Shigeyoshi Fujiwara

Monoclonal antibody specific for capsid antigen of Epstein-Barr virus.

A hybrid cell line (Cl-5l) producing an anti-capsid antibody was obtained by fusion of mouse myeloma cells with spleen cells from mice immunized with purified P3HR-1 Epstein-Barr virus (EBV). Immunofluorescence showed that the Cl-5l antibody reacted with the cytoplasm and the nucleus of P3HR-1 and B95-8 cells, but not with Raji, BJAB, Molt-4, and superinfected Raji cells in the presence of cytosine arabinoside (Ara-C). The viable P3HR-1 and B95-8 cells were not stained nor was the viral infectivity neutralized. The Cl-5l antibody immunoprecipitated 123,000 and 120,000 dalton polypeptides of P3HR-1 and B95-8 cells, respectively, and both were sensitive to phosphonoacetic acid. Specific reactions were not evident with extracts of Raji cells and superinfected Raji cells in the presence of Ara-C. An analysis of the purified virus particles showed that this antibody recognized a capsid component of EBV.

Production of human monoclonal antibodies against Epstein-Barr virus-specific antigens by the virus-immortalized lymphoblastoid cell lines

The possible production of human monoclonal antibodies against Epstein-Barr virus (EBV) was assessed through the EBV immortalization technique. When individual lymphocyte samples from 50 clinical patients and healthy donors were immortalized by EBV, 4 lymphoblastoid lines yielded antibodies to EBV antigens. These positive lines were cloned and each line yielded cultures that secreted monoclonal antibodies against either viral capsid antigen (VCA) or membrane antigen (MA) component. Above all, a clonal line TAKA-SP-8 produced 5 micrograms MA antibody/10(6) cells/ml for more than 12 months. The culture fluid specifically immunoprecipitated a single polypeptide with a size of 93K from both P3HR-1 and B95-8 cell extracts. FUKA-SP-3, on the other hand, secreted 5 micrograms VCA antibody/10(6) cells/ml for at least 8 months. This antibody recognized two polypeptides with sizes of 123K and 120K, from P3HR-1 and B95-8 cell extracts, respectively. When B95-8 and P3HR-1 EBV were treated with the human MA monoclonal, both nuclear antigen (EBNA) synthesis and early antigen (EA) induction were strongly inhibited. All EBV antibody-producing cultures were exclusively achieved from splenic lymphocytes of patients with autoimmune diseases, but not from other donors.

Oncogenes: molecular probes for clinical application in malignant diseases.

Abstract We are now, by means of remarkable genetic engineering techniques, able to utilize retroviral oncogenes to probe for and define a number of homologously associated genes. These genes, the proto-oncogenes, are thought to be involved in the conversion of normal cells to neoplasia. These genes are known to be of normal cellular origin, but many were initially identified in retroviruses. Proto-oncogenes are highly conserved throughout the animal kingdom and are believed to play significant roles in cellular growth and/or differentiation. These proto-oncogenes have been associated recently with a number of chromosomal abnormalities involving certain malignant diseases. Cytogenetic data is accumulating that correlates specific proto-oncogene alterations with known chromosomal insults, such as breaks, rearrangements, and translocation, each associated with certain leukemias and malignancies. These studies have great potential for new approaches in treatment modalities, employing oncogenes as markers in predictive and pathologic diagnosis. For now the goal to use oncogenes as markers of neoplasia is certainly sufficient to justify current research efforts.

Analysis of Epstein-Barr Virus (EBV) of P3HR-1: Isolation of EBV with EBNA induction ability in human cord lymphocytes and without EA induction ability in raji cells

Abstract A subline of P3HR-1 cells was isolated through a prolonged (over 1 year) propagation of the cells at a non-EBV-productive condition followed by cell cloning procedures. Cloned cells thus obtained, designated DHR1, produced EBV when brought back to the EBV-productive condition. Restriction enzyme analysis of the viral DNA revealed that DHR1 EBV is composed of an apparently homogeneous EBV population, and it displays a similar but not identical genome organization compared with HR-1 EBV. The characteristic biological properties of DHR1 EBV included the ability to induce EBV-associated nuclear antigen (EBNA) in human cord lymphocytes and the inability to induce EBV-associated early antigen (EA) in Raji cells. These are in striking contrast to the behavior of the parental HR-1 EBV. Thus, P3HR-1 cultures after a period of nonproductivity reinitiated the production of virus with an apparently homogeneous and unique population of EBV distinguishable from the original HR-1 virus.

Human Epstein-Barr Virus and Cancer

Herpesviruses are ubiquitous in various species, including humans, and some are causally associated with naturally occurring tumors. The Epstein-Barr virus (EBV), which was first discovered in a culture of African Burkitt’s lymphoma in 1964 by Epstein and coworkers Achong and Barr as a previously unknown viral agent, is now considered to be one such oncogenic herpesvirus. EBV can readily convert normal human B lymphocytes in vitro into blast cells with infinite replicative capabilities and is capable of inducing lymphomas in cotton-top marmosets. EBV widely infects humans in early childhood without any serious diseases, followed by lifelong persistence of the virus. EBV is therefore a human ubiquitous viral agent with oncogenic potential. Knowledge of these characteristic features of EBV is essential to understand the functions of this virus in humans.

Multiplicity-dependent induction of viral capsid antigen in raji cells superinfected with epstein-barr virus

A quantitative analysis of Epstein-Barr virus (EBV)-induced early antigen (EA) and viral capsid antigen (VCA) syntheses was carried out in Raji cells superinfected with purified, concentrated P3HR-1 EBV. When the cells were exposed to the virus and assessed by immunofluorescence and immunoprecipitation, EA induction occurred significantly (17%) but not VCA (less than 1%), at a low-input multiplicity of infection (MOI) of 10 EBV DNA copies/cell. In contrast, at a high MOI of 500 EBV DNA copies/cell, the majority of cells were positive for both EA (82%) and VCA (61%). The latter VCA synthesis was accompanied by the replication of EBV DNA. Kinetic studies showed that EA induction was directly proportional to the dilution of the infecting virus, while VCA was made following three-hit kinetics. The implications of these results are discussed in relation to the heterogeneous nature of P3HR-1 EBV and a possible role of EA in VCA synthesis.

Immunologic detection of protein antigens after phenol-chloroform denaturation

The study of gene expression in cells and tissues often begins with phenol-chloroform extraction of the biologic material of interest for the isolation of intact mRNA. In most cases, the proteins denatured by phenol-chloroform are discarded. However, we found that the proteins recovered from phenol-chloroform extractions maintain their antigenicity. Therefore a method was developed for recovering the proteins from phenol-chloroform-denatured extracts that could be saved in lyophilized form until immunologic analysis. In this way, the RNA and the protein analysis can utilize exactly the same sample, and the biologic material can be saved. This is important because often these materials are available only in limited quantities. The method has been used to examine the sea urchin ets-related antigen and sea urchin ets-2 mRNA.

The ets genes in cells and viruses: implications for leukemias and other human diseases.

One of the most interesting scientific challenges of our time has been to understand the spectrum of oncogenic changes necessary to commit normal cells to a neoplastic life-style. On the molecular level, this enigmatic quest centers upon our understanding the essential ingredients needed to control cell growth (and its coordinate processes), as well as to maintain the normal differentiated pattern of expression for specific genes. Another, perhaps simpler, approach to this difficult challenge would be to examine the genetic changes resulting from the introduction of a known acute retroviral transforming sequence capable of eliciting a malignant cellular phenotype in vitro and neoplastic disease in vivo. Through the application of the powerful technique of recombinant technology application in the study of model retrovirus systems, the precise DNA sequences responsible for cellular transformation have been identified and studied. Thus, the employment of acute retroviruses as tools to identify a select cohort of genes capable of malignant transformation has been most fruitful. More important, from this study came a realization that this category of genes, the viral oncogenes, were capable of establishing and maintaining the transformed state, as well as the recognition that these genes were derivatives of a limited population of normal cellular genes, the proto-oncogenes, that can be captured and modified by the viral transduction process. These homologous genes, now numbering several dozen, are found distributed in the genomes of almost all vertebrates and of some invertebrate species, as well as of a few more primitive single-cell species.