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.     

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.     

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.     

An enzyme-linked immunosorbent assay for IgG antibodies to human herpes virus 6

An indirect enzyme-linked immunosorbent assay (ELISA) with human herpes virus 6 (HHV6) membrane antigen was compared with indirect immunofluorescence assay (IFA) for measurement of HHV6 IgG antibodies. Five hundred serum samples from 403 Swedish patients with suspected symptomatic Epstein-Barr virus (EBV) infections were examined. The specificity of the ELISA compared with IFA was 98.7% and the sensitivity was 98.4%. In 90% of the patients, IgG antibodies to HHV6 were detected with both assays. The highest HHV6 IgG titers were found mainly in patients with EBV or CMV infections, but HHV6 mononucleosis was not diagnosed. The same HHV6 antigen was assessed for IgM ELISA but was found to be of limited value due to high IgM reactivity with the control antigen. The HHV6 IgM ELISA requires further investigation. The IgG ELISA described is a reliable alternative to IFA for measurement of HHV6 IgG antibodies and for large scale epidemiological studies.     

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.     

Appearance and some unusual features of Epstein-Barr virus antibody production in infectious mononucleosis and other health disorders.

The indirect immunofluorescence (IF) test and the complement-fixation reaction (CFR) were used in examination of over 1500 sera obtained from patients with infectious mononucleosis (IM) and other health disorders. The evidence obtained supports a direct aetiological relationship between Epstein-Barr virus (EBV) and IM and points on a relationship of EBV to some other lymphadenopathies and health disorders. The incidence of the IgG type antibody against virus capsid antigen (EB-VCA) and soluble antigen (CF-SA) obtained from EBV genome-positive cells among different age groups of patients is described along with results of long-term examinations of serum samples from IM patients. The appearance and dynamics of production of both types of EBV antibody and their persistence in the organism varied. Long-lasting oscillations, in particular of the EB-VCA antibody levels were found in sera of patients with prolonged health disorders following IM. The diagnosis value of the IF test and the CFR is discussed.     

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.     

Virological and serological diagnosis of cytomegalovirus infection in bone marrow allograft recipients

To detect cytomegalovirus (CMV) infections, a total of 1,074 cultures of urine, saliva, or blood were collected weekly from 43 consecutive patients undergoing allogeneic bone marrow transplantation. Twenty-three patients were seronegative before transplant and primary infection occurred in 2 (9%). Twenty patients were initially seropositive and recurrent infections occurred in 5 (25%). Three patients in the recurrent group had proven CMV pneumonitis; viraemia was detected in two recipients, while the third had CMV isolated only from bronchial lavage fluid. The serological response of the 43 patients was defined by testing 559 serial sera for specific IgG and IgM antibodies by radioimmunoassay. Passive acquisition of IgG antibodies from blood products was found in 78% of initially seronegative recipients. One patient with primary infection responded in a pattern typical of immunocompetent individuals with long-term production of specific IgG and transient production of specific IgM antibodies. The second patient also had a typical response, but this was delayed until several weeks after the start of virus excretion. In patients with recurrent infections, specific IgM production did not correlate with episodes of virus excretion. Three of five such patients failed to mount a specific IgM response, and these were the only patients in the study to develop CMV pneumonitis. We conclude that CMV infection in bone marrow recipients can only be diagnosed by detection of virus; therefore, the ability of these patients to mount humoral immune responses should not be relied upon for diagnostic purposes.     

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.     

Epstein-Barr virus-specific serum immunoglobulin A as an acute-phase antibody in infectious mononucleosis.

Immunoglobulin A (IgA) antibodies to Epstein-Barr virus viral capsid antigen were assayed serially in 19 patients with infectious mononucleosis and in 38 controls. Seventy-four percent of infectious mononucleosis patients demonstrated IgA antibody, whereas this was found in 13% of controls. This antibody appeared early in infectious mononucleosis and was virtually gone 10 weeks after onset. Comparison of IgA antibody kinetics was made with IgG and IgM antibodies to viral capsid antigen, heterophile antibody, and antibody to Epstein-Barr virus early antigen and nuclear antigen. Failure to demonstrate IgA antibody was associated with severe illness, prolonged illness, delay in IgG and anti-Epstein-Barr virus nuclear antigen antibody, and low or absent heterophile and anti-early antigen antibody. Assay of IgA antibody to viral capsid antigen is a potentially useful adjunct in the serodiagnosis of infectious mononucleosis or recent Epstein-Barr virus infection, as are the other antibodies tested, but in this study IgM viral capsid antigen antibody was the only acute-phase antibody present in all patients.     

Serodiagnosis of infectious mononucleosis by using recombinant Epstein-Barr virus antigens and enzyme-linked immunosorbent assay technology.

Four recombinant, diagnostically useful Epstein-Barr virus (EBV) proteins representative of the viral capsid antigen (p150), diffuse early antigen (p54), the major DNA-binding protein (p138), and the EBV nuclear antigen (p72) (W. Hinderer, H. Nebel-Schickel, H.H. Sonneborn, M. Motz, R. Kühbeck, and H. Wolf, J. Exp. Clin. Cancer Res. 7[Suppl.]:132, 1988) were used to set up individual enzyme-linked immunosorbent assays (ELISAs) for the qualitative and quantitative detection of immunoglobulin M (IgM) and IgG antibodies. In direct comparison with results obtained by standard immunofluorescence or immunoperoxidase assays, it was then shown that the recombinant EBV ELISAs provide the means for specific and sensitive serodiagnosis of infectious mononucleosis (IM) caused by EBV. The most useful markers in sera from such patients proved to be IgM antibodies against p54, p138, and p150. Additional positive markers for recent or ongoing IM apparently were IgG antibodies against p54 and p138. In contrast, anti-p72 IgG had a high preference for sera from healthy blood donors and, therefore, can be considered indicative of past exposure to the virus. Altogether, the individual ELISAs proved to be as specific and at least as sensitive for the diagnosis of IM as the currently available standard techniques are. Moreover, our findings suggest that, by combining individual test antigens, a workable ELISA system consisting of three assays (IgM against p54, p138, and p150; IgG against p54 and p138; and IgG against p72) can be established for the standardized rapid diagnosis of acute EBV infections.     

Human cytomegalovirus specific IgA antibodies detected by immunoperoxidase assay in serum of patients with cytomegalovirus infections

Cytomegalovirus (CMV)-specific IgA antibodies were determined by an immunoperoxidase assay in sequential serum samples of 10 patients with CMV infection in order to evaluate the feasibility of the use of this technique for diagnosis. In parallel, IgM and IgG antibodies to CMV were studied by enzyme-linked immunosorbent assay (ELISA) and by the immunoperoxidase assay, respectively. CMV IgA antibodies were detected in all 10 CMV patients studied. Specific IgM was detected earlier than IgA in only one of these ten patients. No CMV-specific IgA antibodies (titer less than 2) were detected in 45 medical students. Neither were they found in paired sera of 5 patients with herpes simplex infection, 5 patients with varicella, 6 patients with zoster and 2 patients with Epstein-Barr virus infection. The potential application of the indirect immunoperoxidase IgA assay for serodiagnosis of CMV infections is discussed.     

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.     

Enhancement of Epstein-Barr virus antibody production in systemic lupus erythematosus patients.

Two groups of 22 patients suffering from either systemic lupus erythematosus (SLE) or infectious mononucleosis (IM) were checked for Epstein-Barr virus capsid antigen antibody (EB-VCA) production. The average significant antibody levels as well as the frequency of their occurrence were clearly higher in SLE than in IM patients.     

Infection and persistence of varicella-zoster virus in lymphoblastoid Raji cell line

Infection of Raji cells by varicella-zoster virus (VZV) resulted in permissive infection with establishment of a persistently infected lymphoblastoid cell line. VZV antigens of the membrane and nuclear type, as detected by the indirect immunofluorescence membrane antigen (IFAMA) and anticomplement immunofluorescence (ACIF) tests, were observed. Minute amounts of infectious virus were detected by co-cultivation of VZV-infected Raji cells (Raji-VZV), with permissive human embryo fibroblasts (HEF). The virus isolated was found to be similar to the parent strain. Transient induction of Epstein-Barr viral capsid antigen (EB-VCA) was also observed. The persistently infected Raji-VZV cell line, when free of EB-VCA, was found suitable for measuring antibodies to varicella-zoster virus. The possible interaction in the infected Raji cells between EBV, which is implicated in human malignancy, and VZV which belongs also to the herpes group of viruses, is discussed.     

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.     

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.     

Antibodies against membrane antigens of cytomegalovirus infected cells in sera of patients with a cytomegalovirus infection

The appearance of a new virus specific antigen was demonstrated by an indirect immunofluorescence technique on cell surfaces of CMV infected human fibroblasts, 48–72 hr after inoculation. The development of antibodies to these membrane antigens was followed in thirty-nine serial sera from twelve patients with a virologically and/or serologically confirmed CMV infection. In all patients except one, membrane antibodies could be detected. Sera from four patients collected before infection were negative, as were sera taken 1–13 days after onset of symptoms. From the end of the second week of illness CMV-membrane antibodies as well as CMV macroglobulin antibodies to intracellular antigens were detectable. The membrane antibodies persisted until 335 days after onset of illness. They were mainly of the IgM class. Most positive sera also reacted with non-infected fibroblasts but to a lesser degree. This reaction became negative after prior absorption of the sera with non-infected fibroblasts, whereas the reaction with CMV-infected cells remained positive.Controls consisted of forty-five sera from healthy persons and nine sera from patients suffering from other herpes virus infections with antibodies to herpes simplex, zoster/varicella and Epstein-Barr virus. All but two sera were negative in the membrane immunofluorescence test.     

Antibodies to Epstein-Barr virus and cytomegalovirus in relation to CD4 cell number in human immunodeficiency virus 1 infection.

Interaction between herpesviruses and human immunodeficiency virus (HIV)1 is postulated in the progression of HIV disease. In order to evaluate the specific antibody responses directed to Epstein-Barr virus (EBV) and cytomegalovirus (CMV) and to provide serological evidence suggesting reactivation of these viruses able to accelerate the immunodeficiency, we studied IgA and IgG titres to EBV and CMV in the serum of HIV positive patients in relation to the CD4 cell number. The titres of IgG antibodies to EBV and the prevalence of IgG to CMV were significantly higher in HIV positive patients compared to control high risk HIV negative subjects. In HIV infected patients, anti-VCA IgG antibodies increased and anti-EBNA IgG antibodies decreased progressively in relation to the decline of CD4 cell number whereas anti-CMV IgG antibodies did not varied significantly at the same time. Anti-VCA IgA and anti-EA IgG antibodies were found uncommonly and with low titres. IgA antibodies to EA and CMV were not detected in any patient. The variations in EBV antibody response that we describe in HIV infection were previously reported in other immunodeficiency states and could be distinctive of these diseases.