Epstein-Barr viral antigens used in the diagnosis of nasopharyngeal carcinoma

The major antigen complexes of Epstein-Barr virus (EBV) include the latent infectious proteins, early antigens, membrane antigens and viral capsid antigens. The various polypeptides within each antigen complex have been identified and isolated through gene-cloning technology. These polypeptides are exploited to be used as serological markers for the diagnosis of nasopharyngeal carcinoma (NPC) through enzyme-linked immunosorbent assay. This paper reviews the recent studies on the profile of antibodies in patients with NPC towards these EBV polypeptides of each antigen complex. The sensitivity and specificity of each polypeptide when used as serological markers to NPC patients' sera are summarized.     

Sequential detection of different antigens induced by Epstein-Barr virus and herpes simplex virus in the same Western blot by using dual antibody probes

A dual antibody probing technique that permitted a color-coded identification of polypeptides representing different classes of Epstein-Barr virus (EBV) antigens as well as differentiation of the polypeptides induced by different herpesviruses in the same Western blot was developed. When the nitrocellulose sheet was probed first with monoclonal antibody against EBV early antigen diffuse component (EA-D) and then stained with 4-chloro-1-naphthol, four polypeptides specific for EA-D were identified by purple bands. Subsequently, the same nitrocellulose sheet was reprobed with human serum containing antibodies against EBV early antigen, viral capsid antigen, and nuclear antigen and stained with 3,3'-diaminobenzidine. Several brown bands corresponding to early, viral capsid, and nuclear antigen polypeptides were detected. The dual antibody probing technique was used in an analysis to differentiate polypeptides resulting from either EBV or herpes simplex virus infection, either in cells infected by individual virus or in a cell line dually infected by both viruses. On the basis of different colored bands in different lanes of the same gel, 20 polypeptides with molecular weights ranging from 31,000 to 165,000 were identified as herpes simplex virus-specific proteins. These results suggested that the dual antibody probing technique may be applicable in clinical diagnosis for detecting antigens and antibodies derived from different pathogens.     

Identification of polypeptide components of the Epstein-Barr virus early antigen complex with monoclonal antibodies.

Three monoclonal antibodies were produced against the Epstein-Barr virus-induced early antigen complex. These antibodies were shown to be specific for the early antigen complex by the fact that they only reacted with cells supporting a permissive or abortive Epstein-Barr virus infection and their synthesis was not affected by inhibitors of viral DNA synthesis. One monoclonal antibody, designated R3, was directed against a diffuse component of the early antigen complex since it reacted by immunofluorescence with cells fixed in acetone or methanol. The other two monoclonal antibodies, designated K8 and K9, reacted with a methanol-sensitive restricted component of this complex. The appearance of the R3 antigen in P3HR-1 superinfected Raji cells occurred approximately 4 h earlier than the antigen detected by K8. By both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioimmunoelectrophoresis, it was determined that the R3 monoclonal antibody recognized two major polypeptides with molecular weights of approximately 50,000 to 52,000, whereas K8 and K9 precipitated a protein of approximately 85,000. The R3 monoclonal antibody also immunoprecipitated an in vitro primary translation product. It was, therefore, possible to map this product to the Epstein-Barr virus DNA BamH1 M fragment. These in vitro products were slightly smaller than the in vivo proteins, suggesting that these proteins probably undergo posttranslational modification during the virus replication cycle.     

Identification of multiple Epstein-Barr virus-induced nuclear antigens with sera from patients with rheumatoid arthritis.

By means of the protein immunoblot technique, the Epstein-Barr virus (EBV) nuclear antigen (EBNA) could be identified in a variety of EBV-transformed cell lines with anti-EBNA-positive sera from normal donors. The molecular weight of EBNA expressed in each of the cell lines varied between 70,000 and 75,000 and was dependent upon the strain of infecting virus. In contrast, 15 of 21 sera from patients with rheumatoid arthritis identified antigens in addition to EBNA. The most prominent of these antigens had molecular weights of 110,000 to 115,000 and 92,000. All of the EBV genome-positive cell lines except for QIMR-GOR and cell lines containing the P3HR-1 virus expressed these antigens. The antigens were not present in the EBV genome-negative Ramos and BJAB cell lines, nor were they identified with EBV seronegative sera, indicating that they were EBV related. There was no direct correlation between the presence of antibodies in sera to EBNA, viral capsid antigen or early antigen, and reaction with the 92,000-molecular-weight antigen in immunoblots, indicating that this antigen was distinct from previously described EBV-related antigens.     

Isolation and characterization of monoclonal antibodies to structural and nonstructural herpesvirus saimiri proteins

An improved screening procedure was applied to identify hybridomas secreting antibodies to herpesvirus saimiri-specified polypeptides among the products of fusions between SP2/0 myeloma cells and spleen cells from mice immunized with purified virus particles or virus-specific DNA-binding proteins. Twenty-four monoclonal antibodies were isolated with specificities for 13 different virus-specified polypeptides (or complexes of polypeptides), including the major capsid protein of the virus (150K), the 160K and 130K structural proteins, a 108K structural phosphoprotein, structural glycoproteins, the nonstructural early 76K protein, early nonstructural DNA-binding proteins of 48 to 51K and 110K and the major immediate-early protein of 52K. Antibody to the virus 76K protein precipitated a host protein of 62K, and a number of antibodies specific for host proteins were also isolated. Antibody to the 52K immediate-early polypeptide precipitated the delayed-early 76K protein, whereas the antibody to the 76K protein did not precipitate the 52K polypeptide. These observations suggest the presence of epitopes common to virus and host proteins and an antigenic site common to an immediate-early and a delayed-early virus protein. The antibodies were used to examine the sites of intracellular accumulation of virus polypeptides, the formation of complexes of structural proteins, and the postsynthetic processing of virus proteins. The present collection of monoclonal antibodies provides a set of reagents with specificities for members of each of the major kinetically or functionally distinct classes of virus gene products.     

Characterization of a major protein with a molecular weight of 160,000 associated with the viral capsid of Epstein-Barr virus.

A monoclonal antibody designated V3 was produced against a late protein associated with the Epstein-Barr virus-induced viral capsid antigen complex. The antibody reacted with discrete patches in the nuclei of infected cells as well as with virus particles, as shown by immunofluorescence and ultrastructural immunoperoxidase staining. The molecular weight of the protein precipitated by this monoclonal antibody was ca. 160,000. All anti-viral capsid antigen antibody-positive sera tested in an enzyme-linked immunosorbent assay reacted with this purified protein. The synthesis of the antigen was inhibited by phosphonoacetic acid but was not affected by tunicamycin, indicating that this was a late nonglycosylated viral protein. No differences were noted between the protein isolated from the P3HR-1 and B-95-8 cell lines as determined by immunoprecipitation and peptide mapping. By isoelectric focusing, this protein had a pI on the basic side ranging from 7.5 to 9.0.     

Double immunoenzymatic staining for the simultaneous detection of Epstein-Barr virus induced antigens

Summary A double indirect immunoenzymatic staining was developed for the simultaneous visualization of Epstein-Barr virus-induced early antigens and virus capsid antigens in P3HR1 lymphoblastoid cell line.The double immunocytochemical staining was performed with a four-stage and a two-stage procedure employing human sera and monoclonal antibodies against Epstein-Barr virus-induced antigens, followed by the addition of specific alkaline phosphatase and peroxidase labeled antisera.The selection of substrates yielding reaction products of contrasting colours enabled the observer to distinguish cells expressing Epstein-Barr virus capsid antigens (blue) from cells expressing Epstein-Barr virus early antigens (brown).     

Double immunoenzymatic staining for the simultaneous detection of Epstein-Barr virus induced antigens

A double indirect immunoenzymatic staining was developed for the simultaneous visualization of Epstein-Barr virus-induced early antigens and virus capsid antigens in P3HR1 lymphoblastoid cell line. The double immunocytochemical staining was performed with a four-stage and a two-stage procedure employing human sera and monoclonal antibodies against Epstein-Barr virus-induced antigens, followed by the addition of specific alkaline phosphatase and peroxidase labeled antisera. The selection of substrates yielding reaction products of contrasting colours enabled the observer to distinguish cells expressing Epstein-Barr virus capsid antigens (blue) from cells expressing Epstein-Barr virus early antigens (brown).     

Epstein-barr virus (EBV)-associated antibodies in a case of Burkitt's lymphoma during 14 years of sustained remission

Summary A Burkitt's lymphoma (BL) patient who sustained remission for more than 14 years after chemotherapy was monitored by means of serial serum samplings. The sera were titrated for antibodies against the Epstein-Barr virus (EBV)-associated cell membrane antigen (MA), viral capsid antigens (VCA), early antigen complex (EA R/D), and nuclear antigen (EBNA), and also for reactivity in the antibody-dependent cellular cytotoxicity (ADCC) test. The initial serological profile corresponded to that of most BL patients with active disease. During remission, it changed to resemble that of normal persons with persistent, latent EBV infection, at least qualitatively. The prognostic and biological implications of the titer levels and their changes are discussed.Abbreviations ADCC antibody-dependent cellular cytotoxicity - BCG Bacillus Calmette-Guérin - BL Burkitt's lymphoma - D diffuse component of EA - EA Epstein-Barr virus-induced early antigens - EBNA Epstein-Barr virus-specific nuclear antigen - EBV Epstein-Barr virus - KCC Kenya Cancer Council Registry - MA EBV-associated cell membrane antigen complex - NK natural killer - R restricted component of EA - VCA EB viral capsid antigen complex     

Identification and isolation of the main component (gp350-gp220) of Epstein-Barr virus responsible for generating neutralizing antibodies in vivo.

The majority of hybridomas we have characterized against Epstein-Barr virions react with the major glycoproteins gp350 and gp220 (gp350/220). One of these antibodies, ID4C-1, neutralizes virus infection in vitro. The presence of gp350/220 on the viral envelope could be confirmed directly by immunoelectron microscopy. We used lectin affinity (ricin) and immunoaffinity (ID4C-1) to purify gp350/220 and show that this material is able to induce potent virus-neutralizing antibodies. Absorption of four human and one rabbit anti-Epstein-Barr virus sera with purified gp350/220 suggests that this is the primary component responsible for generating neutralizing antibodies in vivo.     

IgM antibodies to Epstein-Barr virus-associated membrane antigen in sera of infectious mononucleosis patients

By the indirect immunofluorescence technique, IgM antibodies to the cell surface of an Epstein-Barr virus (EBV) producer cell line, P3HR-1, were detected in sera from infectious mononucleosis (IM) patients but not in sera from patients with Burkitt lymphoma or nasopharyngeal carcinoma nor in sera from healthy adult donors having antibodies to EBV-specific viral capsid antigen (VCA). Titers of the IgM antibodies were higher in the earlier stages of IM, a pattern similar to that for IgM antibodies to VCA. The IgM antibodies to the cell surface were identified as being those against the EBV-specific membrane antigen (MA) by the following criteria: (1) The antibodies were reactive to MA-positive cell preparations but to MA-negative cell preparations. (2) Titers of the IgM antibodies were not significantly affected after absorption of sera with sheep red blood cells which could completely eliminate heterophil antibodies in the same sera. Detection of the IgM antibodies to MA may have a particular diagnostic value for providing evidence of a recent EBV infection.     

Immunobiochemical characterization with monoclonal antibodies of Epstein-Barr virus-associated early antigens in chemically induced cells.

Five monoclonal antibodies which are reactive to early antigens of Epstein-Barr virus have been produced by using somatic cell hybridization techniques. The specificity of the monoclonal antibodies to early antigens was demonstrated by indirect immunofluorescence, which showed that the antigens were localized to the nucleus of early antigen-induced Raji cells. Additional indirect immunofluorescence studies showed that like patient antisera to diffuse-staining early antigen, the monoclonal antibodies gave positive staining reactions after methanol fixation. One of the antibodies, 1150-4, was positive by the anti-complement immunofluorescence technique but differed with Epstein-Barr virus-associated nuclear antigen-positive patient sera in that it only stained induced cells. Different fixation methods were found to alter dramatically the appearance of the nuclear staining reactions produced by the monoclonal antibodies. Immunoprecipitation and immunoblot experiments revealed that monoclonal antibodies 1108-1 and 1129-1 recognized two polypeptides of 55,000 and 50,000 daltons (p55;50), 1173-6 and 1180-2 recognized just p50, and 1150-4 identified a 65,000-dalton nuclear protein. Immunobiochemical characterization of these viral antigens showed that p55 is a phosphoprotein, and p55;50 has strong DNA-binding activity preferentially to single-stranded DNA. Elucidation of the role of these nuclear proteins in Epstein-Barr virus infection and the events associated with Epstein-Barr virus-directed lymphocyte transformation may provide significant information on the pathogenicity of this important human virus.     

Production and characterization of monoclonal antibodies against the Epstein-Barr virus membrane antigen.

Five murine hybridoma lines that produce monoclonal antibodies against Epstein-Barr virus membrane antigen (MA) were established. Immunoprecipitation experiments demonstrated that three of the antibodies precipitated both the 236,000 (236K) MA and the 212K MA. The other two antibodies precipitated the 86K MA. Antibodies against the 236K-212K MA and the 86K MA mediated complement-dependent cytolysis of Epstein-Barr-virus-infected cells. The antibodies against the 86K MA neutralized both the B95-8 and P3HR-1 viruses.     

Mechanisms of infection with Epstein-Barr virus. I. Viral DNA replication and formation of noninfectious virus particles in superinfected Raji cells.

Human lymphoblastoid Raji cells, which do not produce virus, supported replication of Epstein-Barr virus (EBV) upon superinfection. Early antigen, viral capsid antigen, and virions were produced in Raji cells superinfected with EBV. Viral DNA replicated under complete inhibition of host cell DNA synthesis to the extent that a few micrograms of EBV DNA were recovered from 107 superinfected Raji cells, corresponding to 5,000 viral genomes/cell. Homology of the synthesized viral DNA to parental EBV DNA was more than 90%. Virions produced by the Raji cells contained a 55S DNA but failed to induce early antigen, viral capsid antigen, and viral DNA synthesis after a second superinfection of Raji cells.     

Primary central nervous system lymphoma expressing the human neurotropic polyomavirus, JC virus, genome

B lymphocytes are known as a potential site for latency and reactivation of the human neurotropic polyomavirus, JC virus (JCV). In light of recent studies on the oncogenicity of JCV and the transforming ability of the JCV early protein, T antigen, we investigated the association of JCV with B-cell lymphomas of the central nervous system. Examination of 27 well-characterized clinical specimens by gene amplification and immunohistochemistry revealed the presence of DNA sequences corresponding to the JCV early genome and the late Agnoprotein in 22 samples and the JCV late genome encoding the viral capsid proteins in 8 samples. Expression of T antigen and that of Agnoprotein by immunohistochemistry were each detected in six specimens. No evidence of the production of viral capsid proteins was observed, ruling out productive infection of JCV in the tumor cells. The results from laser capture microdissection verified the presence of JCV T-antigen sequences in tumor cells with positive immunoreactivity to antibodies against the viral proteins T antigen and Agnoprotein. Due to previous reports demonstrating an association of the Epstein-Barr virus (EBV) with transformation of B lymphocytes, EBV DNA sequences and the EBV transforming protein, latent membrane protein 1 (LMP1), were analyzed in parallel. EBV LMP1 DNA sequences were detected in 16 of 23 samples, and LMP1 expression was detected in 16 samples, 5 of which exhibited positive immunoreactivity to JCV proteins. Double labeling demonstrated coexpression of JCV T antigen and EBV LMP1 in the same cells. The detection of the JCV genome in large numbers of B-cell lymphomas and its coexistence with EBV suggest a potential role for JCV in the pathogenesis of primary CNS lymphoma.     

A simian virus 40-encoded protein of Mr 74,000 daltons is structurally related to the capsid proteins of the virus

We have demonstrated the synthesis of a 74,000-dalton protein (74K protein) in African green monkey kidney cells infected with simian virus (SV)40. The 74K protein was detected late during the lytic cycle. Its synthesis was inhibited by arabinosyl cytosine as was the synthesis of the capsid proteins. Monospecific antibodies raised against VP1 and VP3 precipitated the structural proteins and the 74K protein. The 74K protein was not found in purified virions. Tryptic peptide analysis demonstrated that the 74K protein shares methionine- and serine-containing peptides with VP1 and VP3 and thus is structurally related to the capsid proteins.     

Viral attributes and host factors in carcinogenesis: herpesviruses.

This paper describes two different experiments of nature: 1) the persistence of unusual virus strains of Epstein-Barr virus (EBV) (which proved oncogenic in vitro) and cytomegalovirus (CMV) in lymphoid cells following congenital or early acquired infection; 2) the occurence of multiple cases of Burkitt's lymphoma and nasopharyngeal carcinoma in one family. All the members of this family were EBV viral capsid antigen (VCA) and nuclear antigen (EBNA) antibody positive. The two patients with nasopharyngeal carcinoma had high titers of EBV-VCA, EA, and EBNA antibodies. The only member of this family having EBV early antigen (EA antibodies in addition to the patients with tumors was the mother. Borderline IgA deficiency was documented in 3 members of this family. These findings illustrate the importance of host factors (intracellular resistance to transformation and secondarily, immunological surveillance) in the outcome of the host-virus challenge whether cancer or infectious disease is the outcome. Extensive studies of these cases may provide the best insight into the mysteries of viral oncogenesis.     

Effects of S-adenosylhomocysteine and analogs on Epstein-Barr virus-induced transformation, expression of the Epstein-Barr virus capsid antigen, and methylation of Epstein-Barr virus DNA.

S-Adenosylhomocysteine was found to have no effect on Epstein-Barr virus-induced transformation of B-lymphocytes and to stimulate viral capsid antigen expression only slightly in the FF41-1 cell line. In contrast, the S-adenosylhomocysteine analogs sinefungin and S-isobutyladenosine inhibited Epstein-Barr virus transformation and induced a significant increase in the numbers of cells expressing the viral capsid antigen. An inverse relationship between levels of viral DNA methylation and gene expression was demonstrated.     

Hematologic and immunologic responses in common marmosets (Callithrix jacchus) infected with Plasmodium knowlesi and Epstein-Barr virus

The hematologic and immunologic responses to infection with either the Epstein-Barr virus alone or infection with Epstein-Barr virus and Plasmodium knowlesi were studied using common marmosets (Callithrix jacchus). The assays performed included complete blood cell counts, determinations of natural killer cell activity, and determinations of antibody titers to Epstein-Barr virus early antigen, virus capsid antigen and the nuclear antigen. While no animal showed signs of lymphoproliferative disease, it was found that animals infected with Epstein-Barr virus became positive for early antigen, virus capsid antigen and nuclear antigen at low levels. No difference in antibody titers between Epstein-Barr virus infected animals and co-infected animals was observed. An increase also was found in the number of leukocytes in all groups, and an increase in natural killer cells following infection with Epstein-Barr virus. Some depression in natural killer cells was observed in the co-infected animals when compared to Epstein-Barr virus infected animals.     

Biological properties and viral surface antigens of Burkitt lymphoma- and mononucleosis- derived strains of Epstein-Barr virus released from transformed marmoset cells.

Three strains of Epstein-Barr virus (EBV), two from Burkitt lymphoma (BL) and one from infectious mononucleosis (IM) were used to transform separate cultures of the same batch of primary marmoset leukocytes, and the viruses released from the transformants were compared. The three viruses shared properties of the transforming biotype of EBV, namely, stimulation of DNA synthesis and immortalization of cord blood leukocytes, and failure to induce "early antigen" in lymphoblast lines. All viruses produced more virus in transformed marmoset cells than in transformed human cells, as measured by the number of EBV genomes detected by complementary RNA/DNA hybridization, by virus capsid antigen expression, or by released virions and biologically active virus. Reference human sera and sera from primary EBV infections were used to compare the three virus strains in a virus neutralization test based on inhibition of stimulation of DNA synthesis. Specimens taken late in convalescence from patients with mononucleosis and sera from marmosets experimentally infected with virus from a patient with mononucleosis neutralized the homologous virus, as well as the two virus strains isolated from patients with BL. This finding indicates that viral antigens that elicit neutralizing antibodies are shared among the strains. However, in certain sera the neutralizing-antibody titer against one strain was consistently higher than against another strain. Furthermore, sera taken early after onset of IM contained low levels of neutralizing antibody against IM-derived virus, but failed to neutralize BL-derived virus. These latter findings suggest the existence of heterogeneity among surface antigens of EBVs. The results emphasize the biological and antigenic similarity of EBV isolates from BL and IM and do not suggest major subtype variations. It remains to be determined whether antigenic diversity such as described or virus genome variation detectable by other means is epidemiologically significant.