Gary R. Pearson

Persisting Illness and Fatigue in Adults with Evidence of Epstein-Barr Virus Infection

Clinical, serologic, virologic, and immunologic evaluations for 31 adults with chronic illness and fatigue suggested that 23 had persisting Epstein-Barr virus infection. Among these 23 patients, cellular immune mechanisms were generally normal, but 4 had mild immunoglobulin deficiencies. However, 20 patients had abnormal serologic profiles specific for Epstein-Barr virus shown by significantly elevated titers of antibodies to the viral capsid antigen or early antigen, or by a deficiency of late-appearing antibodies. In 11 of 15 patients tested, circulating immune complexes were found. Circulating interferon was not found in 18 patients tested, but the activity of 2-5 oligoadenylate synthetase, an interferon-induced enzyme, was increased in 5 patients studied. Of 19 patients, 18 had persisting suppressor T-cell activity typically found in patients recovering from acute infectious mononucleosis. We believe that the Epstein-Barr virus may be associated with chronic illness in adults.

Stress-related activation of Epstein-Barr virus

Herpesviruses characteristically persist in a latent state in the body over the lifetime of an individual. Under certain conditions, any one of the herpesviruses can be reactivated. The mechanisms underlying the establishment of latent virus infection or viral reactivation are not well understood; however, it is known that the cellular immune response plays a very important role in the maintenance of latency and in virus reactivation. One of the factors thought to be associated with the reactivation of latent herpes-viruses is psychological stress. Using an examination stress model with medical student subjects, we previously demonstrated the reactivation of latent Epstein-Barr virus (EBV), as measured by increases in antibody titers. In this follow-up study using the same group of medical students, we found evidence for incomplete reactivation of latent EBV, with only selective expression of the latent virus genome.

Identification of an Epstein-Barr virus early gene encoding a second component of the restricted early antigen complex

When the latent Epstein-Barr virus (EBV) genome in B95-8 cells is induced into a replicative phase, two abundant early RNAs are transcribed rightward from the EBV BamHI H DNA fragment into BamHI F. Analysis of cDNA clones prepared from the RNA of cells replicating EBV revealed that both RNAs contain the BHRF1 open reading frame. Part of BHRF1, cloned into a prokaryotic fusion protein expression vector, expressed a fusion protein in Escherichia coli and the purified fusion protein was used to generate a monoclonal antibody against BHRF1. This antibody was then employed to characterize the protein encoded by BHRF1 in cells replicating EBV. The monoclonal antibody reacted with a 17-kDa protein component of the restricted early antigen (EA) complex. The distribution of the protein in cells was similar to that noted when sera from patients with African Burkitts lymphoma were used to stain these cells. The protein was synthesized before the major 47-56 kDa protein associated with the diffuse component of EA in superinfected Raji cells. All human sera containing antibodies to EA as determined by immunofluorescence (IF) reacted with the protein as did some sera determined to be anti-VCA positive and anti-EA negative by IF. The predicted amino acid sequence of the protein has characteristics which suggest that it is a membrane protein. It also has significant homology with both the anchor region of polyoma middle T antigen and with the predicted protein product of the bcl-2 mRNA activated by the 14/18 chromosome translocation characteristic of follicular lymphomas. This latter homology is extensive and colinear, suggesting common evolution and function. However, neither a mRNA which could efficiently translate the BHRF1 protein nor the BHRF1 protein could be detected in latently infected cells. Thus, the bcl-2 predicted protein is similar to an EBV protein synthesized in the early phase of virus infection.

Epstein-Barr virus-induced diseases in boys with the X-linked lymphoproliferative syndrome (XLP): Update on studies of the registry

Analyses of 100 subjects with the X-linked lymphoproliferative syndrome (XLP) in 25 kindreds revealed four major interrelated phenotypes: infectious mononucleosis, malignant B-cell lymphoma, aplastic anemia, and hypogammaglobulinemia. Eighty-one of the patients died. Two male subjects were asymptomatic but showed immunodeficiency to Epstein-Barr virus (EBV). Seventy-five subjects had the infectious mononucleosis phenotype and concurrently, 17 subjects of this group had aplastic anemia. All subjects with aplastic anemia died within a week. Aplastic anemia did not accompany hypogammaglobulinemia or malignant lymphoma phenotypes. Hypogammaglobulinemia had been detected before infectious mononucleosis in three subjects, after infectious mononucleosis in five subjects, and was not associated with infectious mononucleosis in 11 boys with hypogammaglobulinemia. In nine subjects infectious mononucleosis appeared to have evolved into malignant lymphoma; however, the majority of patients with malignant lymphoma showed no obvious antecedent infectious mononucleosis. One subject had infectious mononucleosis following recurrent malignant lymphoma. Twenty-six of 35 lymphomas were in the terminal ileum. Results of immunologic and virologic studies of 15 survivors revealed combined variable immunodeficiency and deficient antibody responses to EBV-specific antigens. Mothers of boys with XLP exhibited abnormally elevated titers of antibodies of EBV. Subjects of both sexes with phenotypes of XLP should be investigated for immunodeficiency to EBV. Persons with inherited or acquired immunodeficiency may be vulnerable to life-threatening EBV-induced diseases.

Stress and the memory T-cell response to the Epstein-Barr virus in healthy medical students.

This study investigated the memory T-cell proliferative response to several early and late Epstein-Barr virus (EBV) polypeptides. Blood samples were collected twice, 1 month before a 3-day block of examinations and again on the last day of the exam series. Ss were 25 healthy, EBV seropositive medical students. The proliferative response to 5 of the 6 EBV polypeptides significantly decreased during examinations. In addition, Ss high (above the median) in seeking support, as measured by the COPE, had lower proliferative responses to 3 EBV polypeptides (p17, p52/50, and p85), as well as higher levels of antibody to EBV virus capsid antigen. The data provide further evidence that psychological stress can modulate the cellular immune response to latent EBV.

Monoclonal Antibody for Rapid Laboratory Detection of Cytomegalovirus Infections: Characterization and Diagnostic Application

Monoclonal antibodies to early (2H2.4, molecular weight 72,000 daltons) and late (2F3.0, molecular weight 68,000 daltons) antigens of the AD-169 strain of cytomegalovirus (CMV) were prepared by fusing mouse spleen cells with NS-1 mouse myeloma cells. The 2H2.4 monoclonal antibody produced a dense immunofluorescence with prominent lobular staining within the nucleus of CMV-infected substrate cells, whereas the reaction of 2F3.0 was more diffuse and generally involved the entire nucleus of the cells. Both monoclonal antibodies had little or no neutralizing activity against CMV in plaque-reduction assays. No cross-reactions were observed between these monoclonal antibodies and other members of the herpesvirus group. The 2H2.4 monoclonal antibody to early CMV antigen was used in a shell vial assay with a low-speed centrifugation step for the rapid (within 16 hours after inoculation) diagnosis of CMV infections. Optimal conditions for the test included centrifugation of shell vials at 700 X g for 45 minutes at 36 degrees C. An inoculum volume of 0.2 ml provided a reasonable balance between the optimal sensitivity for detecting specific viral fluorescence and the easy discrimination of the specific immunofluorescence from the background debris. Because of the commercial availability of the monoclonal antibody and the simplicity of the procedures used in the shell vial assay and subsequent fluorescence techniques, this rapid assay can be done in any laboratory that is familiar with cell culture manipulations.

Human Herpesvirus-6 (HHV-6) infection in multiple sclerosis: a preliminary report

We examined cerebra! spinal fluid (CSF) from multiple sclerosis (MS) patients and patients with other neurological diseases (OND) for antibody specific for Human Herpesvirus-6 (HHV-6) and for HHV-6 DNA detectable by PCR. CSF from MS patients had a higher frequency of IgG antibody to HHV-6 late antigens (39.4%) compared with CSF from OND (7.4%). In contrast, the frequency of detectable IgG antibody in CSF from MS patients specific for Epstein-Barr Virus (EBV) (12.1%) and Human Cytomegalovirus (HCMV) (6.1%) was much lower. Two of 12 MS CSFs (16.7%) also contained HHV-6 DNA detected by PCR. None of four OND CSF were positive for HHV-6 DNA. Plasma from 16 patients with MS, eight with OND and 72 healthy donors were tested for antibodies by ELISA to HHV-6 early (p41/38) and late (gp 110) proteins. Although no differences in ant-gp 110 IgG antibody were detected between MS patients, patients with other neurological diseases, and normals, IgG antibody to early protein p41/38 was detected in > 68% of the plasma from MS patients, 12.5% from OND patient and 27.8% of the controls. IgM antibody to p41/38 was present in >56% of MS patients, 12.5% of OND patients, and 19% of controls. These data suggest that more than half of the MS patients had active, ongoing HHV-6 infections. HHV-6 was also isolated from peripheral blood mononuclear cells (PBMC) from 3/5 MS patients who were in relapse or had progressive disease and was identified as HHV-6 Variant B. These preliminary results support the hypothesis that HHV-6 may be a co-factor in the pathogenesis of some cases of MS.

In Vitro and in Vivo Investigations on Antibody-Dependent Cellular Cytotoxicity

It is now well established that target cell destruction can be mediated through the interaction of antibody with normal lymphoid cells as well as through the classic T-cell immunity mechanism (Cerottini and Brunner, 1974). This phenomenon, recently reviewed by Perlmann, Perlmann, and Wigzell (1972), was first described by Moller in 1965 and has received a number of designations, including antibody-dependent cell-mediated (or cellular) cytotoxicity (ADCC), antibody-dependent lymphocyte cytotoxicity (ADLC), cell dependent antibody-mediated cytotoxicity (CDAMC), and lymphocyte-dependent antibody (LDA). For the purpose of this review, this immunologic reaction will be referred to as ADCC.

Rheumatoid arthritis synovial membrane contains a 62,000-molecular-weight protein that shares an antigenic epitope with the Epstein-Barr virus-encoded associated nuclear antigen.

A monoclonal antibody, selected for reactivity with the Epstein-Barr virus (EBV)-encoded antigen EBNA-1, exhibited strong reactivity with the synovial lining cells in joint biopsies from 10 of 12 patients with rheumatoid arthritis (RA) and adherent cells eluted from these tissues. No staining of RA synovial membrane frozen tissue sections or eluted synovial-lining cells was obtained with monoclonal antibodies directed against other EBV-encoded antigens (anti-p160, anti-gp200/350) or with monoclonal antibodies directed against antigens encoded by cytomegalovirus, herpes simplex viruses, or human T cell leukemia virus type I. Among 12 osteoarthritis and normal synovial biopsies only rare reactive cells were noted. Characterization of the antigen(s) in RA synovium by the Western immunoblotting technique revealed a 62,000-molecular-weight (mol-wt) protein, in contrast to the 70,000-85,000-mol-wt EBNA-1 antigen found in EBV-transformed cells. The structural basis for the cross-reactivity of the RA synovial membrane 62,000-mol-wt protein and the EBNA-1 antigen appears to reside in the glycine-alanine rich region of these molecules. A rabbit antibody directed against a synthetic peptide (IR3-VI-2) derived from the glycine-alanine-rich region of EBNA-1 reacted with the 70,000-85,000-mol-wt EBNA-1 antigen in EBV-infected cells and with the 62,000-mol-wt molecule in RA synovial membrane extracts. Since strong antibody responses to EBNA-1 are known to exist in RA patients, these results suggest that immune responses to a cross-reactive antigen may play a role in the pathogenesis of RA.

Application of Epstein-Barr Virus Serology to the Diagnosis and Staging of North American Patients with Nasopharyngeal Carcinoma

From 1978 to 1981, 151 patients with nasopharyngeal carcinoma (NPC) were enrolled in a prospective, collaborative study of North American patients, most of them white. Thirty-seven had World Health Organization (WHO) type 1 tumors, and 114 had WHO types 2 and 3 tumors. The anti-Epstein-Barr virus (EBV) profile of elevated antibody titers directed against viral capsid antigen and early antigen was seen in 85% of the patients with WHO types 2 and 3 tumors but in only 16% of the patients with WHO type 1 tumors. Geometric mean titers tended to be higher in higher stages of the disease in several staging systems. Low antibody-dependent cellular cytotoxicity at diagnosis appears to reflect a poorer prognosis, and the determination of antibody titers by this assay may prove to be useful for identifying persons in whom recurrent disease is likely to develop after conventional therapy. Anti-EBV titers can aid in diagnosis and treatment planning in patients with NPC, particularly those with occult primary NPC.