Ocrelizumab Alters Cytotoxic Lymphocyte Function While Reducing EBV-Specific CD8+ T-Cell Proliferation in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: The role of B cells in the pathogenic events leading to relapsing multiple sclerosis (R-MS) has only been recently elucidated. A pivotal step in defining this role has been provided by therapeutic efficacy of anti-CD20 monoclonal antibodies. Indeed, treatment with anti-CD20 can also alter number and function of other immune cells not directly expressing CD20 on their cell surface, whose activities can contribute to unknown aspects influencing therapeutic efficacy. We examined the phenotype and function of cytotoxic lymphocytes and Epstein-Barr virus (EBV)-specific immune responses in people with R-MS before and after ocrelizumab treatment. METHODS: In this prospective study, we collected blood samples from people with R-MS (n = 41) before and 6 and 12 months after initiating ocrelizumab to assess the immune phenotype and the indirect impact on cytotoxic functions of CD8+ T and NK cells. In addition, we evaluated the specific anti-EBV proliferative responses of both CD8+ T and NK lymphocytes as surrogate markers of anti-EBV activity. RESULTS: We observed that while ocrelizumab depleted circulating B cells, it also reduced the expression of activation and migratory markers on both CD8+ T and NK cells as well as their in vitro cytotoxic activity. A comparable pattern in the modulation of immune molecules by ocrelizumab was observed in cytotoxic cells even when patients with R-MS were divided into groups based on their prior disease-modifying treatment. These effects were accompanied by a significant and selective reduction of CD8+ T-cell proliferation in response to EBV antigenic peptides. DISCUSSION: Taken together, our findings suggest that ocrelizumab-while depleting B cells-affects the cytotoxic function of CD8+ and NK cells, whose reduced cross-activity against myelin antigens might also contribute to its therapeutic efficacy during MS.

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.

Infectious mononucleosis: new concepts in clinical presentation, epidemiology, and host response.

PURPOSE OF REVIEW: Infectious mononucleosis (IM) is an infectious disease that presents clinically in only a small percentage of individuals despite almost universal infection with the causative agent. Here, we review the latest concepts in the clinical presentation, epidemiology, and host response of this disease. RECENT FINDINGS: Several recently published papers/reviews describe IM as a condition caused by one of several etiologic agents including, cytomegalovirus (HHV-5), Roseola virus (HHV-6) and Toxoplasmosis amongst others; this review focuses on IM as solely caused by the human herpes virus 4 (HHV-4). Since the initial discovery of the virus in the 1960s and its subsequent discovery as the primary etiologic agent for IM it has been associated with several human cancers and autoimmune disorders. Recent published findings show a correlation between HHV-4 and the autoimmune disorder, multiple sclerosis (MS), suggesting earlier IM could possibly act as a causative factor. Considering the important links being made with IM to so many cancers and autoimmune disorders it is surprising that a standard investigative procedure has yet to be determined for this disease. A standard approach to the investigation of IM would ensure more cases are diagnosed, particularly atypical cases, this would benefit epidemiological studies, and more immediately help practitioners distinguish viral from bacterial throat infections, enabling them to treat accordingly. SUMMARY: The understanding of the latest concepts in clinical presentation, epidemiology and host response to IM would benefit greatly from the introduction of a standard procedure for its investigation and diagnosis.

Distinguishing features of long COVID identified through immune profiling.

Post-acute infection syndromes may develop after acute viral disease1. Infection with SARS-CoV-2 can result in the development of a post-acute infection syndrome known as long COVID. Individuals with long COVID frequently report unremitting fatigue, post-exertional malaise, and a variety of cognitive and autonomic dysfunctions2-4. However, the biological processes that are associated with the development and persistence of these symptoms are unclear. Here 275 individuals with or without long COVID were enrolled in a cross-sectional study that included multidimensional immune phenotyping and unbiased machine learning methods to identify biological features associated with long COVID. Marked differences were noted in circulating myeloid and lymphocyte populations relative to the matched controls, as well as evidence of exaggerated humoral responses directed against SARS-CoV-2 among participants with long COVID. Furthermore, higher antibody responses directed against non-SARS-CoV-2 viral pathogens were observed among individuals with long COVID, particularly Epstein-Barr virus. Levels of soluble immune mediators and hormones varied among groups, with cortisol levels being lower among participants with long COVID. Integration of immune phenotyping data into unbiased machine learning models identified the key features that are most strongly associated with long COVID status. Collectively, these findings may help to guide future studies into the pathobiology of long COVID and help with developing relevant biomarkers.

Anti-Epstein-Barr Virus BNLF2b for Mass Screening for Nasopharyngeal Cancer.

BACKGROUND: Population screening of asymptomatic persons with Epstein-Barr virus (EBV) DNA or antibodies has improved the diagnosis of nasopharyngeal carcinoma and survival among affected persons. However, the positive predictive value of current screening strategies is unsatisfactory even in areas where nasopharyngeal carcinoma is endemic. METHODS: We designed a peptide library representing highly ranked B-cell epitopes of EBV coding sequences to identify novel serologic biomarkers for nasopharyngeal carcinoma. After a retrospective case-control study, the performance of the novel biomarker anti-BNLF2b total antibody (P85-Ab) was validated through a large-scale prospective screening program and compared with that of the standard two-antibody-based screening method (EBV nuclear antigen 1 [EBNA1]-IgA and EBV-specific viral capsid antigen [VCA]-IgA). RESULTS: P85-Ab was the most promising biomarker for nasopharyngeal carcinoma screening, with high sensitivity (94.4%; 95% confidence interval [CI], 86.4 to 97.8) and specificity (99.6%; 95% CI, 97.8 to 99.9) in the retrospective case-control study. Among the 24,852 eligible participants in the prospective cohort, 47 cases of nasopharyngeal carcinoma (38 at an early stage) were identified. P85-Ab showed higher sensitivity than the two-antibody method (97.9% vs. 72.3%; ratio, 1.4 [95% CI, 1.1 to 1.6]), higher specificity (98.3% vs. 97.0%; ratio, 1.01 [95% CI, 1.01 to 1.02]), and a higher positive predictive value (10.0% vs. 4.3%; ratio, 2.3 [95% CI, 1.8 to 2.8]). The combination of P85-Ab and the two-antibody method markedly increased the positive predictive value to 44.6% (95% CI, 33.8 to 55.9), with sensitivity of 70.2% (95% CI, 56.0 to 81.4). CONCLUSIONS: Our results suggest that P85-Ab is a promising novel biomarker for nasopharyngeal carcinoma screening, with higher sensitivity, specificity, and positive predictive value than the standard two-antibody method. (Funded by the National Key Research and Development Program of China and others; ClinicalTrials.gov number, NCT04085900.).

An ACE2-Based Bimodular Fusion Protein Enables Reorientation of Endogenous Anti-Epstein-Barr Virus Antibodies Toward SARS-CoV-2 Spike.

The use of soluble recombinant angiotensin-converting enzyme 2 (rACE2) as a decoy capable of blocking SARS-CoV-2 entry into cells has been envisaged as a therapeutic strategy to reduce viral loads in patients with severe COVID-19. We engineered a novel form of rACE2, fused to the Epstein-Barr virus antigen P18F3 (rACE2-P18F3), to reorient a preexisting humoral response toward Epstein-Barr virus against SARS-CoV-2 particles. Recombinant ACE2-P18F3 was able to bind to the SARS-CoV-2 spike protein, neutralize viral entry into cells, and promote the phagocytosis of spheres coated with different spike variants by monocytic cells. The results position rACE2-P18F3 as a promising therapeutic candidate to universally block coronavirus cell entry and clear viral particles.

Protective anti-gB neutralizing antibodies targeting two vulnerable sites for EBV-cell membrane fusion.

Epstein-Barr virus (EBV) infects more than 90% of the world's adult population and accounts for a significant cancer burden of epithelial and B cell origins. Glycoprotein B (gB) is the primary fusogen essential for EBV entry into host cells. Here, we isolated two EBV gB-specific neutralizing antibodies, 3A3 and 3A5; both effectively neutralized the dual-tropic EBV infection of B and epithelial cells. In humanized mice, both antibodies showed effective protection from EBV-induced lymphoproliferative disorders. Cryoelectron microscopy analyses identified that 3A3 and 3A5 bind to nonoverlapping sites on domains D-II and D-IV, respectively. Structure-based mutagenesis revealed that 3A3 and 3A5 inhibit membrane fusion through different mechanisms involving the interference with gB-cell interaction and gB activation. Importantly, the 3A3 and 3A5 epitopes are major targets of protective gB-specific neutralizing antibodies elicited by natural EBV infection in humans, providing potential targets for antiviral therapies and vaccines.

EBV miRNAs BART11 and BART17-3p promote immune escape through the enhancer-mediated transcription of PD-L1.

Epstein-Barr virus (EBV) is reportedly the first identified human tumor virus, and is closely related to the occurrence and development of nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), and several lymphomas. PD-L1 expression is elevated in EBV-positive NPC and GC tissues; however, the specific mechanisms underlying the EBV-dependent promotion of PD-L1 expression to induce immune escape warrant clarification. EBV encodes 44 mature miRNAs. In this study, we find that EBV-miR-BART11 and EBV-miR-BART17-3p upregulate the expression of PD-L1 in EBV-associated NPC and GC. Furthermore, EBV-miR-BART11 targets FOXP1, EBV-miR-BART17-3p targets PBRM1, and FOXP1 and PBRM1 bind to the enhancer region of PD-L1 to inhibit its expression. Therefore, EBV-miR-BART11 and EBV-miR-BART17-3p inhibit FOXP1 and PBRM1, respectively, and enhance the transcription of PD-L1 (CD274, http://www.ncbi.nlm.nih.gov/gene/29126 ), resulting in the promotion of tumor immune escape, which provides insights into potential targets for EBV-related tumor immunotherapy.

Epstein-Barr Virus (EBV) Genotypes Associated with the Immunopathological Profile of People Living with HIV-1: Immunological Aspects of Primary EBV Infection.

BACKGROUND: The aim of the present study was to evaluate the immunological profile of adult HIV-1+ patients coinfected with primary Epstein-Barr virus (EBV) infection who were free of antiretroviral drugs and inhabitants of the Brazilian Amazon region. MATERIALS AND METHODS: Primary EBV infection was screened by the semiquantitative detection of IgM and IgG anti-VCA. Genotypes were determined by conventional PCR. EBV and HIV viral load (VL) were quantified by real-time PCR. Cytokine dosage and cell quantification were performed by cytometry. RESULTS: Only HIV-1+ individuals had primary EBV infection (7.12%). The EBV-1 genotype was the most prevalent (47.37%). The VL of HIV-1 was lower in the HIV/EBV-2 group. CD4+ T lymphocytes were inversely proportional to the VL of EBV in HIV/EBV-1/2 multi-infected patients. The HIV/EBV-2 group had the lowest cytokine levels, especially IFN-γ and IL-4. Different correlations were proposed for each coinfection. The late search for specific care related to HIV infection directly affected the cytokine profile and the number of CD8+ T lymphocytes. Symptoms were associated with the increase in VL of both viruses and cytokine profile. CONCLUSIONS: Different immunological profiles were associated with EBV genotypes in primary infection, with EBV-2 being more frequent in patients with low levels of HIV viral load. With late infection monitoring and consequent delay in the initiation of HAART, clinical changes and effects on the maintenance of the immune response were observed.

Killer Cell Immunoglobulin-Like Receptor Haplotype B Modulates Susceptibility to EBV-Associated Classic Hodgkin Lymphoma.

Tumor cells of classic Hodgkin lymphoma (cHL) are derived from antigen presenting B cells that are infected by Epstein Barr virus (EBV) in ~30% of patients. Polymorphic Killer cell immunoglobulin-like receptors (KIRs) expressed on NK cells interact with human leukocyte antigen (HLA) class I and play a key role in immune surveillance against virally infected cells and tumor cells. We investigated the effect of KIR types on cHL susceptibility overall (n=211) and in EBV-stratified subgroups using the Dutch GoNL cohort as controls (n=498). The frequency of the KIR haplotype B subgroup was significantly different between EBV+ and EBV- cHL patients (62% vs. 77%, p=0.04) and this difference was more pronounced in nodular sclerosis (NS) cHL (49% vs. 79%, p=0.0003). The frequency of KIR haplotype B subgroup was significantly lower in EBV+ NS cHL compared to controls (49% vs. 67%, p=0.01). Analyses of known KIR - HLA interaction pairs revealed lower carrier frequencies of KIR2DS2 - HLA-C1 (29% vs. 46%, p=0.03) and KIR2DL2 - HLA-C1 (29% vs. 45%, p=0.04) in EBV+ NS cHL patients compared to controls. Carriers of the KIR haplotype B subgroup are less likely to develop EBV+ NS cHL, probably because of a more efficient control over EBV-infected B cells.

The three-dimensional structure of Epstein-Barr virus genome varies by latency type and is regulated by PARP1 enzymatic activity.

Epstein-Barr virus (EBV) persists in human B-cells by maintaining its chromatinized episomes within the nucleus. We have previously shown that cellular factor Poly [ADP-ribose] polymerase 1 (PARP1) binds the EBV genome, stabilizes CTCF binding at specific loci, and that PARP1 enzymatic activity correlates with maintaining a transcriptionally active latency program. To better understand PARP1's role in regulating EBV latency, here we functionally characterize the effect of PARP enzymatic inhibition on episomal structure through in situ HiC mapping, generating a complete 3D structure of the EBV genome. We also map intragenomic contact changes after PARP inhibition to global binding of chromatin looping factors CTCF and cohesin across the EBV genome. We find that PARP inhibition leads to fewer total unique intragenomic interactions within the EBV episome, yet new chromatin loops distinct from the untreated episome are also formed. This study also illustrates that PARP inhibition alters gene expression at the regions where chromatin looping is most effected. We observe that PARP1 inhibition does not alter cohesin binding sites but does increase its frequency of binding at those sites. Taken together, these findings demonstrate that PARP has an essential role in regulating global EBV chromatin structure and latent gene expression.

Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system of unknown etiology. We tested the hypothesis that MS is caused by Epstein-Barr virus (EBV) in a cohort comprising more than 10 million young adults on active duty in the US military, 955 of whom were diagnosed with MS during their period of service. Risk of MS increased 32-fold after infection with EBV but was not increased after infection with other viruses, including the similarly transmitted cytomegalovirus. Serum levels of neurofilament light chain, a biomarker of neuroaxonal degeneration, increased only after EBV seroconversion. These findings cannot be explained by any known risk factor for MS and suggest EBV as the leading cause of MS.

Programmed death 1 monoclonal antibody helped to treat mixed chimeric and reactivation of Epstein-Barr virus in a patient with adult-onset chronic active Epstein-Barr virus infection after allogeneic hematopoietic stem cell transplantation: A case report.

RATIONALE: Systemic forms of chronic active Epstein-Barr virus infection (CAEBV) can predispose a patient to a protracted course of fulminant hemophagocytic lymphohistiocytosis, which has a poor prognosis. Epstein-Barr virus (EBV) infection may persist even after theoretically curative hematopoietic stem cell transplantation. PATIENT CONCERNS: A female patient with CAEBV underwent chemotherapy followed by allogeneic hematopoietic stem cell transplantation from her human leukocyte antigen-matched sister. Neutrophil and platelet engraftment was observed on day +12 and +10. Full donor chimerism (DC) was achieved on Day +21. DIAGNOSES: From day +38, EBV-DNA in the blood was persistently positive, and DC declined. We attempted empirical interventions such as withdrawal of immune suppression, multiple donor lymphocyte infusion, stem cell boost, and interferon-α treatment. However, EBV-DNA copies continued to increase aggressively, whereas DC decreased rapidly and then reached a nadir of 63.27%. INTERVENTIONS: Salvage programmed death 1 (PD-1) antibody treatment was administered as salvage therapy at +69 and +84. OUTCOMES: EBV-DNA was negative on day +97 and was ultimately undetectable. Equivalently, a full and stable DC was obtained at +97. LESSONS: We summarize a case of PD-1 antibody used as salvage treatment in a post-transplant patient with CAEBV, which was eradicated and full DC was obtained. This case suggests that the PD-1 antibody appears to be a promising option for fighting EBV and mixed DCs.