The action of DNA antagonists on Epstein-Barr virus (EBV)-associated early antigen (EA) in Burkitt lymphoma lines

The effect of two DNA antagonists (IUDR and Ara C) on EBV-associated antigens was studied in two BL-derived carrier culture lines (P3HR-1 and Onesmas). Both drugs led to an accumulation of the early antigen (EA) from 2% in the untreated to 5–40% in the treated cultures. Ara C blocked the production of viral capsid antigen (VCA) whereas a small number of VCA + cells were still present after IUDR treatment. Reversion of Ara C-induced DNA inhibition led to the appearance of VCA + cells, reaching a higher level than in the untreated control samples. Combined immunofluorescence and autoradiography showed that the majority of VCA + cells incorporated H³-thymidine. These facts are in line with the hypothesis that EA can be made in the absence of cellular DNA synthesis, whereas VCA production is dependent on the DNA synthesis. The relationship between EA and two other EBV-associated antigens, MA (membrane antigen) and VCA (viral capsid antigen) was studied by the two-color immuno-fluorescence technique. VCA + cells were EA+ and MA+. EA + VCA - cells were partly MA + and partly MA-. This is in good agreement with the corresponding findings on the EBV-infected Raji cell system (Gergely et al., 1970a).     

Early and late components of Epstein-Barr virus-associated membrane antigen in superinfected Daudi cells and their reactivity with sera from nasopharyngeal carcinoma patients.

Investigations were carried out on newly synthesized MA, VCA and EA in Daudi cells superinfected with the P3HR-1 strain of EBV and treated with trypsin to remove previously adsorbed MA-positive material from the cell surface. Synthesis of MA, VCA and EA was completely blocked by puromycin. A marked reduction in the frequency of MA-positive cells was observed in the infected cell cultures in the presence of either Ara-C or PA, but a fraction of the MA-positive cells was insensitive to the inhibitors. Differential absorption of an EBV antibody-positive human serum with Ara-C-treated or -untreated infected cells revealed two antigenically different components of MA: early (Ara-C-insensitive) and late (Ara-C-sensitive) MA. Three of five sera from patients with NPC, but none of five sera from normal adults, showed an apparent 'prozone' phenomenon in their reactivity against late but not early MA.     

Somatic cell hybrids between human lymphoma cell lines. V. IdUrd inducibility and P3HR-1 superinfectability of Daudi/HeLa (DAD) and Daudi/P3HR-1 (DIP-1) cell lines.

We have studied two types of somatic cell hybrid with regard to expression of the Epstein-Barr virus (EBV) cycle and its regulation. The first, DIP-1, a hybrid formed between two human lymphoma EBV producers (Daudi and P3HR-1), contained EBV DNA, expressed the virus-determined nuclear antigen (EBNA), andwas a producer of the EBV-associated antigens EA (early antigen) and VCA (viral capsid antigen). The second, DAD, a hybrid series of clones formed between Daudi and a HeLa cell derivative (D98), differed with regard to the expression of EBNA, EA, VCA and the content of EBV DNA. EA was regularly induced in the EBV DNA-containing hybrids following treatment with iododeoxyuridine (IdUrd). This induction was greater in lines spontaneously expressing EA. In two hybrids, DIP-1 and DAD10, VCA and virus DNA synthesis were also induced in the presence of IdUrd, the latter being detected by in situ hybridization with P3HR-1 EBV complementary RNA. Finally, while DIP-1 was superinfectable by the P3HR-1 EBV strain, the DAD series of hybrids were refractory to P3HR-1 superinfection and lacked EBV receptors.     

Abortive expression of the Epstein-Barr virus (EBV) cycle in a variety of EBV DNA-containing cell lines, as reflected by nucleic acid hybridization in situ.

A variety of Epstein-Barr virus (EBV) DNA-containing cell lines have been tested for the expression of the EBV-associated antigens EBNA (nuclear antigen), EA (early antigen), and VCA (viral capsid antigen), and for the presence of cells containing disproportionate amounts of EBV DNA. The antigen tests utilized immunofluorescence and 125I-labelled antibodies combined with autoradiography. EBV-DNA was detected by in situ hybridization with 3H-labelled EBV RNA complementary to P3HR-1 EBV DNA (P-EBVcRNA). The P-EBVcRNA has been shown to represent the majority of the P3HR-1 EBV DNA sequences. It was concluded that EBV DNA-containing cell lines can be divided into those that express only EBNA, those that express EBNA and EA and those that express EBNA, EA and VCA and also contain cells that undergo disproportionate EBV DNA synthesis. Consequently, in some cell lines there is an abortive expression of the EBV cycle in that some cells spontaneously express EA but fail to continue further to viral DNA synthesis. A similar pattern can be found after experimental induction of the EBV cycle, suggesting that related mechanisms govern the spontaneous expression of the EBV cycle and the extent of its inducibility.     

Immunofluorescence and anti-complement immunofluorescence absorption tests for quantitation of Epstein-Barr virus-associated antigens.

Immunofluorescence absorption methods are described which permit quantitative estimation and differentiation of Epstein-Barr virus (EBV)-associated antigens (virus capsid antigen, VCA, early antigen, EA and EBV-determined nuclear antigen, EBNA) in cell extracts. EBNA was present in all cell lines (producer and non-producer) which carried the EBV-genome, while VCA and EA were present in producer lines only. All the antigens were absent from a lymphoid cell line (MOLT-4) which lacked the EBV-genome, as well as from leukemia cells from peripheral blood. The techniques demonstrated antigenic identity of the various antigens when prepared from different cell lines.     

Comparative study of anti EBV antibodies in non-Hodgkin's lymphomams and angio-immunoblastic lymphadenopathies (author's transl)]

Antibody titers to Epstein-Barr virus (EBV)--related antigens (viral capsid antigen : VCA; early antigen : EA; and EBV associated nuclear antigen : EBNA) were determined in the sera of 86 patients and 150 matched control subjects. The patients belonged to four histological groups : diffuse and nodular non-hodgkin's lymphomas angio-immunoblastic lymphadenopathies and apparented syndroms. The incidence of antibodies to other herpes-viruses (cytomégalovirus, herpes simplex virus, and varicella zoster virus) was compared. There was a significantly higher incidence of anti VCA and anti EA titers in some patients, not associated with an increase in titres of antibodies to other herpes viruses.     

Epstein-Barr virus-induced malignant lymphoma in a white-lipped marmoset

One of six white-lipped marmosets inoculated with cell-free B95-8 virus developed diffuse malignant lymphoma. Epstein-Barr virus (EBV) DNA was detected in a pathologically enlarged mandibular lymph node by DNA-DNA hybridization. The affected animal at the time of killing had EBV antibody titers of 1:320 for viral capsid antigen (VCA) and 1:20 for early antigen (EA) while all non-diseased animals had less than or equal to 1:80 VCA antibody and no detectable EA antibody. This is the first report of lymphoma development in a white-lipped marmoset following EBV inoculation.     

Differential inhibition of epstein-barr virus induction by the amino acid analogue,L-canavanine

The effect of an amino acid analogue, L-canavanine, on the synthesis of Epstein-Barr virus (EBV) antigens was investigated in lymphoblastoid cells. The analysis revealed that after infection of BJAB and NC-37 cells with P3HR-I EBV synthesis of early antigen (EA) was not affected by canavanine in concentrations up to 8.4 mM. The synthesis of EBV-determined nuclear antigen (EBNA) and of viral capsid antigen (VCA) was significantly inhibited at concentrations higher than 2.8 mM. Spontaneous induction of EA in P3HR-I cells was not affected by canavanine. On the other hand, EA induction by the tumor promoter TPA, by iododeoxyuridine (IdUrd), by antiserum to human IgM and by n-butyric acid was clearly inhibited by this treatment. Application of 0.3 mM canavanine resulted in more than 95% inhibition of EA induction by TPA. Under these conditions cell growth and incorporation of radiolabelled amino acids into an acid-insoluble fraction was significantly impaired. Differential treatment of the cells with canavanine established that EA induction was completely suppressed when the cells were treated concomitantly with canavanine and TPA. Subsequent treatment with canavanine after prior exposure to TPA resulted in some viral antigen induction depending on the time period of TPA exposure. Pretreatment of the cells overnight with canavanine followed by washing and addition of the tumor promoter did not suppress EA induction by TPA. These data support the concept that EA induction by superinfection follows a different pathway from antigen induction by chemical inducers.     

Detection of antibodies against virally-induced tumor-associated antigens by mixed hemadsorption

Antigens were solubilized and attached to glass slides. The coated glass slides were then used to detect relevant antibodies by a mixed hemadsorption technique. These studies focused primarily on solubilizing the Epstein-Barr virus-associated antigens VCA, EA and MA. Well-characterized, discordant sera from patients with Burkitt's lymphoma and nasopharyngeal carcinoma were then used to evaluate the antigenicity of slides coated with the solubilized antigenic extracts. Good correlations were observed between titers of these sera for VCA, EA and MA as determined by immunofluorescence methods and by mixed hemadsorption. Most antigenic extracts could be stored directly or as coated-glass slides for many months with only moderate decreases in antigenicity. Antibodies to Epstein-Barr-associated nuclear antigen (EBNA) were not detectable in these studies.     

Segregation of the EBV-determined nuclear antigen (EBNA) in somatic cell hybrids derived from the fusion of a mouse fibroblast and a human Burkitt lymphoma line

Four independently derived hybrids between the mouse fibroblast line A9 and the human, Burkitt-lymphoma-derived lymphoblastoid cell line Daudi were studied for the presence of the Epstein-Barr virus (EBV) genome, the EBV-determined nuclear antigen (EBNA), other EBV-associated antigens, human surface immunoglobulin and the presence of human chromosomes. The four lines differed in the number of their EBV genomes. There was a parallelism between this number, as detected by c/RNA/DNA hybridization, and the frequency of EBNA-positive nuclei. None of the other EBV-antigens, EA, VCA or MA, was expressed at any time, either in the untreated hybrid cells or after IUDR-treatment. The hybrids did not carry detectable surface-associated immunoglobulin or EBV-receptors. The presence of the EBV genome was coincident with the maintenance of human chromosomes, but the hybrids that have lost detectable viral genomes and EBNA still contained a considerable number of human chromosomes, suggesting that the viral genome may be associated with a few chromosomes only.     

Productive Epstein-Barr viral infection of the human lymphoblastoid cell line 6410 with release of early antigen inducing and transforming virus.

The human lymphoblastoid cell line 6410 was superinfected with P3HR-1 derived Epstein-Barr virus. Subsequent to the first cycle of infection, characterized by early (EA) and virus capsid (VCA) antigen synthesis, the culture, designated 6410-EBV, continued to synthesize VCA. Further immunofluorescence and electron microscopic examination over 18-24 months showed the 6410-EBV culture to be productively infected with EBV. Virus was recovered, in quantity, from the culture fluids and assayed for ability to induce EA synthesis in Raji cells and to transform human umbilical cord lymphocytes. The virus was found to possess both properties, in contrast to P3HR-1 virus which only induces EA. HLA and karyologic analyses of P3HR-1, 6410 and 6410-EBV cultures showed relatedness of 6410-EBV to 6410 cells and dissimilarity to P3HR-1. The biologic activity data coupled with the cytologic analyses confirm a productive infection of the target cells. The reason for differences in biologic activity between the infecting (P3HR-1) and progeny virus is unresolved. It may be speculated that the endogenous EBV genome of 6410 cells was activated and gave rise to a different strain of EBV or to a mixed progeny-parent population as a result of dual infection. Alternatively, the parent strain of virus may have been modfied as a result of interaction with the new host cell.     

Recovery of transforming EBV from non-producer cells after superinfection with non-transforming P3HR-1 EBV.

Cells of the Raji and NC37 lines can be induced by chemical inducers, such as BrdUrd and IdUrd, or the tumor-promoter TPA to EA-expression only, but do not reveal any VCA synthesis. After superinfection by nontransforming P3HR-1 EBV, however, a varying percentage of the cell population shows VCA synthesis and releases infectious viral particles. The recovered virus differs biologically from P3HR-1 EBV since it transforms human umbilical cord blood lymphocytes into EBNA-positive lymphoblastoid cell lines. Cells of these established lines are susceptible to renewed infection by P3HR-1 EBV which results in EA induction and VCA synthesis. Only cells of one line, NC37-R1, spontaneously produce VCA and EBV particles, which reveal transforming properties and do not induce EA upon superinfection of Raji cells. Infection of P3HR-1 EBV-converted BJA-B cells also leads to EA and VCA induction and the release of viral particles. In contrast to particles recovered from Raji and NC37 cells, no transforming activity was detectable in these virus preparations. According to these data, we propose that viral genomes persisting within Raji and NC37 cells are defective and become complemented by the superinfecting P3HR-1 virus.     

Epstein-Barr virus antibodies in tonsillar carcinoma patients;.

Sera from 18 tonsillar carcinoma patients and from 18 matched control subjects were examined for the presence of antibodies to viral capsid antigen (VCA), early antigen (EA) and nuclear antigen (EBNA) of EB virus. Antibodies to all three antigens were found more frequently and in significantly higher titres in the tonsillar carcinoma patients than in control subjects.     

Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines

Anti-complement immunofluorescence (ACIF) was used to study the complementfixing antigens of human lymphoblastoid cell lines. These cell lines carry the Epstein-Barr virus (EBV) genome although only producer cultures synthetize EBV-specific antigens (virus capsid antigen, VCA and early antigen, EA) detectable by direct and indirect immunofluorescence, usually in less than 5% of the cells. The ACIF test revealed an antigen localized in the nucleus of the lymphoblastoid cells. In contrast to EA and VCA, this antigen was present in over 90% of the cells of both producer and non-producer cultures. The antigen was shown to be specific for EBV by comparing the reactions of 52 sera in the ACIF test. Sera giving the nuclear reaction contained antibodies to VCA, EA or antigens detectable by complement fixation tests on cell extracts, but sera without EBV antibodies failed to give the reaction. Weak, equivocal or discordant reactions occurred with six sera with low titres in VCA, EA or complement fixation tests. Cell lines derived by transformation of human and primate lymphocytes by EBV gave the nuclear reaction. Control cells with no known association with EBV were non-reactive. These included foetal lymphocytes transformed by phytohaemagglutinin, cell lines derived from breast cancer, glioma, normal glia, pleuritis maligna and myeloma, and two marmoset lymphoid lines carrying Herpesvirus saimiri (HVS). In preliminary experiments, the ACIF test was used as a tool to trace the EBV genome at the cellular level. Cells from two Burkitt lymphoma biopsies, one tested after biopsy and one after passaging in nude mice, contained an EBV-specific antigen. Three clones of cells derived from hybrids of mouse somatic cells and a human lymphoblastoid cell line also contained such an antigen, but the number of reactive cells varied from clone to clone. A fourth clone was non-reactive.     

Induction of epstein-barr virus by a new tumor promoter,teleocidin, compared to induction by TPA

The effect of teleocidin, a new, naturally occurring tumor promoter, on induction of Epstein-Barr virus (EBV), was compared with that of a known tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Early antigen (EA) and/or capsid antigen (VCA) of EBV was induced in the EBV genome-carrying cell lines C-6 and P3HR-I cells by teleocidin, its effect being maximal at a concentration of 12.5 ng/ml. The production of infectious EBV from P3HR-I cells was enhanced by teleocidin maximally at a concentration of 0.5 to 2.5 ng/ml. The outgrowth of EBV-transformed cells from peripheral lymphocytes of seropositive healthy donors was also enhanced by teleocidin at a concentration of 0.02 to 0.5 ng/ml. TPA tested simultaneously in all experiments exhibited the same activities as teleocidin, and was effective at similar concentrations. Teleocidin enhanced both EA and VCA synthesis in P3HR-I cells additively with n-butyrate, but not with TPA. This suggests that teleocidin and TPA have a common mechanism of action, although their chemical structures are different.     

Growth and antigenic properties of a biopsy-derived Burkitt's lymphoma in thymus-less (nude) mice

A Burkitt's lymphoma was transferred to the congenitally athymic mouse mutant nude with biopsy material from a 7-year-old Kenyan girl. The tumor grows locally at the site of inoculation with no distant metastases. The established tumor has been maintained for six passages so far with preservation of histological and cytological appearance. The mouse-passaged tumor has a normal human diploid female chromosome complement. Isozyme studies have shown tumor to be of the same glucose-6-phosphatedehydrogenase (G-6-PD) and phosphoglucomutase₁ phenotype (B and 2-1 respectively) as tumor biopsy from the patient. The mouse-passaged line maintained surface IgM, similarly to the original tumor and the derived tissue culture line, but lost the IgG coating characteristic for the original tumor but absent from two subsequent biopsies and from the derived tissue culture line. This is in line with previous observations indicating that surface-associated IgG on Burkitt biopsies is due to coating from the outside, whereas surface-associated IgM is a cell marker. Whereas the biopsy cells of the patient were positive for EBV-associated membrane antigen (MA), but not for early antigen (EA) and viral capsid antigen (VCA), the mouse-passaged line was positive for all three. This suggests that the restrictive influence of the human host on the production of EA and VCA in the Burkitt tumor is raised in the mouse host. The serum of the tumor-bearing nude mice contained anti-human antibodies, but no detectable EBV (anti-MA, EA and VCA)-antibodies.     

Prognostic value of serum Epstein–Barr virus antibodies in patients with nasopharyngeal carcinoma and undetectable pretreatment Epstein–Barr virus DNA

Epstein–Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC). Serum IgA antibodies against early antigen (EA‐IgA) and viral capsid antigen (VCA‐IgA) are the most commonly used to screen for NPC in endemic areas. However, the prognostic value of serum EA‐IgA and VCA‐IgA in patients with NPC is less clear. We hypothesize that serum EA‐IgA and VCA‐IgA levels have prognostic impact for survival outcomes in NPC patients with undetectable pretreatment EBV (pEBV) DNA. In this series, 334 patients with non‐metastatic NPC and undetectable pEBV DNA were included. Serum EA‐IgA and VCA‐IgA were determined by ELISA. After analysis, serum EA‐IgA and VCA‐IgA loads correlated positively with T, N, and overall stage (all P < 0.05). Serum EA‐IgA was not associated with survival outcome in univariable analyses. But patients with serum VCA‐IgA >1:120 had significantly inferior 5‐year progression‐free survival (80.4% vs 89.6%, P = 0.025), distant metastasis‐free survival (88.4% vs 94.8%, P = 0.050), and locoregional relapse‐free survival (88.4% vs 95.6%, P = 0.023; log–rank test). Multivariable analyses revealed that N stage was the only independent prognostic factor (all P < 0.05), but the VCA‐IgA became insignificant. Further analyses revealed that serum VCA‐IgA was not an independent prognostic factor in early N (N0–1) or advanced N (N2–3) stage NPC. In summary, although both EA‐IgA and VCA‐IgA correlate strongly with TNM stage, our analyses do not suggest that these antibodies are prognostic biomarkers in patients with NPC and undetectable pEBV DNA.     

Expression of the Epstein-Barr virus genome in a nasopharyngeal carcinoma epithelial tumor cell line

An epithelial tumor cell line was recently established from a biopsy specimen of a nasopharyngeal carcinoma (NPC), and designated HONE-I. Uncloned (parental) HONE-I and HONE-I clone (C)-40 cells were found to contain latent Epstein-Barr virus (EBV). Expression of the latent EBV genome in HONE-I C-40 cells has been examined. It was possible to detect a small percentage of cells spontaneously synthesizing EBV early antigen (EA) and virus capsid antigen (VCA) by immunofluorescence (IF). In addition, the EBV nuclear antigens (EBNA-I and EBNA-2), as well as the EBV latent membrane protein (LMP) were detected in the HONE-I cells. Attempts were made to induce the latent EBV genome in these cells with iododeoxyuridine (IUdR). We observed a significant increase in the number of EA/VCA-positive cells, an increase in EBV DNA, the synthesis of virus particles, and the rescue of infectious virus after treatment of HONE-I C-40 cells with IUdR. The HONE-I C-40 cells should facilitate studies of the expression and regulation of the EBV genome in NPC epithelial tumor cells, which have not previously been available.     

Antibody to Epstein-Barr virus and cellular immunity in Hodgkin's disease and chronic lymphatic leukemia

Antibodies to Epstein-Barr virus capsid antigen (VCA), early antigen (EA) and cellular immunity as measured by skin reactivity to keyhole limpet hemocyanin (KLH) and Brucella antigen (BA) were measured in 15 cases of Hodgkin's disease (HD) and 14 cases of chronic lymphatic leukemia (CLL) before treatment and one year later. Both groups of diseases were associated with elevated VCA and EA antibody levels when compared with 18 controls, and in both groups the mean titers were unchanged after therapy. A negative skin test to KLH was associated with a short survival in the HD group and was found more frequently in CLL patients with active disease. In the young HD patients, a higher EBV titer was found in pretreatment sera of non-survivors compared with those who are still alive. There was no correlation, however, between tests for cell-mediated immunity and humoral antibodies against EBV. The finding of similar titers in patients with depressed and normal skin reactivity indicates that the elevated titers are probably not the result of a non-specifically depressed immune defense.     

Significance of Plasma IgA and IgG Antibodies to Epstein-Barr Virus Early and Viral Capsid Antigens in Thai Nasopharyngeal Carcinoma

Epstein-Barr virus (EBV) is an important causal factor of human nasopharyngeal carcinoma (NPC). High levels ‍of serum IgA and IgG antibodies to EBV early and viral capsid antigens (IgA/EA, IgA/VCA, IgG/EA and IgG/VCA) ‍have been reported in NPC patients. Since specific serum IgA/EA, IgA/VCA and IgG/EA are claimed to be useful ‍serological markers for NPC. In order to evaluate whether plasma IgA/EA, IgA/VCA, IgG/EA and IgG/VCA antibody ‍levels are useful markers for diagnosis and prognosis of Thai NPC, we examined the prevalence of these antibodies ‍in 79 NPC patients, and 127 age-matched controls (47 healthy subjects (HS), 32 cases of other disease (OD) and 48 ‍cases of other cancer (OC)) by using an indirect immunofluorescence assay. The prevalence of plasma IgA/EA, IgA/ ‍VCA, and IgG/EA in NPC patients (55.7, 68.4 and 68.4%) was significantly higher than in the HS (0.0, 0.0 and ‍20.5%,), OD (0.0, 0.0 and 3.1%) and OC (0.0, 0.0 and 20.8%) groups (p<0.05). The prevalence of plasma IgG/VCA ‍in NPC patients (93.7%) was significantly different from those for the OD and OC groups (71.9 and 43.8%) but not ‍for the HS group (89.4%). In NPC patients, the geometric mean titers (GMT) of plasma IgA/EA, IgA/VCA and IgG/ ‍EA were increased with an advanced clinical stage of disease but not IgG/VCA. In contrast, GMT of IgG/VCA was ‍increased with aggressive type of disease (histological type) but not IgA/EA, IgA/VCA, and IgG/VCA. The results of ‍our study suggest that plasma IgA/EA, IgA/VCA and IgG/EA antibodies may be useful markers for diagnosis and ‍assessing prognosis of Thai NPC. ‍