Relation between Epstein-Barr viral and cell membrane immunofluorescence in Burkitt tumor cells. IV. Differentiation between antibodies responsible for membrane and viral immunofluorescence.

Previous reports (1, 2) have established that the expression of certain distinctive membrane antigen(s) on the surface of Burkitt's lymphoma (BL) and infectious mononucleosis (IM) cells is dependent on the presence of Epstein-Barr virus (EBV) in the cell line. The investigations reported here provide evidence that antibodies directed against EBV antigens, as revealed by the immunofluorescence test on acetone-fixed smears (8), and the membrane reactive antibodies, although often present in the same serum, are nevertheless distinctly different. Absorption of Mutua serum, the standard reference serum for demonstrating membrane antigen(s) on BL and IM cells, with BL cells completely removed anti-membrane activity without significantly affecting the anti-EBV antibody titer. Furthermore, sera were found which contained one type of antibody but not the other. Sera with high anti-membrane but low anti-EBV activity were found among relatives of BL patients. These sera reacted with the membranes of EBV-carrying BL and IM cells in essentially the same way, i.e., against the same spectrum of target cells, as the EBV-positive Mutua serum. They were unable to block the membrane reactivity of FITC-conjugated Mutua serum, however. In some cases they showed weak but incomplete blocking. One such EBV-negative, membrane-positive BL relative serum (Robert) was conjugated with FITC and used for direct staining of BL and IM cells. Again, this conjugate reacted against the same target cell spectrum as a Mutua conjugate, but its reactivity was completely blocked by a number of Burkitt patients' sera, although unconjugated Robert serum did not block the Mutua-conjugate. A number of other membrane-positive BL relative sera also failed to block Mutua, but completely blocked the Robert conjugate. A number of Swedish and African control sera and an isoantiserum gave no blocking against Robert conjugate. It therefore appears that the Mutua conjugate contains at least two antibody specificities against the EBV-determined membrane antigens. One, but not the other, is shared with the antibody specificity present in Robert's serum and a number of other sera from relatives of BL patients.

Relation between Epstein-- Barr viral and cell membrane immunofluorescence in Burkitt tumor cells. II. Comparison of cells and sera from patients with Burkitt's lymphoma and infectious mononucleosis.

Blastoid cell cultures derived from leukocytes of patients in the acute stage of infectious mononucleosis (IM) and harboring Epstein-Barr (EB) virus in at least 1% of the cells were found to possess antigens in their membranes which presently are indistinguishable from those detected in Burkitt's lymphoma (BL) cells by the techniques employed. It was noted that, in the course of IM, antibodies are formed which react in indirect immunofluorescence tests with membrane antigens of live cells from Burkitt tumor lines as well as from IM leukocyte cultures, including an autochthonous line in the case of one patient. Sets of sera from IM patients were tested which included a serum collected weeks to years before onset of illness. In the majority of these the pre-IM serum failed to react in membrane immunofluorescence (MIF) tests with any of several Burkitt tumor cell lines employed, but in some cases the presera reacted with cells of some but not others of the lines. Possible explanations for these discrepant results have been discussed. The antibodies involved in the MIF test are evidently distinct from those responsible for the EBV and heterophile reactions. Maximal MIF activity is attained long after the other two antibodies have reached maximal titers and antibodies to EBV and membrane antigens seem to persist for years whereas the heterophile reaction turns negative within a few months.

Relation between Epstein-Barr viral and cell membrane immunofluorescence in Burkitt tumor cells. 3. Comparison of blocking of direct membrane immunofluorescence and anti-EBV reactivities of different sera.

Sera from patients with Burkitt's lymphoma (BL), infectious mononucleosis (IM), carcinoma of the postnasal space (Ca PNS), and various controls were investigated for antibodies against the Epstein-Barr virus (EBV) by immunofluorescence on acetone-fixed smears (5) and for antibodies against the distinctive antigenic sites expressed on the surface of viable lymphoblastoid cells within EBV-carrying culture lines (1). The latter were studied by the blocking of direct membrane staining with FITC-conjugated Mutua serum. This serum has been derived from a Burkitt's lymphoma patient in long-term regression after chemotherapy and is free from detectable isoantibodies. It has been used previously as a standard of reference to demonstrate the presence of the membrane antigen(s) on all lines derived from BL biopsies and leukocytes from IM patients. It was found that 102 of 279 (37%) of the sera tested had high anti-EBV titers (>==80) and high membrane-blocking (Bl > 0.5) activity, 124 of 279 (44%) of the sera were low in both tests, 22 of 279 (8%) had low EBV titers (<==80), in spite of a high blocking index, and 31 of 279 (11%) of the sera were low in blocking activity (<0.5), in spite of a high EBV titer. The two tests thus gave concordant results with 81% and discordant with 19% of the sera. The majority of sera from BL patients were high in both tests. IM sera also showed a relationship between the two antibody activities but, in general, both activities were lower than in BL cases. Ca PNS sera seemed to fall into two main groups: (a) high anti-EBV, high blocking or (b) low anti-EBV, low blocking. Control sera, including four isoantisera, showed predominantly low reactivities in both tests.

Relation between Epstein-Barr viral and cell membrane immunofluorescence of Burkitt tumor cells. I. Dependence of cell membrane immunofluorescence on presence of EB virus.

A comparison was made of the immunofluorescence tests for detection of cell membrane and Epstein-Barr virus antigens in cells from Burkitt tumor biopsies or continuous cultures derived therefrom. On the whole, cell membrane fluorescence in established lines appeared to depend not only upon the presence of EBV but to a considerable degree also upon the extent of the persistent viral infection. There was no constant relationship, however, between the results of the two tests and exceptions to the rule were noted. These observations indicate that different antigens are involved in the two tests. Biopsy cells in general and young cultures may reveal strong MIF activity but few, if any, EBV-positive cells. The reverse, the presence of relatively large numbers of EBV antigen-containing cells in the absence of significant MIF reactions, was also noted on occasion in a few established cultures. The possible interpretations of these findings have been discussed.

Herpes-type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells.

Cultured cells derived from male patients with Burkitt's lymphoma and harboring herpes-type virus particles were lethally irradiated. These irradiated cells induced normal peripheral leukocytes of female infants to grow within 2 to 4 weeks after mixed cultivation. Cells of a line free of this agent failed to stimulate growth. If either type of cell was cultured separately, it did not survive under the experimental conditions. Herpes-type viral antigen and C-group chromosomal marker previously described in cultured Burkitt cells were found in all of the female cell cultures that were obtained.

Differential reactivity of human serums with early antigens induced by Epstein-Barr virus.

Inoculation of 64-10 or Raji cultures with Epstein-Barr virus derived from the HRI-K clone of the P3J Burkitt's lymphoma line caused abortive infections in most of the lymphoblastoid cells with synthesis of "early antigens" but few, if any, capsids. Antibodies to early antigens were detected by indirect immunofluorescence in serums of many patients with infectious mononucleosis, Burkitt's lymphoma, or nasopharyngeal carcinoma. These antibodies were rarely present in other serums even though some of them showed high titers of antibodies to Epstein-Barr virus when assayed on EB3 Burkitt tumor cells; they also prevented synthesis of early antigens, provided the serums were mixed with the virus prior to inoculation. Antibodies to early antigens possibly reflect current or recent disease processes that are associated with the virus.

Elevated levels of antibodies to Epstein-Barr virus antigens in sera and synovial fluids of patients with rheumatoid arthritis.

The frequencies and levels of antibodies to Epstein-Barr virus (EBV)-specific antigens were determined in paired sera and synovial fluids from patients with rheumatoid arthritis (RA) and in sera from patients with other connective tissue diseases; i.e., systemic lupus erythematosus, progressive systemic sclerosis, and osteoarthritis (OA). The specimens were also tested for the presence of antibodies to RA-associated nuclear antigen. Compared to healthy controls, the patients' sera showed increased frequencies of elevated antibody titers (>/=320) to Epstein-Barr viral capsid antigen, a correspondingly enhanced (twofold to threefold) geometric mean titer, and an increased frequency of antibodies at elevated titers (>/=10), usually to the restricted component and rarely the diffuse component of the early antigen complex. Levels of antibody to the EBV-associated nuclear antigen were within the normal range. Enhancement of antibody titers was more pronounced in seropositive RA patients (i.e., positive for rheumatoid factor) than in those who were not. Enhancement was also found in systemic lupus erythematosus and progressive systemic sclerosis. Antibody to RA-associated nuclear antigen was detected at an increased frequency only in the group of seropositive RA patients (90%), as compared to 8-15% in the other connective tissue diseases and 6-8% in healthy controls. The antibody titers in the synovial fluids equaled or were at most twofold higher or lower than those in the sera. In addition, levels of EBV-specific antibodies were studied serially over a period of 6-10 mo in patients with RA and OA. Parameters of disease activity were determined and compared to antibody levels. EBV-specific antibodies in sera of OA patients remained constant and within normal limits throughout the study. Although EBV-specific antibodies were often elevated in RA patients, they also remained constant, with the exception of three patients, who showed gradual increases in one of the four antibodies, which did not correlate with disease activity.

Attachment of antinuclear antibodies to nasopharyngeal carcinoma or other cells during preparation of biopsy imprints.

The presence of Epstein-Barr virus (EBV) genomes in nasopharyngeal and other carcinomas or Burkitt's and other B-cell lymphomas can be established by the demonstration of viral nucleic acid sequences in DNA extracts from biopsy specimens, the detection of EBV-associated nuclear antigen (EBNA) in biopsy imprints, and the inhibition of leukocyte migration by tumor extracts. Of these techniques, the detection of EBNA-positive tumor cells can be performed most readily in the laboratory. This report shows that a patient's antibodies to nuclear antigens can gain access to cell nuclei during the preparation of imprints. If the antibodies are directed against EBNA, nuclear immunofluorescence is elicited solely in the tumor cells when only complement (C') and fluorescein-labeled antibodies to C' are applied to the imprints without prior exposure to anti-EBNA-positive sera. If nonspecific antinuclear antibodies (ANA) are involved, the nuclear immunofluorescence seen in the EBNA-specific and control assays is not limited to the tumor cells but extends to any normal cells that may be present in the imprints. Furthermore, nuclear fluorescence is elicited when solely an anti-human IgG conjugate is applied because ANA is measurable by indirect immunofluorescence, whereas detection of EBNA requires augmentation of the antigen-antibody complexes by C', which differentiates further between EBNA-specific and nonspecific staining. Attachment of antibodies to nuclei can be avoided by minimizing the deposit of blood during imprint preparation and by rapid drying of the imprints. Similar results are obtained experimentally when smears of lymphoblasts are made in the presence of anti-EBNA or ANA.

Burkitt's lymphoma: its clinical course in relation to immunologic reactivities to Epstein-Barr virus and tumor-related antigens.

In 141 patients with African Burkitt's lymphoma, the relationship between Epstein-Barr virus (EBV)-related antibody titers and the clinical course of this disease was presented. Antiviral capsid antigen tests gave positive results in all patients, siblings, and control neighbors; but the geometric mean antibody titers to viral capsid antigen were significantly higher in patients than in siblings or neighbors (P less than 0.001). No control neighbors or siblings had antibodies to restricted (EA-R) or diffuse (EA-D) early antigen. Mean geometric anti-EA-R titers at admssion and at last visit were significantly lower in patients with stage (I and II) than in those with stage (III and IV) disease; this most likely reflected the degree of tumor burden. Patients who relapsed after 1 year of sustained remission had significantly higher anti-EA-R titers than did those who did not. The increase in the probability of relapse was sixfold for those patients with an anti-EA-R titer of greater than 160 after 1 year of sustained remission. Survivors and nonsurvivors differed significantly in the final EA-R and Epstein-Barr virus nuclear antigen (EBNA) titers (P less than 0.05 and P less than 0.001, respectively). Anti-EA-D titers were particularly likely to be positive in patients with multiple relapses. When skin reactivity to an antigen from RAJI cells was compared to EBV-related serologic reactions in the same patient, a significant inverse correlation (P less than 0.001) between skin reactivity and EBNA titers appeared. Pretreatment sera from patients with high EBNA titers did not block skin reactivity to the RAJI antigen.

Relation between neutralization of Epstein-Barr virus and antibodies to cell-membrane antigens-induced by the virus.

The neutralization of Epstein-Barr virus (EBV) infectivity by sera with known antibody activities against EBV-associated antigens was investigated in experimentally infected Raji and RPMI 64-10 cells. Antibodies against EBV-induced cell-membrane antigens were apparently responsible for neutralization of viral infectivity. Antibodies against EBV capsid antigens and EBV-induced early antigens were not involved. The implication of these results on the relationship of the membrane antigens to the infectious virus particle is discussed.

Epstein-Barr virus-associated and other antiviral antibodies during intense BCG administration to patients with Burkitt's lymphoma in remission.

Patients with Burkitt's lymphoma in chemotherapy-induced remission received through dermal scarifications one or two doses per week of approximately 3 X 10(8) living BCG organisms (Pasteur Institute vaccine). This treatment was always followed by usually rapid increases by 1--4 log2 steps in the antibody titers to Epstein-Barr virus (EBV)-associated cell membrane antigens. Titer increases of less than 2.5 log2 steps within the first month after the start of BCG treatment correlated with a significantly elevated frequency of extradural relapse as compared to that seen in patients with larger titer rises. During this time, antibodies to EBV-associated viral capsid antigens and early antigens of D and R specificity, as well as antibodies against herpes simplex, varicella, cytomegalovirus, measles, and respiratory syncytial virus antigens, did not show any consistent or impressive changes.

Epstein-Barr virus-specific serology in immunologically compromised individuals.

Since B-lymphocytes are targets and a continuing habitat of Epstein-Barr virus (EBV) and the cell-mediated immune system becomes secondarily involved, one may anticipate that primary and persistent EBV infections in immunologically compromised individuals take unusual courses. Depending on the immunological defect, the clinical, hematological, and serological responses to primary EBV infections may be more or less pronounced than in immunologically competent patients. Infectious mononucleosis has per se an immunosuppressive effect which may enhance a preexisting immune defect. The persistent latent viral carrier state which regularly ensues after the primary EBV infection may become decontrolled by immunosuppressive diseases or therapy, leading rarely to illnesses referable to the virus but often to increases in the titers of antibodies to viral capsid and early antigens and/or declines in the antibody titer to EBV-associated nuclear antigen. Absence or dysfunction of different leukocyte subpopulations may account for the differential changes in antibody patterns.

Epstein-Barr virus-specific leukocyte migration inhibition reaction with tumor biopsy extracts: correlation with the presence of viral DNA.

The Epstein-Barr virus (EBV)-specific leukocyte migration inhibition (LMI) reaction was used to detect EBV antigens in human tumor biopsies in parallel with nuclei acid hybridization for EBV DNA. None of six EBV DNA-negative tumors gave any significant LMI reaction. Fourteen of 17 EBV DNA-positive tumors gave a significant difference between the migration of leukocytes from EBV-seropositive versus -seronegative donors. One tumor gave a borderline reaction. The two-LMI-negatives in this group had only a marginal EBV DNA content. It is suggested that the EBV-specific LMI test may be useful for detecting EBV genomes in tissue and tumor extracts.

Immunological characterization of Hodgkin's and non-Hodgkin's lymphoma patients with high antibody titers against Epstein-Barr virus-associated antigens.

We have studied nine Hodgkin's lymphoma (HD) and ten non-Hodgkin's lymphoma (NHL) patients with extraordinarily high anti-viral capsid antigen (VCA) titers (greater than 5120). Controls were 13 HD and 23 NHL patients with anti-VCA titers between 40 and 2560. High anti-VCA titers were present in NHL patients at the time of diagnosis or within 16 months, whereas the rise of anti-VCA titers in HD patients appeared to be a late event during the clinical course of the disease (mean time from diagnosis, 68 months). In particular, we have asked whether the exceptionally high anti-Epstein-Barr virus (EBV) titers in some HD and NHL patients can be correlated to some of the EBV-specific and -nonspecific parameters of cell-mediated immunity. The battery of non-EBV-specific immunological tests included the assessment of natural killer cell activity and the analysis of T-lymphocyte subclasses according to surface markers, together with spontaneous and mitogen-induced DNA synthesis and their helper or suppressor activity on PWM-generated immunoglobulin synthesis. Outgrowth inhibition (Ol) and leukocyte migration inhibition were used to assess EBV-specific cell-mediated immunity. The majority of the high-titer HD and NHL patients showed a drastically reduced OKT4:OKT8 ratio in their peripheral lymphocyte population. Low-titer HD and NHL patients showed no such reduction. There was no strict correlation between the number of OKT8-positive cells and suppressor activity in the functional PWM-induced immunoglobulin production test. Part of the high-titer HD patients showed defective cellular responses in the outgrowth inhibition test, directed against the proliferation of EBV-transformed (EBV-determined nuclear antigen-positive) cells. Some of them showed also a deficient leukocyte migration inhibition response to EBV-determined nuclear antigen but, interestingly, not to early antigen-VCA. In the NHL group, only one of the high-titer patients showed a similar defect. None of the low-titer HD and NHL patients showed such defects.

Cell-mediated immune reactions in three patients with malignant lymphoproliferative diseases in remission and abnormally high Epstein-Barr virus antibody titers.

Two patients with Hodgkin's disease in remission and one chronic lymphatic leukemia patient with extraordinarily high anti-Epstein-Barr virus (EBV) (viral capsid antigen) antibody titers (greater than 10,000) were selected to study a spectrum of cell-mediated immune responses, including natural killer, interferon-boosted killer, antibody-dependent lymphocytotoxicity, and T-cell-mediated reactions. The purpose was to compare these reactions in patients with immunosuppression and a high EBV load who can hold their EBV-carrying cells under control with the corresponding reactions in patients with EBV-carrying lymphoproliferative disease. In contrast to the latter group, the three patients of the present study showed a less profound and less general suppression of the immune responses. Multiple effector mechanisms probably safeguard against the proliferation of EBV-transformed B-cells. Clinically manifest EBV-carrying lymphoproliferative disease occurs only in very severe immunodeficiencies effecting multiple effectors.

Cellular immune defects to Epstein-Barr virus-determined antigens in young males.

Three males with the X-linked lymphoproliferative syndrome (XLP) with hypo- or agammaglobulinemia following Epstein-Barr virus (EBV) infection and two males with the chronic mononucleosis syndrome were investigated for immune responses to EBV-determined antigens. Males with XLP showed profound cellular immune defects. Markedly diminished responses of natural killer cell and interferon-activated killer cell activities and impaired leukocyte migration inhibition responses to phytohemagglutinin were determined in patients with XLP. The two patients with chronic mononucleosis showed less severe defects. All patients showed partial or complete impairment of their EBV-specific immune responses as measured by leukocyte migration inhibition. EBV-specific antibodies were markedly diminished against EBV-associated nuclear antigen, early antigen, and viral capsid antigen in males with XLP. In contrast, patients with chronic mononucleosis had elevated antibodies to most EBV-specific antigens. Individuals with life-threatening EBV-induced lymphoproliferative disorders may exhibit multiple defective immune mechanisms against the virus.

Marked hyperbilirubinemia in infectious mononucleosis. Analysis of laboratory data in seven patients.

While mild to moderate hepatic dysfunction is commonly encountered in infectious mononucleosis induced by Epstein-Barr virus (EBV), clinical jaundice with high bilirubin levels (greater than or equal to 6.0 mg/dL [greater than or equal to 103 mumol/L] is only occasionally encountered. In this study, seven patients with primary EBV infections had peak bilirubin levels of 10.2 to 23.0 mg/dL (174 to 393 mumol/L) and, for the most part, presented initial diagnostic problems. Complications included the virus-associated hemophagocytic syndrome and acute respiratory distress syndrome in one patient and transient renal failure in another. The laboratory data suggested that a combination of hemolysis and viral-induced cholestasis was responsible for the intense hyperbilirubinemia in at least five patients. Physicians should be aware that marked hyperbilirubinemia can occur with EBV-induced infectious mononucleosis and, thereby, obviate the need for costly diagnostic laboratory tests and, occasionally, invasive procedures.

Infectious mononucleosis in patients aged 40 to 72 years: report of 27 cases, including 3 without heterophil-antibody responses.

Epstein-Barr-virus (EBV)-induced infectious mononucleosis usually occurs in young adults between the ages of 15 and 30. When it occurs in older individuals, it frequently presents diagnostic problems. This report describes data from 27 such patients aged 40 to 72, all of whom had definitive evidence of a current EBV primary infection. Protracted fever, jaundice, pleural effusion, anemia, or the Guillain-Barré syndrome were dominant clinical findings among these patients. Fourteen patients were hospitalized and numerous diagnostic procedures were performed, including bone-marrow aspirations (8 patients), abdominal CAT scan procedures (4 patients), and liver (2 patients) or lymph-node biopsies (1 patient). Overall, the laboratory data in these patients were similar to those seen in young adults, with the exception of more marked hepatic dysfunction and more prominent antibody responses to the restricted (R) component of the early antigen complex. Particularly difficult were the diagnostic problems encountered in three patients in this study (3/27) who failed to develop heterophil antibodies.

Clinical and laboratory evaluation of cytomegalovirus-induced mononucleosis in previously healthy individuals. Report of 82 cases.

The present report describes the clinical and laboratory profile of 82 previously healthy individuals who developed cytomegalovirus (CMV)-induced mononucleosis. Many of these patients posed initial diagnostic problems and were hospitalized with diagnoses such as fever of undetermined origin, active viral hepatitis, acute leukemia, probable systemic lupus erythematosus, autoimmune hemolytic anemia, and severe pancytopenia. These patients underwent a variety of diagnostic biopsies, including liver biopsies (6) and bone marrow aspirations (9). Four patients had exploratory laparotomies, 1 for a ruptured spleen, and another had a splenectomy following an erroneous initial diagnosis of agnogenic myeloid metaplasia. There was no apparent clinical response to a short course of steroid therapy in 3 of 5 cases and acyclovir in another. The vast majority of these patients demonstrated infectious mononucleosis-type reactive blood smears, negative heterophil antibody studies, mildly or moderately elevated aspartate aminotransferase activity, and evidence for subclinical hemolysis on serial specimens. The peak serum bilirubin levels were above 2.0 mg/dl in only 2 of 71 cases tested, both of the latter patients having significant hemolysis (hemoglobin values 8.6-9.3 g/dl). The CMV-IgM test had a high sensitivity for detection of CMV macroglobulins (positive in 81 of 82 cases). In contrast, complement-fixing antibodies to CMV showed diagnostic four-fold titer changes in only 39/82 cases (47.6%). Despite its great sensitivity, the CMV-IgM test is limited by a one-way crossreaction of acute Epstein-Barr virus (EBV)-IM sera and spurious positive reactions in some sera due to the presence of rheumatoid factors. Based on EBV-specific serologic studies, the 82 patients with CMV-IM could be divided into 4 groups: 3 patients without antibodies to EBV; 2) 69 patients with uncomplicated serologic data indicative of long-past EBV infections; (3) 6 patients with unusual antibody profiles, e.g., anti-D responses; and (4) 5 patients, including 1 originally susceptible to EBV, with apparent dual CMV/EBV infections. At the conclusion of our study, final diagnoses and initial hematologic data were correlated in 750 cases in which CMV macroglobulins were searched for. The vast majority of patients with active CMV infections initially demonstrated either markedly or moderately reactive peripheral blood smears. These data support our impression that diagnostic tests for CMV, as well as for EBV, are seldom indicated in symptomatic previously healthy patients whose blood smears during the acute phase (first several weeks) of their illnesses are either nonreactive or minimally reactive.

Enzyme-linked immunosorbent assay for the detection of Epstein-Barr virus-induced antigens and antibodies.

Two Epstein-Barr virus (EBV)-specific ELISA tests were developed. One, based on the use of crude extracts from virus producer cells highly induced in the presence of Ara C (providing EA + VCA- cells) or in the absence of the drug (providing EA + VCA + cells) is suitable for the detection of antibodies directed against antigen complexes associated with the lytic virus cycle; i.e., EA, VCA and presumably also MA. The second, performed with purified EBNA, can be used for the detection of antibodies to the transformation-associated nuclear antigen. The tests are expected to find application in the dissection of antibody responses of patients to various antigenic subcomponents, the monitoring of EBV-coded antigens during biochemical purification, and the screening of spent media from hybridoma cultures for EBV-specific antibodies.