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.     

An immunoperoxidase assay for the detection of specific IgA antibody in Epstein-Barr virus infections.

A technique using indirect immunoperoxidase antibody was developed for the detection of specific serum IgA antibody to Epstein-Barr virus capsid antigen and early antigen. The IgA technique was compared with an immunofluorescence antibody method. Epstein-Barr virus IgA antibody against viral capsid antigen was detected in all nine patients with Epstein-Barr virus associated undifferentiated nasopharyngeal carcinoma, in 13 (72.2%) of 18 patients with infectious mononucleosis, in 21 (28.3%) of 74 patients with acute lymphoblastic leukaemia, and in six (20%) of 30 patients who had recently had kidney transplants. Epstein-Barr virus IgA antibody against viral capsid antigen was also detected in four (10%) of 40 healthy subjects, but it was not found in any of 20 cord blood samples. Epstein-Barr virus IgA antibody to early antigen was detected in six (66.6%) patients with nasopharyngeal carcinoma and in two (2.7%) patients with acute lymphoblastic leukaemia. The immunoperoxidase assay for Epstein-Barr virus specific IgA was simple, reliable, and rapid and correlated well (r = 0.94) with the immunofluorescence antibody technique.     

Contribution to rapid diagnosis of infectious mononucleosis

By indirect immunofluorescence (IF) technique humoral antibodies to Epstein-Barr virus capsid antigen (EB-VCA) and to cytomegalovirus (CMV) were detected in 47% and 9% of persons with infectious mononucleosis (IM), respectively. In 23% of the patients examined, IgM antibodies to both viruses were detected, while in 8% of them high titres of IgG only were found in the absence of IgM class antibodies to EB-VCA or to CMV. The finding of IgM antibody to EB-VCA was in good correlation with the persisting symptoms of the disease. Discrepancy between the presence of specific IgM and the absence of heterophilic antibodies was observed in some children and in all persons with persistent or recurrent signs of IM. In the latter, specific IgM was found only during exacerbation of the disease, but during remissions IgG antibodies persisted in high levels. Antibodies to Epstein-Barr virus nuclear antigen (EBNA) were detected in all chronically ill persons and antibodies to the R-component of Epstein-Barr virus early antigen (EA) were present in the majority of them.     

Comparison of tests for heterophile antibodies with a test for specific IgM-antibodies to Epstein-Barr virus. Brief report

Seventy serum samples from 54 patients with clinically suspected infectious mononucleosis were examined, using the Paul-Bunnell-Davidsohn test, a rapid slide test and an ELISA for the detection of IgM antibodies against the viral capsid antigen (VCA) of Epstein-Barr virus. The ELISA technique was the most sensitive, detecting IgM in 29 sera, whereas only 19 sera were positive in the Paul-Bunnell-Davidsohn test (titre greater than or equal to 64). However, using a titre of greater than or equal to 32 as a diagnostic level, the number of positive sera was 23, the same as for the rapid slide test. There was a high agreement (91%) between the heterophile antibodies tests and the VCA-IgM ELISA. The Paul-Bunnell-Davidsohn test still holds a place in the serologic diagnosis of infectious mononucleosis, but sera should always be tested for VCA-IgM in cases of heterophile antibodies negative mononucleosis.     

A systematic study of Epstein-Barr virus serologic assays following acute infection.

We determined the presence of IgG and IgM antibody to viral capsid antigen (VCA-IgG, VCA-IgM) and IgG antibody to the Epstein-Barr virus nuclear antigen (EBNA) by indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) during the acute illness and at 1, 2, 6, and 48 months in a prospective population-based case series of 95 persons with an acute illness serologically confirmed as Epstein-Barr virus infection. The acute illness was characterized by the presence of VCA-IgG and VCA-IgM (by ELISA) and by the absence of EBNA in most, but not all, patients. During follow-up, VCA-IgG antibodies remained detectable in all patients, while the proportion with VCA-IgM declined and the number with detectable EBNA antibodies steadily increased. The primary differences between the 2 serologic test methods were the increased persistence of VCA-IgM during follow-up by ELISA and the earlier detection of EBNA by IFA. Clinicians should consider the illness stage and the laboratory technique to appropriately interpret serologic test results in suspected cases of mononucleosis caused by the Epstein-Barr virus.     

The response to Epstein-Barr virus infection in Sj?gren's syndrome

To assess the response to Epstein-Barr virus (EBV) infection in patients with primary Sj?gren's syndrome (SS), the frequency of detection of EBV DNA was studied in salivary gland biopsies and the antibody and idiotypic response to the virus was compared with healthy controls and infectious mononucleosis (IM). Viral DNA, detected by in-situ hybridization, was found in biopsies from two out of 12 patients with SS and six out of 10 controls. IgG, IgA and IgM antibodies to the virus, measured by ELISA using synthetic peptides (early antigen and EBNA-1) and a cloned fusion protein (EBNA-1), were normal in sera from 20 patients with SS, whereas infectious mononucleosis patients showed an increase in IgM antibodies to EBNA-1 and IgG antibodies to early antigen. One similarity between infectious mononucleosis and Sj?gren's syndrome was a significant increase in the germline heavy chain idiotype G6 in both diseases, suggesting activation of similar B-cell subsets. It is possible that this is due to EBV, though the low frequency of EBV DNA in biopsies and the normal levels of EBV antibodies in SS does not lend any evidence that the virus itself is the causative agent.     

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.     

Humoral immune response in Epstein-Barr virus infections. I. Elevated serum concentration of the IgG1 subclass in infectious mononucleosis and nasopharyngeal carcinoma

Using radial immunodiffusion we measured IgG subclass concentrations and studied their distribution in serum samples from patients with infectious mononucleosis (IM) and nasopharyngeal carcinoma (NPC), two Epstein-Barr virus (EBV)-associated diseases, in comparison with two control groups [completely anti-EBV negative persons and subjects carrying antibodies to the viral capsid antigen (VCA)]. Antibody titres to VCA and to the early antigen (EA) were determined by indirect immunofluorescence and revealed characteristic patterns for the respective diagnostic groups. Nephelometric assays served for quantitating total protein, albumin, total IgG, IgA and IgM in all the sera. In the IM and NPC groups the concentration of IgG1 was significantly elevated by more than 50% whereas the other three subclasses remained unchanged as compared with the controls. Correspondingly, we found a significant increase of total IgG in IM and NPC. In IM, the only disease where VCA-specific IgM antibodies have been reported to occur, IgM levels were markedly elevated. Our data suggest that the IgG1 subclass plays an important role in the humoral immune response to EBV-determined antigens and that it is possibly involved in the control of virus infection.     

Hodgkin's disease and Epstein-Barr virus. Altered antibody pattern before diagnosis

In patients with Hodgkin's disease, titers of IgG antibody against viral capsid antigen of Epstein-Barr virus and the prevalence of antibodies against early antigen are higher than expected. To evaluate whether this condition antedates diagnosis, we identified 43 persons with Hodgkin's disease, from whom blood had been drawn and stored for an average of 50.5 months before diagnosis, and 96 controls from the same populations, from whom blood had been drawn at the same time. The relative risks of Hodgkin's disease associated with elevated levels of IgG and IgA antibodies against capsid antigen were 2.6 (90 percent confidence interval, 1.1 to 6.1) and 3.7 (1.4 to 9.3), respectively. For Epstein-Barr nuclear antigen, the relative risk was 4.0 (1.4 to 11.4), and for early antigen D it was 2.6 (1.1 to 6.1). However, the prevalence of IgM antibody against capsid antigen was substantially lower in patients with Hodgkin's disease (0.22 [0.04 to 1.3]). These associations were stronger in serum samples obtained at least three years before diagnosis than in serum samples obtained closer to diagnosis. We conclude that the development of Hodgkin's disease may in some patients be preceded by enhanced activation of Epstein-Barr virus. Whether Epstein-Barr virus has a direct role in the pathogenesis of the disease or is simply a marker for a more fundamental factor affecting the immune control of latent infections is unknown.     

Detection of immunoglobulin M antibody to Epstein-Barr virus by use of an enzyme-labeled antigen.

Immunoglobulin M (IgM) antibodies to Epstein-Barr virus were detected by using microtiter plates coated with anti-mu-chain antiserum and enzyme-labeled Epstein-Barr virus antigen. Optimum conditions for labeling were determined. The addition of unlabeled control antigen to the enzyme-labeled antigen was effective in reducing background reactions. Rheumatoid factor no longer interfered. Blocking of specific IgM antibody by IgG also was no longer observed. Four different methods for the detection of acute Epstein-Barr virus infections were compared. A combination of the enzyme-labeled antigen-IgM test with the detection of antibodies to either Epstein-Barr nuclear antigen or heterophile antigen was highly sensitive and specific. By changing the solid phase, IgA antibodies to Epstein-Barr virus could be detected.     

Immunoglobulins and EB virus antibodies in infectious mononucleosis

Antibodies to EB virus capsid, EB virus complement-fixing antibodies and IgM, IgA and IgG immunoglobulins were estimated in sera from seventy-four patients with infectious mononucleosis, eighty-nine control patients (mostly with infectious diseases) and 232 healthy medical students and nurses. Complement fixing (but not virus capsid) antibodies were much lower in patients tested during active infectious mononucleosis than in patients tested during late convalescence or in controls, a disparity which could form the basis of a diagnostic test. Immunoglobulin levels were higher in patients with active infectious mononucleosis than in controls and, when tested 6 months to 2 yr later, were still higher than those in healthy individuals.     

Evaluation of 12 commercial tests for detection of Epstein-Barr virus-specific and heterophile antibodies

Ten microbiological departments in Norway have participated in a multicenter evaluation of the following commercial tests for detection of Epstein-Barr virus (EBV)-specific and heterophile antibodies: CAPTIA Select viral capsid antigen (VCA)-M/G/EBNA (Centocor Inc.), Enzygnost anti-EBV/immunoglobulin M (IgM) and IgG (Dade Behring), Vironostika EBV VCA IgM/IgG/EBNA enzyme-linked immunosorbent assay (ELISA) (Organon Teknika), SEROFLUOR immunofluorescence assay and EBV Combi-Test (Institute Virion Ltd.), anti-EBV recombinant IgM- and IgG-early antigen/EBNA IgG ELISA (Biotest Diagnostics), EBV IgM/IgG/EBNA ELISA (Gull Laboratories), Paul-Bunnell-Davidsohn test (Sanofi Diagnostics Pasteur), Monosticon Dri-Dot (Organon Teknika), Avitex-IM (Omega Diagnostics Ltd.), Alexon Serascan infectious mononucleosis test (Alexon Biomedical Inc. ), Clearview IM (Unipath Ltd.), and Cards+/-OS Mono (Pacific Biotech, Inc.). The test panel included sera from patients with primary EBV infection, immunocompromised patients with recent cytomegalovirus infection, healthy persons (blood donors), and EBV-seronegative persons. Among the tests for EBV-specific antibodies the sensitivity was good, with only small differences between the different assays. However, there was a greater variation in specificity, which varied between 100% (Enzygnost) and 86% (Biotest). Tests for detection of heterophile antibodies based on purified or selected antigen (Avitex, Alexon, Clearview IM, and Cards+/-OS Mono) were more sensitive than the Paul-Bunnell-Davidsohn and Monosticon tests.     

Serological diagnosis of Epstein-Barr virus infection: Problems and solutions

Serological tests for antibodies specific for Epstein-Barr virus (EBV) antigens are frequently used to define infection status and for the differential diagnosis of other pathogens responsible for mononucleosis syndrome. Using only three parameters [viral capsid antigen (VCA) IgG, VCA IgM and EBV nuclear antigen (EBNA)-1 IgG],it is normally possible to distinguish acute from past infection: the presence of VCA IgM and VCA IgG without EBNA-1 IgG indicates acute infection, whereas the presence of VCA IgG and EBNA-1 IgG without VCA IgM is typical of past infection. However, serological findings may sometimes be difficult to interpret as VCA IgG can be present without VCA IgM or EBNA-1 IgG in cases of acute or past infection, or all the three parameters may be detected simultaneously in the case of recent infection or during the course of reactivation. A profile of isolated EBNA-1 IgG may also create some doubts. In order to interpret these patterns correctly, it is necessary to determine IgG avidity, identify anti-EBV IgG and IgM antibodies by immunoblotting, and look for heterophile antibodies, anti-EA (D) antibodies or viral genome using molecular biology methods. These tests make it possible to define the status of the infection and solve any problems that may arise in routine laboratory practice.     

Evaluation of 12 Commercial Tests for Detection of Epstein-Barr Virus-Specific and Heterophile Antibodies

Ten microbiological departments in Norway have participated in a multicenter evaluation of the following commercial tests for detection of Epstein-Barr virus (EBV)-specific and heterophile antibodies: CAPTIA Select viral capsid antigen (VCA)-M/G/EBNA (Centocor Inc.), Enzygnost anti-EBV/immunoglobulin M (IgM) and IgG (Dade Behring), Vironostika EBV VCA IgM/IgG/EBNA enzyme-linked immunosorbent assay (ELISA) (Organon Teknika), SEROFLUOR immunofluorescence assay and EBV Combi-Test (Institute Virion Ltd.), anti-EBV recombinant IgM- and IgG-early antigen/EBNA IgG ELISA (Biotest Diagnostics), EBV IgM/IgG/EBNA ELISA (Gull Laboratories), Paul-Bunnell-Davidsohn test (Sanofi Diagnostics Pasteur), Monosticon Dri-Dot (Organon Teknika), Avitex-IM (Omega Diagnostics Ltd.), Alexon Serascan infectious mononucleosis test (Alexon Biomedical Inc.), Clearview IM (Unipath Ltd.), and Cards±OS Mono (Pacific Biotech, Inc.). The test panel included sera from patients with primary EBV infection, immunocompromised patients with recent cytomegalovirus infection, healthy persons (blood donors), and EBV-seronegative persons. Among the tests for EBV-specific antibodies the sensitivity was good, with only small differences between the different assays. However, there was a greater variation in specificity, which varied between 100% (Enzygnost) and 86% (Biotest). Tests for detection of heterophile antibodies based on purified or selected antigen (Avitex, Alexon, Clearview IM, and Cards±OS Mono) were more sensitive than the Paul-Bunnell-Davidsohn and Monosticon tests.     

Performance of the Architect EBV Antibody Panel for Determination of Epstein-Barr Virus Infection Stage in Immunocompetent Adolescents and Young Adults with Clinical Suspicion of Infectious Mononucleosis

The Architect EBV antibody panel is a new chemiluminescence immunoassay system used to determine the stage of Epstein-Barr virus (EBV) infection based on the detection of IgM and IgG antibodies to viral capsid antigen (VCA) and IgG antibodies against Epstein-Barr nuclear antigen 1 (EBNA-1). We evaluated its diagnostic accuracy in immunocompetent adolescents and young adults with clinical suspicion of infectious mononucleosis (IM) using the RecomLine EBV IgM and IgG immunoblots as the reference standard. In addition, the use of the antibody panel in a sequential testing algorithm based on initial EBNA-1 IgG analysis was assessed for cost-effectiveness. Finally, we investigated the degree of cross-reactivity of the VCA IgM marker during other primary viral infections that may present with an EBV IM-like picture. High sensitivity (98.3% [95% confidence interval {CI}, 90.7 to 99.7%]) and specificity (94.2% [95% CI, 87.9 to 97.8%]) were found after testing 162 precharacterized archived serum samples. There was perfect agreement between the use of the antibody panel in sequential and parallel testing algorithms, but substantial cost savings (23%) were obtained with the sequential strategy. A high rate of reactive VCA IgM results was found in primary cytomegalovirus (CMV) infections (60.7%). In summary, the Architect EBV antibody panel performs satisfactorily in the investigation of EBV IM in immunocompetent adolescents and young adults, and the application of an EBNA-1 IgG-based sequential testing algorithm is cost-effective in this diagnostic setting. Concomitant testing for CMV is strongly recommended to aid in the interpretation of EBV serological patterns.     

Expression of a germline human kappa chain-associated cross-reactive idiotype after in vitro and in vivo infection with Epstein-Barr virus

The mouse monoclonal antibody 17.109 recognizes a cross-reactive idiotype (CRI) associated with kappa IIIb light chains of human IgM-rheumatoid factor (RF) paraproteins. The 17.109 idiotypic determinant is encoded by one or a group of closely related V kappa genes. The association of the idiotype with IgM- and IgA-rheumatoid factors in certain autoimmune diseases necessitates an understanding of how human B lymphocytes can be induced to express the idiotype. To investigate the cellular expression of the 17.109 CRI, peripheral blood lymphocytes from normal donors were stimulated in vitro with Epstein-Barr virus (EBV) and pokeweed mitogen (PWM). EBV induced greater expression of IgM-associated 17.109 CRI than did PWM. The 17.109 CRI was preferentially associated with IgM rather than with IgG. In vivo EBV infection was studied in college students with infectious mononucleosis and displayed similar elevation of IgM-associated 17.109 CRI in sera obtained at presentation of clinical illness. Later, IgM levels declined while IgG-associated 17.109 CRI rose. The 17.109 idiotype was unrelated to antibodies against the Epstein-Barr virus nuclear antigen and the viral capsid antigen and was probably due to generalized activation of early B cells. These observations support the hypothesis that the 17.109 CRI is expressed by in vitro and in vivo EBV-infected cells. The 17.109 idiotype identifies a highly conserved V kappa gene product, which is expressed preferentially after EBV infection, but not exclusively with RF autoantibodies.     

Incidence of anti-intermediate filament antibody in serum samples of students with suspected glandular fever.

Serum samples from 40 students with suspected infectious mononucleosis were tested for the presence of antibodies to intermediate filaments (AIFA) of the cytoskeleton. Twenty had antibodies to the Epstein-Barr virus capsid antigen before their illness, and during it their sera remained negative by the Paul-Bunnell test. The other 20 patients did not have antibodies to the Epstein-Barr virus capsid antigen before their illness and seroconverted during the illness. These patients (true infectious mononucleosis group) developed positive Paul-Bunnell tests. Sera from normal subjects (blood donors) were also tested for AIFA. AIFA was present in titres greater than 1/10 in 80% of the infectious mononucleosis group (mean titre 1/40-1/80), 10% of the Paul-Bunnell negative glandular fever group, and 8.5% of the normal blood donors.     

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.     

Anti-human cytomegalovirus nuclear antigen antibodies of different immunoglobulin classes

IgG, IgM and IgA immunoglobulin classes of antibodies to human cytomegalovirus nuclear antigens (CMNA) were studied by the acid-fixed nuclear binding technique (AFNB) and combined anti-complement immunofluorescence (combined ACIF). In acute cases of infectious mononucleosis (IM) of human cytomegalovirus (HCMV) origin and in the so-called double virus infections (HCMV + Epstein-Barr virus), anti-CMNA IgM antibodies were detected. They were absent from both anti-HCMV positive sera of healthy donors and sera of patients suffering of IM caused by EBV used as controls. The presence of anti-CMNA IgM may thus serve as an additional evidence of acute HCMV infection. Non-complement-fixing IgA classes of the anti-CMNA antibodies were not found in some of the sera gathered during the acute phase of IM of EBV origin: in one fourth of the HCMV seropositive donors and in a number of late serum samples. But non-complement-fixing and complement-fixing anti-CMNA components of the IgG class were detected.     

Epstein-Barr virus infections in childhood.

IgM and IgG antibodies against Epstein-Barr virus (EBV) capsid antigen and antibodies against EBV nuclear antigen and heterophil antibodies were investigated in 115 paired sera of children with acute infections and in 100 sera of healthy controls of the same age and sex. EBV-specific IgM antibodies could be recognized in 13.7% of the patients and in 7% of the controls. Antibodies against EBV nuclear antigen were not detected in the IgM-positive sera.