anti-EBNA1 Antibody Research Articles

A High Prevalence of Anti-EBNA1 Heteroantibodies in Systemic Lupus Erythematosus (SLE) Supports Anti-EBNA1 as an Origin for SLE Autoantibodies.

BackgroundThat Epstein–Barr virus (EBV) infection is associated with systemic lupus erythematosus (SLE) is established. The challenge is to explain mechanistic roles EBV has in SLE pathogenesis. Previous studies identify four examples of autoantibody cross-reactions between SLE autoantigens and Epstein–Barr nuclear antigen 1 (EBNA1). For two of these examples, the earliest detected autoantibody specifically cross-reacts with EBNA1; thereby, defined EBNA1 epitopes induce a robust autoantibody response in animals. These results suggest that the autoantibodies initiating the process leading to SLE may emerge from the anti-EBNA1 heteroimmune response. If this hypothesis is true, then anti-EBNA1 responses would be more frequent in EBV-infected SLE patients than in EBV-infected controls. We tested this prediction.MethodsWe evaluated published East Asian data by selecting those with a positive anti-viral capsid antigen (VCA) antibody immunoglobulin G (IgG) test and determining whether anti-EBNA1 was more common among the EBV-infected SLE cases than among matched EBV-infected controls with conditional logistic regression analysis.ResultsAll the qualifying SLE patients (100%) in this dataset were EBV-infected compared to age- and sex-matched controls (92.2%) [odds ratio (OR) = 28.6, 95% CI 6.4–∞, p = 8.83 × 10-8], confirming the known close association of EBV infection with SLE. Furthermore, virtually all the SLE cases have both anti-VCA IgG and anti-EBNA1 IgG antibodies [124 of 125 (99.2%)], which are more frequently present than in age- and sex-matched EBV-infected controls [232 of 250 (93.2%)] (OR = 9.7, 95% CI 1.5–414, p = 0.0078) for an 89.7% SLE attributable risk from anti-EBNA1, which is in addition to the 100% SLE risk attributable to EBV infection in these data.ConclusionsThe association of EBV infection with SLE is reconfirmed. The prediction that anti-EBNA1 is more frequent in these SLE cases than in EBV-infected controls is true, consistent with the hypothesis that anti-EBNA1 contributes to SLE. This second EBV-dependent risk factor is consistent with a molecular mimicry model for the generation of SLE, starting with EBV infection, progressing to anti-EBNA1 response; then molecular mimicry leads to anti-EBNA1 antibodies cross-reacting with an SLE autoantigen, causing autoantibody epitope spreading, and culminating in clinical SLE. These results support the anti-EBNA1 heteroimmune response being a foundation from which pathogenic SLE autoimmunity emerges.

Molecular mimicry between Anoctamin 2 and Epstein-Barr virus nuclear antigen 1 associates with multiple sclerosis risk

Multiple sclerosis (MS) is a chronic inflammatory, likely autoimmune disease of the central nervous system with a combination of genetic and environmental risk factors, among which Epstein-Barr virus (EBV) infection is a strong suspect. We have previously identified increased autoantibody levels toward the chloride-channel protein Anoctamin 2 (ANO2) in MS. Here, IgG antibody reactivity toward ANO2 and EBV nuclear antigen 1 (EBNA1) was measured using bead-based multiplex serology in plasma samples from 8,746 MS cases and 7,228 controls. We detected increased anti-ANO2 antibody levels in MS (P = 3.5 × 10-36) with 14.6% of cases and 7.8% of controls being ANO2 seropositive (odds ratio [OR] = 1.6; 95% confidence intervals [95%CI]: 1.5 to 1.8). The MS risk increase in ANO2-seropositive individuals was dramatic when also exposed to 3 known risk factors for MS: HLA-DRB1*15:01 carriage, absence of HLA-A*02:01, and high anti-EBNA1 antibody levels (OR = 24.9; 95%CI: 17.9 to 34.8). Reciprocal blocking experiments with ANO2 and EBNA1 peptides demonstrated antibody cross-reactivity, mapping to ANO2 [aa 140 to 149] and EBNA1 [aa 431 to 440]. HLA gene region was associated with anti-ANO2 antibody levels and HLA-DRB1*04:01 haplotype was negatively associated with ANO2 seropositivity (OR = 0.6; 95%CI: 0.5 to 0.7). Anti-ANO2 antibody levels were not increased in patients from 3 other inflammatory disease cohorts. The HLA influence and the fact that specific IgG production usually needs T cell help provides indirect evidence for a T cell ANO2 autoreactivity in MS. We propose a hypothesis where immune reactivity toward EBNA1 through molecular mimicry with ANO2 contributes to the etiopathogenesis of MS.

The Engagement Between Vitamin D and the Immune System: Is Consolidation by a Marriage to Be Expected?

The Engagement Between Vitamin D and the Immune System: Is Consolidation by a Marriage to Be Expected?

Idiopathic nephrotic syndrome: the EBV hypothesis.

Steroid sensitive nephrotic syndrome is marked by a massive proteinuria and loss of podocytes foot processes. The mechanism of the disease remains debated but recent publications suggest a primary role of Epstein-Barr Virus (EBV). EBV replication in the peripheral blood is found in 50% of patients during the first flare of the disease. The genetic locus of steroid sensitive nephrotic syndrome was also identified as influencing antibodies directed against EBNA1. EBV is able to establish, latent benign infection in memory B cells that display phenotypes similar to antigen-selected memory B cells. Consistently, memory B cells reconstitution after rituximab infusion is a predictor of the relapse of proteinuria. We suggest that a specific anti-EBNA1 antibody internalized in the podocytes via the neonatal Fc receptor might cross-react with a major protein present in the same cell trafficking compartment. The diversion of this major podocyte protein in the urinary space and the subsequent depletion is supposed to result in podocyte damages with loss of foot processes and massive proteinuria. Immunosuppression of B cells and subsequent clearance of anti-EBNA1 antibodies would lead to a restoration of the normal level of the protein allowing recovery of proteinuria and of normal podocyte morphology.

High-Density Peptide Microarray Analysis of IgG Autoantibody Reactivities in Serum and Cerebrospinal Fluid of Multiple Sclerosis Patients

Intrathecal immunoglobulin G (IgG) synthesis and oligoclonal IgG bands in cerebrospinal fluid (CSF) are hallmarks of multiple sclerosis (MS), but the antigen specificities remain enigmatic. Our study is the first investigating the autoantibody repertoire in paired serum and CSF samples from patients with relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and other neurological diseases by the use of high-density peptide microarrays. Protein sequences of 45 presumed MS autoantigens (e.g.MOG, MBP, and MAG) were represented on the microarrays by overlapping 15mer peptides. IgG reactivities were screened against a total of 3991 peptides, including also selected viral epitopes. The measured antibody reactivities were highly individual but correlated for matched serum and CSF samples. We found 54 peptides to be recognized significantly more often by serum or CSF antibodies from MS patients compared with controls (pvalues <0.05). The results for RRMS and PPMS clearly overlapped. However, PPMS patients presented a broader peptide-antibody signature. The highest signals were detected for a peptide mapping to a region of the Epstein-Barr virus protein EBNA1 (amino acids 392-411), which is homologous to the N-terminal part of human crystallin alpha-B. Our data confirmed several known MS-associated antigens and epitopes, and they delivered additional potential linear epitopes, which await further validation. The peripheral and intrathecal humoral immune response in MS is polyspecific and includes antibodies that are also found in serum of patients with other diseases. Further studies are required to assess the pathogenic relevance of autoreactive and anti-EBNA1 antibodies as well as their combinatorial value as biomarkers for MS.

Reduced Transplacental Transfer of a Subset of Epstein-Barr Virus-Specific Antibodies to Neonates of Mothers Infected with Plasmodium falciparum Malaria during Pregnancy.

Over 35% of children in a region of malaria endemicity are infected with Epstein-Barr virus (EBV) by 6 months of age. This susceptibility may be linked to impaired transplacental transfer of antibodies. In this study, we determined the effect of malaria exposure during pregnancy on the transfer of EBV-specific maternal antibodies in a region of western Kenya that experiences endemic malaria. Pregnant mothers were recruited and followed up until delivery to determine levels of neonatal malaria exposure. Levels of EBV lytic (viral capsid antigen [VCA], Z transcriptional activator [Zta], and early diffuse antigen complex [EAd]) and EBV latent (EBV nuclear antigen-1 (EBNA1]) and tetanus-specific IgG antibodies were measured in 70 paired maternal and cord blood samples using a Luminex-bead-based assay. A high proportion (63%) of the infants were exposed to malaria in utero. Levels of EBV- and tetanus-specific antibodies were similar in malaria-infected mothers and in mothers who had no detectable malaria infection. Malaria-exposed neonates had significantly lower levels of anti-EBNA1, anti-Zta, and anti-EAd antibodies than were seen in their mothers. In utero malaria exposure resulted in significant reductions in transplacental transfer of anti-VCA-p18 and anti-EBNA1 antibodies of 13% and 22%, respectively. Neonates received significantly low levels of anti-Zta and anti-EAd antibodies irrespective of malaria exposure levels. In multivariate analysis, in utero malaria exposure was associated with a significant reduction in the transfer of anti-VCA-p18 and anti-EBNA1 antibodies to the neonates (P = 0.0234 and P = 0.0017, respectively). Malaria during pregnancy results in differential levels of transfer of EBV-specific antibodies from the mother to the fetus. The impaired transplacental transfer of some antibodies may lead to the malaria-exposed neonates being susceptible to early EBV infection.

Epstein-Barr Virus (HHV-4) Inoculation to Rabbits by Intranasal and Oral Routes Results in Subacute and/or Persistent Infection Dissimilar to Human Disease

Objective: We report the infection of New Zealand white rabbits with Epstein-Barr virus (EBV). Methods: EBV prepared in B95-8 (producer) cells was inoculated to rabbits by combined intranasal and oral routes. Blood and white blood cell (WBC) samples were taken before infection, then on days 8, 28 and 98 post-infection (p.i.). Results: Administration of either 3 × 10⁸ (group A, 11 rabbits) or 1 × 10⁹ (group B, 10 rabbits) EBV DNA copies per animal induced subacute and/or persistent infection. The IgG antibodies in plasma were detected by ELISA as well as by immunoblot (IB). The IB bands showed mainly antibodies to the BZRF1/Zta transactivation polypeptide (69.2%), the p54 early protein (53.4%) and to the p23 capsid protein (35.8%). No anti-EBNA1 antibody was detected throughout. Viral DNA could be detected by PCR in WBCs and/or spleen of 7 out of 21 infected rabbits (30%), while 60-80% of them showed serologic response. The transiently present EBV DNA was accompanied by LMP1 antigen. Conclusions: Rabbits developed persistent EBV infection in the absence of EBNA1 antibodies and by the lack of typical infectious mononucleosis-like syndrome. The absence of EBNA1 antibody may reflect the lack of EBNA1 in B cells of EBV-inoculated rabbits.

AB0155 Anti-VCA and EBNA1 antibodies are produced in the rheumatoid synovium in the presence of ectopic lymphoid structures and correlate with ACPA production

BackgroundEctopic lymphoid structures (ELS) are preferential sites for Epstein-Barr virus (EBV) latency and reactivation in the brain of multiple sclerosis (1) and are functional sites of autoreactive B cell activation...

Epstein-Barr Virus, Hygiene, and Multiple Sclerosis in Mexico City

Background/Aims: We studied whether serological antibody titers against Epstein-Barr virus (EBV) were related to multiple sclerosis (MS) risk in Mexico City, a population undergoing an epidemiological transition. Additionally, we explored the association between markers of hygiene and intestinal parasite infection and MS prevalence. Methods: We conducted a hospital based case-control study in Mexico City; 52 incident MS cases. Childhood and adult hygiene scores were created using information from questionnaires. 51 cases and 51 controls provided blood samples for biomarker analyses. Results: There was a 3-fold increase in risk of MS with each 1 unit increase in anti-EBNA1 titer (OR 3.02, 95% CI: 1.01-9.02). Increasing childhood and adult hygiene scores were associated with a modestly increased risk of MS (ORchildhood 1.20, 95% CI: 1.04-1.38; ORadult 1.28, 95% CI: 1.04-1.58). We found no association between antibody titer to neither Stronglyloides nor Ascaris and MS risk. Conclusion: High anti-EBNA1 antibody titers appears to be associated with increased MS risk even in a region where MS had historically low incidence and infectious mononucleosis is rare, providing further support for the postulated role of EBV in MS etiology. We also found moderate support for a role of higher ‘hygiene’ being associated with susceptibility to MS.

About making a CHO production cell line “research-friendly” by genetic engineering

The use of Chinese Hamster Ovary (CHO) cells for transient gene expression is gaining importance steadily, as it has been shown that both quality and quantity of proteins derived from CHO cells differs from and can even be superior to material derived from Human Embryonic Kidney (HEK293) cells [1-4]. In order to augment yields HEK cell lines have been genetically modified, by e.g. stable integration of an expression cassette coding for the Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) in conjunction with expression plasmid vectors containing the EBNA1 interaction site oriP. Here we describe the generation of a CHO cell line stably expressing the EBNA1 gene to enhance yields after large scale transient transfection with polyethylenimine (PEI). An expression plasmid featuring the EBNA1 gene was transfected into an in-house available CHO wild type cell line by nucleofection and several stable pools were established by antibiotic selection. In a first approach, 133 clones were then isolated by limiting dilution cloning and subsequently expanded for further analysis. The approach aimed at identifying clones showing both the presence of EBNA1 protein and enhanced yields after PEI-mediated transient expression of a reporter protein at the same time. Cell lysates or nuclear and cytoplasmic protein extracts from these clones were tested for presence of EBNA1 protein by Western Blot. An in-licensed cell line, CHO EBNALT85 (Icosagen AS, Tartu, Estonia) expressing the full length EBNA1 gene, and the HEK293-6E cell line (from the group of Y. Durocher, NRC, Canada) expressing a truncated EBNA1 gene served as positive controls. A number of commercially available anti-EBNA1 antibodies were tested in Western blotting, but most antibodies failed to detect the EBNA1 protein produced by these positive control cells. Only one antibody (Antibody 1EB12, Santa Cruz Biotechnology) was identified that detected the EBNA1 proteins in both positive control cell lines. However, by applying this antibody on the 133 generated clones it became obvious that in most of them the EBNA1 protein appeared to be largely degraded. The functionality of the EBNA1 protein in the cells was probed by PEI-mediated transient transfection of expression plasmids encoding the gene for the reporter protein Secreted Alkaline Phosphatase (SeAP). SeAP expression levels were compared with respect to presence or absence of the EBNA1 interaction sequence oriP on the SeAP expression plasmid. Only one clone showed a twofold enhanced production level of the reporter protein compared to the non-engineered parental CHO cell line. However, in this specific clone no EBNA1 protein could be detected by Western Blot, and the enhanced expression level was independent from the presence or absence of the oriP sequence on the SeAP expression plasmid. We therefore conclude that this clone does not possess a functional EBNA1 protein and an EBNA1/oriP based enhancement of productivity. The mechanism underlying the enhanced yield of the reporter protein in this particular clone remains currently elusive. In an alternative approach flow cytometric cell sorting was applied to generate CHO clones producing a functional EBNA1 protein. Using the EBNA1 positive CHO cell line CHO EBNALT85 it could be shown that expressing eGFP from a transiently transfected expression plasmid with oriP sequence resulted in a 30- to 40-fold increase of highly fluorescent cells in comparison to an expression plasmid lacking the oriP sequence. Thus, the already generated CHO pools stably transfected with the EBNA1 gene were transiently supertransfected with an eGFP expression plasmid containing the oriP sequence. Highly fluorescent cells were collected by cell sorting and expanded. Analysis by Western Blot revealed that this sub-pool was enriched for cells exhibiting the presence of a full length EBNA1 protein besides a major breakdown product. Transient transfection of this sub-pool with SeAP expression vectors led to the conclusion that the pool contained cells with a functional EBNA1 protein capable of enhancing SeAP yields in dependence on the presence of the oriP sequence on the SeAP expression plasmid. Clones were again generated from this sub-pool by limiting dilution cloning and characterized in analogy to the first approach. 9 out of 12 clones that were analyzed further showed increased levels of reporter protein production in dependence on the presence of the oriP element on the expression plasmid. The series of clones exhibiting highest expression levels will now be further evaluated by expression of other protein candidates.

Low intrathecal immune response of anti–EBNA-1 antibodies and EBV DNA from multiple sclerosis patients

Numerous studies have been carried out to determine whether an Epstein–Barr virus (EBV) infection can be considered a risk factor for multiple sclerosis (MS), following the evidence of an increase in IgG response to nuclear antigen-1 (EBNA-1) in both serum and cerebrospinal fluid (CSF) from MS patients. However, the possible interaction between EBV and MS has still not been well characterized, and the possible pathogenic role is yet to be determined. A case-control study (76 cases and 75 controls) was conducted to investigate anti-EBV antibodies synthesis in serum and CSF through intrathecal specific IgG synthesis to EBNA-1, as well as the presence of EBV DNA in plasma, peripheral blood mononuclear cells, and CSF from MS patients. Intrathecal EBNA-1 specific IgG synthesis was detected in 6.6% MS patients and in 17.3% controls. No EBV DNA was found in plasma or CSF, and our findings showed no evidence of high intrathecal EBNA-1 specific IgG synthesis or of significant EBV DNA in CSF in MS patients.

Detection of Epstein-Barr Virus DNA in Plasma and Lymph Node Biopsy Samples of Pediatric and Adult Patients with Hodgkin Lymphoma

Hodgkin lymphoma (HL) is commonly associated with latent Epstein-Barr virus (EBV) infection. The aim of this study is a molecular analysis of the EBV status in both involved lymph node biopsies and plasma samples of patients with HL. Plasma and lymph node biopsy samples obtained from 15 pediatric and 10 adult HL patients were examined for EBV DNA using conventional polymerase chain reaction (PCR). The control group consisted of 30 healthy pediatrics and adults. In addition, immunoglobulin G (IgG) anti-EBV nuclear antigen (EBNA)-1 antibody was determined in sera of patients and controls using enzyme-linked immunosorbent assay (ELISA). IgG Anti EBNA-1 antibody was detected in 21 (84%) and 8 (26%) of patients and controls, respectively (P < .05). EBV DNA was detected in 12 (48%) and 1 (3%) plasma samples of patient and control cases, respectively. Significant difference was observed in plasma DNA detection between patients and controls (P < .05). Comparison of EBV DNA detection in plasma and biopsy samples between children and adult patients was only significant for plasma samples (P = .025). Significant correlation was observed in positive detection of EBV DNA between plasma and biopsy samples of the same individual (P < .001, r = .923). Frequency of EBV DNA in plasma and biopsy samples obtained from mixed-cellularity subgroup was higher than the nodular sclerosis; however, no significant difference was observed between these 2 subgroups. EBV detection in plasma of childhood Hodgkin lymphoma in a population with EBV seroconversion might be of value as a biomarker for EBV-associated Hodgkin lymphoma.

Targeting the nuclear antigen 1 of Epstein-Barr virus to the human endocytic receptor DEC-205 stimulates protective T-cell responses

AbstractDendritic cells (DCs) express many endocytic receptors that deliver antigens for major histocompatibility class (MHC) I and II presentation to CD8+ and CD4+ T cells, respectively. Here, we show that targeting Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) to one of them, the human multilectin DEC-205 receptor, in the presence of the DC maturation stimulus poly(I:C), expanded EBNA1-specific CD4+ and CD8+ memory T cells, and these lymphocytes could control the outgrowth of autologous EBV-infected B cells in vitro. In addition, using a novel mouse model with reconstituted human immune system components, we demonstrated that vaccination with αDEC-205-EBNA1 antibodies primed EBNA1-specific IFN-γ–secreting T cells and also induced anti-EBNA1 antibodies in a subset of immunized mice. Because EBNA1 is the one EBV antigen that is expressed in all proliferating cells infected with this virus, our data suggest that DEC-205 targeting should be explored as a vaccination approach against symptomatic primary EBV infection and against EBV-associated malignancies.

Epstein-Barr virus nuclear antigen type 1 binding: electron microscopy

Epstein-Barr virus (EBV) nuclear antigen type-1 (EBNA-1) was extracted and purified from Raji cells by chromatography on DNA-Sepharose and Blue-dextran Sepharose. Its complexes with plasmid pM765-10 derived from EBV (strain MABA) DNA were visualized by electron microscopy. The criteria of specificity were as follows: (1) preferential binding of EBNA-1 to the ori-P region of pM765-10; (2) specific enlargement of EBV DNA/EBNA-1 complexes with anti-EBNA-1 (IR-3) IgG antibody; and (3) resistance of the resulting EBV DNA/EBNA-1/antiEBNA-1 antibody complexes to treatment with 1.5 M NaCl. The optimal conditions for the formation of EBV DNA/EBNA-1 complexes were 50 to 150 mM NaCl and pH 6.O. A balanced equilibrium of EBNA-1 and pM765-10 was necessary to achieve both a high yield and specificity of EBV DNA/EBNA-1 complexes. EBV DNA/EBNA-1 specific binding, EM; EBV DNA/EBNA-1/antibody complexes,