seattle Genetics: Speakers Bureau.

Difficult Balance Between EBV Treatment and Posttransplant Immunosuppression: A Successful Transplant in a Child With Recurrent Epstein-Barr Virus–Induced Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening clinical syndrome. HLH can be classified into 2 major forms: primary and secondary. Viral infections are frequently implicated in the onset of active HLH episodes. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for primary HLH and refractory/relapsed HLH after proper chemoimmunotherapy, although following immunosuppressive therapy may lead to infectious complications, including viral infections. We report a case of a 6-year-old boy with Epstein-Barr virus (EBV)-induced hemophagocytic lymphohistiocytosis. The patient underwent an allo-HSCT from a 10/10 HLA-matched unrelated donor. Because he received myeloablative and immunosuppressive treatment, another EBV reactivation occurred, as well as cytomegalovirus (CMV) reactivation. After antiviral therapy, on day +27, elimination of EBV and CMV was achieved. Repeated chimerism tests evaluated decreasing donor chimerism; graft-versus-host disease prophylaxis was reduced from day +32 and eventually withdrawn. Later on, the patient developed acute graft-versus-host disease (skin rush, gastrointestinal dysfunction). Immunosuppressive agents (methylprednisolone, cyclosporine) were applied once again, which led to an increase of CMV viremia and polyomavirus (BK virus) primary infection. Virus infection can induct a severe disorder, such as HLH, and recur after its treatment. We believe our case represents dynamic changes in immunologic reaction to viral infection, which depend on modifications in treatment after allo-HSCT. These observations underscore the importance and difficulty of balancing immunosuppressive therapy and infection control.

Chapter XX Antiviral Treatment and Cancer Control.

Hepatitis B virus (HBV), hepatitis C virus (HCV), human papilloma virus (HPV), Epstein-Barr virus (EBV), human T-cell lymphotropic virus type 1 (HTLV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), and Merkel cell polyomavirus (MCV) contribute to about 10-15% global burden of human cancers. Conventional chemotherapy or molecular target therapies have been used to treat virus-associated cancers. However, a more proactive approach would be the use of antiviral treatment to suppress or eliminate viral infections to prevent the occurrence of cancer in the first place. Antiviral treatments against chronic HBV and HCV infection have achieved this goal, with significant reduction in the incidence of hepatocellular carcinoma in treated patients. Antiviral treatments for EBV, KSHV, and HTLV-1 had limited success in treating refractory EBV-associated lymphoma and post-transplant lymphoproliferative disorder, KSHV-associated Kaposi's sarcoma in AIDS patients, and HTLV-1-associated acute, chronic, and smoldering subtypes of adult T-cell lymphoma, respectively. Therapeutic HPV vaccine and RNA interference-based therapies for treating HPV-associated infection or cervical cancers also showed some encouraging results. Taken together, antiviral therapies have yielded promising results in cancer prevention and treatment. More large-scale studies in a real-world setting are necessary to confirm the efficacy of antiviral therapy. Further investigation for more effective and convenient antiviral regimens warrants more attention.

Off-the-shelf EBV-specific T-cell Immunotherapy for EBV-associated PTLD.

Off-the-shelf EBV-specific T-cell Immunotherapy for EBV-associated PTLD.

Identification of Residues Controlling dUTPase Enzyme Activity from Epstein‐Barr Virus

Epstein Barr‐Virus (EBV) is primarily known for causing infectious mononucleosis, but also increases the incidence of lymphomas and autoimmune disorders. There is currently no vaccine or treatment once infected by EBV. The EBV genome encodes a dUTPase enzyme. This enzyme catalyzes the reaction of dUTP to dUMP which is a precursor of the thymidine nucleotide. The enzyme activity regulates the dUTP/dTTP ratio and decreases misincorporation of uracil into newly synthesized DNA. A dUTPase knockout is lethal in mice, E. coli., and S. cerevisiae. The Murine gammaherpesvirus lacking its viral dUTPase has reduced viral replication. It is hypothesized that inhibition of EBV dUTPase would result in uracil misincorporation, double stranded DNA breaks, and a reduction in replication of the viral genome. Characterization of residues required for enzyme activity in EBV dUTPase was performed using sequence alignments and structural data. Mutagenesis and comparative enzyme kinetics on these residues identified aspartate 76 as being critical for enzyme activity. Analysis of structural data identified a histidine residue hypothesized to regulate substrate specificity. A small molecule binding site was identified using computational mapping of two EBV dUTPase 3D structures. We hypothesis that this allosteric binding site contains a serine that post transcriptionally regulates dUTPase activity. This research will guide the development of inhibitors that bind to critical dUTPase residues offering a potential treatment for EBV.Support or Funding InformationThis work was supported by, UWL College of Science and Health, UWL Research and Creativity, and Toce Deans Distinguished Fellowship

Tumor Microenvironment Conditioning by Abortive Lytic Replication of Oncogenic γ-Herpesviruses.

Epstein Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) constitute the human γ-herpesviruses and two of the seven human tumor viruses. In addition to their viral oncogenes that primarily belong to the latent infection programs of these viruses, they encode proteins that condition the microenvironment. Many of these are early lytic gene products and are only expressed in a subset of infected cells of the tumor mass. In this chapter I will describe their function and the evidence that targeting them in addition to the latent oncogenes could be beneficial for the treatment of EBV- and KSHV-associated malignancies.

Epstein-Barr Virus Latent Membrane Protein 1 Regulates Host B Cell MicroRNA-155 and Its Target FOXO3a via PI3K p110α Activation.

Epstein-Barr Virus (EBV) is associated with potentially fatal lymphoproliferations such as post-transplant lymphoproliferative disorder (PTLD), a serious complication of transplantation. The viral mechanisms underlying the development and maintenance of EBV+ B cell lymphomas remain elusive but represent attractive therapeutic targets. EBV modulates the expression of host microRNAs (miRs), non-coding RNAs that regulate gene expression, to promote survival of EBV+ B cell lymphomas. Here, we examined how the primary oncogene of EBV, latent membrane protein 1 (LMP1), regulates host miRs using an established model of inducible LMP1 signaling. LMP1 derived from the B95.8 lab strain or PTLD induced expression of the oncogene miR-155. However, PTLD variant LMP1 lost the ability to upregulate the tumor suppressor miR-193. Small molecule inhibitors (SMI) of p38 MAPK, NF-κB, and PI3K p110α inhibited upregulation of miR-155 by B95.8 LMP1; no individual SMI significantly reduced upregulation of miR-155 by PTLD variant LMP1. miR-155 was significantly elevated in EBV+ B cell lymphoma cell lines and associated exosomes and inversely correlated with expression of the miR-155 target FOXO3a in cell lines. Finally, LMP1 reduced expression of FOXO3a, which was rescued by a PI3K p110α SMI. Our data indicate that tumor variant LMP1 differentially regulates host B cell miR expression, suggesting viral genotype as an important consideration for the treatment of EBV+ B cell lymphomas. Notably, we demonstrate a novel mechanism in which LMP1 supports the regulation of miR-155 and its target FOXO3a in B cells through activation of PI3K p110α. This mechanism expands on the previously established mechanisms by which LMP1 regulates miR-155 and FOXO3a and may represent both rational therapeutic targets and biomarkers for EBV+ B cell lymphomas.

Administration of Third-Party Virus-Specific T-Cells (VST) at the Time of Initial Therapy for Infection after Haemopoietic Stem Cell Transplant Is Safe and Associated with Favourable Clinical Outcomes (the R3ACT-Quickly trial)

Background Administration of partially HLA-matched third party virus-specific T cells (VST) from a cryopreserved cell bank is safe and effective after failure of standard antiviral therapy to resolve viral infection occurring after allogeneic stem cell transplantation (HSCT). Aim In this phase I trial, we assessed the safety and efficacy of administering partially HLA-matched third party VST at the time of initial antiviral therapy following HSCT rather than waiting for failure of at least two weeks of standard antiviral treatment. Methods A cryopreserved cell bank of VST directed at cytomegalovirus (CMV), Epstein Barr virus (EBV) or adenovirus (Adv) was established using G-CSF mobilised peripheral blood from healthy stem cell donors. After stimulation with peptide mixes, VST were selected by expression of CD137+ cells and cultured with cytokines. HSCT recipients were treated with up to 4 doses of 2x107 of VST/m2, the first commencing within 7 days of initial antiviral treatment for viral reactivation. Results A total of 188 doses of VST were manufactured from 7 donors with 12 product manufacturing runs (CMV n=3, EBV n=4 and Adv n=5). Median virus specificity was 75% for CMV, 83% for EBV and 37% for Adv. Thirty HSCT recipients were treated with VST a median of 55 days post-transplant. Data from 25 patients treated for initial viral reactivation were available for analysis (CMV n=22, EBV n=2, Adv n=1). Median age was 58 years (0-71). Patients underwent transplant for myeloid malignancies (n=16), lymphoid malignancies (n=5) and non-malignant conditions (n=4). Patients with malignant disease were transplanted in CR1 (n=8), CR2 (n=3), >CR2 (n=3) or with active disease (n=7). Conditioning was myeloablative in 11 patients and reduced intensity in 14 patients. Donors were matched unrelated (n=20), haploidentical (n=4) or siblings (n=1). 21 patients received some form of T cell depletion (most commonly pre-transplant thymoglobuline in vivo). All patients received VST within 7 days of commencing initial antiviral therapy. 18 patients received a single VST infusion, 6 received 2 and 1 received 4 VST infusions. There were 3 mild infusion related adverse events (vomiting, hypertension, fever). 3 patients had aGVHD pre-infusion (2 grade 1 skin, 1 grade 3 GI). Two patients died of acute GVHD (1 patient with resolved grade 3 GI GVHD pre-VST infusion developed grade 4 GI GVHD 89 days post- infusion as immunosuppression was weaned; the other patient developed de novo liver and GI GVHD 30 days post infusion in the context of a rapid wean in immunosuppression for severe BK virus haemorrhagic cystitis). 2 patients developed de novo grade 1 GVHD post-infusion. 2 patients developed mild limited cGVHD and 1 patient developed extensive cGVHD after VST infusion. 23/25 patients (92%) had complete viral clearance of the infection for which VST were given, 2 had a partial viral response. Median time to best viral response was 20 days. There were 5 deaths (refractory aGVHD in 2 patients, pulmonary VOD/CMV pneumonitis, disease relapse, and sepsis/aspiration pneumonia). 4 of 25 patients died within 12 months of transplant for a 1 year NRM of 12%. At a median follow up of 431 days (112-1391) post-transplant, 20 of 25 patients (80%) remain alive (Figure 1). Conclusion Infusion of third party partially HLA-matched donor-derived VST at the time of first antiviral treatment for CMV, EBV and Adv post HSCT is associated with minimal infusion toxicity, a low rate of moderate to severe GVHD and complete viral clearance in 92% of recipients. Overall survival in this group of high-risk patients requiring treatment for viral reactivation after HSCT is high. A randomised trial will be performed to determine whether administration of third-party VST in addition to standard anti-viral treatment improves transplant outcomes. Figure 1 Disclosures Gottlieb: Haemalogix P/L: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy; Gilead: Consultancy; AbbVie: Consultancy; University of Sydney: Employment; Merck: Consultancy. Ritchie:Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; BMS: Research Funding; Takeda: Research Funding; Beigene: Research Funding; Imago: Research Funding; Novartis: Honoraria; Sanofi: Honoraria.

2090 EBV Colitis With Fulminate Ulcerative Colitis: A Clinical Dilemma

INTRODUCTION: Epstein-Barr virus (EBV) is a virus of the herpes family. EBV can be detected in the gastrointestinal tissue of patients with inflammatory bowel disease(IBD). However, the clinical significance of EBV colitis in ulcerative colitis (UC) is unclear. We present an interesting and rare case of EBV colitis in a patient with fulminant UC. CASE DESCRIPTION/METHODS: A 73-year-old male with history of hypertension and recent diagnosis of UC who was doing well on mesalamine developed a flare with acute onset diarrhea, abdominal pain, and rectal bleeding. Stool tests were negative for C difficile and common bacterial and viral pathogens. Fecal calprotectin was >2000 μg/mg, and CT abdomen/pelvis was notable for thickened colon walls. Colonoscopy showed moderate inflammation with aphthous ulcerations throughout the colon with sparing of the cecum. Patient was started on oral steroids and transitioned to adalimumab. He continued to have weight loss and diarrhea and was switched to vedolizumab after two months. Due to continued symptoms, repeat colonoscopy 6 months later showed severe inflammation with confluent ulcerations in continuous and circumferential pattern from rectum to transverse colon (Figure 1). Laboratory workup was notable for anemia (hgb 9.0 g/dL), hypoalbuminemia (2.9 g/dL) and high C-reactive protein (3.22 mg/dL). Patient was admitted for IV steroids. Infliximab was started on day 5 due to no improvement in symptoms. At this time biopsies obtained during colonoscopy were reported to be positive for EBV-encoded small RNA by in-situ hybridization (Figure 2). EBV was also detected in whole blood by PCR. IV gancyclovir was started. After 7 days of treatment with gancyclovir, repeat flexible sigmoidoscopy showed no significant improvement. Immunostains for EBV, CMV, and HSV were negative. EBV PCR declined (60,000 to 30,000 IU/ml). A subtotal abdominal colectomy with end ileostomy and rectal stump was eventually performed without any complications. Patient gained weight and did well post operatively. DISCUSSION: EBV colitis with fulminant UC is rare. The clinical implication of EBV colitis and treatment benefits are unclear. In addition, there is lack of a standard therapy for EBV infection with case reports showing improvement with gancyclovir. Our case of true EBV colitis with fulminant UC illustrates the importance of considering a superimposed infection in patients with IBD refractory to steroids and immunosuppressive therapy early in the course of the disease to prevent progression.

Establishment and verification of immortalized lymphoblastic cell lines derived from platelets CD36 deficient individuals

Objective To investigate the establishment and verification of immortal lymphoblastic cell lines from platelets CD36 deficient individuals. Methods From January 2012 to January 2018, eight individuals with platelet CD36 deficiency were selected as the study subjects. Among them, six cases were blood donors from Nanning Blood Center, one case was patient with platelet transfusion refractoriness (PTR) hospitalized in Guangxi 923 Hospital, and 1 case was mother of a child with fetal/neonatal alloimmune thrombocytopenia (FNAIT) hospitalized in Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region. All the subjects′ ages ranged from 15 to 66 years. The subjects were identified as platelet CD36 deficiency by flow cytometry, monoclonal antibody immobilization of platelet (MAIPA) and CD36 genotyping. Among them, one case was a CD36 mutant heterozygote of 538T>C, three cases were mutant heterozygote of 380C>T, one case was mutant homozygous of 329-330del, and 3 cases were mutant heterozygote of 329-330del. Volume of 5 mL heparin anticoagulant blood was collected from participants and their lymphocytes were isolated. Epstein-Barr virus (EBV) and cyclosporine were used to treat peripheral blood lymphocytes of participants with platelet CD36 deficiency to obtain immortal lymphoblast cell lines, which were frozen after stable passages. Resuscitation activity and mycoplasma detection of these cell lines were performed, and the CD36 gene was sequenced for verification. The procedure of this study is accordance with the requirement of the revised World Medical Association Declaration of Helsinki in 2013. Informed consent was obtained from each participant. Results ① After EBV and cyclosporine treatment, the peripheral blood lymphocytes of the participants were cultured for 7 d, and blastoformation of the cells with prickly edges were observed, and some of the cells were agglutinated in clusters. After continuous replacement of the complete culture medium for about 1 month, a large number of cells proliferated and grew vigorously, and more clonal spheres could be seen to form by naked eyes, and immortalized lymphoblastic cell line was successfully obtained. ② CD36 deficient immortalized lymphoblastic cell lines were subcultured for 20 generations, then frozen in liquid nitrogen. All of them were successfully resuscitated, and the cell lines were observed growing well under inverted phase contrast microscope. ③ Mycoplasma test results of the CD36 deficient immortalized lymphoblastic cell lines showed negative. ④ DNA of CD36 deficient immortalized lymphoblastic cell lines were amplified and sequenced by PCR, and comparison results showed that the DNA of immortalized lymphoblastic cell lines were identical to those of individuals with CD36 deficiency, and no gene mutation occurred. The genotypes of CD36 deficient immortalized lymphoblastic cell lines established in this study included 1 case of CD36 gene 538T>C mutant heterozygote, three cases of 380C>T mutant heterozygotes, one case of 329-330del mutant homozygote and 3 cases of 329-330del mutant heterozygotes. Conclusions CD36 deficient immortalized lymphoblastic cell lines passaged stably, the genotypes of these platelets CD36 deficient individuals are permanently preserved and can be used as long-term experimental reliable materials for the study of CD36 related platelet immunology. Key words: Antigens, CD36; Platelets; Herpesvirus 4, human; Cell line; Lymphoblast cell lines

Biomarkers for PTLD diagnosis and therapies.

Post-transplant lymphoproliferative disorder (PTLD) represents a spectrum of lymphoproliferative disorders and is a serious complication of pediatric transplantation. The majority of PTLD are associated with Epstein Barr virus (EBV) and the characteristic EBV+ B cell lymphomas are the leading post-transplant malignancy in children. EBV+ PTLD remains a formidable issue in pediatric transplantation and is thought to result from impaired immunity to EBV as a result of immunosuppression. However, the key viral and immune factors that determine whether EBV+ PTLD develops remain unknown. Recently, there has been much interest in developing biomarkers in order to improve and achieve more personalized approaches, in the clinical diagnosis, management, and treatment of EBV+ PTLD. Here, we review the status of immune-, viral-, and B cell lymphoma-derived candidates for biomarkers of EBV+ PTLD.

LATENT MEMBRANE PROTEIN 2A (LMP2A) MIMICS B‐CELL RECEPTOR SIGNALING AND PROMOTES IMMUNE ESCAPE IN EPSTEIN‐BARR VIRUS (EBV)‐POSITIVE DIFFUSE LARGE B‐CELL LYMPHOMA (DLBCL)

Background: EBV+ DLBCL is an aggressive malignancy that is largely resistant to current therapeutic regimens. LMP2A is expressed during different latency stages of EBV-infected B cells in which it triggers activation of cytoplasmic protein tyrosine kinases. Early studies have revealed that an immunoreceptor tyrosine-based activation motif in the cytoplasmic N-terminus of LMP2A can trigger a transient increase of the cytosolic Ca2+ concentration similar to that observed in activated B cells. Meanwhile, tumor cells often express PD-L1 in EBV+ DLBCL, providing a possible mechanism for immune escape. We thus explored the correlations between LMP2A expression, B-cell receptor (BCR) signaling, and PD-L1 expression in EBV+ DLBCL. Design: Two cohorts of DLBCL cases, respectively EBV-positive (n=28) and EBV-negative (n=32), were selected. LMP2A, PD-L1 and the BCR signaling-related molecule, phosphorylated form of SYK (pSYK), were immunohistochemically evaluated on formalin-fixed, paraffin-embedded tumor tissues. The expression status of LMP2A, pSYK and BCR were further validated by a flow cytometry analysis using fresh tissues from 3 EBV-positive and 5 EBV-negative DLBCL patients. Results: The EBV-positive cases, with a median age of 61yrs, were more frequently associated with a high-risk IPI score and a non-GCB phenotype (P=0.029). Compared with EBV-negative ones, patients with EBV-positive tumors showed a worse response to the RCHOP therapy and a shorter median survival (P = 0.041). Twenty-one EBV-positive cases (75 %) expressed LMP2A, whereas none of the EBV-negative cohort expressed this protein. The expression level of pSYK was significantly lower (P = 0.0313) in EBV+ DLBCL cases than those EBV-negative ones, and the pSYK level correlated negatively with LMP2A level (P = 0.119). The expression level of PD-L1 was significantly higher (P = 0.042) in EBV+ DLBCL, which correlated positively with the LMP2A level (P=0.184). Flow cytometry confirmed the LMP2A expression in EBV+ DLBCL, which correlated with a down-regulated BCR and pSYK expression. Conclusions: EBV+ DLBCL seems to feature an inactive BCR signaling, which may be related to the expression of LMP2A. Besides, LMP2A may function and promote the PD-L1 expression. These findings indicate that targeting immune checkpoints including PD-1/PD-L1 instead of BCR signaling molecules seems to be more reasonable and promising for the treatment of EBV+ DLBCL. Keywords: diffuse large B-cell lymphoma (DLBCL); Epstein-Barr virus (EBV).