Interactions between High Seroreactivity to Human Herpes Virus 6A and Epstein-Barr Virus in MS Development: A Presymptomatic Case-Control Study.

Synergistic interactions between human herpesvirus 6A (HHV-6A) and Epstein-Barr virus (EBV) are hypothesized in the etiopathogenesis of multiple sclerosis (MS). This study investigated if HHV-6A and EBV seroreactivities interact regarding the risk of developing MS. Antibodies against viral antigens were analyzed in biobank samples from 670 individuals who later developed MS and matched controls. Additive interactions were analyzed. A significant interaction between HHV-6A and EBNA-1 seroreactivities was observed in study participants above the median age of 24.9 years (attributable proportion due to interaction = 0.45). This finding supports the hypothesis that HHV-6A and EBV infections interact in MS development. ANN NEUROL 2024;96:302-305.

A two-years real-word study with fingolimod: early predictors of efficacy and an association between EBNA-1 IgG titers and multiple sclerosis progression.

Background: Although fingolimod, a sphingosine 1-phosphate receptor agonist, has shown to be an effective treatment reducing relapse rate and also slowing down the disability progression in relapsing-remitting multiple sclerosis (RRMS) patients, it is important to quickly identify those suboptimal responders. Objective: The main objective was to assess different clinical, radiological, genetic and environmental factors as possible early predictors of response in MS patients treated with fingolimod for 24 months. The secondary objective was to analyze the possible contribution of the environmental factors analyzed to the progression and activity of the disease along the 2-years of follow-up. Methods: A retrospective study with 151 patients diagnosed with MS, under fingolimod treatment for 24 months, with serum samples at initiation and six months later, and with clinical and radiological data at initiation and 24 months later, were included in the study. Clinical and radiological variables were collected to establish NEDA-3 (no evidence of disease activity: patients without relapses, disability progression and new T2 lesions or Gd+ lesions) and EDA (evidence of disease activity: patients with relapses and/or progression and/or new T2 lesions or gadolinium-positive [Gd+] lesions) conditions. Human leukocyte antigen II (HLA-II), EBNA-1 IgG and VCA IgG from Epstein-Barr virus (EBV) and antibody titers against Human herpesvirus 6A/B (HHV-6A/B) were also analyzed. Results: A total of 151 MS patients fulfilled the inclusion criteria: 27.8% was NEDA-3 (37.5% among those previously treated with high efficacy therapies >24 months). The following early predictors were statistically significantly associated with NEDA-3 condition: sex (male; p=0.002), age at baseline (older; p=0.009), relapses 2-years before fingolimod initiation ≤1 (p=0.010), and absence of Gd+ lesions at baseline (p=0.006). Regarding the possible contribution of the environmental factors included in the study to the activity or the progression of the disease, we only found that EBNA-1 IgG titers decreased in 20.0% of PIRA (progression independent from relapse activity) patients vs. 73.3% of RAW (relapse-associated worsening) patients (p=0.006; O.R. = 11.0). Conclusion: MS patients that are male, older, and with a low clinical and radiological activity at fingolimod initiation have a greater probability to reach NEDA-3 condition after two years with this therapy. An intriguing association of EBV with the progression of the disease has also been described, but it should be further study in a larger cohort to confirm these results.

Alzheimer's disease risk associated with changes in Epstein-Barr virus nuclear antigen 1-specific epitope targeting antibody levels.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder influenced by age, sex, genetic factors, immune alterations, and infections. Multiple lines of evidence suggest that changes in antibody response are linked to AD pathology. METHODS: To elucidate the mechanisms underlying AD development, we investigated antibodies that target autoimmune epitopes using high-resolution epitope microarrays. Our study compared two groups: individuals with AD (n = 19) and non-demented (ND) controls (n = 19). To validate the results, we measured antibody levels in plasma samples from AD patients (n = 96), mild cognitive impairment (MCI; n = 91), and ND controls (n = 97). To further explore the invlovement of EBV, we performed epitope masking immunofluorescence microscopy analysis and tests to induce lytic replication using the B95-8 cell line. RESULTS: In this study, we analyzed high-resolution epitope-specific serum antibody levels in AD, revealing significant disparities in antibodies targeting multiple epitopes between the AD and control groups. Particularly noteworthy was the significant down-regulation of antibody (anti-DG#29) targeting an epitope of Epstein-Barr virus nuclear antigen 1 (EBNA1). This down-regulation increased AD risk in female patients (odds ratio up to 6.6), but not in male patients. Our investigation further revealed that the down-regulation of the antibody (anti-DG#29) is associated with EBV reactivation in AD, as indicated by the analysis of EBV VCA IgG or IgM levels. Additionally, our data demonstrated that the epitope region on EBNA1 for the antibody is hidden during the EBV lytic reactivation of B95-8 cells. CONCLUSION: Our findings suggest a potential relationship of EBV in the development of AD in female. Moreover, we propose that antibodies targeting the epitope (DG#29) of EBNA1 could serve as valuable indicators of AD risk in female.

Heightened Epstein-Barr virus immunity and potential cross-reactivities in multiple sclerosis.

BACKGROUND: Epstein-Barr virus (EBV) is a likely prerequisite for multiple sclerosis (MS) but the underlying mechanisms are unknown. We investigated antibody and T cell responses to EBV in persons with MS (pwMS), healthy EBV-seropositive controls (HC) and post-infectious mononucleosis (POST-IM) individuals up to 6 months after disease resolution. The ability of EBV-specific T cell responses to target antigens from the central nervous system (CNS) was also investigated. METHODS: Untreated persons with relapsing-remitting MS, POST-IM individuals and HC were, as far as possible, matched for gender, age and HLA-DRB1*15:01. EBV load was determined by qPCR, and IgG responses to key EBV antigens were determined by ELISA, immunofluorescence and Western blot, and tetanus toxoid antibody responses by multiplex bead array. EBV-specific T cell responses were determined ex vivo by intracellular cytokine staining (ICS) and cross-reactivity of in vitro-expanded responses probed against 9 novel Modified Vaccinia Ankara (MVA) viruses expressing candidate CNS autoantigens. RESULTS: EBV load in peripheral blood mononuclear cells (PBMC) was unchanged in pwMS compared to HC. Serologically, while tetanus toxoid responses were unchanged between groups, IgG responses to EBNA1 and virus capsid antigen (VCA) were significantly elevated (EBNA1 p = 0.0079, VCA p = 0.0298) but, importantly, IgG responses to EBNA2 and the EBNA3 family antigens were also more frequently detected in pwMS (EBNA2 p = 0.042 and EBNA3 p = 0.005). In ex vivo assays, T cell responses to autologous EBV-transformed B cells and to EBNA1 were largely unchanged numerically, but significantly increased IL-2 production was observed in response to certain stimuli in pwMS. EBV-specific polyclonal T cell lines from both MS and HC showed high levels of autoantigen recognition by ICS, and several neuronal proteins emerged as common targets including MOG, MBP, PLP and MOBP. DISCUSSION: Elevated serum EBV-specific antibody responses in the MS group were found to extend beyond EBNA1, suggesting a larger dysregulation of EBV-specific antibody responses than previously recognised. Differences in T cell responses to EBV were more difficult to discern, however stimulating EBV-expanded polyclonal T cell lines with 9 candidate CNS autoantigens revealed a high level of autoreactivity and indicate a far-reaching ability of the virus-induced T cell compartment to damage the CNS.

Hitting Epstein Barr virus where it hurts: computational methods exploration for siRNA therapy in alleviating Epstein Barr virus-induced multiple sclerosis.

Multiple sclerosis (MS), an intricate neurological disorder, continues to challenge our understanding of the pivotal interplay between the immune system and the central nervous system (CNS). This condition arises from the immune system's misdirected attack on nerve fiber protection, known as myelin sheath, alongside nerve fibers themselves. This enigmatic condition, characterized by demyelination and varied clinical manifestations, prompts exploration into its multifaceted etiology and potential therapeutic avenues. Research has revealed a potential connection between Epstein Barr virus (EBV), specifically Epstein Barr Nuclear Antigen 1 (EBNA-1), and MS. The immune response to EBNA-1 antigen triggers the production of anti-EBNA-1 molecules, including IgG that identify a similar amino acid sequence to EBNA-1 in myelin, inadvertently targeting myelin sheath and contributing to MS progression. Currently, no treatment exists for EBNA-1-induced MS apart from symptom management. Addressing this, a novel potential therapeutic avenue utilizing small interference RNAs (siRNA) has been designed. By targeting the conserved EBNA-1 gene sequences in EBV types 1 and 2, five potential siRNAs were identified in our analysis. Thorough evaluations encompassing off-target binding, thermodynamics and secondary structure elucidation, efficacy prediction, siRNA-mRNA sequence binding affinity exploration, melting temperature, and docking of siRNAs with human argonaute protein 2 (AGO2) were conducted to elucidate the siRNAs efficiency. These designed siRNA molecules harnessed promising silencing activity in the EBNA-1 gene encoding the EBNA-1 antigen protein and thus have the potential to mitigate the severity of this dangerous virus.

A metabolic dependency of EBV can be targeted to hinder B cell transformation.

After infection of B cells, Epstein-Barr virus (EBV) engages host pathways that mediate cell proliferation and transformation, contributing to the propensity of the virus to drive immune dysregulation and lymphomagenesis. We found that the EBV protein EBNA2 initiates nicotinamide adenine dinucleotide (NAD) de novo biosynthesis by driving expression of the metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in infected B cells. Virus-enforced NAD production sustained mitochondrial complex I activity, to match adenosine triphosphate (ATP) production with bioenergetic requirements of proliferation and transformation. In transplant patients, IDO1 expression in EBV-infected B cells, and a serum signature of increased IDO1 activity, preceded development of lymphoma. In humanized mice infected with EBV, IDO1 inhibition reduced both viremia and lymphomagenesis. Virus-orchestrated NAD biosynthesis is therefore a druggable metabolic vulnerability of EBV-driven B cell transformation, opening therapeutic possibilities for EBV-related diseases.

A Simple Nonviral Method to Generate Human Induced Pluripotent Stem Cells Using SMAR DNA Vectors.

Induced pluripotent stem cells (iPSCs) are a powerful tool for biomedical research, but their production presents challenges and safety concerns. Yamanaka and Takahashi revolutionised the field by demonstrating that somatic cells could be reprogrammed into pluripotent cells by overexpressing four key factors for a sufficient time. iPSCs are typically generated using viruses or virus-based methods, which have drawbacks such as vector persistence, risk of insertional mutagenesis, and oncogenesis. The application of less harmful nonviral vectors is limited as conventional plasmids cannot deliver the levels or duration of the factors necessary from a single transfection. Hence, plasmids that are most often used for reprogramming employ the potentially oncogenic Epstein-Barr nuclear antigen 1 (EBNA-1) system to ensure adequate levels and persistence of expression. In this study, we explored the use of nonviral SMAR DNA vectors to reprogram human fibroblasts into iPSCs. We show for the first time that iPSCs can be generated using nonviral plasmids without the use of EBNA-1 and that these DNA vectors can provide sufficient expression to induce pluripotency. We describe an optimised reprogramming protocol using these vectors that can produce high-quality iPSCs with comparable pluripotency and cellular function to those generated with viruses or EBNA-1 vectors.

Latent Epstein-Barr virus infection collaborates with Myc over-expression in normal human B cells to induce Burkitt-like Lymphomas in mice.

Epstein-Barr virus (EBV) is an important cause of human lymphomas, including Burkitt lymphoma (BL). EBV+ BLs are driven by Myc translocation and have stringent forms of viral latency that do not express either of the two major EBV oncoproteins, EBNA2 (which mimics Notch signaling) and LMP1 (which activates NF-κB signaling). Suppression of Myc-induced apoptosis, often through mutation of the TP53 (p53) gene or inhibition of pro-apoptotic BCL2L11 (BIM) gene expression, is required for development of Myc-driven BLs. EBV+ BLs contain fewer cellular mutations in apoptotic pathways compared to EBV-negative BLs, suggesting that latent EBV infection inhibits Myc-induced apoptosis. Here we use an EBNA2-deleted EBV virus (ΔEBNA2 EBV) to create the first in vivo model for EBV+ BL-like lymphomas derived from primary human B cells. We show that cord blood B cells infected with both ΔEBNA2 EBV and a Myc-expressing vector proliferate indefinitely on a CD40L/IL21 expressing feeder layer in vitro and cause rapid onset EBV+ BL-like tumors in NSG mice. These LMP1/EBNA2-negative Myc-driven lymphomas have wild type p53 and very low BIM, and express numerous germinal center B cell proteins (including TCF3, BACH2, Myb, CD10, CCDN3, and GCSAM) in the absence of BCL6 expression. Myc-induced activation of Myb mediates expression of many of these BL-associated proteins. We demonstrate that Myc blocks LMP1 expression both by inhibiting expression of cellular factors (STAT3 and Src) that activate LMP1 transcription and by increasing expression of proteins (DNMT3B and UHRF1) known to enhance DNA methylation of the LMP1 promoters in human BLs. These results show that latent EBV infection collaborates with Myc over-expression to induce BL-like human B-cell lymphomas in mice. As NF-κB signaling retards the growth of EBV-negative BLs, Myc-mediated repression of LMP1 may be essential for latent EBV infection and Myc translocation to collaboratively induce human BLs.

The Level of Anti-Viral Antigen-Specific Antibodies to EBNA-1 in the Serum of MS Patients Does not Depend on the Severity of the Disease.

Multiple sclerosis (MS) is an autoimmune neurodegenerative disease leading to inevitable disability and primarily affecting the young and middle-aged population. Recent studies have shown a direct correlation between the risk of MS development and Epstein-Barr virus (EBV) infection. Analysis of the titer of EBV-specific antibodies among patients with MS and healthy donors among Russian population confirmed that MS is characterized by an increased level of serum IgG binding EBNA-1 (EBV nuclear antigen 1). The number of patients with elevated levels of EBNA-1-specific antibodies does not differ statistically significantly between two groups with diametrically opposite courses of MS: benign MS or highly active MS. It can be assumed that the primary link between EBV and the development of MS is restricted to the initiation of the disease and does not impact its severity.

Disease-Modifying Treatments for Multiple Sclerosis Affect Measures of Cellular Immune Responses to EBNA-1 Peptides.

BACKGROUND AND OBJECTIVES: Epstein-Barr virus (EBV) has been strongly implicated in the pathogenesis of multiple sclerosis (MS). Despite this, there are no routinely used tests to measure cellular response to EBV. In this study, we analyzed the cellular response to EBV nuclear antigen-1 (EBNA-1) in people with MS (pwMS) using a whole blood assay. METHODS: This cross-sectional study took place in a dedicated MS clinic in a university hospital. We recruited healthy controls, people with epilepsy (PWE), and pwMS taking a range of disease-modifying treatments (DMTs) including natalizumab, anti-CD20 monoclonal antibodies (mAbs), dimethyl fumarate (DMF), and also treatment naïve. Whole blood samples were stimulated with commercially available PepTivator EBNA1 peptides and a control virus-cytomegalovirus (CMV) peptide. We recorded the cellular response to stimulation with both interferon gamma (IFN-γ) and interleukin-2 (IL-2). We also compared the cellular responses to EBNA1 with IgG responses to EBNA1, viral capsid antigen (VCA), and EBV viral load. RESULTS: We recruited 86 pwMS, with relapsing remitting MS, in this group, and we observed a higher level of cellular response recorded with IFN-γ (0.79 IU/mL ± 1.36) vs healthy controls (0.29 IU/mL ± 0.90, p = 0.0048) and PWE (0.17 IU/mL ± 0.33, p = 0.0088). Treatment with either anti-CD20 mAbs (0.28 IU/mL ± 0.57) or DMF (0.07 IU/mL ± 0.15) resulted in a cellular response equivalent to control levels or in PWE (p = 0.26). The results of recording IL-2 response were concordant with IFN-γ: with suppression also seen with anti-CD20 mAbs and DMF. By contrast, we did not record any differential effect of DMTs on the levels of IgG to either EBNA-1 or VCA. Nor did we observe differences in cellular response to cytomegalovirus between groups. DISCUSSION: This study demonstrates how testing and recording the cellular response to EBNA-1 in pwMS may be beneficial. EBNA-1 stimulation of whole blood samples produced higher levels of IFN-γ and IL-2 in pwMS compared with controls and PWE. In addition, we show a differential effect of currently available DMTs on this response. The functional assay deployed uses whole blood samples with minimal preprocessing suggesting that employment as a treatment response measure in clinical trials targeting EBV may be possible.

Shared and distinct interactions of type 1 and type 2 Epstein-Barr Nuclear Antigen 2 with the human genome.

BACKGROUND: There are two major genetic types of Epstein-Barr Virus (EBV): type 1 (EBV-1) and type 2 (EBV-2). EBV functions by manipulating gene expression in host B cells, using virus-encoded gene regulatory proteins including Epstein-Barr Nuclear Antigen 2 (EBNA2). While type 1 EBNA2 is known to interact with human transcription factors (hTFs) such as RBPJ, EBF1, and SPI1 (PU.1), type 2 EBNA2 shares only ~ 50% amino acid identity with type 1 and thus may have distinct binding partners, human genome binding locations, and functions. RESULTS: In this study, we examined genome-wide EBNA2 binding in EBV-1 and EBV-2 transformed human B cells to identify shared and unique EBNA2 interactions with the human genome, revealing thousands of type-specific EBNA2 ChIP-seq peaks. Computational predictions based on hTF motifs and subsequent ChIP-seq experiments revealed that both type 1 and 2 EBNA2 co-occupy the genome with SPI1 and AP-1 (BATF and JUNB) hTFs. However, type 1 EBNA2 showed preferential co-occupancy with EBF1, and type 2 EBNA2 preferred RBPJ. These differences in hTF co-occupancy revealed possible mechanisms underlying type-specific gene expression of known EBNA2 human target genes: MYC (shared), CXCR7 (type 1 specific), and CD21 (type 2 specific). Both type 1 and 2 EBNA2 binding events were enriched at systemic lupus erythematosus (SLE) and multiple sclerosis (MS) risk loci, while primary biliary cholangitis (PBC) risk loci were specifically enriched for type 2 peaks. CONCLUSIONS: This study reveals extensive type-specific EBNA2 interactions with the human genome, possible differences in EBNA2 interaction partners, and a possible new role for type 2 EBNA2 in autoimmune disorders. Our results highlight the importance of considering EBV type in the control of human gene expression and disease-related investigations.

Epstein-Barr virus latency patterns in polymorphic lymphoproliferative disorders and lymphomas in immunodeficiency settings: Diagnostic implications.

Epstein-Barr virus (EBV) is responsible for many B cell lymphoproliferative disorders (LPD) spanning subclinical infection to immunodeficiency-related neoplasms. EBV establishes a latent infection in the host B cell as defined histologically by the expression of EBV latent membrane proteins and nuclear antigens. Herein, we characterize the latency patterns of immunodeficiency-related neoplasms including post-transplant lymphoproliferative disorders (PTLD) and therapy-related LPD (formerly iatrogenic) with latent membrane protein-1 (LMP-1) and EBV nuclear antigen-2 (EBNA-2) immunohistochemistry. The latency pattern was correlated with immunodeficiency and dysregulation (IDD) status and time from transplant procedure. 38 cases of EBV+ PTLD in comparison to 27 cases of classic Hodgkin lymphoma (CHL) and diffuse large B cell lymphoma (DLBCL) arising in either the therapy-related immunodeficiency setting (n = 12) or without an identified immunodeficiency (n = 15) were evaluated for EBV-encoded small RNAs by in situ hybridization (EBER-ISH) and for LMP-1 and EBNA-2 by immunohistochemistry. A full spectrum of EBV latency patterns was observed across PTLD in contrast to CHL and DLBCL arising in the therapy-related immunodeficiency setting. Polymorphic-PTLD (12 of 16 cases, 75 %) and DLBCL-PTLD (9 of 11 cases, 82 %) showed the greatest proportion of cases with latency III pattern. Whereas, EBV+ CHL in an immunocompetent patient showed exclusively latency II pattern (13 of 13 cases, 100 %). The majority of EBV+ PTLD occurred by three years of transplant procedure date and were enriched for latency III pattern (21 of 22 cases, 95 %). Immunohistochemical identification of EBV latency by LMP-1 and EBNA-2 can help classify PTLD in comparison to other EBV+ B cell LPD and lymphomas arising in therapy-related immunodeficiency and non-immunodeficiency settings.

Serologic Response to the Epstein-Barr Virus Peptidome and the Risk for Multiple Sclerosis.

Importance: It remains unclear why only a small proportion of individuals infected with the Epstein-Barr virus (EBV) develop multiple sclerosis (MS) and what the underlying mechanisms are. Objective: To assess the serologic response to all EBV peptides before the first symptoms of MS occur, determine whether the disease is associated with a distinct immune response to EBV, and evaluate whether specific EBV epitopes drive this response. Design, Setting, and Participants: In this prospective, nested case-control study, individuals were selected among US military personnel with serum samples stored in the US Department of Defense Serum Repository. Individuals with MS had serum collected at a median 1 year before onset (reported to the military in 2000-2011) and were matched to controls for age, sex, race and ethnicity, blood collection, and military branch. No individuals were excluded. The data were analyzed between September 1, 2022, and August 31, 2023. Exposure: Antibodies (enrichment z scores) to the human virome measured using VirScan (phage-displayed immunoprecipitation and sequencing). Main Outcome and Measure: Rate ratios (RRs) for MS for antibodies to 2263 EBV peptides (the EBV peptidome) were estimated using conditional logistic regression, adjusting for total anti-EBV nuclear antigen 1 (EBNA-1) antibodies, which have consistently been associated with a higher MS risk. The role of antibodies against other viral peptides was also explored. Results: A total of 30 individuals with MS were matched with 30 controls. Mean (SD) age at sample collection was 27.8 (6.5) years; 46 of 60 participants (76.7%) were male. The antibody response to the EBV peptidome was stronger in individuals with MS, but without a discernible pattern. The antibody responses to 66 EBV peptides, the majority mapping to EBNA antigens, were significantly higher in preonset sera from individuals with MS (RR of highest vs lowest tertile of antibody enrichment, 33.4; 95% CI, 2.5-448.4; P for trend = .008). Higher total anti-EBNA-1 antibodies were also associated with an elevated MS risk (top vs bottom tertile: RR, 27.6; 95% CI, 2.3-327.6; P for trend = .008). After adjusting for total anti-EBNA-1 antibodies, risk estimates from most EBV peptides analyses were attenuated, with 4 remaining significantly associated with MS, the strongest within EBNA-6/EBNA-3C, while the association between total anti-EBNA-1 antibodies and MS persisted. Conclusion and Relevance: These findings suggest that antibody response to EBNA-1 may be the strongest serologic risk factor for MS. No single EBV peptide stood out as being selectively targeted in individuals with MS but not controls. Larger investigations are needed to explore possible heterogeneity of anti-EBV humoral immunity in MS.

Evaluating the Clinical Utility of Epstein-Barr Virus Antibodies as Biomarkers in Multiple Sclerosis: A Systematic Review.

BACKGROUND: EBV is a necessary but not sufficient factor in the pathophysiology of multiple sclerosis (MS). EBV antibodies to the nuclear antigen (EBNA1) and viral capsid antigen (VCA) rise rapidly prior to MS disease manifestations, and their absence has clinical utility with a high negative predictive value. It remains unclear whether EBV levels act as prognostic, monitoring, or pharmacodynamic/response biomarkers. Substantial literature on this topic exists but has not been systematically reviewed. We hypothesized that EBV levels against EBNA1 and VCA are potential prognostic and monitoring biomarkers in MS, and that patient population, MS clinical phenotype, and EBV assay method may play important roles in explaining variation among study outcomes. METHODS: We systematically searched PubMed and EMBASE from inception to April 1, 2022. After removal of duplicates, records were screened by abstract. Remaining full-text articles were reviewed. Clinical and MRI data were extracted from full-text articles for comparison and synthesis. RESULTS: Searches yielded 696 unique results; 285 were reviewed in full, and 36 met criteria for data extraction. Heterogeneity in sample population, clinical outcome measures, assay methods and statistical analyses precluded a meta-analysis. EBV levels were not consistently associated with clinical disease markers including conversion from CIS to RRMS, neurological disability, or disease phenotype. Studies using repeated-measures design suggest that EBNA1 levels may temporarily reflect inflammatory disease activity as assessed by gadolinium-enhancing Magnetic Resonance Imaging (MRI) lesions. Limited data also suggest a decrease in EBV levels following initiation of certain disease-modifying therapies. CONCLUSION: Heterogeneous methodology limited generalization and meta-analysis. EBV antibody levels are unlikely to represent prognostic biomarkers in MS. The areas of highest ongoing promise relate to diagnostic exclusion and pharmacodynamic/disease response. Use of EBV antibodies as biomarkers in clinical practice remains additionally limited by lack of methodological precision, reliability, and validation.

Epstein-Barr virus induces germinal center light zone chromatin architecture and promotes survival through enhancer looping at the BCL2A1 locus.

IMPORTANCE: Epstein-Barr virus has evolved with its human host leading to an intimate relationship where infection of antibody-producing B cells mimics the process by which these cells normally recognize foreign antigens and become activated. Virtually everyone in the world is infected by adulthood and controls this virus pushing it into life-long latency. However, immune-suppressed individuals are at high risk for EBV+ cancers. Here, we isolated B cells from tonsils and compare the underlying molecular genetic differences between these cells and those infected with EBV. We find similar regulatory mechanism for expression of an important cellular protein that enables B cells to survive in lymphoid tissue. These findings link an underlying relationship at the molecular level between EBV-infected B cells in vitro with normally activated B cells in vivo. Our studies also characterize the role of a key viral control mechanism for B cell survival involved in long-term infection.

Validation of a robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of VK-2019, a selective EBNA1 inhibitor.

EBNA1 is an Epstein Barr virus (EBV) protein expressed in all EBV-associated cancers. EBNA1 plays a critical role in the replication and maintenance of EBV episomes in latently infected cells. VK-2019 was developed as a highly specific inhibitor of EBNA1 DNA binding activity and is currently in phase 1 development as a treatment for EBV-associated carcinomas. A sensitive and reliable method was developed to quantify VK-2019 in human plasma using liquid chromatography with tandem mass spectrometry to perform detailed pharmacokinetic studies. VK-2019 was extracted from plasma using protein precipitation with acetonitrile. Separation of VK-2019, two purported metabolites, and the internal standard, VK-2019-d6, was achieved with a Zorbax XDB C18 column using a gradient flow over 6 min. VK-2019 was detected using a SCIEX 4500 triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The assay range was 0.5-500 ng/mL and proved to be accurate and precise. Dilutions of 1:10 were accurately quantified. VK-2019 was stable in plasma at -70°C for approximately 18 months. The method was applied to assess the total plasma concentrations of VK-2019 in a patient who received a single and multiple oral daily doses of 120 mg.

Seroreactivity against lytic, latent and possible cross-reactive EBV antigens appears on average 10 years before MS induced preclinical neuroaxonal damage.

BACKGROUND: Multiple sclerosis (MS) and presymptomatic axonal injury appear to develop only after an Epstein-Barr virus (EBV) infection. This association remains to be confirmed across a broad preclinical time range, for lytic and latent EBV seroreactivity, and for potential cross-reacting antigens. METHODS: We performed a case-control study with 669 individual serum samples obtained before clinical MS onset, identified through cross-linkage with the Swedish MS register. We assayed antibodies against EBV nuclear antigen 1 (EBNA1), viral capsid antigen p18, glycoprotein 350 (gp350), the potential cross-reacting protein anoctamin 2 (ANO2) and the level of sNfL, a marker of axonal injury. RESULTS: EBNA1 (latency) seroreactivity increased in the pre-MS group, at 15-20 years before clinical MS onset, followed by gp350 (lytic) seroreactivity (p=0.001-0.009), ANO2 seropositivity appeared shortly after EBNA1-seropositivity in 16.7% of pre-MS cases and 10.0% of controls (p=0.001).With an average lag of almost a decade after EBV, sNfL gradually increased, mainly in the increasing subgroup of seropositive pre-MS cases (p=8.10-5 compared with non-MS controls). Seropositive pre-MS cases reached higher sNfL levels than seronegative pre-MS (p=0.038). In the EBNA1-seropositive pre-MS group, ANO2 seropositive cases had 26% higher sNfL level (p=0.0026). CONCLUSIONS: Seroreactivity against latent and lytic EBV antigens, and in a subset ANO2, was detectable on average a decade before the appearance of a gradually increasing axonal injury occurring in the last decade before the onset of clinical MS. These findings strengthen the hypothesis of latent EBV involvement in the pathogenesis of MS.

Epstein-Barr virus-encoded EBNA2 downregulates ICOSL by inducing miR-24 in B-cell lymphoma.

ABSTRACT: Hematological malignancies such as Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B-cell lymphoma (DLBCL) cause significant morbidity in humans. A substantial number of these lymphomas, particularly HL and DLBCLs have poorer prognosis because of their association with Epstein-Barr virus (EBV). Our earlier studies have shown that EBV-encoded nuclear antigen (EBNA2) upregulates programmed cell death ligand 1 in DLBCL and BLs by downregulating microRNA-34a. Here, we investigated whether EBNA2 affects the inducible costimulator (ICOS) ligand (ICOSL), a molecule required for efficient recognition of tumor cells by T cells through the engagement of ICOS on the latter. In virus-infected and EBNA2-transfected B-lymphoma cells, ICOSL expression was reduced. Our investigation of the molecular mechanisms revealed that this was due to an increase in microRNA-24 (miR-24) by EBNA2. By using ICOSL 3' untranslated region-luciferase reporter system, we validated that ICOSL is an authentic miR-24 target. Transfection of anti-miR-24 molecules in EBNA2-expressing lymphoma cells reconstituted ICOSL expression and increased tumor immunogenicity in mixed lymphocyte reactions. Because miR-24 is known to target c-MYC, an oncoprotein positively regulated by EBNA2, we analyzed its expression in anti-miR-24 transfected lymphoma cells. Indeed, the reduction of miR-24 in EBNA2-expressing DLBCL further elevated c-MYC and increased apoptosis. Consistent with the in vitro data, EBNA2-positive DLBCL biopsies expressed low ICOSL and high miR-24. We suggest that EBV evades host immune responses through EBNA2 by inducing miR-24 to reduce ICOSL expression, and for simultaneous rheostatic maintenance of proproliferative c-MYC levels. Overall, these data identify miR-24 as a potential therapeutically relevant target in EBV-associated lymphomas.

Sequence variation of the Epstein-Barr virus nuclear antigen 1 (EBNA1) gene in chronic lymphocytic leukemia and healthy volunteer subjects.

Epstein-Barr virus-related malignancies have been linked to variations in the sequences of EBV genes, notably EBNA1. Therefore, the purpose of this study was to examine the DBD/DD domain and USP7 binding domain sequences at the C-terminus of the EBNA1 gene in patients with chronic lymphocytic leukemia (CLL). This study included 40 CLL patients and 21 healthy volunteers. Using commercial kits, total DNA was extracted from buffy coat samples, and each sample was tested for the presence of the EBV genome. The C-terminus of EBNA1 was then amplified from positive samples, using nested PCR. Sanger sequencing was used to identify mutations in the PCR products, and the results were analyzed using MEGA11 software. The mean ages of CLL patients and healthy individuals were 61.07 ± 10.2 and 59.08 ± 10.3, respectively. In the EBNA-1 amplicons from CLL patients and healthy individuals, 38.5% and 16.7%, respectively, harbored mutations in the DBD/DD domain of the C-terminal region of the EBNA1 gene (P = 0.378). The mutation frequency at locus 97,320 was significantly higher in CLL patients than in healthy individuals (P = 0.039). Three EBV subtypes based on residue 487 were detected. The frequency of alanine, threonine, and valine in both groups was 88, 8, and 4 percent, respectively (P = 0.207). Moreover, all of the isolates from healthy donors had alanine at this position. The findings indicated that the presence of threonine or valine at residue 487 as well as a synonymous substitution at residue 553 in the C-terminal region of EBNA1 might be involved in the pathogenesis of EBV in CLL patients.