Antibodies to Epstein-Barr virus in nasopharyngeal carcinoma, tonsillar carcinoma and control groups

In the sera of 17 patients with nasopharyngeal carcinoma (NPC) and of 19 patients with tonsillar carcinoma (TC) the titres of IgA, IgG and IgM antibodies to EBV VCA (viral capsid antigen) and of IgG antibodies to EBV EA (early antigen) were determined by the indirect immunofluorescence (IF) method. Significant difference was observed in the frequency of IgA antibodies to EBV VCA and IgG antibodies to EBV EA between NPC patients and controls. There was also a significant difference between the frequency of IgM antibody to EBV VCA and EBV EA antibody titres in TC patients and controls. The geometric mean titre (GMT) of IgG antibodies to EBV VCA was significantly higher in the NPC and TC patients as compared to controls.     

Antibody response to Epstein-Barr virus antigens in patients with chronic viral infection

We tested antibody titres against Epstein-Barr virus (EBV) antigens in patients suffering from chronic viral disease and compared them with those determined in sex- and age-matched healthy controls. Patient sera showed signs of active EBV infection [antibodies against early antigen (EA) and/or viral capsid antigen (VCA) in the IgM or IgA classes] significantly more frequently than the control group. Correspondingly, geometric mean titres (GMT) of antibodies against all viral antigens were elevated in the patients. The strongest association with EBV was observed in patients whose clinical symptoms closely resembled infectious mononucleosis: 92% of the subjects in this subgroup possessed anti-EA and 41 and 25% had IgM and IgA anti-VCA antibody, respectively. In patients with signs of lymphoproliferation only and in those suffering from frequent respiratory infections the association with EBV was less marked but still significant. Patients with transient defects in humoral and cellular immunity mounted higher titres against VCA in the IgG class than those without immune defects.     

Decreased antibody reactivity to Epstein-Barr virus capsid antigen in type 1 (insulin-dependent) diabetes mellitus.

In this study, antibody levels to Epstein-Barr virus (EBV) capsid antigen (VCA) and EBV early antigens (EA) were analysed by enzyme immunoassay in 54 newly diagnosed type 1 diabetic children and in matched controls. The patients had significantly lower EBV VCA IgG-class antibody levels (p less than 0.02). This was true particularly in young patients and in boys (p less than 0.005). VCA IgA-class antibody levels were also decreased in young patients (p less than 0.02). VCA IgM-class antibodies were observed in two of the patients only. IgG- and IgA-class antibodies to EBV EA or rubella virus antigen showed no differences between patients and controls. The results suggest that EBV infections coincide with the onset of clinical diabetes relatively rarely. However, the abnormally low antibody response to EBV VCA in diabetic children suggests abnormalities in the EBV-specific immune response.     

Epstein-Barr virus (EBV) antibodies in children with non-Hodgkin's lymphomas

Antibody titres against Epstein-Barr virus (EBV) antigens in children suffering from non-Hodgkin's lymphoma (NHL) were determined. IgG antibody titres against the viral capsid antigen (VCA) and early antigen (EA) exceeded those found in healthy control subjects. On the other hand, antibody titres against EBV-determined nuclear antigen (EBNA complex) were generally lower than in the control group. The most striking phenomenon observed in the patient group was the frequent activation of latent virus infection as revealed by the periodical appearance of anti-EA and IgM class anti-VCA antibodies. Antibody titres against EBV antigens were generally lower among patients with progressing disease than in those with a more favourable course of the illness. The closest relation to EBV based on serological findings, was detected in lymphoblastic lymphomas of Burkitt-type histology, poorly differentiated lymphocytic lymphomas, and in lymphomas localized in the abdomen. The question whether EBV might be involved in a certain proportion of the cases examined is discussed and further approaches to elucidate this problem are suggested.     

Improved sensitivity of detection by avidin-biotin complex (ABC) immunocytochemistry in Epstein-Barr virus serology

Serum antibodies against Epstein-Barr virus (EBV)-determined antigens have traditionally been titrated by the indirect immunofluorescence (IIF) technique. The avidin-biotin complex (ABC) immunocytochemical technique was used to determine the serum levels of IgA against EBV viral capsid antigen (IgA/VCA) and IgA against EBV early antigen (IgA/EA) in sera of 106 nasopharyngeal carcinoma (NPC) patients prior to treatment and 100 normal individuals. The sensitivity of the ABC technique is enhanced by an amplification of the antigen-antibody reaction, which involves the binding of the enzyme-linked ABC to the second biotinylated antibody. There was a good correlation (r = 0.9988) between ABC and IIF-determined IgA/VCA-positive titres, with the ABC technique being more sensitive than IIF in the detection of IgA/VCA in NPC sera: 94% (99/106) and 76% (80/106), respectively. The frequency of IgA/EA reactivity in NPC sera was also markedly increased by immunodetection with the ABC technique as compared with IIF technique: 63% (69/106) and 28% (30/106) respectively. Both the immunocytochemical techniques were equally specific in discriminating between elevated serum titres of IgA/VCA and IgA/EA in NPC sera from normal human sera.     

Herpesvirus group antibodies in children with Hodgkin's disease

Summary During the period of three years (1972–1974), serum samples from 60 patients (children and adolescents) with lympho-hematopoietic system diseases were examined for antibodies to all four human herpesviruses. Among these were 26 active Hodgkin's disease (AHD) patients and 6 HD patients with a minimum five years' remission. Simultaneously matched controls (age, sex) of AHD patients were examined. Antibody levels against the viral capsid antigen of Epstein-Barr virus (EBV/VCA) in AHD patients were significantly higher, with overrepresentation of higher titres (1:160), than in matched controls. The lowest EBV/VCA antibody titres were in the leukemia-non-Hodgkin's lymphoma patients. We could not prove any significant relationship between cytomegalovirus or herpes simplex virus type 1 antibody titres and AHD or any other disease of lymphohematopoietic system. The varicella-zoster virus antibody titres in AHD patients were significantly higher than in matched controls. No significant differences in antibodies against EBV/VCA and the other human herpesviruses between the evolution and remission period of AHD patients could be detected. No differences in EBV/VCA antibody titres were observed between the healthy school-children aged 10 to 15 years who were and who were not in contact with a HD patient.     

Observations on the resistance of Fpstein-Barr virus DNA synthesis to hydroxyurea

The EBV genome was expressed in EBV “negative” D98/HR-1 somatic cell hybrids following treatment with IUdR as monitored by induction of early antigen (EA), virus capsid antigen (VCA), and virus particles. When the hybrid cells were treated with hydroxyurea (HU) following induction, total DNA synthesis was dramatically reduced but expression of both EA and VCA was not altered. The number of EBV genome equivalents increased following induction with IUdR in the presence of 5 mM HU. Synthesis of EBV specific DNA proceeded to the same degree with or without treatment with HU in lymphoblastoid cells infected with EBV while HU treatment reduced total DNA synthesis by 85–90%. Cytosine arabinoside (ara-C) used in the same manner as HU in IUdR induced hybrid cells dramatically reduced cellular DNA synthesis and totally inhibited EBV DNA synthesis.Of all viruses tested thus far, EBV is unique in that EBV specific DNA synthesis is refractile to inhibition by treatment with HU. Several possible explanations for this unique phenomenon are presented.     

High incidence of elevated antibody titers to Epstein-Barr virus in patients with uveitis

Summary. We assayed Epstein-Barr virus (EBV) antibody titers in patients’ sera using indirect immunofluorescence and tested for the presence of antibody to EBV immediate-early BZLF1 protein ZEBRA by Western blotting to explore the association of EBV infection with uveitis. IgG and IgA antibodies to viral capsid antigen (VCA), IgG antibodies to early antigen (EA), and antibodies to EBV nuclear antigen were detected at higher titers in sera of patients with uveitis than in the sera of healthy controls. Neither IgM antibody to VCA nor EA was detected in the patients’ sera. Anti-ZEBRA-IgG antibodies were detected in most patients’ sera, but not in those of healthy controls. These results suggest that uveitis might be a disease accompanied by EBV reactivation.     

Epstein-Barr virus-related antibody patterns in ataxia-telangiectasia.

Epstein-Barr virus-related antibody titres were determined in twenty-seven patients with ataxia-telangiectasia (AT) and twenty-two healthy members of their families, in twenty-two patients with other diseases, among them ten with Behçet''s disease and ten with various primary immune deficiencies, and fifteen healthy members of their families, and in twenty-three unrelated healthy controls. The AT patients showed an increased incidence (55.6%) of high antibody titres (greater than or equal to 1:320) to viral capsid antigen (VCA), and also a high incidence (48.2%) of antibodies to Epstein-Barr virus (EBV) induced early antigens (EA), but low titres (less than 1:10) of antibodies to the EBV-associated nuclear antigen (EBNA) in 44.2% of the cases. The geometric means of anti-VCA were three-to four-fold higher, and of anti-EBNA six-fold lower, than those of the control groups. The patients with the other diseases did not differ significantly from the controls except for a higher incidence of anti-EBNA titres of less than 1:10 (38.1% vs 4--5%). AT patients with low anti-EBNA titres tended to have more advanced T cell deficiencies than AT patients with moderate anti-EBNA titres, as detected by counts of total lymphocytes and E-rosetting cells, and skin test responses. The results support the hypothesis that a functioning T cell system is required to release EBNA from EBV genome-carrying cells for initial and maintained production of anti-EBNA.     

Complexity of EBV homologous DNA in continuous lymphoblastoid cell lines

The complexity of Epstein-Barr Virus (EBV) homologous DNA in 11 EBV-infected lymphoblastoid cell lines which have been passaged for several years in culture was determined by hybridization of lymphoblastoid cell DNA to DNA extracted from EBV purified from HR-1 cells and labeled in vitro. Five of the cell lines analyzed contained no early (EA) or viral capsid (VCA) antigens which have been associated with the replication of EBV. Two other cell lines contained EA but not VCA. Of the seven VCA-negative cell lines, those which contained some early antigen or in which early antigen could be induced with IUDR had 56, 48, and 25 copies per diploid cell genome of more than 90% of the sequences of EBV DNA. Five cell lines which did not contain EA even after induction had 23, 8, 8, 6, and 2 copies per cell of the EBV genome. The data indicate that there is a correlation between the number of copies of EBV DNA in nonpermissive cells and the ability of these cells to express EA. Three of the five EA and VCA negative nonpermissive cell lines contained DNA homologous to more than 90% of the sequences of EBV DNA. The kinetics of hybridization of the DNA of two other nonpermissive cell lines, Namalwa and SKL, which contain two and eight copies, respectively, of some EBV DNA sequences, suggest but do not prove that these cell lines may contain incomplete viral genomes. The retention of the full complexity of viral DNA in most nonpermissive lymphoblastoid cell lines may be related to the relatively large number of copies of the viral genome in these cells.     

Production of human monoclonal antibodies against Epstein-Barr virus-specific antigens by the virus-immortalized lymphoblastoid cell lines

The possible production of human monoclonal antibodies against Epstein-Barr virus (EBV) was assessed through the EBV immortalization technique. When individual lymphocyte samples from 50 clinical patients and healthy donors were immortalized by EBV, 4 lymphoblastoid lines yielded antibodies to EBV antigens. These positive lines were cloned and each line yielded cultures that secreted monoclonal antibodies against either viral capsid antigen (VCA) or membrane antigen (MA) component. Above all, a clonal line TAKA-SP-8 produced 5 micrograms MA antibody/10(6) cells/ml for more than 12 months. The culture fluid specifically immunoprecipitated a single polypeptide with a size of 93K from both P3HR-1 and B95-8 cell extracts. FUKA-SP-3, on the other hand, secreted 5 micrograms VCA antibody/10(6) cells/ml for at least 8 months. This antibody recognized two polypeptides with sizes of 123K and 120K, from P3HR-1 and B95-8 cell extracts, respectively. When B95-8 and P3HR-1 EBV were treated with the human MA monoclonal, both nuclear antigen (EBNA) synthesis and early antigen (EA) induction were strongly inhibited. All EBV antibody-producing cultures were exclusively achieved from splenic lymphocytes of patients with autoimmune diseases, but not from other donors.     

The seroepidemiology of infection due to Epstein-Barr virus in southern India

We investigated the seroepidemiology of infection due to Epstein-Barr virus (EBV) in 181 south Indian subjects aged 0-25 years using the indirect immunofluorescence method to titrate antibodies to viral capsid antigen (VCA), nuclear antigen (EBNA), and early antigen (EA). The age-specific prevalence of IgG antibodies to VCA rose rapidly to 90% by the age of 5 years. The prevalence of VCA-specific IgM and the geometric mean titre of VCA-specific IgG antibodies were highest between the ages of 6 months and 2 years, the median age of primary infection being 1.4 years. Thus primary EBV infection occurs early in life. EA antibody prevalence was highest (55%) in the third year of life and remained between 30% and 40% thereafter. This pattern of EA antibody prevalence suggests that the latent EBV infection that persists lifelong after primary infection may be reactivated in many individuals. EBNA antibody prevalence was low until the age of 2 years but rose to 80% in the fourth year. Geometric mean titres of antibodies to EA and EBNA were low and stable at all ages. These results are similar to data from areas where EBV-associated Burkitt's lymphoma is endemic and indicate a high EBV infection load early in life.     

Immune responses to Epstein-Barr virus lytic proteins in patients with nasopharyngeal carcinoma

Immune responses to three Epstein-Barr virus (EBV) lytic proteins, DNase, thymidine kinase (TK), and BMRF-1 gene products (50/52 kDa diffused early antigen, EA-D complex) were determined in EBV-infected control individuals and patients with nasopharyngeal carcinoma (NPC). Immunofluorescence assays (IFA) were used to detect their humoral immune responses using recombinant EBV lytic proteins expressed in a baculovirus system as antigens. Cell proliferation assays were performed to evaluate their cellular immune responses by monitoring 3H-thymidine incorporation. Seventy patients with NPC and 32 non-cancer controls were analyzed. The results of IFA showed antibody titers to all three EBV lytic proteins to be higher in the patients with NPC especially for the IgA class. Positivity rates of the three IgA antibodies also were higher in the patients with NPC population. Furthermore, the profiles of the IgA antibodies correlated with those to total early antigens (EA) expressed in the early phase and viral capsid antigen (VCA) expressed in the late phase, of EBV replication. The most interesting finding was that antibody titers to the three EBV lytic proteins were associated significantly with metastases of cervical lymph nodes in patients with NPC. As for cellular immunity to the EA-D complex and DNase, weak responses were observed in the cell proliferation assays. Peripheral blood cells from most individuals could not be stimulated to proliferate, except for a few patients with NPC whose antibody titers against the EA-D complex and DNase also were very high.     

Epstein-Barr nuclear antigen (EBNA) carrying lymphocytes in human palatine tonsils.

The presence of Epstein-Barr virus (EBV) antigens in human palatine tonsilderived lymphocytes (TDL) was investigated using the indirect fluorescent antibody (FA) technique. The TDL were screened for the presence of EBV early antigen (EA), virus capsid antigen (VCA), and EBV nuclear antigen (EBNA). In 76% of the patients diagnosed as recurrent exudative tonsillitis, and in 33% diagnosed as recurrent tonsillitis and/or serous otitis media, EBNA was demonstrated in the purified TDLs. No EA- or VCA-producing cells were found in either the glass adsorbed or TDL cell preparations from all of the patients. These data suggest that in our patient sample, the tonsils may serve as a reservoir for EBV carrying lymphocytes and a basis for recurrent disease.     

Comparison of three different serological techniques for primary diagnosis and monitoring of nasopharyngeal carcinoma in two age groups from Tunisia

Nasopharyngeal carcinoma (NPC) in Tunisia is characterized by its bimodal age distribution involving juvenile patients of 10-24 years and adult patients of 40-60 years. Three serological techniques were compared for primary diagnosis (N = 117) and post-treatment monitoring (N = 21) of NPC patients separated in two age groups. Immunofluorescence assay (IFA) was used as the "gold standard" for detection of IgG and IgA antibodies reactive with Epstein-Barr virus (EBV) early (EA) and viral capsid (VCA) antigens. Results were compared with ELISA measuring IgG and IgA antibody reactivity to defined EBNA1, EA, and VCA antigens. Immunoblot was used to reveal the molecular diversity underlying the anti-EBV IgG and IgA antibody responses. The results indicate that young NPC patients have significantly more restricted anti-EBV IgG and IgA antibody responses with aberrant IgG VCA/EA levels in 78% compared to 91.7% in elder patients. IgA VCA/EA was detected in 50% of young patients versus 89.4% for the elder group (P < 0.001). Immunoblot revealed a reduced overall diversity of EBV antigen recognition for both IgG and IgA in young patients. A good concordance was observed between ELISA and IFA for primary NPC diagnosis with 81-91% overall agreement. Even better agreement (95-100%) was found for antibody changes during follow-up monitoring, showing declining reactivity in patients in remission and increasing reactivity in patients with persistent disease or relapse. ELISA for IgA anti-VCA-p18 and immunoblot proved most sensitive for predicting tumor relapse. VCA-p18 IgA ELISA seems suitable for routine diagnosis and early detection of NPC complication.     

Epstein-Barr Virus in Normal Pregnant Women*

ABSTRACT: Acquired immune suppression accompanying normal pregnancy may be associated with reactivation of Epstein-Barr virus (EBV). Pregnant women with reactivated EBV having anti-EA antibodies show high titers of antiviral capsid antigen (VCA) geometric mean titers (GMT) of 522 versus 170 in those lacking anti-early antigen (EA). Among twenty-seven seropositive women at parturition, 17 (63%) had generated antibody to EA, and all 27 (100%) demonstrated significant increases in antibody to VCA (p < 0.01). In contrast, antibody titers to cytomegalovirus, herpes hominis, varicella-zoster, and rubella viruses in the pregnant women were comparable to those found in nonpregnant controls.     

IgA directed against early antigen of Epstein-Barr virus is no specific marker for the diagnosis of nasopharyngeal carcinoma

The aim of this study was to evaluate the significance and specificity of IgA directed against Epstein-Barr virus (EBV)-specific early antigens (EA) for the unequivocal diagnosis of nasopharyngeal carcinoma (NPC). Therefore, sera from patients with diseases other than NPC, selected on the basis of elevated antibody titres against EBV antigens, were compared to sera from NPC patients with regard to the presence of IgA directed against EBV viral capsid antigen (VCA-IgA) and IgA directed against EA (EA-IgA). Four hundred forty-seven out of 7,508 non-NPC sera tested showed high titres (>512) of IgG directed against Epstein Barr viral capsid antigen (VCA-IgG) and positive VCA-lgA (?32). Two hundred twenty-seven of these sera were compared to 51 VCA-IgA-positive sera from NPC patients regarding the titre of EA-lgA. 60.7% of VCA-lgA-positive NPC sera showed positive EA-lgA, however 33% of VCA-IgA-positive non-NPC patients also exhibited EA-lgA. This result demonstrates that EA-lgA is not specific for NPC and does not allow an unequivocal serological diagnosis of NPC in individual cases. It seems therefore to be of questionable use for screening programs in NPC low-risk areas. The data do not contradict the usefulness of this marker for monitoring of patients treated for NPC and for screening programmes in high-risk areas. © 1994 Wiley-Liss, Inc.     

HLA-DR antigens and the antibody response against Epstein-Barr virus

Antibodies against Epstein-Barr virus (EBV) antigens, i.e. the viral capsid antigen (VCA) and the Epstein-Barr nuclear antigen (EBNA), were determined in two independent populations with known HLA-DR phenotypes. The first population consisted of 151 patients with rheumatoid arthritis; the second one of 88 healthy parents of leukemic children. Although the group of patients with rheumatoid arthritis differed significantly in the frequency of 4 DR antigens from the second group, both groups had the same correlation between HLA-DR antigens and the antibody response to EBV antigens. There was a significant correlation between HLA-DR1 and reduced titers of antibodies to VCA, whereas the persons with only one identifiable DR antigen had higher anti-VCA titers. The persons with HLA-DR5 had significantly higher anti-EBNA titers than those without DR5.     

Epstein-Barr virus serology in bone marrow transplantations: a one-year retrospective study with detection of EBV IgM-VCA-specific antibodies

The specific antibody response to Epstein-Barr virus (EBV) antigens of 41 bone marrow transplant recipients with leukemia or aplastic anemia was examined retrospectively by immunofluorescence test (IF) over 1 year. We observed high titers (greater than 640) of IgG-viral capsid antigen (VCA) with emergence of IgG-early antigen (EA) and frequent absence or low levels of Epstein-Barr nuclear antigen (EBNA) antibodies. After absorption to remove rheumatoid factor (RF), five of the 41 recipients had IgM-VCA antibody to EBV, which appeared between weeks 26 and 48 after BMT and persisted for 1-4 months. No heterophil antibodies were detected in these sera, and none of the five recipients had a history of infectious mononucleosis.     

Evaluation of a multiplex fluorescent microsphere immunoassay for the determination of epstein-barr virus serologic status

Epstein-Barr virus (EBV), a human herpesvirus, affects up to 95% of adults. Diagnosis of acute EBV infection can be challenging and often relies on the serologic antibody pattern to 3 distinct antigens, most often determined by indirect fluorescent antibody (IFA), enzyme-linked immunosorbent assays (ELISAs), and, more recently, multiplex assays. We compared a multiplex assay for the simultaneous detection of antibodies to viral capsid (VCA), nuclear (EBNA), and early (EA) EBV antigens with ELISAs using IFA for discrepancy resolution. Concordance of the multiplex assay was good for all 4 antigens: VCA IgM, 86.6% vs ELISA and 92.9% vs IFA; VCA IgG, 92.8% vs ELISA and 98.0% vs IFA; EBNA IgG, 90.3% vs ELISA and 98.1% vs IFA; and EA IgG, 83.8% vs ELISA and 92.8% vs IFA. After IFA resolution, correlation between the multiplex assay and ELISA for serologic disease stage, based on the antibody profile of all 4 analytes, was 90%. The multiplex assay showed good correlation with an established ELISA and even better correlation with the "gold standard" IFA. Advantages of the multiplex assay over traditional methods include multiple results per assay, inclusion of internal controls for each assay, and well-to-well monitoring of assay drift.