EBV-IgA antibody responses in endemic and nonendemic populations with nasopharyngeal carcinoma: tumour marker prognostication study and a cross-sectional study.

Nasopharyngeal carcinoma (NPC) is the most prevalent head and neck cancer in Indonesia, with 100% Epstein-Barr virus (EBV) infection in tumor cells. NPC is rare in the Netherlands. The involvement of EBV in NPC pathogenesis is reflected by early onset aberrant IgA antibody responses to various EBV proteins. Screening for elevated EBV-IgA levels is proposed for NPC risk assessment in endemic countries but is poorly studied in nonendemic regions. This study analyzed the overall diversity (immunoblot) as well as the prevalence and normalized levels of IgA responses to immunodominant peptide epitopes of EBV proteins VCA P18, EBNA 1, and Zebra (Zta) (N-terminus, P 125, P 130, full-length recombinant Zebra) in Indonesian (n=50) and Dutch (n=50) patients with NPC. The results confirmed that elevated levels of IgA-VCA P18 and IgA-EBNA 1 were found in both NPC populations, but that IgA-Zta was more variable. IgA-Zta responses were more pronounced in Indonesian NPC cases, reflecting more frequent EBV reactivation overall. IgA-VCA P18 and IgA-EBNA are independent tumor markers and are both necessary for NPC risk assessment. Overall, these results confirmed the diagnostic benefit of combined IgA-VCA P18/-EBNA 1 testing for NPC risk assessment in endemic and nonendemic populations.

Comparative Analysis of the Humoral Immune Response to the EBV Proteome across EBV-Related Malignancies.

BACKGROUND: Epstein-Barr virus (EBV) is linked to multiple cancers, including classical Hodgkin lymphoma (cHL), endemic Burkitt lymphoma (eBL), nasopharyngeal carcinoma (NPC), and extranodal natural killer/T-cell lymphoma (NKTCL). METHODS: Anti-EBV IgG and IgA antibody responses targeting 202 sequences from 86 EBV proteins were measured using the same EBV whole proteome array across four case-control studies investigating EBV-positive cHL, eBL, NPC, and NKTCL (407 cases/620 controls). We grouped EBV-targeted antibodies into pathways by immunoglobulin type (IgA and IgG) and life-cycle stage (latent, immediate early lytic, early lytic, late lytic, and glycoprotein) and evaluated their association with each cancer type. In an additional analysis, we focused on the subset of 46 individual antibodies representing the top candidates for each cancer and compared their associations across the four cancer types using multivariable linear regression models. RESULTS: IgA antibody responses targeting all EBV life-cycle stages were associated with NPC but limited to anti-early lytic stage for cHL. NPC and eBL were associated with IgG antibodies across the viral life cycle; cHL with antibodies in the early lytic, late lytic and glycoprotein stages; and NKTCL with antibodies in the latent, immediate early lytic and early lytic phases. EBNA3A, BBLF1, BDLF4, and BLRF2 IgG antibodies were associated with all cancer types. CONCLUSIONS: Our observed similarities and differences across four EBV-associated cancers may inform EBV-related oncogenesis. IMPACT: Understanding the comparative humoral immune response across EBV-related cancers may aid in identifying shared etiologic roles of EBV proteins and inform unique pathogenic processes for each cancer.

Subcellular Distribution of BALF2 and the Role of Rab1 in the Formation of Epstein-Barr Virus Cytoplasmic Assembly Compartment and Virion Release.

Epstein-Barr virus (EBV) replicates its genome in the nucleus and undergoes tegumentation and envelopment in the cytoplasm. We are interested in how the single-stranded DNA binding protein BALF2, which executes its function and distributes predominantly in the nucleus, is packaged into the tegument of virions. At the mid-stage of virus replication in epithelial TW01-EBV cells, a small pool of BALF2 colocalizes with tegument protein BBLF1, BGLF4 protein kinase, and the cis-Golgi marker GM130 at the perinuclear viral assembly compartment (AC). A possible nuclear localization signal (NLS) between amino acids 1100 and 1128 (C29), which contains positive charged amino acid 1113RRKRR1117, is able to promote yellow fluorescent protein (YFP)-LacZ into the nucleus. In addition, BALF2 interacts with the nucleocapsid-associated protein BVRF1, suggesting that BALF2 may be transported into the cytoplasm with nucleocapsids in a nuclear egress complex (NEC)-dependent manner. A group of proteins involved in intracellular transport were identified to interact with BALF2 in a proteomic analysis. Among them, the small GTPase Rab1A functioning in bi-directional trafficking at the ER-Golgi interface is also a tegument component. In reactivated TW01-EBV cells, BALF2 colocalizes with Rab1A in the cytoplasmic AC. Expression of dominant-negative GFP-Rab1A(N124I) diminished the accumulation of BALF2 in the AC, coupling with attenuation of gp350/220 glycosylation. Virion release was significantly downregulated by expressing dominant-negative GFP-Rab1A(N124I). Overall, the subcellular distribution of BALF2 is regulated through its complex interaction with various proteins. Rab1 activity is required for proper gp350/220 glycosylation and the maturation of EBV. IMPORTANCE Upon EBV lytic reactivation, the virus-encoded DNA replication machinery functions in the nucleus, while the newly synthesized DNA is encapsidated and transported to the cytoplasm for final virus assembly. The single-stranded DNA binding protein BALF2 executing functions within the nucleus was also identified in the tegument layer of mature virions. Here, we studied the functional domain of BALF2 that contributes to the nuclear targeting and used a proteomic approach to identify novel BALF2-interacting cellular proteins that may contribute to virion morphogenesis. The GTPase Rab1, a master regulator of anterograde and retrograde endoplasmic reticulum (ER)-Golgi trafficking, colocalizes with BALF2 in the juxtanuclear concave region at the midstage of EBV reactivation. Rab1 activity is required for BALF2 targeting to the cytoplasmic assembly compartment (AC) and for gp350/220 targeting to cis-Golgi for proper glycosylation and virion release. Our study hints that EBV hijacks the bi-directional ER-Golgi trafficking machinery to complete virus assembly.

Evolution of functional antibodies following acute Epstein-Barr virus infection.

While Epstein-Barr virus causes mostly asymptomatic infection, associated malignancies, and autoimmune and lymphoproliferative diseases occur. To dissect the evolution of humoral immune responses over the course of EBV infection and to gain a better understanding of the potential contribution of antibody (Ab) function to viral control, we comprehensively profiled Ab specificities and Fc-functionalities using systems serology and VirScan. Ab functions against latent (EBNA1), early (p47/54) and two late (gp350/220 and VCA-p18) EBV proteins were overall modest and/or short-lived, differing from humoral responses induced during acute infection by other viruses such as HIV. In the first year post infection, only p18 elicited robust IgM-driven complement deposition and IgG-driven neutrophil phagocytosis while responses against EBNA-1 were largely Fc-functionally silent and only matured during chronic infection to drive phagocytosis. In contrast, Abs against Influenza virus readily mediated broad Fc-activity in all participants. These data suggest that EBV evades the induction of robust Fc-functional Abs, potentially due to the virus' life cycle, switching from lytic to latent stages during infection.

Impact of Epstein-Barr virus co-infection on natural acquired Plasmodium vivax antibody response.

BACKGROUND: The simultaneous infection of Plasmodium falciparum and Epstein-Barr virus (EBV) could promote the development of the aggressive endemic Burkitt's Lymphoma (eBL) in children living in P. falciparum holoendemic areas. While it is well-established that eBL is not related to other human malaria parasites, the impact of EBV infection on the generation of human malaria immunity remains largely unexplored. Considering that this highly prevalent herpesvirus establishes a lifelong persistent infection on B-cells with possible influence on malaria immunity, we hypothesized that EBV co-infection could have impact on the naturally acquired antibody responses to P. vivax, the most widespread human malaria parasite. METHODOLOGY/PRINCIPAL FINDINGS: The study design involved three cross-sectional surveys at six-month intervals (baseline, 6 and 12 months) among long-term P. vivax exposed individuals living in the Amazon rainforest. The approach focused on a group of malaria-exposed individuals whose EBV-DNA (amplification of balf-5 gene) was persistently detected in the peripheral blood (PersVDNA, n = 27), and an age-matched malaria-exposed group whose EBV-DNA could never be detected during the follow-up (NegVDNA, n = 29). During the follow-up period, the serological detection of EBV antibodies to lytic/ latent viral antigens showed that IgG antibodies to viral capsid antigen (VCA-p18) were significantly different between groups (PersVDNA > NegVDNA). A panel of blood-stage P. vivax antigens covering a wide range of immunogenicity confirmed that in general PersVDNA group showed low levels of antibodies as compared with NegVDNA. Interestingly, more significant differences were observed to a novel DBPII immunogen, named DEKnull-2, which has been associated with long-term neutralizing antibody response. Differences between groups were less pronounced with blood-stage antigens (such as MSP1-19) whose levels can fluctuate according to malaria transmission. CONCLUSIONS/SIGNIFICANCE: In a proof-of-concept study we provide evidence that a persistent detection of EBV-DNA in peripheral blood of adults in a P. vivax semi-immune population may impact the long-term immune response to major malaria vaccine candidates.

Utility of Epstein-Barr Virus DNA in Nasopharynx Swabs as a Reflex Test to Triage Seropositive Individuals in Nasopharyngeal Carcinoma Screening Programs.

BACKGROUND: Epstein-Barr virus (EBV) DNA detection in the nasopharynx is considered a biomarker for nasopharyngeal carcinoma (NPC). We evaluated its performance as a reflex test to triage EBV seropositives within an NPC screening program in China. METHODS: The study population was embedded within an ongoing NPC screening trial and included 1111 participants who screened positive for anti-EBV VCA (antibodies against EBV capsid antigens)/EBNA1 (EBV nuclear antigen1)-IgA antibodies (of 18 237 screened). Nasopharynx swabs were collected/tested for EBNA1 gene EBV DNA load. We evaluated performance of EBV DNA in the nasopharynx swab as a reflex test to triage EBV serological high-risk (those referred to endoscopy/MRI) and medium-risk (those referred to accelerated screening) individuals. RESULTS: By the end of 2019, we detected 20 NPC cases from 317 serological high-risk individuals and 4 NPC cases from 794 medium-risk individuals. When used to triage serological high-risk individuals, nasopharynx swab EBV DNA was detected in 19/20 cases (positivity rate among cases: 95.0%; 95% CI, 75.1%-99.9%), with a referral rate of 63.4% (201/317, 95% CI, 57.8%-68.7%) and NPC detection rate among positives of 9.5% (19/201, 95% CI, 5.8%-14.4%). The performance of an algorithm that combined serology with triage of serology high-risk individuals using EBV DNA testing yielded a sensitivity of 72.4% (95% CI, 3.0%-81.4%) and specificity of 97.6% (95% CI, 97.2%-97.9%). When used to triage EBV serological medium-risk individuals, the positivity rate among cases was 75.0% (95% CI, 19.4%-99.4%), with a referral rate of 61.8% (95% CI, 58.4%-65.2%) and NPC detection rate among positives of 0.6% (95% CI, 0.1%-1.8%). CONCLUSIONS: Nasopharynx swab EBV DNA showed promise as a reflex test to triage serology high-risk individuals, reducing referral by ca. 40% with little reduction in sensitivity compared to a serology-only screening program.

Quantitative cytokine level of TNF-α, IFN-γ, IL-10, TGF-ß and circulating Epstein-Barr virus DNA load in individuals with acute Malaria due to P. falciparum or P. vivax or double infection in a Malaria endemic region in Indonesia.

Plasmodium falciparum Malaria and Epstein-Barr Virus (EBV) infection are risk factors in the development of Burkitt's lymphoma. In Indonesia, 100% of the population is persistently infected with EBV early in life and at risk of developing EBV-linked cancers. Currently, 10.7 million people in Indonesia are living in Malaria-endemic areas. This cross-sectional study was initiated to investigate how acute Malaria dysregulates immune control over latent EBV infection. Using blood and plasma samples of 68 patients with acute Malaria and 27 healthy controls, we measured the level of parasitemia for each plasmodium type (P. falciparum, P. vivax, and mixed) by microscopy and rapid test. The level of 4 regulatory cytokines was determined by quantitative ELISA and the level of circulating EBV genome by real-time PCR targeting the single copy EBNA-1 sequence. All Plasmodium-infected cases had high-level parasitemia (>1000 parasites/ul blood) except for one case. EBV-DNA levels were significantly more elevated in P. falciparum and P. vivax infections (P<0.05) compared to controls. EBV-DNA levels were not related to age, gender, Malaria symptoms, or plasmodium type. TNF-α and IL-10 levels were increased in Malaria cases versus controls, but IFN-γ and TGF- ß levels were comparable between the groups. Only TNF-α levels in P. falciparum cases showed a clear correlation with elevated EBV DNA levels (R2 = 0.8915). This is the first study addressing the relation between EBV (re)activation and cytokine responses during acute Malaria, revealing a clear correlation between pro-inflammatory cytokine TNF-α and EBV-DNA levels, specifically in P. falciparum cases, suggesting this cytokine to be key in dysregulating EBV homeostasis during acute P. falciparum Malaria.

Cumulative Roles for Epstein-Barr Virus, Human Endogenous Retroviruses, and Human Herpes Virus-6 in Driving an Inflammatory Cascade Underlying MS Pathogenesis.

Roles for viral infections and aberrant immune responses in driving localized neuroinflammation and neurodegeneration in multiple sclerosis (MS) are the focus of intense research. Epstein-Barr virus (EBV), as a persistent and frequently reactivating virus with major immunogenic influences and a near 100% epidemiological association with MS, is considered to play a leading role in MS pathogenesis, triggering localized inflammation near or within the central nervous system (CNS). This triggering may occur directly via viral products (RNA and protein) and/or indirectly via antigenic mimicry involving B-cells, T-cells and cytokine-activated astrocytes and microglia cells damaging the myelin sheath of neurons. The genetic MS-risk factor HLA-DR2b (DRB1*1501ß, DRA1*0101α) may contribute to aberrant EBV antigen-presentation and anti-EBV reactivity but also to mimicry-induced autoimmune responses characteristic of MS. A central role is proposed for inflammatory EBER1, EBV-miRNA and LMP1 containing exosomes secreted by viable reactivating EBV+ B-cells and repetitive release of EBNA1-DNA complexes from apoptotic EBV+ B-cells, forming reactive immune complexes with EBNA1-IgG and complement. This may be accompanied by cytokine- or EBV-induced expression of human endogenous retrovirus-W/-K (HERV-W/-K) elements and possibly by activation of human herpesvirus-6A (HHV-6A) in early-stage CNS lesions, each contributing to an inflammatory cascade causing the relapsing-remitting neuro-inflammatory and/or progressive features characteristic of MS. Elimination of EBV-carrying B-cells by antibody- and EBV-specific T-cell therapy may hold the promise of reducing EBV activity in the CNS, thereby limiting CNS inflammation, MS symptoms and possibly reversing disease. Other approaches targeting HHV-6 and HERV-W and limiting inflammatory kinase-signaling to treat MS are also being tested with promising results. This article presents an overview of the evidence that EBV, HHV-6, and HERV-W may have a pathogenic role in initiating and promoting MS and possible approaches to mitigate development of the disease.

PCR-Free Shallow Whole Genome Sequencing for Chromosomal Copy Number Detection from Plasma of Cancer Patients Is an Efficient Alternative to the Conventional PCR-Based Approach.

Somatic copy number alterations can be detected in cell-free DNA (cfDNA) by shallow whole genome sequencing (sWGS). PCR is typically included in library preparations, but a PCR-free method could serve as a high-throughput alternative. To evaluate a PCR-free method for research and diagnostics, archival peripheral blood or bone marrow plasma samples, collected in EDTA- or lithium-heparin-containing tubes, were collected from patients with non-small-cell lung cancer (n = 10 longitudinal samples; 4 patients), B-cell lymphoma (n = 31), and acute myeloid leukemia (n = 15), or from healthy donors (n = 14). sWGS was performed on PCR-free and PCR library preparations, and the mapping quality, percentage of unique reads, genome coverage, fragment lengths, and copy number profiles were compared. The percentage of unique reads was significantly higher for PCR-free method compared with PCR method, independent of the type of collection tube: EDTA PCR-free method, 96.4% (n = 35); EDTA PCR method, 85.1% (n = 32); heparin PCR-free method, 94.5% (n = 25); and heparin PCR method, 89.4% (n = 10). All other evaluated metrics were highly comparable for PCR-free and PCR library preparations. These results demonstrate the feasibility of somatic copy number alteration detection by PCR-free sWGS using cfDNA from plasma collected in EDTA- or lithium-heparin-containing tubes and pave the way for an automated cfDNA analysis workflow for samples from cancer patients.

Epidemiology of Epstein-Barr virus infection and infectious mononucleosis in the United Kingdom.

BACKGROUND: Epstein-Barr Virus (EBV) is a ubiquitous gamma-herpesvirus with which ~ 95% of the healthy population is infected. EBV infection has been implicated in a range of haematological malignancies and autoimmune diseases. Delayed primary EBV infection increases the risk of subsequent complications. Contemporaneous seroepidemiological data is needed to establish best approaches for successful vaccination strategies in the future. METHODS: We conducted a sero-epidemiological survey using serum samples from 2325 individuals between 0 and 25 years old to assess prevalence of detectable anti-EBV antibodies. Second, we conducted a retrospective review of Hospital Episode Statistics to examine changes in Infectious Mononucleosis (IM) incidence over time. We then conducted a large case-control study of 6306 prevalent IM cases and 1,009,971 unmatched controls extracted from an East London GP database to determine exposures associated with IM. RESULTS: 1982/2325 individuals (85.3%) were EBV seropositive. EBV seropositivity increased more rapidly in females than males during adolescence (age 10-15). Between 2002 and 2013, the incidence of IM (derived from hospital admissions data) increased. Exposures associated with an increased risk of IM were lower BMI, White ethnicity, and not smoking. CONCLUSIONS: We report that overall EBV seroprevalence in the UK appears to have increased, and that a sharp increase in EBV seropositivity is seen in adolescent females, but not males. The incidence of IM requiring hospitalisation is increasing. Exposures associated with prevalent IM in a diverse population include white ethnicity, lower BMI, and never-smoking, and these exposures interact with each other. Lastly, we provide pilot evidence suggesting that antibody responses to vaccine and commonly encountered pathogens do not appear to be diminished among EBV-seronegative individuals. Our findings could help to inform vaccine study designs in efforts to prevent IM and late complications of EBV infection, such as Multiple Sclerosis.

Reactivation of Epstein-Barr virus by a dual-responsive fluorescent EBNA1-targeting agent with Zn2+-chelating function.

Epstein-Barr nuclear antigen 1 (EBNA1) plays a vital role in the maintenance of the viral genome and is the only viral protein expressed in nearly all forms of Epstein-Barr virus (EBV) latency and EBV-associated diseases, including numerous cancer types. To our knowledge, no specific agent against EBV genes or proteins has been established to target EBV lytic reactivation. Here we report an EBNA1- and Zn2+-responsive probe (ZRL5P4) which alone could reactivate the EBV lytic cycle through specific disruption of EBNA1. We have utilized the Zn2+ chelator to further interfere with the higher order of EBNA1 self-association. The bioprobe ZRL5P4 can respond independently to its interactions with Zn2+ and EBNA1 with different fluorescence changes. It can selectively enter the nuclei of EBV-positive cells and disrupt the oligomerization and oriP-enhanced transactivation of EBNA1. ZRL5P4 can also specifically enhance Dicer1 and PML expression, molecular events which had been reported to occur after the depletion of EBNA1 expression. Importantly, we found that treatment with ZRL5P4 alone could reactivate EBV lytic induction by expressing the early and late EBV lytic genes/proteins. Lytic induction is likely mediated by disruption of EBNA1 oligomerization and the subsequent change of Dicer1 expression. Our probe ZRL5P4 is an EBV protein-specific agent that potently reactivates EBV from latency, leading to the shrinkage of EBV-positive tumors, and our study also suggests the association of EBNA1 oligomerization with the maintenance of EBV latency.

Multilaboratory Assessment of Epstein-Barr Virus Serologic Assays: the Case for Standardization.

IgA antibodies targeting Epstein-Barr virus (EBV) have been proposed for screening for nasopharyngeal carcinoma (NPC). However, methods differ, and the antigens used in these assays differ considerably between laboratories. To enable formal comparisons across a range of established EBV serology assays, we created a panel of 66 pooled serum samples and 66 pooled plasma samples generated from individuals with a broad range of IgA antibody levels. Aliquots from these panels were distributed to six laboratories and were tested by 26 assays measuring antibodies against VCA, EBNA1, EA-EBNA1, Zta, or EAd antigens. We estimated the correlation between assay pairs using Spearman coefficients (continuous measures) and percentages of agreement (positive versus negative, using predefined positivity cutoffs by each assay developer/manufacturer). While strong correlations were observed between some assays, considerable differences were also noted, even for assays that targeted the same protein. For VCA-IgA assays in serum, two distinct clusters were identified, with a median Spearman coefficient of 0.41 (range, 0.20 to 0.66) across these two clusters. EBNA1-IgA assays in serum grouped into a single cluster with a median Spearman coefficient of 0.79 (range, 0.71 to 0.89). Percentages of agreement differed broadly for both VCA-IgA (12% to 98%) and EBNA1-IgA (29% to 95%) assays in serum. Moderate-to-strong correlations were observed across assays in serum that targeted other proteins (correlations ranged from 0.44 to 0.76). Similar results were noted for plasma. We conclude that standardization of EBV serology assays is needed to allow for comparability of results obtained in different translational research studies across laboratories and populations.

Differences in the Epstein-Barr Virus gp350 IgA Antibody Response Are Associated With Increased Risk for Coinfection With a Second Strain of Epstein-Barr Virus.

BACKGROUND: The Epstein-Barr virus (EBV) viral glycoprotein gp350 has been proposed as a candidate antigen for an EBV vaccine. However, the proposed formulations of these vaccines have not taken into account the presence of 2 unique EBV strains (EBV-1 and EBV-2) present in areas of high incidence of the EBV-associated cancer, Burkitt lymphoma. METHODS: In this study, we analyze the kinetics of EBV-1 and EBV-2 infection in an asymptomatic infant cohort from Kisumu, Kenya. We also analyzed the kinetics of the antibody response against 5 EBV antigens, gp350 (IgG and IgA), VCA (IgG), EBNA-1 (IgG), EAd (IgG), and Zta (IgG). RESULTS: We observed a high frequency of coinfection with both EBV types over time, with the only observable defect in the antibody response in infants coinfected being a significantly lower level of anti-gp350 IgA at peak response. Gp350 IgA levels were also significantly lower in coinfected infants 2.5 months postinfection and at the time of coinfection. CONCLUSIONS: These results suggest that anti-gp350 IgA antibodies may be important for sterilizing immunity against secondary infection. These findings have implications for the development of an efficacious EBV vaccine to prevent both EBV-1 and EBV-2 infection in a population at high risk for Burkitt lymphoma.

Vesicle-bound EBV-BART13-3p miRNA in circulation distinguishes nasopharyngeal from other head and neck cancer and asymptomatic EBV-infections.

Cell-free microRNA (miRNA) in biofluids released by tumors in either protein or vesicle-bound form, represent promising minimally-invasive cancer biomarkers. However, a highly abundant non-tumor background in human plasma and serum complicates the discovery and detection of tumor-selective circulating miRNAs. We performed small RNA sequencing on serum and plasma RNA from Nasopharyngeal Carcinoma (NPC) patients. Collectively, Epstein Barr virus-encoded miRNAs, more so than endogenous miRNAs, signify presence of NPC. However, RNAseq-based EBV miRNA profiles differ between NPC patients, suggesting inter-tumor heterogeneity or divergent secretory characteristics. We determined with sensitive qRT-PCR assays that EBV miRNAs BART7-3p, BART9-3p and BART13-3p are actively secreted by C666.1 NPC cells bound to extracellular vesicles (EVs) and soluble ribonucleoprotein complexes. Importantly, these miRNAs are expressed in all primary NPC tumor biopsies and readily detected in nasopharyngeal brushings from both early and late-stage NPC patients. Increased levels of BART7-3p, BART9-3p and particularly BART13-3p, distinguish NPC patient sera from healthy controls. Receiver operating characteristic curve analysis using sera from endemic NPC patients, other head and neck cancers and individuals with asymptomatic EBV-infections reveals a superior diagnostic performance of EBV miRNAs over anti-EBNA1 IgA serology and EBV-DNA load (AUC 0.87-0.96 vs 0.86 and 0.66 respectively). The high specificity of circulating EBV-BART13-3p (97%) for NPC detection is in agreement with active secretion from NPC tumor cells. We conclude EV-bound BART13-3p in circulation is a promising, NPC-selective, biomarker that should be considered as part of a screening strategy to identify NPC in endemic regions.

Establishment and characterization of new tumor xenografts and cancer cell lines from EBV-positive nasopharyngeal carcinoma.

The lack of representative nasopharyngeal carcinoma (NPC) models has seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. Here we report the successful growth of five NPC patient-derived xenografts (PDXs) from fifty-eight attempts of transplantation of NPC specimens into NOD/SCID mice. The take rates for primary and recurrent NPC are 4.9% and 17.6%, respectively. Successful establishment of a new EBV-positive NPC cell line, NPC43, is achieved directly from patient NPC tissues by including Rho-associated coiled-coil containing kinases inhibitor (Y-27632) in culture medium. Spontaneous lytic reactivation of EBV can be observed in NPC43 upon withdrawal of Y-27632. Whole-exome sequencing (WES) reveals a close similarity in mutational profiles of these NPC PDXs with their corresponding patient NPC. Whole-genome sequencing (WGS) further delineates the genomic landscape and sequences of EBV genomes in these newly established NPC models, which supports their potential use in future studies of NPC.

Sequence Variation of Epstein-Barr Virus: Viral Types, Geography, Codon Usage, and Diseases.

One hundred thirty-eight new Epstein-Barr virus (EBV) genome sequences have been determined. One hundred twenty-five of these and 116 from previous reports were combined to produce a multiple-sequence alignment of 241 EBV genomes, which we have used to analyze variation within the viral genome. The type 1/type 2 classification of EBV remains the major form of variation and is defined mostly by EBNA2 and EBNA3, but the type 2 single-nucleotide polymorphisms (SNPs) at the EBNA3 locus extend into the adjacent gp350 and gp42 genes, whose products mediate infection of B cells by EBV. A small insertion within the BART microRNA region of the genome was present in 21 EBV strains. EBV from saliva of U.S. patients with chronic active EBV infection aligned with the wild-type EBV genome with no evidence of WZhet rearrangements. The V3 polymorphism in the Zp promoter for BZLF1 was found to be frequent in nasopharyngeal carcinoma cases from both Hong Kong and Indonesia. Codon usage was found to differ between latent and lytic cycle EBV genes, and the main forms of variation of the EBNA1 protein have been identified.IMPORTANCE Epstein-Barr virus causes most cases of infectious mononucleosis and posttransplant lymphoproliferative disease. It contributes to several types of cancer, including Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B cell lymphoma, nasopharyngeal carcinoma, and gastric carcinoma. EBV genome variation is important because some of the diseases associated with EBV have very different incidences in different populations and geographic regions, and differences in the EBV genome might contribute to these diseases. Some specific EBV genome alterations that appear to be significant in EBV-associated cancers are already known, and current efforts to make an EBV vaccine and antiviral drugs should also take account of sequence differences in the proteins used as targets.

Epstein-Barr Virus Gene BARF1 Expression is Regulated by the Epithelial Differentiation Factor ΔNp63α in Undifferentiated Nasopharyngeal Carcinoma.

Epstein-Barr Virus (EBV) BamHI-A rightward frame 1 (BARF1) protein is considered a viral oncogene in epithelial cells and has immune-modulating properties. During viral lytic replication BARF1 is expressed as an early gene, regulated by the immediate early EBV protein R. However, in viral latency BARF1 is exclusively expressed in epithelial tumors such as nasopharyngeal (NPC) and gastric carcinoma (GC) but not in lymphomas, indicating that activation of the BARF1 promoter is cell type specific. Undifferentiated NPC is characterized by high expression of ΔNp63 isoforms of the epithelial differentiation marker p63, a member of the p53 family of transcription factors. Transcription factor binding site analysis indicated potential p53 family binding sites within the BARF1 promoter region. This study investigated ability of various p53 family members to transactivate the BARF1 promoter. Using BARF1 promoter luciferase reporter constructs we demonstrate that only p63 isoform ΔNp63α is capable of transactivating the BARF1 promoter, but not the TAp63 isoforms, p53 or p73. Direct promoter binding of ΔNp63α was confirmed by Chromatin Immune Precipitation (ChIP) analysis. Deletion mutants of the BARF1 promoter revealed multiple ΔNp63 response elements to be responsible for BARF1 promoter transactivation. However, ΔNp63α alone was not sufficient to induce BARF1 in tumor cells harboring full EBV genomes, indicating that additional cofactors might be required for full BARF1 regulation. In conclusion, in EBV positive NPC and GC, BARF1 expression might be induced by the epithelial differentiation marker ΔNp63α, explaining BARF1 expression in the absence of lytic reactivation.

Curcuminoids as EBV Lytic Activators for Adjuvant Treatment in EBV-Positive Carcinomas.

Epstein-Barr virus (EBV) persists in nasopharyngeal (NPC) and gastric carcinomas (EBVaGC) in a tightly latent form. Cytolytic virus activation (CLVA) therapy employs gemcitabine and valproic acid (GCb+VPA) to reactivate latent EBV into the lytic phase and antiviral valganciclovir to enhance cell death and prevent virus production. CLVA treatment has proven safe in phase-I/II trials with promising clinical responses in patients with recurrent NPC. However, a major challenge is to maximize EBV lytic reactivation by CLVA. Curcumin, a dietary spice used in Asian countries, is known for its antitumor property and therapeutic potential. Novel curcuminoids that were developed to increase efficacy and bioavailability may serve as oral CLVA adjuvants. We investigated the potential of curcumin and its analogs (curcuminoids) to trigger the EBV lytic cycle in EBVaGC and NPC cells. EBV-reactivating effects were measured by immunoblot and immunofluorescence using monoclonal antibodies specific for EBV lytic proteins. Two of the hit compounds (41, EF24) with high lytic inducing activity were further studied for their synergistic or antagonistic effects when combined with GCb+VPA and analyzed by cytotoxicity and mRNA profiling assays to measure the EBV reactivation. Curcuminoid as a single agent significantly induced EBV reactivation in recombinant GC and NPC lines. The drug effects were dose- and time-dependent. Micromolar concentration of curcuminoid EF24 enhanced the CLVA effect in all cell systems except SNU719, a naturally infected EBVaGC cell that carries a more tightly latent viral genome. These findings indicated that EF24 has potential as EBV lytic activator and may serve as an adjuvant in CLVA treatment.

Patterns of Interindividual Variability in the Antibody Repertoire Targeting Proteins Across the Epstein-Barr Virus Proteome.

Background: Little is known about variation in antibody responses targeting the full spectrum of Epstein-Barr virus (EBV) proteins and how such patterns inform disease risk. Methods: We used a microarray to measure immunoglobulin G (IgG) and immunoglobulin A (IgA) antibody responses against 199 EBV protein sequences from 5 EBV strains recovered from 289 healthy adults from Taiwan. We described positivity patterns, estimated the correlation between antibodies, and investigated the associations between environmental and genetic risk factors and variations in antibody responses. Results: Healthy adults were more likely to mount IgG antibody responses to EBV proteins (median positivity frequency, 46.5% for IgG and 17.3% for IgA; P = 1.6 × 10-46, by the Wilcoxon rank sum test). Responses against glycoproteins were particularly prevalent. The correlations between antibody responses of the same class were higher than correlations across classes. The mucosal exposure to proteins involved in EBV reactivation (as determined by the IgA response) was associated with smoking (P = .002, by the sequence kernel association test-combined), and approximately one quarter of adults displayed antibody responses associated with EBV-related cancer risk. Conclusions: These data comprehensively define the variability in human IgG and IgA antibody responses to the EBV proteome. Patterns observed can serve as the foundation for elucidating which individuals are at highest risk of EBV-associated clinical conditions and for identifying targets for effective immunodiagnostic tests.