Characteristics of Viral Protein Expression by Epstein-Barr Virus

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Jun 20, 1995 - The frequency of Epstein-Barr virus (EBV) antigen-positive B cells in the peripheral .... mononucleosis had positive IgG and IgM titers against EB-VCA (EB-VCA–IgG ..... Klein, G., E. Svedmayr, M. Jondal, and P. O. Persson.
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Nov. 1995, p. 696–699 1071-412X/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 2, No. 6

Characteristics of Viral Protein Expression by Epstein-Barr Virus-Infected B Cells in Peripheral Blood of Patients with Infectious Mononucleosis H. J. WAGNER,1† M. HORNEF,1 J. MIDDELDORP,2

AND

H. KIRCHNER1*

Institute of Immunology and Transfusion Medicine, University of Lu ¨beck School of Medicine, Lu ¨beck, Germany,1 and Organon Teknika, Boxtel, The Netherlands2 Received 10 May 1995/Returned for modification 20 June 1995/Accepted 21 August 1995

The frequency of Epstein-Barr virus (EBV) antigen-positive B cells in the peripheral blood of patients with infectious mononucleosis compared with that for latently EBV-infected individuals was examined by immunocytochemistry. B cells positive for Epstein-Barr nuclear antigen (EBNA) 1, EBNA2, and latent membrane protein were frequently found in all peripheral B lymphocyte preparations from 25 patients suffering for 3 to 28 days from infectious mononucleosis by using monoclonal antibodies and the alkaline phosphatase antialkaline technique. There was a significant decrease in the number of positive B cells during the course of disease. EBNA1-positive B cells were detected in 0.01 to 2.5% of total B cells (median, 0.8%), EBNA2-positive B cells were detected in 0.01 to 4.5% of total B cells (median, 0.9%), and latent membrane protein-positive B cells were detected in 0.01 to 1.8% of total B cells (median, 0.5%), depending on the duration of clinical signs. In contrast, we did not find any EBNA1- or EBNA2-positive B cells in 2 3 106 peripheral blood B lymphocytes of 10 latently EBV-infected individuals, whereas aliquots of the same cell preparations were EBV DNA positive by a PCR assay. Therefore, it appears to be possible to detect infectious mononucleosis by immunocytochemical determination of latent EBV products, which might be of relevance for the diagnosis of EBV reactivations in immunosuppressed patients. phatase (APAAP) technique. We focused on the question of whether EBNA1-, EBNA2-, and LMP-positive cells were immunocytochemically detectable in freshly isolated peripheral B cells during the course of infectious mononucleosis compared with the latent state of infection. The detection of active infection by immunostaining of EBV antigen-positive peripheral blood B cells might be of importance for the diagnosis of reactivated EBV infections in immunosuppressed individuals.

The Epstein-Barr virus (EBV), a ubiquitous human herpesvirus with tropism for B lymphocytes, is the causative agent of infectious mononucleosis and has been associated with the development of Burkitt’s lymphoma, nasopharyngeal carcinoma, and B-cell lymphoma in immunosuppressed individuals (for a review, see reference 13). Furthermore, the virus has been detected in cases of Hodgkin’s disease (6) and peripheral T-cell lymphomas (21). The virus is transmitted primarily through the saliva to epithelial cells of the oropharynx, where all steps of virus multiplication are completed. From this initial site, the virus infects circulating B cells (13). After primary infection, the virus persists in the host for life, being detectable in a minimal number of peripheral blood B cells (24). Three distinct forms of latency depending on the different expression of six Epstein-Barr nuclear antigens (EBNA1, -2, -3A, -3B, -3C, and leader protein) and three latent membrane proteins (LMP1, LMP2A, and LMP2B) have been described for EBV-positive B-cell lines in vitro (18). Among these antigens, EBNA1 mediates DNA replication from the EBV latent origin, oriP, whereas EBNA2 and LMP play important roles in B-cell immortalization (12, 13). By means of an anticomplement indirect immunofluorescence assay, EBNA-positive cells were found in cytocentrifuge preparations of peripheral blood B lymphocytes from patients with infectious mononucleosis (10). The aim of our study was to modify this test to a sensitive one by using monoclonal antibodies and the alkaline phosphatase antialkaline phos-

MATERIALS AND METHODS Samples and controls. Twenty-five samples of heparinized blood (10 to 30 ml each) from 25 patients (18 males and 7 females, 2.5 to 42 years of age, median age of 20 years) with infectious mononucleosis were kindly provided by the Department of Otorhinolaryngology, University of Lu ¨beck Medical School, as well as by the hospital of the German Federal Armed Forces, Hamburg, Germany. The diagnosis of infectious mononucleosis was based on clinical signs, including pharyngitis, cervical lymphadenopathy, and fever as well as atypical lymphocytes in the blood smear, and was confirmed by serology. The duration of illness was 3 to 28 days. Blood samples (450 ml each) from 10 latently EBV-infected blood donors were collected in standard blood bags containing 63 ml of CPD anticoagulant solution (15.56 mM citric acid, 89.43 mM sodium citrate, 18.18 mM sodium dihydrogen phosphate, 130.03 mM glucose). After centrifugation at 4,000 3 g for 10 min, the buffy coat (about 60 ml) was recovered by an automatic separation device (Fenwall Optipress; Baxter S.A., Deerfield, Ill.). Cytocentrifuge preparations of cell line Raji, an EBV-infected cell line established from a Burkitt’s lymphoma (American Type Culture Collection CCL 86), served as positive controls for immunostaining of EBNA1, EBNA2, and LMP. A cytocentrifuge preparation of EBV-negative cell line BJAB, which was derived from an EBV-negative Burkitt’s lymphoma (11), was used as a negative control for immunostaining of EBV antigens. Serology. Serological testing for the detection of immunoglobulin G (IgG) and IgM antibodies to the EBV capsid antigen (EB-VCA) was performed by standard indirect immunofluorescence assays (Fresenius, Oberursel, Germany) and that for the detection of IgG antibodies to EBNA was performed by an anticomplement immunofluorescence assay (Fresenius). Patients with infectious mononucleosis had positive IgG and IgM titers against EB-VCA (EB-VCA–IgG $ 1:80 and EB-VCA–IgM $ 1:10) and were negative for EBNA-IgG (EBNAIgG , 1:10). Latently EBV-infected individuals were characterized with the

* Corresponding author. Mailing address: Institute of Immunology and Transfusion Medicine, University of Lu ¨beck School of Medicine, Ratzeburger Allee 160, D-23538 Lu ¨beck, Germany. Phone: 0049 451 500 2840. Fax: 0049 451 500 2857. † Present address: Department of Pediatrics, University of Lu ¨beck School of Medicine, Lu ¨beck, Germany. 696

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FIG. 1. EBNA1- (A), EBNA2- (B), and LMP-positive (C) cells in B-cell preparations of patients with infectious mononucleosis. Staining was done with a mouse monoclonal antibody by the APAAP technique. The counterstain was hematoxylin. Magnification, 31,000.

following diagnostic criteria: EB-VCA–IgG $ 1:40, EBNA-IgG $ 1:10, and EB-VCA–IgM , 1:10. Sample preparation. Mononuclear cells from whole blood of patients with infectious mononucleosis as well as from buffy coats of latently EBV-infected individuals were isolated by standard density centrifugation (Ficoll separation solution; Biochrom, Berlin, Germany). In order to enrich B cells from this fraction, other cells were lysed with a cocktail of monoclonal antibodies and complement (rabbit complement; Behring, Marburg, Germany). Specific monoclonal antibodies used in this cocktail were directed against erythrocytes, granulocytes, monocytes, T lymphocytes, and platelets. The lysed cells were separated from the B cells by density centrifugation as recommended by the manufacturer (Lympho-Kwik Isolation Reagent B-Cell; One Lambda, Los Angeles, Calif.) (15). Small aliquots of isolated B cells from each individual were analyzed by flow cytometry (Epics 2 flow cytometer; Coulter, Krefeld, Germany) after the cells were stained with a fluorescein isothiocyanate-conjugated anti-CD19 monoclonal antibody (Coulter Clone). The purity of the isolated B cells was 85 to 95% CD19 positive, with a median of 88%. B cells were suspended in 0.01 M phosphate-buffered saline, pH 7.2, and centrifuged onto acetone-cleaned glass slides with a cytocentrifuge (Cytospin-2; Shandon, Astmoor, United Kingdom) at 550 rpm for 5 min, giving a density of 105 cells per spot. Cytocentrifuge preparations were air dried for 8 to 12 h and fixed in acetone-methanol (1:1) for 90 s at 48C. Immunostaining (APAAP). As primary antibodies, an anti-EBNA1 mouse monoclonal antibody (2) was used in a dilution of 1:100, whereas an anti-EBNA2 mouse monoclonal antibody (PE 2; Dako, Hamburg, Germany) (26) and an anti-LMP mouse monoclonal antibody (CS 1-4; Dako) (17) were diluted 1:50 in 0.05 M Tris-buffered saline, pH 7.6. Slides were preincubated with normal rabbit serum (Dako) (30 min, room temperature [RT]) in order to prevent nonspecific binding of antibodies, incubated with 20 ml of primary antibody (30 min, RT), and washed twice in Tris-buffered saline. Further steps of the staining were carried out with reagents of the Universal Dako APAAP Kit (Dako) according to the manufacturer’s protocol. Briefly, slides were incubated with 25 ml of rabbit anti-mouse Ig (30 min, RT), washed twice in Tris-buffered saline, and subsequently incubated with APAAP complex (30 min, RT). These two steps were repeated once. The enzyme label was developed with Naphthol AS-MX Phosphate and Fast Red TR (Dako). Slides were counterstained with Mayer’s hematoxylin (Sigma, Deisenhofen, Germany), mounted in Immunomount (Shandon), and examined at a magnification of 3400 with a Leica Diaplan microscope (Leitz, Wetzlar, Germany). A total of 1,000 cells were counted for each antigen. Detection of EBV DNA in latently infected individuals by PCR. DNA was isolated from the B cells of 10 latently EBV-infected individuals by the procedure published by Miller et al. (14). In order to detect EBV DNA in these samples, we used a PCR method to amplify the repetitive BamHI-W region of EBV as previously described (24). This assay allows the detection of about 1 EBV

genome in 150,000 cells. The DNA of about 106 B cells was examined in eight separate reactions for each individual. In each reaction, 1 mg of DNA, representing approximately 150,000 cells, was assessed. Statistical analysis. The Spearman rank test was employed for the statistical evaluation of the correlation between the number of EBV antigen-positive lymphocytes in patients with infectious mononucleosis and the duration of clinical symptoms. A probability of ,0.05 was considered to be significant.

RESULTS Using the APAAP immunostaining technique described above, we found EBNA1-, EBNA2-, and LMP-positive cells in B-cell preparations of all samples from patients with infectious mononucleosis. EBNA1- and EBNA2-positive B cells showed a strong nuclear staining, and a granular cytoplasmic staining was found for LMP-positive B cells. These EBV antigen-positive B cells could be easily distinguished from negative cells, which did not show any specific staining (Fig. 1). During the course of infectious mononucleosis, we found that 0.8% of the cells in the fraction of enriched peripheral B cells were positive for EBNA1 (range, 0.01 to 2.5%), 0.9% were positive for EBNA2 (range, 0.01 to 5%), and 0.5% were positive for LMP (range, 0.01 to 1.8%). As can be seen in Fig. 2, a significant correlation between the number of EBV-positive B cells in the peripheral blood of patients with infectious mononucleosis and the duration of clinical illness was found for EBNA1 (P , 0.05 and r 5 20.44), with 1.2% 6 0.5% positive B cells during the first week and 0.6% 6 0.7% during the second and third weeks of illness. For EBNA2 (P , 0.05 and r 5 20.48), 1.5% 6 0.4% B cells were positive during the first week, 0.9% 6 1.4% were positive during the second week, and 0.6% 6 0.7% were positive during the third week of illness. For LMP (P , 0.01% and r 5 20.59), 1.0% 6 0.3% B cells were positive during the first week and 0.4% 6 0.3% were positive during the second and third weeks of illness. In all 10 latently EBV-infected individuals, EBV-related se-

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FIG. 2. Percentages of EBNA1-, EBNA2-, and LMP-positive B cells in the peripheral blood of patients with infectious mononucleosis in relation to the duration of illness. The number of samples for EBNA1 was 25, the number for EBNA2 was 23, and that for LMP was 22. Some subject values are obscured by identical values. The correlation coefficient and probability values were calculated by the Spearman rank test.

quences were found in the DNA of isolated B cells by means of PCR assay and subsequent Southern hybridization. Of the eight separate PCR reactions performed per individual, we found eight positive signals in one individual, seven positive signals in five individuals, six positive signals in two individuals, five positive signals in one individual, and one positive signal in one individual (Fig. 3; data for individuals 3 to 10 are not shown). Furthermore, we investigated parallel samples of 2 3 106 uncultured B cells of each EBV DNA-positive, latently EBVinfected individual for either EBNA1 or EBNA2 by means of replicate testing by the APAAP immunostaining technique (10 slides with 105 B cells each for both EBNA1 and EBNA2 were examined, giving a total of 200 slides for 10 latently EBVinfected individuals). Neither EBNA1- nor EBNA2-positive B cells were found in these cytocentrifuge preparations when the same staining procedure and appropriate positive controls consisting of B cells from patients with infectious mononucleosis and Raji cells were used. Thus, EBV-infected B cells appear to be present in the peripheral blood of EBV-seropositive healthy individuals who do not show protein expression of EBNA1 or EBNA2 at an immunocytochemically detectable level.

CLIN. DIAGN. LAB. IMMUNOL.

and LMP-positive B lymphocytes in cytocentrifuge preparations of all specimens from 25 patients with acute EBV mononucleosis. All three antigens were expressed in these positive B cells in their typical nuclear or cytoplasmic distribution as described by Jiang et al. (8). Thus, the EBNA-positive B cells circulating during infectious mononucleosis in vivo showed phenotypic characteristics of EBV-transformed lymphoblastoid cell lines established in vitro (18). As EBV antigens were found in all samples of patients with infectious mononucleosis and staining of positive B cells was strong, the immunostaining technique seems to be more sensitive to EBV proteins in B-cell preparations of EBV mononucleosis patients than the anticomplement indirect immunofluorescence assay (4, 9, 10). The significant decrease in the number of EBNA1-, EBNA2-, and LMP-positive B cells during the course of infectious mononucleosis might be explained by the development of a primary cytotoxic T-cell response during the active stage of the disease and the targeting of growth-transformed, lymphoblastoid cell line-like cells (20). Discussion of the expression of latent EBV genes on the mRNA level in peripheral blood lymphocytes in the lifelong carrier state has provoked controversy. Qu and Rowe (16) failed to find EBNA mRNA expression in uncultured peripheral blood cells of latently EBV-infected individuals by means of reverse transcription PCR analysis. The authors of this study did, however, detect mRNA expression of LMP2A in all four subjects investigated (16). As a consequence, a new type of latency, namely, latency IV, was proposed by Wolf et al. for the expression of EBV gene products in peripheral blood B lymphocytes of latently EBV-infected individuals in which LMP2A is exclusively expressed (25). In contrast, Tierney et al. found EBNA1 mRNA in three of six healthy EBV-seropositive persons and in another two individuals after a second round of amplification, LMP2A mRNA in four of six individuals, and Epstein-Barr virus early RNA1 in all six persons investigated by RT-PCR (22). At the protein level, we did not find any EBNA1- or

DISCUSSION The EBV-cell interactions during the active and latent stages of EBV infection remain largely unexplored, since virusinfected cells constitute a very small fraction of the total B-cell pool in vivo. During the acute phase of infectious mononucleosis, EBNA-positive B lymphocytes were detected in the blood by Klein et al. (10). The authors of this study used an anticomplement indirect immunofluorescence assay and sera of patients with positive anti-EBNA-IgG titers for detection of the EBNA complex in the cells and found 0.5 to 2.0% EBNApositive cells in a fraction of enriched B cells from five patients who had been ill for 11 to 60 days. Subsequent investigations either failed to reproduce these findings (4) or mentioned problems with the reproducibility of this method (9). We modified this method by using monoclonal antibodies (2, 17, 26) and the APAAP technique (3) and detected EBNA1-, EBNA2-,

FIG. 3. Detection of EBV DNA in latently EBV-infected individuals by a PCR assay. In one set of experiments, the DNAs of two individuals were examined together with three negative and one positive control DNAs. The 296-bp band shows the part of the BamHI-W region of EBV amplified by PCR. Aliquots of the same cell preparations were analyzed for EBNA1 and EBNA2 expression by means of monoclonal antibodies and the APAAP immunostaining method.

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EBNA2-positive B cells by immunostaining of cytocentrifuge preparations of 2 3 106 B cells from 10 latently infected individuals, although EBV DNA was frequently detectable in aliquots of the same cell preparation by means of PCR analysis. Our data indicate that EBNA1 and EBNA2 are more weakly expressed in EBV-infected cells during latency in vivo than they are during primary infection; if EBNA1 or EBNA2 mRNAs are expressed at all, their corresponding proteins are not detectable as antigens by means of the described immunocytochemical method. Therefore, it appears to be possible to specifically detect infectious mononucleosis by immunostaining of latent EBV proteins in cytocentrifuge preparations of peripheral blood B cells. Recently, we reported that a patient under immunosuppressive therapy after renal transplantation experienced an EBV reactivation that was demonstrated by immunocytochemical detection of EBV proteins in his peripheral B cells (7). The immunostaining method described above provides an estimation of viral activity directly at the level of protein expression and may be superior to serological methods for the detection of EBV reactivation, as serological methods require repeated determinations in order to confirm increasing antibody titers and cannot rule out heterotypic IgM antibodies. Further prospective studies are necessary to evaluate this new diagnostic method. The determination of EBV antigens may become a useful tool for the detection of patients at high risk of developing posttransplant lymphoproliferative disorder, just as the determination of cytomegalovirus antigens in peripheral leukocytes (23) serves as a diagnostic parameter (1). This is of special importance because early diagnosis may facilitate successful treatment of EBV-associated lymphoproliferative disease (5, 19). In addition, immunocytochemical determination of EBV antigens may provide new information concerning the clinical importance of EBV reactivation in immunosuppressed patients.

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ACKNOWLEDGMENTS We thank G. W. Bornkamm, Munich, for advice and B. Wustrow and K. Siegers, Department of Otorhinolaryngology, University of Lu ¨beck School of Medicine, as well as D. Georgi, hospital of the German Federal Armed Forces, Hamburg, for providing samples from infectious mononucleosis patients. REFERENCES 1. Bein, G., J. Hoyer, J. Steinhoff, L. Fricke, H. Machnik, and H. Kirchner. 1993. A longitudinal prospective study of cytomegalovirus pp65 antigenemia in renal transplant recipients. Transplant Int. 6:185–190. 2. Chen, M.-R., J. M. Middeldorp, and S. D. Hayward. 1993. Separation of the complex DNA binding domain of EBNA-1 into DNA recognition and dimerization subdomains of novel structure. J. Virol. 67:4875–4885. 3. Cordell, J. L., B. Falini, W. N. Erber, A. K. Ghosh, Z. Abdulaziz, S. Macdonald, K. A. F. Pulford, H. Stein, and D. Y. Mason. 1984. Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J. Histochem. Cytochem. 32:219–229. 4. Crawford, D. H., A. B. Rickinson, S. Finerty, and M. A. Epstein. 1978. Epstein-Barr virus genome-containing, EB nuclear antigen-negative B-lymphocyte populations in blood in acute infectious mononucleosis. J. Gen. Virol. 38:449–460. 5. Fischer, A., S. Blanche, and J. Le Bidois. 1991. Anti-B-cell monoclonal

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