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Antibodies to Epstein-Barr virus (EBV) nuclear antigen family (EBNA) and three of its individual members, EBNA 1, EBNA 2 (A and B) and EBNA 6, were measured by anticomplement immunofluorescence (ACIF) in sera of 75 healthy controls, 13 patients with chronic EBV infection, 38 with non-Hodgkin lymphoma (NHL), 23 with Hodgkin's disease (HD), 105 with nasopharyngeal carcinoma (NPC) and 7 patients with infectious mononucleosis (IM). Their anti-EBV lytic antigens were also measured. We observed that: (1) anti-EBNA 2A and E6 rose in parallel 4-6 weeks after IM, followed by anti-EBNA 1 at 3-6 months, (2) all seropositive individuals had anti-EBNA 1; 74% also had anti-EBNA 2A and E6, (3) anti-EBNA 1 accounted for most of the anti-EBNA reactivity in non-IM sera. Striking disease-associated differences were noted on the humoral responses to the lytic and transformation-associated antigens. Compared to the controls, anti-EBNA 1, -EBNA 2A and -EBNA 6 were simultaneously four to 10 times higher in chronic reactivations, whereas only anti-EBNA 1 was elevated (10 times) in NPC. Individual EBNA titres were normal in NHL or HD patients.

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.

It was shown by double immunofluorescence studies that Epstein-Barr viral nuclear antigen (EBNA) was preserved in EBV-infected cells after they had entered the productive viral cycle, as signalled by the appearance of the early antigen (EA)complex. A nuclear component of the EA comples could be clearly distinguished from EBNA with regard to antigenic specificity.

Using the indirect leukocyte migration inhibition technique T cells have been identified as being responsible for Epstein-Barr virus nuclear antigen-induced specific leukocyte migration inhibitory factor production. The response was dependent on the presence of macrophages or their product, T-lymphocyte activating factor.

After Epstein-Barr virus (EBV) infection in vivo, B-cells with latent virus infection persist indefinitely through life. These cells grow in vitro on explanation and can be established as immortal B-cell lines. To reconcile the unlimited growth potential in vitro with the maintenance of a low proportion of B-cells infected by EBV in vivo, a strict in vivo control mechanism has to be postulated. Certain aspects of this control are apparent when the primary infection is followed by infectious mononucleosis. This is characterized by lymphocytosis and the presence of activated T-cells. The T-cell proliferation is probably the manifestation of the immune response against EBV antigens. However, the reaction of T-cells upon encounter of B-blasts is also likely to contribute to the events. At present, it is difficult to detect an EBV-specific component in the action of the T-cells in the acute phase of mononucleosis exerted on B-cells. However, for the clinical course of the disease the activation of T-cells is important. The activated T-cells may control and also eliminate the B-cells infected by EBV. In addition to the immunity which develops during the disease, th immunoregulatory mechanism is likely to have a role in the inhibition of B-cell proliferation.

Non-Hodgkin lymphoma (NHL) patients with centroblastic (Cb) or centroblastic-centrocytic (Cb/Cc)-diffuse lymphomas, immunocytoma (IC) and chronic lymphocytic leukemia (CLL) in clinical stages III-IV and with active disease (highly malignant group) were compared to NHL patients with CLL, IC, and centrocytic (Cc) or centroblastic-centrocytic (Cb-Cc)-diffuse/follicular lymphomas, in clinical stages I-II and with inactive disease (low malignant group) based on the presence of antibodies to Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) and 2 (EBNA-2). In the highly malignant group, anti-EBNA-1 geometric mean titers (GMT) were 13.2 (range less than 2-80) and anti-EBNA-2 60.6 (range: 20-320). The ratio between the logarithms of anti-EBNA-1 and anti-EBNA-2 antibody titers was less than 1.0 (mean: 0.32) in all the patients examined. In 6 out of 8 patients of the low malignant group, anti-EBNA-1 titers were higher (mean: 30.1; range 10-160) than anti-EBNA-2 titers (mean: 4.3; range less than 2-80) and the EBNA 1/2 ratio was greater than 1.0. In healthy EBV-seropositive individuals, anti-EBNA-1 GMT were 49 (range: 10-320) and only 5 out of 17 individuals had detectable anti-EBNA-2 titers (GMT: 3; range less than 5-20). The EBNA-1/2 ratio was in all cases greater than 1. Among patients of the highly and low malignant groups, patients with follicular-cell-derived lymphomas had elevated antibody titers against the restricted component of early antigens (EA-R), whereas all patients with IC and 2 out of 4 CLL patients had elevated antibody titers against the diffuse component of early antigens (EA-D). The results indicate that the ratio between anti-EBNA-1 and anti-EBNA-2 antibody titers may be of diagnostic importance in patients with immunodeficiencies.

An Italian case of nasopharyngeal carcinoma was studied clinically; the immunologic study showed the correlation with the Epstein-Barr virus specific antigen.