Persistent Epstein-Barr Virus Infection Research Articles

X-Linked Lymphoproliferative Syndrome: A Spectrum of Clinical and Immunological Profile and Novel Pathogenic Variants from Chandigarh, India

Introduction: X-linked lymphoproliferative syndrome (XLP) is a rare primary immune deficiency. Two types of XLP have been described: XLP-1 and XLP-2. Methods: We found 7 patients with XLP (3 had XLP-1 and 4 had XLP-2) after reviewing the data from Pediatric Immunodeficiency Clinic from 1997 to 2021. Results: Mean age at diagnosis was 3.8 years, and mean delay in diagnosis was 2.6 years. Five patients had recurrent episodes of infections. Four patients developed at least one episode of hemophagocytic lymphohistiocytosis (HLH) (2 with XLP-1 and 2 with XLP-2). Of these, 2 had recurrent HLH (both with XLP-2). Epstein-Barr virus (EBV) infection was detected in 2 (1 with XLP-1 and 1 with XLP-2). Both these patients had HLH. One child with XLP-2 had inflammatory bowel disease. Hypogammaglobulinemia was seen in 3 (2 with XLP-1 and 1 with XLP-2). Genetic analysis showed previously reported variants in 5, while 2 had novel variants (one in exon 7 of XIAP gene [c.1370dup p.Asn457Lysfs Ter16] and other had splice site variant in intron 1 of SH2D1A gene [c.138-2_138-1insG]). Episodes of HLH were managed with intravenous immunoglobulin (IVIg), methylprednisolone, oral prednisolone, cyclosporine, and rituximab. Inflammatory bowel disease was managed using oral prednisolone and azathioprine. One patient underwent haploidentical hematopoietic stem cell transplantation. One child with XLP-2 and WAS died because of fulminant pneumonia. Discussion/Conclusions: XLP should be considered as a strong possibility in any patient with features of HLH, repeated infections with hypogammaglobulinemia, persistent EBV infection, and early-onset IBD.

Mechanisms of T cell evasion by Epstein-Barr virus and implications for tumor survival.

Epstein-Barr virus (EBV) is a prevalent oncogenic virus estimated to infect greater than 90% of the world's population. Following initial infection, it establishes latency in host B cells. EBV has developed a multitude of techniques to avoid detection by the host immune system and establish lifelong infection. T cells, as important contributors to cell-mediated immunity, make an attractive target for these immunoevasive strategies. Indeed, EBV has evolved numerous mechanisms to modulate T cell responses. For example, it can augment expression of programmed cell death ligand-1 (PD-L1), which inhibits T cell function, and downregulates the interferon response, which has a strong impact on T cell regulation. It also modulates interleukin secretion and can influence major histocompatibility complex (MHC) expression and presentation. In addition to facilitating persistent EBV infection, these immunoregulatory mechanisms have significant implications for evasion of the immune response by tumor cells. This review dissects the mechanisms through which EBV avoids detection by host T cells and discusses how these mechanisms play into tumor survival. It concludes with an overview of cancer treatments targeting T cells in the setting of EBV-associated malignancy.

Caebv Disease Originates from Infected Hematopoietic Stem Cells

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

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

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

EBV-induced T-cell responses in EBV-specific and nonspecific cancers.

Epstein-Barr virus (EBV) is a ubiquitous human tumor virus associated with various malignancies, including B-lymphoma, NK and T-lymphoma, and epithelial carcinoma. It infects B lymphocytes and epithelial cells within the oropharynx and establishes persistent infection in memory B cells. With a balanced virus-host interaction, most individuals carry EBV asymptomatically because of the lifelong surveillance by T cell immunity against EBV. A stable anti-EBV T cell repertoire is maintained in memory at high frequency in the blood throughout persistent EBV infection. Patients with impaired T cell immunity are more likely to develop life-threatening lymphoproliferative disorders, highlighting the critical role of T cells in achieving the EBV-host balance. Recent studies reveal that the EBV protein, LMP1, triggers robust T-cell responses against multiple tumor-associated antigens (TAAs) in B cells. Additionally, EBV-specific T cells have been identified in EBV-unrelated cancers, raising questions about their role in antitumor immunity. Herein, we summarize T-cell responses in EBV-related cancers, considering latency patterns, host immune status, and factors like human leukocyte antigen (HLA) susceptibility, which may affect immune outcomes. We discuss EBV-induced TAA-specific T cell responses and explore the potential roles of EBV-specific T cell subsets in tumor microenvironments. We also describe T-cell immunotherapy strategies that harness EBV antigens, ranging from EBV-specific T cells to T cell receptor-engineered T cells. Lastly, we discuss the involvement of γδ T-cells in EBV infection and associated diseases, aiming to elucidate the comprehensive interplay between EBV and T-cell immunity.

No Additional Risk of Ampicillin Rash Among Pediatric Liver Transplant Recipients With Concurrent Epstein-Barr Virus Infection.

Epstein-Barr virus (EBV) infection frequently develops in children undergoing liver transplantation (LT) because of mandated immunosuppressive therapy. There is a risk of ampicillin rash when penicillin derivatives are used in patients with EBV-associated infectious mononucleosis. Hence, the administration of penicillin derivatives may raise concerns about ampicillin rash in patients with high EBV loads. However, no studies confirmed the risk of administering penicillin derivatives to EBV-infected children after LT. This retrospective study was conducted at the largest pediatric transplantation center in Japan. We investigated all pediatric liver transplant recipients who received penicillin derivatives within 2 years of LT between 2014 and 2020. We separated the cohort into EBV-positive and EBV-negative groups to assess the frequency of ampicillin and antibiotic-associated rash. Two hundred eighty-six liver transplant recipients were enrolled. There were 111 recipients in the EBV-positive group and 175 recipients in the EBV-negative group. In the EBV-positive group, 49 patients had high EBV DNA loads (≥1000 copies/µg DNA). None of the patients in either group developed ampicillin rash, and the frequency of antibiotic-associated rash did not differ [8/111 (7.2%) vs. 10/175 (5.7%), P = 0.797]. Additional subgroup analysis revealed no difference in the frequency of antibiotic-associated rashes regardless of the presence or absence of high EBV loads. In this study, ampicillin rash was not observed, and antibiotic-associated rash was not associated with concurrent EBV infection. Penicillin derivatives can be used safely, even in liver transplant recipients with persistent asymptomatic EBV infection.

Modification of the course of post-COVID syndrome associated with reactivation of chronic persistent Epstein−Barr virus infection and chronic nasopharyngotonsillitis of mixed etiology

A wide range of symptoms that occur in patients with post-COVID syndrome can include both manifestations of the clinical consequences of COVID-19 and symptoms of an exacerbation of a chronic pathology that they had before the development of coronavirus infection. Correct assessment of clinical data can help determine the origin of the existing manifestations of the disease and build a plan for adequate diagnostic and therapeutic measures.
 In the article, on the example of a clinical case, a variant of the modified (exacerbation of chronic pathology) course of the post-COVID syndrome was analyzed. An example of modern laboratory diagnostics of reactivation of chronic persistent EpsteinBarr virus infection and exacerbation of chronic nasopharyngotonsillitis of mixed viral-bacterial-mycotic etiology, developed against the background of post-COVID syndrome, is given. The principles of therapy for this comorbid condition are presented and explained.

Challenges to allogeneic hematopoietic stem cell transplantation in a patient with GATA2 deficiency and persistent Epstein-Barr virus infection

The germline Guanine-adenine-thymine-adenine 2 (GATA2) heterozygous mutations (GATA2 deficiency) are identified as a complex immunodeficiency, which entails susceptibility to non-tuberculous mycobacterium (NTM), fungus, or Epstein-Barr virus (EBV) infection, and hematological disorders including myelodysplastic syndrome (MDS)/acute myelogenous leukemia, accompanied by characteristic sighs such as lymphedema or deafness. A 21-year-old woman with persistent EBV infection was diagnosed with GATA2 deficiency based on the onset of MDS and her family history. The patient underwent bone marrow transplantation from an unrelated donor. She developed steroid-resistant graft-versus-host disease (GVHD), leading to an NTM infection, the deterioration of lymphedema, and life-threatening gut bleeding caused by not only GVHD but also thrombotic microangiopathy. Although allogeneic hematopoietic stem cell transplantation is a potential therapeutic option in patients with GATA2 deficiency, GATA2 haploinsufficiency in non-hematopoietic cells may affect the post-transplant complications. Therefore, the establishment of treatment guidelines on donor and graft selection, the intensity of conditioning regimens, GVHD prophylaxis, and prevention of post-transplantation infections based on the history of infection and degree of organ damage is necessary for patients with GATA2 deficiency.

An in-silico insight into the predictive interaction of Apolipoprotein-E with Epstein-Barr virus proteins and their probable role in mediating Alzheimer’s disease

Recent reports suggest that persistent Epstein-Barr virus (EBV) infection and its recurrent reactivation could instigate the formation of proteinaceous plaques in the brain: a hallmark of Alzheimer’s disease (AD). Interestingly, a major genetic risk factor of AD, the apolipoprotein E (ApoE), could also influence the outcome of EBV infection in an individual. The ApoE is believed to influence the proteinaceous plaque clearance from the brain, and its defective functioning could result in the aggregate deposition. The persistent presence of EBV infection in a genetically predisposed individual could create a perfect recipe for severe neurodegenerative consequences. Therefore, in the present study, we investigated the possible interactions between ApoE and various EBV proteins using computational tools. Our results showed possibly stable de-novo interactions between the C-terminal domain of ApoE3 and EBV proteins: EBV nuclear antigen-1 (EBNA1) and BamHI Z fragment leftward open reading frame-1 (BZLF1). The EBNA1 protein of EBV plays a crucial role in establishing latency and replication of the virus. Whereas BZLF1 is involved in the lytic replication cycle. The proposed interaction of EBV proteins at the ligand-binding site of ApoE3 on CTD could interfere with- its capability to sequester amyloid fragments and, hence their clearance from the brain giving rise to AD pathology. This study provides a new outlook on EBV’s underexplored role in AD development and paves the way for novel avenues of investigation which could further our understanding of AD pathogenesis. Communicated by Ramaswamy H. Sarma

Nasopharyngeal Carcinoma Progression: Accumulating Genomic Instability and Persistent Epstein-Barr Virus Infection.

Genomic instability facilitates the evolution of cells, tissues, organs, and species. The progression of human malignancies can be regarded as the accumulation of genomic instability, which confers a high evolutionary potential for tumor cells to adapt to continuous changes in the tumor microenvironment. Nasopharyngeal carcinoma (NPC) is a head-and-neck squamous-cell carcinoma closely associated with Epstein–Barr virus (EBV) infection. NPC progression is driven by a combination of accumulated genomic instability and persistent EBV infection. Here, we present a review of the key characteristics of genomic instability in NPC and the profound implications of EBV infection. We further discuss the significance of profiling genomic instability for the assessment of disease progression and treatment efficacy, as well as the opportunities and challenges of targeted therapies for NPC based on its unique genomic instability.

Herpesviruses and their genetic diversity in the blood virome of healthy individuals: effect of aging

BackgroundAs we age, the functioning of the human immune system declines. The results of this are increases in morbidity and mortality associated with infectious diseases, cancer, cardiovascular disease, and neurodegenerative disease in elderly individuals, as well as a weakened vaccination response. The aging of the immune system is thought to affect and be affected by the human virome, the collection of all viruses present in an individual. Persistent viral infections, such as those caused by certain herpesviruses, can be present in an individual for long periods of time without any overt pathology, yet are associated with disease in states of compromised immune function. To better understand the effects on human health of such persistent viral infections, we must first understand how the human virome changes with age. We have now analyzed the composition of the whole blood virome of 317 individuals, 21–70 years old, using a metatranscriptomic approach. Use of RNA sequencing data allows for the unbiased detection of RNA viruses and active DNA viruses.ResultsThe data obtained showed that Epstein-Barr virus (EBV) was the most frequently expressed virus, with other detected viruses being herpes simplex virus 1, human cytomegalovirus, torque teno viruses, and papillomaviruses. Of the 317 studied blood samples, 68 (21%) had EBV expression, whereas the other detected viruses were only detected in at most 6 samples (2%). We therefore focused on EBV in our further analyses. Frequency of EBV detection, relative EBV RNA abundance and the genetic diversity of EBV was not significantly different between age groups (21–59 and 60–70 years old). No significant correlation was seen between EBV RNA abundance and age. Deconvolution analysis revealed a significant difference in proportions of activated dendritic cells, macrophages M1, and activated mast cells between EBV expression positive and negative individuals.ConclusionsAs it is likely that the EBV RNA quantified in this work is derived from reactivation of the latent EBV virus, these data suggest that age does not affect the rate of reactivation nor the genetic landscape of EBV. These findings offer new insight on the genetic diversity of a persistent EBV infection in the long-term.

Effectiveness of treatment of patients with systemic autoimmune diseases on the background of reactivation of persistent Epstein-Barr virus infection

The article presents the study of effectiveness of inosine pranobex (IP) in patients with systemic autoimmune diseases (SAD) on the background of reactivation of persistent Epstein-Barr (EBV) infection. Among 380 patients with SAD (systemic lupus erythematosus, systemic vasculitides, rheumatoid arthritis, psoriasis), in 144 patients (37.9%) the reactivation of persistent EBV infection was detected through virus DNA identification using polymerase chain reaction (PCR) in three biological matrices (blood, saliva, scraping from the lesion site). 48 patients were receiving inosine pranobex at a dose of 50 mg/kg per day for three months. Treatment efficacy was controlled by studying the levels of expression of miR-146а, miR-155, miR EBV (BART-13 and BART-15), TLR9, the quantity of lymphocytes populations and subpopulations. After treatment, PCR results showed a decrease in viral replication in 66.7% of cases. The use of IP contributed to a significant decrease in the level of IgM, IgG specific antibodies, an increase in the level of expression of anti-inflammatory miR-146a, a decrease in the level of expression of pro-inflammatory miR-155 which may signify the strengthening of antiviral control. The study data demonstrated the decrease in the expression of miR EBV (BART-13 and BART-15) and TLR9 on the immunocompetent cells that can also be attributed to the criteria for IP effectiveness. The effectiveness of IP was also proved by the stabilization of cell mechanisms, namely the tendency to normalizing T and B cell populations, decrease in the number of natural killer cells and activated cells (CD25+, CD3+ HLA DR+). On the other hand, the number of lymphocytes with suppressor activity (CD4+25+) remained significantly high mitigating autoimmune aggression. The results of the study show that the use of IP for treating the acute phase of EBV infection contributed to the decrease of repliсative activity of the virus; suppressing the aggressiveness of autoimmune reactions. The decrease in the expression of miR EBV (BART-13 and BART-15) can be recommended as a criterion for the IP effectiveness; the decrease in the expression of TLR9 on immunocompetent cells –as a criterion for suppressing autoimmune reactions.

The CD8 T Cell-Epstein-Barr Virus-B Cell Trialogue: A Central Issue in Multiple Sclerosis Pathogenesis.

The cause and the pathogenic mechanisms leading to multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), are still under scrutiny. During the last decade, awareness has increased that multiple genetic and environmental factors act in concert to modulate MS risk. Likewise, the landscape of cells of the adaptive immune system that are believed to play a role in MS immunopathogenesis has expanded by including not only CD4 T helper cells but also cytotoxic CD8 T cells and B cells. Once the key cellular players are identified, the main challenge is to define precisely how they act and interact to induce neuroinflammation and the neurodegenerative cascade in MS. CD8 T cells have been implicated in MS pathogenesis since the 80’s when it was shown that CD8 T cells predominate in MS brain lesions. Interest in the role of CD8 T cells in MS was revived in 2000 and the years thereafter by studies showing that CNS-recruited CD8 T cells are clonally expanded and have a memory effector phenotype indicating in situ antigen-driven reactivation. The association of certain MHC class I alleles with MS genetic risk implicates CD8 T cells in disease pathogenesis. Moreover, experimental studies have highlighted the detrimental effects of CD8 T cell activation on neural cells. While the antigens responsible for T cell recruitment and activation in the CNS remain elusive, the high efficacy of B-cell depleting drugs in MS and a growing number of studies implicate B cells and Epstein-Barr virus (EBV), a B-lymphotropic herpesvirus that is strongly associated with MS, in the activation of pathogenic T cells. This article reviews the results of human studies that have contributed to elucidate the role of CD8 T cells in MS immunopathogenesis, and discusses them in light of current understanding of autoreactivity, B-cell and EBV involvement in MS, and mechanism of action of different MS treatments. Based on the available evidences, an immunopathological model of MS is proposed that entails a persistent EBV infection of CNS-infiltrating B cells as the target of a dysregulated cytotoxic CD8 T cell response causing CNS tissue damage.

T cell epitope screening of Epstein-Barr virus fusion protein gB.

Glycoprotein B (gB) is an essential fusion protein for the Epstein-Barr virus (EBV) infection of both B cells and epithelial cells and is thus a promising target antigen for a prophylactic vaccine to prevent or reduce EBV-associated disease. T cell responses play key roles in the control of persistent EBV infection and in the efficacy of a vaccine. However, to date, T cell responses to gB have been characterized for only a limited number of human leukocyte antigen (HLA) alleles. Here, we screened gB T cell epitopes in 23 healthy EBV carriers and ten patients with nasopharyngeal cancer (NPC) using a peptide library spanning the entire gB sequence. We identified twelve novel epitopes in the context of seven new HLA restrictions that are common in Asian populations. Two epitopes, gB214-223 and gB840-849, restricted by HLA-B*58:01 and B*38:02, respectively, elicited specific CD8+ T cell responses to inhibit EBV-driven B cell transformation. Interestingly, gB-specific CD8+ T cells were more frequent in healthy viral carriers with EBV reactivation than in those without EBV reactivation, indicating that EBV reactivation in vivo stimulates both humoral (VCA-gp125-IgA) and cellular responses to gB. We further found that most gB epitopes are conserved among different EBV strains. Our study broadens the diversity and HLA restrictions of gB epitopes and suggests that gB is a common target of T cell responses in healthy viral carriers with EBV reactivation. In particular, the precisely mapped and conserved gB epitopes provide valuable information for prophylactic vaccine development.ImportanceT cells are crucial for the control of persistent EBV infection and the development of EBV-associated diseases. The EBV gB protein is essential for virus entry into B cells and epithelial cells and is thus a target antigen for vaccine development. Understanding T cell responses to gB is important for subunit vaccine design. Herein, we comprehensively characterized T cell responses to full-length gB. Our results expand the available gB epitopes and HLA restrictions, particularly those common in Asian populations. Furthermore, we showed that gB-specific CD8+ T cells inhibit B cell transformation ex vivo and that gB-specific CD8+ T cell responses in vivo may be associated with intermittent EBV reactivation in asymptomatic viral carriers. These gB epitopes are highly conserved among geographically separated EBV strains. Precisely mapped and conserved T cell epitopes may contribute to immune monitoring and to the development of a gB subunit vaccine.

532 - A Multicenter, Multicohort, Open-Label, Single Arm per Cohort, Phase II Study to Assess the Efficacy and Safety of Tabelecleucel in Patients with EBV-Associated Diseases Using an Adaptive Two-Stage Study Design

Background Epstein-Barr virus infection is associated with a variety of life-threatening malignant and non-malignant diseases. In immunocompromised individuals where T-cell function is impaired, loss of immune control of Epstein-Barr virus (EBV) can lead to proliferation of EBV-transformed B cells and result in immunodeficiency-associated lymphoproliferative diseases (IA-LPD). EBV-driven IA-LPDs are a heterogenous group of diseases with variable clinical and pathologic features, including primary immunodeficiency-associated LPD (EBV+ PID-LPD), acquired immunodeficiency-associated LPD (EBV+ AID-LPD), and post-transplant lymphoproliferative disorder (EBV+ PTLD). In addition, EBV-infection of mesenchymal cells can cause sarcomas, including leiomyosarcoma (EBV+ sarcoma). In rare instances, EBV viremia (ie, persistent EBV infection) can lead to non-malignant yet life-threatening complications such as chronic active EBV (CAEBV) or hemophagocytic lymphohistiocytosis (HLH). During the transition from primary EBV infection to latent state, only a subset of viral genes is expressed. The resulting latency proteins are a target of CD8+ T cells, making EBV+ malignancies good candidates for treatment with EBV-targeted cytotoxic lymphocytes. Tabelecleucel (tab-cel) is an off-the-shelf, partially human leukocyte antigen (HLA)-matched, allogeneic EBV-specific T-cell immunotherapy generated from screened EBV+ donors. Tabelecleucel is characterized by both HLA genotype and HLA restriction, the HLA allele(s) through which tab-cel exerts cytotoxic activity. Tab-cel is selected for each patient from an inventory based on the HLA restriction(s) and partial HLA matching. Initial safety and efficacy data support that tab-cel was well tolerated and showed clinical activity in patients with each of the EBV-associated diseases being proposed in study 205 [Nikiforow S et al. ESMO 2020; Kurlander LS et al. Ann Oncol 2018; Prockop S et al. JCI 2020]. These data encourage investigation to further evaluate the safety and efficacy of tab-cel in these populations. Study design and objectives This is a multicenter, multicohort, open-label, single-arm per cohort, phase II study to assess the efficacy and safety of tab-cel in patients with EBV-associated diseases (ATA129-EBV-205). The primary objective of the study is to assess the clinical benefit of tab-cel as measured by objective response rate. This study will also evaluate clinically relevant disease-specific outcomes and will characterize the safety profile of tab-cel in these patient populations. This study will enroll patients who are relapsed/refractory or newly diagnosed and ineligible for first-line therapy in the following cohorts: EBV+ AID-LPD, EBV+ PID-LPD, EBV+ sarcoma, including LMS, CAEBV/HLH and EBV+ PTLD with central nervous system [CNS]-involvement. In addition, a cohort of patients with previously untreated EBV+ PTLD where first-line therapy is not appropriate is planned to start enrollment in 2021. A Phase III study of tab-cel for solid organ or allogeneic hematopoietic cell transplant patients with EBV+ PTLD after failure of rituximab or rituximab plus chemotherapy (NCT03394365) is ongoing. Patients in each cohort will receive tab-cel in 35-day cycles. During each cycle, patients will receive intravenous tab-cel at a dose of 2 × 106 cells/kg on days 1, 8, and 15, followed by an observation period through day 35 (Figure 1). An adaptive two-stage design will be used for each cohort in this study, and for each, a maximum of eight patients will be enrolled in stage 1. The decision to move to stage 2 enrollment for any given cohort will be based on an analysis of the first eight evaluable patients in the cohort using investigator's assessment (per defined radiologic, clinical, and/or laboratory response criteria). If at least two among the first eight evaluable patients in stage 1 are responders W(ie, complete response or partial response), additional patients may be enrolled in stage 2 of that cohort. The number of patients enrolled in stage 2 will depend on the number of observed responders in stage 1 (Table 1). This design will allow for minimization of the expected number of patients required to evaluate the efficacy of tab-cel in each disease cohort independently. Figure 1. Study design schema Download : Download high-res image (589KB) Download : Download full-size image Disclosures Prockop: Jasper Pharmaceuticals: Research Funding; Mesoblast: Consultancy, Research Funding; Atara Biotherapeutics: Research Funding; Memorial Sloan Kettering: Patents & Royalties: IP related to the development of third party viral specific T cells with all of my interests assigned to MSK. Dinavahi: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Navarro: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Guzman-Becerra: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Sun: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Gamelin: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company.

A Multicenter, Multicohort, Open-Label, Single-Arm per Cohort, Phase II Study to Assess the Efficacy and Safety of Tabelecleucel in Patients with EBV-Associated Diseases Using an Adaptive Two-Stage Study Design

Background Epstein-Barr virus infection is associated with a variety of life-threatening malignant and non-malignant diseases. In immunocompromised individuals where T-cell function is impaired, loss of immune control of Epstein-Barr virus (EBV) can lead to proliferation of EBV-transformed B cells and result in immunodeficiency-associated lymphoproliferative diseases (IA-LPD). EBV-driven IA-LPDs are a heterogenous group of diseases with variable clinical and pathologic features, including primary immunodeficiency-associated LPD (EBV+ PID-LPD), acquired immunodeficiency-associated LPD (EBV+ AID-LPD), and post-transplant lymphoproliferative disorder (EBV+ PTLD). In addition, EBV-infection of mesenchymal cells can cause sarcomas, including leiomyosarcoma (EBV+ sarcoma). In rare instances, EBV viremia (ie, persistent EBV infection) can lead to non-malignant yet life-threatening complications such as chronic active EBV (CAEBV) or hemophagocytic lymphohistiocytosis (HLH). During the transition from primary EBV infection to latent state, only a subset of viral genes is expressed. The resulting latency proteins are a target of CD8+ T cells, making EBV+ malignancies good candidates for treatment with EBV-targeted cytotoxic lymphocytes. Tabelecleucel (tab-cel) is an off-the-shelf, partially human leukocyte antigen (HLA)-matched, allogeneic EBV-specific T-cell immunotherapy generated from screened EBV+ donors. Tabelecleucel is characterized by both HLA genotype and HLA restriction, the HLA allele(s) through which tab-cel exerts cytotoxic activity. Tab-cel is selected for each patient from an inventory based on the HLA restriction(s) and partial HLA matching. Initial safety and efficacy data support that tab-cel was well tolerated and showed clinical activity in patients with each of the EBV-associated diseases being proposed in study 205 [Nikiforow S et al. ESMO 2020; Kurlander LS et al. Ann Oncol 2018; Prockop S et al.JCI 2020]. These data encourage investigation to further evaluate the safety and efficacy of tab-cel in these populations. Study design and objectives This is a multicenter, multicohort, open-label, single-arm per cohort, phase II study to assess the efficacy and safety of tab-cel in patients with EBV-associated diseases (ATA129-EBV-205). The primary objective of the study is to assess the clinical benefit of tab-cel as measured by objective response rate. This study will also evaluate clinically relevant disease-specific outcomes and will characterize the safety profile of tab-cel in these patient populations. This study will enroll patients who are relapsed/refractory or newly diagnosed and ineligible for first-line therapy in the following cohorts: EBV+ AID-LPD, EBV+ PID-LPD, EBV+ sarcoma, including LMS, CAEBV/HLH and EBV+ PTLD with central nervous system [CNS]-involvement. In addition, a cohort of patients with previously untreated EBV+ PTLD where first-line therapy is not appropriate is planned to start enrollment in 2021. A Phase III study of tab-cel for solid organ or allogeneic hematopoietic cell transplant patients with EBV+ PTLD after failure of rituximab or rituximab plus chemotherapy (NCT03394365) is ongoing. Patients in each cohort will receive tab-cel in 35-day cycles. During each cycle, patients will receive intravenous tab-cel at a dose of 2 × 106 cells/kg on days 1, 8, and 15, followed by an observation period through day 35 (Figure 1). An adaptive two-stage design will be used for each cohort in this study, and for each, a maximum of eight patients will be enrolled in stage 1. The decision to move to stage 2 enrollment for any given cohort will be based on an analysis of the first eight evaluable patients in the cohort using investigator's assessment (per defined radiologic, clinical, and/or laboratory response criteria). If at least two among the first eight evaluable patients in stage 1 are responders W(ie, complete response or partial response), additional patients may be enrolled in stage 2 of that cohort. The number of patients enrolled in stage 2 will depend on the number of observed responders in stage 1 (Table 1). This design will allow for minimization of the expected number of patients required to evaluate the efficacy of tab-cel in each disease cohort independently. Figure 1. Study design schema Disclosures Prockop: Jasper Pharmaceuticals: Research Funding; Mesoblast: Consultancy, Research Funding; Atara Biotherapeutics: Research Funding; Memorial Sloan Kettering: Patents & Royalties: IP related to the development of third party viral specific T cells with all of my interests assigned to MSK. Dinavahi:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Navarro:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Guzman-Becerra:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Sun:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Gamelin:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company.

<p>Pulmonary Lymphoepithelioma-Like Carcinoma: A Mini-Review</p>

Pulmonary lymphoepithelioma-like carcinoma (PLELC) is a rare and distinct subtype of non-small-cell lung carcinoma associated with Epstein–Barr virus (EBV) infection. We systematically reviewed the recent research that expands our knowledge about PLELC, with main focus on its genetic profile, tumor-infiltrating environment, PD-L1 expression, circulating EBV-DNA, clinical utility of 18F-FDG PET/CT, and treatment strategy. A low frequency of typical driver mutations and widespread existence of copy number variations was detected in PLELC. Persistent EBV infection may trigger intense infiltration of lymphocytes, representing enhanced tumor immunity and possibly resulting in a better prognosis. Circulating EBV-DNA in the plasma of patients with PLELC may predict disease progression and response to therapy. PLELC is 18F-FDG avid, and 18F-FDG PET may help refine palliation strategies and subsequently improve the prognosis. Most of the reported patients present at early and resectable stage, and surgical resection with curative intent is the preferred approach. There is currently no consensus on the regimen of chemotherapy for patients with advanced stages. EGFR-targeted therapies seem to have no therapeutic effect, and the clinical impact of PD-1/PD-L1 therapy is uncertain but worthy of further research.

Latent Membrane Protein 1 Contributes to Deficient Cellular Immunity in NKTCL Patients

Introduction Extranodal NK/T cell lymphoma (NKTCL) is an aggressive malignance that is correlated closely with persistent Epstein-Barr virus (EBV) infection and belongs to a latency II EBV disease. Chronic persistent viral infection is known to result in T cell exhaustion, which will dramatically impede anti-tumor immunity. Delineating mechanisms of immunosuppression contributes to the development of novel immuno-therapeutically strategies. Herein, we described the immune status and possible role of latent membrane protein 1(LMP1) in NKTCL. Methods Peripheral blood mononuclear cells (PBMCs) from newly diagnosed NKTCL patients and age-matched healthy donors (HDs) were isolated and stained with surface marker or intracellular marker after permeabilization. The frequency of EBV-specific cytotoxic T cells (CTLs) was analyzed by staining with HLA-A2 tetramers assembled with synthetic peptides from LMP1. T cell proliferation was detected by dilution of stained CFSE, and apoptosis of T cells was performed via annexinV and 7-AAD dual-staining. Cytotoxicity assay of natural killer cells (NK) was measured by detecting apoptosis of CFSE-labeled K562 cells after co-culturing with PBMCs. All flow cytometry was performed by BD FACS CantoII, and analyzed with FlowJo software. Results PBMCs from 19 NKTCL patients and 18 HDs were analyzed, which showed that the patients had higher ratio of CD4+/CD8+ cells and lower frequency of CD3-CD56+NK cells, higher percentage of immunosuppressive CD4+CD25hiCD127low T regulatory cells (Tregs) and HLA-DR-CD11b+CD33+ myeloid derived suppressor cells (MDSCs). Notably, the ratio of CD8+/Tregs, a parameter representing immune effector cells status, was obviously decreased in NKTCL patients (Figure-a). Given the potential association of T cell exhaustion and NKTCL, we detected expression of T cell exhaustion markers on T cells from NKTCL patients, and found that both CD4+ and CD8+ T cells expressed much higher level of inhibitory molecules PD1, CTLA4, TIGIT and TIM3 in NKTCL patients than that of HDs (Figure-b). To further explore cellular immunity in NKTCL, T cells were divided into four subsets: CD45RA-CCR7+ central memory T cells (Tcm), CD45RA+CCR7+ terminally differentiated effector T cells (Temra), CD45RA+CCR7- naïve T cells (Tn), CD45RA-CCR7- effector memory T cells (Tem) (Figure-c). It showed that CD4+ T cells had lower Tcm, Temra and Tn but much higher proportion of Tem compared with HDs (Figure-d). Moreover, we found that expression of PD1 and CTLA4 were upregulated on all lymphocyte subsets in NKTCL patients than HDs (Figure-e). We collected additional 13 NKTCL patients and 10 HDs with positive HLA-A2 phenotype to measure percentages of EBV-specific CTLs, which showed that frequency of EBV-specific CTLs was remarkably decreased in NKTCL patients (Figure-f). In addition, EBV-specific CTLs from NKTCL patients were more likely to express higher levels of exhaustion markers but produced much less IFN-γ (Figure g-h). To explore the potent mechanism of immunosuppression in NKTCL, LMP1 attracted our attention, which had been proven capable of activating multiple signaling pathways to exert its oncogenesis function. To elucidate effect of LMP1 on immune cells, we constructed two LMP1-derived peptides and found that LMP1-derived peptides suppressed the proliferation of CD4+ and CD8+ T cells and inhibited IFN-γ production of CD8+ T cells and NK cells (Figure i-j). Meanwhile, LMP1 promoted apoptosis of both CD4+ and CD8+ T cells (Figure-k). Subsequently regulation of LMP1 on exhaustion markers of T cells were detected, which showed that PD1, CTLA4, TIGIT and TIM3 were significantly upregulated on both CD4+ and CD8+ T cells after treatment with LMP1 derived peptides (Figure-m). Furthermore, LMP1 impaired NK cytotoxicity ability, evidenced by decrease apoptosis of target cells (Figure-n). Besides its effect on immune effector cells, we also found that LMP1-expressing NKTCL cell lines obviously induced both Tregs and MDSCs after co-culture. Interestingly, the extent of induction by LMP1 of Tregs and MDSCs were in line with the expression level of LMP1 on NKTCL cells (Figure o-p). Conclusion Our study showed that LMP1 contributes to deficient cellular immunity in NKTCL patients, providing us with more insight into immunosuppressive in this entity of disease, which would lead to identification of novel treatment strategies. Figure Disclosures No relevant conflicts of interest to declare.

Defining early events of Epstein-Barr virus (EBV) infection in immortalized nasopharyngeal epithelial cells using cell-free EBV infection.

Epstein-Barr virus (EBV) infection is strongly associated with nasopharyngeal carcinoma, a common cancer in Southeast Asia and certain regions of Africa. However, the dynamics of EBV episome maintenance in infected nasopharyngeal epithelial (NPE) cells remain largely undefined. Here, we report the establishment of a highly efficient cell-free EBV infection method for NPE cells. By using this method, we have defined some of the dynamic events involved in the early stage of EBV infection in NPE cells. We report, for the first time, a rapid loss of EBV copies from infected NPE cells during the first 12-72 h post-infection. The rate of EBV loss slowed at later stages of infection. Live cell imaging revealed that the freshly infected NPE cells were delayed in entry into mitosis compared with uninfected cells. Freshly infected NPE cells transcribed significantly higher levels of lytic EBV genes BZLF1 and BMRF1 yet significantly lower levels of EBER1/2 than stably infected NPE cells. Notably, there were very low or undetectable levels of protein expressions of EBNA1, LMP1, Zta and Rta in freshly infected NPE cells, whereas EBNA1 and LMP1 proteins were readily detected in stable EBV-infected NPE cells. The kinetics of EBV loss and the differential EBV gene expression profiles between freshly and stably infected NPE cells are in line with the suggestion of epigenetic changes in the EBV genome that affect viral gene expression and the adaptation of host cells to EBV infection to maintain persistent EBV infection in NPE cells.

Clinical and Immunological Characterization of ICF Syndrome in Japan

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a rare autosomal recessive primary immunodeficiency. Hypogammaglobulinemia is a major manifestation of ICF syndrome, but immunoglobulin replacement therapy does not seem to be effective for some ICF patients. Therefore, we aimed to reassess the immunological characteristics of this syndrome. Eleven Japanese patients with ICF syndrome were enrolled. We performed whole-exome sequencing in four cases and homozygosity mapping using SNP analysis in two. We evaluated their clinical manifestations and immunological status. We newly diagnosed six ICF patients who had tentatively been diagnosed with common variable immunodeficiency. We identified two novel mutations in the DNMT3B gene and one novel mutation in the ZBTB24 gene. All patients showed low serum IgG and/or IgG2 levels and were treated by periodic immunoglobulin replacement therapy. Three of the six patients showed worse results of the mitogen-induced lymphocyte proliferation test. Analyses of lymphocyte subpopulations revealed that CD19+CD27+ memory B cells were low in seven of nine patients, CD3+ T cells were low in three patients, CD4/8 ratio was inverted in five patients, CD31+ recent thymic emigrant cells were low in two patients, and CD19+ B cells were low in four patients compared with those in the normal controls. ICF2 patients showed lower proportions of CD19+ B cells and CD16+56+ NK cells and significantly higher proportions of CD3+ T cells than ICF1 patients. T cell receptor excision circles were undetectable in two patients. Despite being treated by immunoglobulin replacement therapy, three patients died of influenza virus, fatal viral infection with persistent Epstein-Barr virus infection, or JC virus infection. One of three dead patients showed normal intelligence with mild facial anomaly. Two patients presented with autoimmune or inflammatory manifestations. Infectious episodes decreased in three patients who were started on trimethoprim-sulfamethoxazole and/or antifungal drugs in addition to immunoglobulin replacement therapy. These patients might have suffered from T cell immunodeficiency. These results indicate that patients with ICF syndrome have a phenotype of combined immunodeficiency. Thus, to achieve a better prognosis, these patients should be treated as having combined immunodeficiency in addition to receiving immunoglobulin replacement therapy.