Epstein‒Barr virus–associated cellular immunotherapy

Epstein‒Barr virus (EBV) is a human herpes virus that is saliva-transmissible and universally asymptomatic. It has been confirmed that more than 90% of the population is latently infected with EBV for life. EBV can cause a variety of related cancers, such as nasopharyngeal carcinoma, diffuse large B-cell lymphoma, and Burkitt lymphoma. Currently, many clinical studies have demonstrated that EBV-specific cytotoxic T lymphocytes and other cell therapies can be safely and effectively transfused to prevent and treat some diseases caused by EBV. This review will mainly focus on discussing EBV-specific cytotoxic T lymphocytes and will touch on therapeutic EBV vaccines and chimeric antigen receptor T-cell therapy briefly.     

Molecular detection of Epstein–Barr virus in different types of lymphoma

Molecular Biology Reports - Epstein-Barr virus (EBV) is a member of the γ herpesvirus subfamily. It is widely spread, potentially oncogenic and has been studied in different human cancers such...     

Replication of Epstein–Barr Viral DNA

Epstein–Barr virus (EBV) is a paradigm for human tumor viruses: it is the first virus recognized to cause cancer in people; it causes both lymphomas and carcinomas; yet these tumors arise infrequently given that most people in the world are infected with the virus. EBV is maintained extrachromosomally in infected normal and tumor cells. Eighty-four percent of these viral plasmids replicate each S phase, are licensed, require a single viral protein for their synthesis, and can use two functionally distinct origins of DNA replication, oriP, and Raji ori. Eighty-eight percent of newly synthesized plasmids are segregated faithfully to the daughter cells. Infectious viral particles are not synthesized under these conditions of latent infection. This plasmid replication is consistent with survival of EBV’s host cells. Rare cells in an infected population either spontaneously or following exogenous induction support EBV’s lytic cycle, which is lethal for the cell. In this case, the viral DNA replicates 100-fold or more, uses a third kind of viral origin of DNA replication, oriLyt, and many viral proteins. Here we shall describe the three modes of EBV’s replication as a function of the viral origins used and the viral and cellular proteins that mediate the DNA synthesis from these origins focusing, where practical, on recent advances in our understanding.     

Nucleoside diphosphate kinase/Nm23 and Epstein–Barr virus

Compelling evidence indicates the pro-fibrogenic action of leptin in liver. Peroxisome proliferator-activated receptor-γ (PPARγ) can reverse hepatic stellate cell (HSC) activation and maintain HSC quiescence. HSC activation, a key step in the development of liver fibrosis, is coupled with the up-expression of leptin and the dramatic down-expression of PPARγ. The present study is aimed to assess the effect of leptin on PPARγ gene expression in primary cultured rat HSCs and investigate the related mechanisms by using Western blotting analysis, real-time PCR, transient transfection approach, and cell growth analysis. The results suggest that leptin negatively regulates PPARγ gene expression at mRNA level, protein level and PPARγ gene promoter activity level in HSCs. The inhibitory effect of leptin on PPARγ gene expression contributes to cell growth of activated HSCs in vitro. Phosphatidylinositol 3-kinase/AKT (PI-3 K/AKT) and extracellular signal-regulated kinase (ERK) signaling pathways mediate the leptin-induced inhibition of PPARγ gene expression. In summary, these findings suggest that leptin down-regulates PPARγ gene expression through activation of PI-3 K/AKT or ERK signaling pathway in primary cultured rat HSCs. Our results might provide novel insights into the mechanisms for the pro-fibrogenic action of leptin in liver.     

Epstein–Barr virus and thyroid cancer: The role of viral expressed proteins

Background : Thyroid cancer is a common endocrine malignancy whose incidence has increased in recent years. Several internal and external risk factors are involved in the development of this cancer, such as infectious agents. Evidence supporting the role of viral infection as an etiology for the invasiveness of thyroid cancer is increasing. The aim of this study was to determine the presence of the Epstein–Barr virus (EBV) and the association between viral gene products and thyroid tumor development. Methods : Fifty-seven thyroid cancer specimens were collected from the same number of patients as well as 18 samples from healthy controls. The presence of the EBV genome and the genotyping was examined by polymerase chain reaction (PCR). Also, an enzyme-linked immunosorbent assay and real-time PCR were used to measure the expression levels of viral and cellular genes. Results : The EBV DNA was detected in 71.9% of the samples, and it was also found that the presence of the EBV was associated with increasing development of thyroid tumor. Conclusion : Our results demonstrated that EBV infection may play a role in the development of thyroid tumor.     

Viral DNA contamination is responsible for Epstein–Barr virus detection in cytotoxic T lymphocytes stimulated in vitro with Epstein–Barr virus B-lymphoblastoid cell line

Epstein–Barr virus (EBV)-transformed B-lymphoblastoid cell lines (LCLs) are used to prepare human EBV-specific T lymphocytes (EBV-CTL) in vitro. Within an LCL, up to 5–7% the cells release infectious EBV, and this has fostered safety concerns for therapeutic applications because of the exposure of T cells to EBV. The release of infectious viruses can be prevented by ganciclovir, but this drug may seriously affect LCL growth. In the wake of these concerns, the present work was designed to compile safety data on EBV-CTL preparation for the purpose of submission to a regulatory agency. We showed that further to supernatant exclusion, the number of EBV genome copies (EBVc) associated with the EBV-CTL always made up a constant proportion of the EBVc number detected in the culture supernatant. In addition, such was the case whether infectious virus could be produced by the LCL or not, suggesting that the EBV signal detected was due to a DNA contamination rather than an infection. Furthermore, we demonstrated that the number of EBVc associated with the EBV-CTL was highly sensitive to DNAse treatment, and finally that EBVc could no longer be detected after the EBV-CTL had been amplified in the absence of LCL. Consequently, during in vitro EBV-CTL preparation, either T cells cannot be infected or they die rapidly after EBV infection.     

Targeting the lateral interactions of transmembrane domain 5 of Epstein–Barr virus latent membrane protein 1

The lateral transmembrane protein–protein interaction has been regarded as “undruggable” despite its importance in many biological processes. The homo-trimerization of transmembrane domain 5 (TMD-5) of latent membrane protein 1 (LMP-1) is critical for the constitutive oncogenic activation of the Epstein–Barr virus (EBV). Herein, we report a small molecule agent, NSC 259242 (compound 1), to be a TMD-5 self-association disruptor. Both the positively charged acetimidamide functional groups and the stilbene backbone of compound 1 are essential for its inhibitory activity. Furthermore, cell-based assays revealed that compound 1 inhibits full-length LMP-1 signaling in EBV infected B cells. These studies demonstrated a new strategy for identifying small molecule disruptors for investigating transmembrane protein–protein interactions.     

Epstein–Barr virus DNA quantification and follow-up in Tunisian nasopharyngeal carcinoma patients

The prognostic value of the Epstein–Barr virus (EBV) DNA load in sera of nasopharyngeal carcinoma (NPC) patients measured before any treatment, after treatment and before relapse was assessed. The real-time polymerase chain reaction was used to detect the viral load levels among 74 NPC subjects. Patients were followed up for a period going from 1 to 6 years (median 4 years). Before treatment, the EBV DNA load was correlated with lymph node involvement and advanced stages. After treatment, the viral load level declined significantly and patients presenting a viral load level lower than 1000 copies/ml showed a better overall survival (OS). Moreover, a significant result was found when the 6-year OS rates of patients having fewer or more than 15,000 copies/ml of viral load before relapse were compared. These results suggest that the EBV DNA load quantification after treatment may be a useful predictor of disease progression and survival.     

Epstein–Barr virus and rheumatoid arthritis: is there a link?

Rheumatoid arthritis is a systemic autoimmune disease characterized by chronic, destructive, debilitating arthritis. Its etiology is unknown; it is presumed that environmental factors trigger development in the genetically predisposed. Epstein–Barr virus, a nearly ubiquitous virus in the human population, has generated great interest as a potential trigger. This virus stimulates polyclonal lymphocyte expansion and persists within B lymphocytes for the host's life, inhibited from reactivating by the immune response. In latent and replicating forms, it has immunomodulating actions that could play a role in the development of this autoimmune disease. The evidence linking Epstein–Barr virus and rheumatoid arthritis is reviewed.     

Epstein–Barr virus latent membrane protein 1 increases chemo-resistance of cancer cells via cytoplasmic sequestration of Pim-1

Improved treatment of EBV positive lymphoma depends on the identification of molecular mechanism underlying chemo-resistance. LMP1 is an essential transmembrane protein for EBV-induced immortalization of hematopoietic cells. Herein, we show that an oncogenic Pim-1 is translocated to the cytoplasm by LMP1. Three lines of evidence indicate that cytoplasmic sequestration of Pim-1 may be required for LMP1-induced cancer cell survival. First, Pim-1 enhanced the survival of LMP1-overexpressing cells treated with doxorubicin. Second, nuclear export of Pim-1 was sufficient to increase the survival. Third, knockdown of Pim-1 effectively suppressed LMP-1-induced survival of cancer cells. Collectively, these data suggest that Pim-1 is a downstream target of LMP1, and that it contributes to the chemo-resistance of cancer cells.     

Immune responses to Epstein–Barr virus: molecular interactions in the virus evasion of CD8+ T cell immunity

Persistent viruses have mechanisms for modulating the host immune responses that are essential for achieving a lifelong virus–host balance while minimizing the viral pathogenicity. Here we review some of the immune-modulating mechanisms evolved by the ubiquitous but potentially oncogenic Epstein–Barr virus, with particular emphasis on the molecular mechanisms of genes interfering with HLA class I antigen presentation.     

Translation of intronless RNAs is strongly stimulated by the Epstein–Barr virus mRNA export factor EB2

The Epstein–Barr virus protein (EB2) allows the nuclear export of a particular subset of early and late viral RNAs derived from intronless genes. EB2 is conserved among most herpesvirus members and its presence is essential for the production of infectious particles. Here we show that, besides its role as a nuclear export factor, EB2 strongly stimulates translation of unspliced mRNAs without affecting overall cellular translation. Interestingly, this effect can be reversed by the addition of an intron within the gene. The spliced mRNA is then efficiently exported and translated even in the absence of EB2. Moreover, we show that EB2 associates with translating ribosomes and increases the proportion of its target RNA in the polyribosomal fraction. Finally, testing of EB2 homolog proteins derived from EBV-related herpesviruses, shows that, even if they play similar roles within the replication cycle of their respective virus, their mechanisms of action are different.     

Patients with systemic lupus erythematosus have abnormally elevated Epstein–Barr virus load in blood

Various genetic and environmental factors appear to be involved in systemic lupus erythematosus (SLE). Epstein–Barr virus (EBV) is among the environmental factors that are suspected of predisposing to SLE, based on the characteristics of EBV itself and on sequence homologies between autoantigens and EBV antigens. In addition, higher titers of anti-EBV antibodies and increased EBV seroconversion rates have been observed in SLE patients as compared with healthy control individuals. Serologic responses do not directly reflect EBV status within the body. Clarification of the precise status of EBV infection in SLE patients would help to improve our understanding of the role played by EBV in this disease. In the present study we determined EBV types in SLE patients (n = 66) and normal control individual (n = 63) by direct PCR analysis of mouthwash samples. We also compared EBV load in blood between SLE patients (n = 24) and healthy control individuals (n = 29) using semiquantitative PCR assay. The number of infections and EBV type distribution were similar between adult SLE patients and healthy control individuals (98.5% versus 94%). Interestingly, the EBV burden in peripheral blood mononuclear cells (PBMCs) was over 15-fold greater in SLE patients than in healthy control individuals (mean ± standard deviation: 463 ± 570 EBV genome copies/3 μg PBMC DNA versus 30 ± 29 EBV genome copies/3 μg PBMC DNA; P = 0.001), suggesting that EBV infection is abnormally regulated in SLE. The abnormally increased proportion of EBV-infected B cells in the SLE patients may contribute to enhanced autoantibody production in this disease.     

Immuno-PCR Assay for Quantitation of Antibodies to Epstein–Barr Virus

Successful disease prevention and therapy critically depend on timely diagnosis of infections. Quantitative immuno-PCR (qiPCR) technology improves the sensitivity in the detection of antibodies to pathogens. A qiPCR-based assay was developed to determine IgG antibodies to Epstein–Barr virus (EBV) in the human blood serum. EBV nuclear protein 1 fragment (pEBV) was expressed in Escherichia coli. A synthetic single-stranded deoxyribonucleotide was conjugated to streptavidin, and the conjugate was used to detect рEBV–IgG1–biotin complexes by qiPCR. The IgG1 titers determined by qiPCR were compared to the results of enzyme-linked immunosorbent assay (ELISA). The sensitivity of qiPCR was one order of magnitude higher than that of ELISA. Thus, a highly sensitive qiPCR-based assay was developed to quantitate antibodies specific to the recombinant EBV antigen.     

Comprehensive epitope mapping of the Epstein–Barr virus latent membrane protein-2 in normal, non tumor-bearing individuals

Latent membrane protein (LMP)-2 is one of the Epstein–Barr virus (EBV)-encoded proteins consistently expressed by nasopharyngeal carcinoma (NPC). EBV-transformed lymphoblastoid cell lines (LCL) have been used in patients with NPC to induce LMP-2-recognizing T cell lines which have been in turn utilized for protein-wide mapping of T cell epitopes. However, comprehensive mapping of naturally recognized LMP-2 epitopes in non tumor-bearing individuals has not been reported. Here, we applied a low sensitivity epitope-defining technique for the identification of LMP-2 CTL responses detectable ex vivo in EBV-experienced individuals. This screening tool has been previously validated by analyzing memory CTL responses to Flu, cytomegalovirus (CMV), and the melanoma associated antigen gp100/Mel17. Peripheral blood monocytes (PBMC) from ten Caucasian and ten Chinese individuals were stimulated ex vivo with pools of nonamer (9-mer) peptides overlapping in a stepwise fashion each single amino acid of the LMP-2 sequence. No obvious differences were observed between the immune response of the two ethnic groups save for those related to the divergence in the ethnic prevalence of HLA haplotypes. Several novel and known LMP-2 epitopes were identified. Reactivity toward at least one LMP-2 epitope was detected in 18 of the 20 donors but no prevalent human leukocyte antigen (HLA)/epitope combination was observed confirming that LMP-2 reactivity in the context of common HLA alleles is more pleiotropic than that of FLU and CMV. We believe that the usefulness of these epitopes occurring naturally in non-cancer bearing patients as reagents for the immunization of patients with early or advanced stage NPC deserves further evaluation. Maurizio Provenzano and Silvia Selleri equally contributed to this work.     

The LMP2A protein of Epstein–Barr virus regulates phosphorylation of ITSN1 and Shb adaptors by tyrosine kinases

Latent Membrane Protein 2A (LMP2A) is an Epstein–Barr virus-encoded protein that is important for the maintenance of latent infection. Its activity affects cellular differentiation, migration, proliferation and B cell survival. LMP2A resembles a constitutively activated B cell antigen receptor and exploits host kinases to activate a set of downstream signaling pathways. In the current study we demonstrate the interaction of LMP2A with intersectin 1 (ITSN1), a key endocytic adaptor protein. This interaction occurs via both the N- and C-tails of LMP2A and is mediated by the SH3 domains of ITSN1. Additionally, we identified the Shb adaptor and the Syk kinase as novel binding ligands of ITSN1. The Shb adaptor interacts simultaneously with the phosphorylated tyrosines of LMP2A and the SH3 domains of ITSN1 and mediates indirect interaction of ITSN1 to LMP2A. Syk kinase promotes phosphorylation of both ITSN1 and Shb adaptors in LMP2A-expressing cells. In contrast to ITSN1, Shb phosphorylation depends additionally on Lyn kinase activity.Considering that Shb and ITSN1 are implicated in various receptor tyrosine kinase signaling, our results indicate that LMP2A can affect a number of signaling pathways by regulating the phosphorylation of the ITSN1 and Shb adaptors.