Thomas B. Sculley

New Type B Isolates of Epstein--Barr Virus from Burkitt's Lymphoma and from Normal Individuals in Endemic Areas

All Epstein-Barr virus (EBV) isolates can be classified as type A or type B depending upon the identity of their EBV nuclear antigen (EBNA) 2 protein. The great majority of isolates examined to date encode an EBNA 2A protein like that of the reference type A strain B95-8. Type B virus strains, encoding an antigenically distinct EBNA 2B protein, have as yet only been rescued from rare Burkitts lymphoma (BL) cell lines of African origin (Jijoye, AG876). Our recent finding that type B isolates are less efficient than type A in in vitro transformation assays prompted us to determine (i) the relative contribution the two types of virus make to the incidence of BL in endemic areas of Africa (Kenya) and New Guinea and (ii) the relative incidence of infection with these two types in the normal population in these same areas. On the first point, EBNA 2 gene typing using specific DNA probes showed that four of ten recently established Kenyan BL cell lines and two of four BL cell lines from New Guinea carried type B virus isolates. To address the second point, spontaneous lymphoblastoid cell lines were established from the blood of normal virus carriers and typed for EBNA 2 at the protein level; a significant proportion (greater than 20%) of the normal population in both the above BL-endemic areas were infected with type B isolates. This is the first indication of the widespread nature of type B virus infection in any community and the first isolation of such viruses from a non-BL source. The reproducible size of the EBNA 2B protein encoded by all type B isolates irrespective of their geographical origin, and of the EBNA 1 protein encoded by all type B isolates from one area, contrasted markedly with the extreme variability in the size both of EBNA 2A and of EBNA 1 seen generally among type A isolates. This suggests that the number of type B virus strains in existence worldwide could be quite limited. Most importantly, the data suggest that type B viruses, despite their relatively poor performance in in vitro transformation assays, can contribute at least as efficiently as can type A viruses to the pathogenesis of BL.

Composite response of naive T cells to stimulation with the autologous lymphoblastoid cell line is mediated by CD4 cytotoxic T cell clones and includes an Epstein-Barr virus-specific component

We have approached the challenge of generating a primary T cell response to Epstein-Barr virus (EBV) in vitro by stimulating naive T cells with the autologous EBV-transformed lymphoblastoid cell line (LCL), a rich source of EBV-associated cytotoxic T lymphocyte (CTL) epitopes. Responsive T cells from three EBV-seronegative donors were cloned in agarose, phenotyped for T cell markers by flow cytometry, and their cytotoxic properties analyzed in the 51Cr release assay. Most clones (greater than 95%) expressed the CD4 phenotype and 59% of these clones showed cytotoxic properties. The dominant CTL response was specific for FCS-associated epitopes presented by FCS-grown autologous LCL target cells and was restricted by class II HLA antigens. Other clonal components included: (i) an EBV-specific response by HLA-restricted CD4 CTL clones that did not discriminate between A- and B-type EBV transformants; (ii) an EBV-specific response by an HLA-restricted CD4 CTL clone that discriminated between A- and B-type transformants, and (iii) a nonspecific cytotoxic response by CD3+,4+,8-, CD3+,4-,8-, and CD3-,4-,8- clones that were broadly allotypic or restricted to the lysis of K562 target cells. The EBV-specific CTL clones did not lyse the autologous EBV-negative B or T cell blasts and their specificity patterns of lysis were supported by the cold target competition data. These studies highlight the role of CD4 CTL in the establishment in vitro of a primary immune response to a human virus.

Reactions of sera from patients with rheumatoid arthritis, systemic lupus erythematosus and infectious mononucleosis to Epstein-Barr virus-induced polypeptides.

P3HR-1 and Ramos cells induced with sodium butyrate and 12-O-tetradecanoylphorbol 13-acetate were used in the protein immunoblot technique to identify Epstein-Barr virus (EBV)-specific antibodies present in sera from clinically normal individuals and patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and infectious mononucleosis (IM). Sixteen EBV-specific polypeptides were detected ranging in mol. wt. from 22,000 (22K) to 140K. Many of the sera contained antibodies to different subsets of these antigens, and a high proportion expressed autoantibodies which reacted with cellular components from an EBV genome-negative cell line. About 50% of the sera from each category reacted with the 44K to 48K and 36K and 38K early antigen (EA) components. A high proportion of the SLE sera (64%) were found to contain anti-EA antibodies, suggesting an association between EBV and SLE. Almost all of the EBV-seropositive sera examined contained antibodies against a 22K late antigen, but none of the sera from IM patients reacted with this polypeptide.

Failure of Epstein‐Barr virus‐specific cytotoxic T lymphocytes to lyse B cells transformed with the B95‐8 strain is mapped to an epitope that associates with the HLA‐B8 antigen

There are two types, A and B, of Epstein‐Barr virus (EBV) and B95‐8 represents the common type A laboratory strain. Herein, we show in a family study that paternal EBV‐specific cytotoxic T lymphocytes (CTL) generated in short‐term cultures following stimulation with the autologous B95‐8‐transformed lymphoblastoid cell line (LCL) or B cells freshly infected with the B95‐8 isolate did not lyse haploidentical B95‐8 LCL expressing the HLA‐A1‐B8,‐DR3 paternal haplotype. In contrast, the haploidentical B95‐8 LCL expressing the HLA‐A11,‐B51,‐DR7 paternal haplotype was strongly lysed. Moreover, paternal CTL generated in response to stimulation with the B95‐8 LCL expressing the haploidentical HLA‐A1,‐B8, ‐DR3 paternal haplotype included an allogeneic response against the maternal haplotype but no EBV‐specific response as shown by the poor lysis of the autologous LCL target cells. However, stimulation with the haploidentical HLA‐A11,‐B51, ‐DR7 paternal haplotype resulted in the generation of both an allogeneic and an EBV‐specific response. CTL clones were generated from two HLA‐B8+ donors in response to stimulation with the autologous type A LCL transformed with wildtype EBV. The clones were cross‐reactive for an immunodominant B95‐8‐associalcd peptide epitope that interacted with the HLA‐B8 allele but failed to lyse B95‐8‐transformed LCL largets unless the targets were pre‐coated with the exogenous peptide. A CTL clone that was initially stimulated with the autologous BL74 LCL lysed the spontaneous autologous LCL and spontaneous LCL from an HLA‐B8+ donor, but failed to lyse the B95‐8 LCL from that donor. The observed haplotype preference can be explained in terms of sequence variation between the B95‐8 and the corresponding wildtype epitope. Our findings may help to clarify the role of EBV in the pathogenesis of primary Sjögrens syndrome which is closely associated with HLA‐B8.

Epstein-Barr Virus Nuclear Antigens 1 and 2 in Burkitt Lymphoma Cell Lines Containing either ‘A’- or ‘B’-Type Virus

Epstein-Barr virus (EBV) has previously been classified into two different types according to the organization of the EB nuclear antigen 2 (EBNA2) gene region. Type A virus hybridizes with probes from B95-8 or M-ABA viruses and the B type virus with probes from the Jijoye virus strain. The substituted region in EBV type B codes for a different, but related EBNA2 antigen, named EBNA2B as opposed to EBNA2A. In this study Burkitt lymphoma cell lines, previously typed according to the EBV viral genomes they carry, as well as some matching lymphoblastoid cell lines were examined by immunoblotting for the expression of both EBNA1 and EBNA2 antigens. Variation in the molecular weight of EBNA1 indicated that both A and B virus types contained a variety of different virus isolates. EBNA2A was identified in all lines carrying A type viral genomes, but was not observed in any of the lines harboring B type virus. EBNA2B was identified in 4 of 10 Burkitt lymphoma lines carrying EBV type B.

Cytotoxic T lymphocyte discrimination between type A Epstein-Barr virus transformants is mapped to an immunodominant epitope in EBNA 3

An immunodominant Epstein-Barr virus (EBV)-specific cytotoxic T lymphocyte (CTL) epitope, represented by peptide 68, has been mapped to the EBV nuclear antigen, EBNA 3. The epitope is recognized by class I-restricted CTLs through HLA-B8 and is functionally active on type A but not type B lymphoblastoid cell lines (LCLs). Herein we show that peptide 68 is not expressed as a functional CTL epitope by type A LCLs infected with an EBV B95-8 isolate. CTLs from cultures stimulated with autologous type A IARC-BL74 or QIMR-WIL LCLs lysed autologous cells stimulated with phytohaemagglutinin (PHA blasts) and coated with exogenous peptide 68. No peptide 68-specific CTLs were generated in cultures stimulated with autologous type A B95-8 or type B AG876 LCLs. However, the B95-8 LCL coated with peptide 68 was effective in the induction of a peptide-specific CTL response. A peptide 68-specific CTL clone failed to lyse the B95-8 LCL, type B AG876 LCL and PHA blasts, although such targets were lysed when coated with peptide 68.

Identification of Epstein-Barr Virus-induced Polypeptides in P3HR-1 Cells by Protein Immunoblot

The protein immunoblot technique was used to identify Epstein-Barr virus-specific antigens present in sodium butyrate-induced P3HR-1 cells. Using sera from patients with either nasopharyngeal carcinoma or arthritis, 16 polypeptides were detected ranging in molecular weight from 22K to 140K. Each of the anti-EA-, anti-VCA-positive sera were found to contain antibodies to different subsets of the antigens. A 72K protein was identified which was consistent with the nuclear antigen (EBNA), and culturing cells in the presence of disodium phosphonoacetate allowed identification of 140K and 22K antigens as late viral products. Treatment of cells with sodium butyrate revealed that expression of some antigens increased in parallel with the time of incubation of the cells in butyrate while other antigens either appeared early and then decreased in intensity or were only present after a number of days of butyrate treatment. One of the antigens which decreased with the time cells were treated with butyrate was EBNA.

Characterization of Epstein-Barr Virus Nuclear Antigen(s) in Different Cell Lines by Radioimmunoelectrophoresis

Utilizing radioimmunoelectrophoresis, Epstein-Barr virus nuclear antigens (EBNA) with molecular weights of 67,000 and 70,000 were detected in NC37 cells, while the Raji cell line contained an antigen with a molecular weight of 70,000. Cell lines containing the B-95 strain of Epstein-Barr virus (EBV) expressed antigens with a molecular weight of 73,000. Three different B-lymphocyte cell lines, established by infection with the B-95 virus, also exhibited 73,000 molecular weight EBNA proteins. Cell lines containing the P3HR-1 strain of EBV demonstrated a single antigen with a molecular weight of 72,000. The results indicate that the molecular weight of EBNA is determined by the strain of infecting virus.

Patterns of reactivity of Epstein-Barr virus-specific T cells in A-type donor cultures after reactivation with autologous A- or B-type transformants

Regression endpoints were assessed in cultures from 11 Epstein-Barr (EB) virus A-type seropositive donors and 2 seronegative donors using A- and B-type EB virus preparations. In 9/11 of the seropositive donors, the resulting endpoints using A-type or B-type virus were similar and demonstrated a significant T cell response to both virus types. However, the regression endpoints for 2/11 seropositive donors were reproducibly higher with B-type virus compared with A-type virus, indicating a weak T cell response to the B-type virus compared with that to the A-type virus. Seronegative donor cultures showed no regression. The patterns of reactivity of bulk cultures of EB virus-specific cytotoxic T cells and T cell clones from selected seropositive donors were compared. Four of six donors showed evidence of a cytotoxic T cell response to A- and B-type autologous transformants while cytotoxic T cells from 2/6 donors (corresponding to those identified as lacking regression to B-type virus) lysed autologous targets infected with A-type but not B-type virus. The results show that while most A-type seropositive donors are capable of mounting a T cell response to A- and B-type virus, certain donors apparently lack B-type reactivity.

Western Blot Analysis of Antibody Specificities in Subacute Sclerosing Panencephalitis: Reactivity to Measles Virus Proteins Produced in Persistently Infected Cells

The specificity of serum antibodies from patients with subacute sclerosing panencephalitis (SSPE) and seropositive controls to measles virus proteins produced in acutely and persistently infected human cells was examined by western blot analysis. Sera from both SSPE patients and controls reacted to the H, N, and F1 virus proteins produced in acutely infected AV3 cells. However, while SSPE-derived sera reacted with the same proteins in persistently infected cells (AV3Al/MV), most control sera failed to react with the hemagglutinin protein produced in such cells (Hp). Most sera also reacted poorly with the M protein from either source, and the reactivity to the P protein was variable. Although the exact reason(s) for the different reactivities to the proteins were not determined, differences in antibody concentration did not appear to be responsible. The dramatic differences in the reactivity of SSPE and control sera to the Hp protein suggest that either the protein coevolves in persistent infections or multiple forms of the protein evolve in such infections and SSPE patients develop broad-spectrum humoral immunity as a consequence of exposure to them. Alternatively, over time there may be selective loss of some H-reactive antibody subsets by individuals who contract measles, but do not develop SSPE.