Antibody and Antibody-Dependent Cellular Cytotoxicity Responses against the BamHI A Rightward Open-Reading Frame-1 Protein of Epstein-Barr Virus (EBV) in EBV-Associated Disorders

Abstract

Antibody-dependent cellular cytotoxicity (ADCC) is an important antiviral effector mechanism. ADCC to the protein encoded by the Epstein-Barr virus (EBV) BamHI A rightward open-reading frame-1 (BARF1) was studied by transducing Raji-tk- cells with the BARF1 gene using a retroviral expression vector. The transduced Raji cells expressed BARF1 on the cell surface, as determined by flow cytometry. Sera from chronic and acute infectious mononucleosis and nasopharyngeal carcinoma patients were found to contain antibodies that react with the BARF1 protein. When BARF1-expressing Raji cells were used as targets for ADCC, sera from several nasopharyngeal carcinoma patients demonstrated significant ADCC reactivity, whereas sera from healthy EBV-seronegative and -seropositive persons lacked such reactivity. BARF1-specific ADCC activity could be competitively inhibited with recombinant BARF1 protein. The level of anti-BARF1 antibody activity in sera of patients with EBV-associated diseases suggests that the BARF1 protein may serve as a target on EBV-infected cells for ADCC.

Establishment of a monkey kidney epithelial cell line with the BARF1 open reading frame from Epstein-Barr Virus

“…Since antibody dependent cell cytotoxicity (ADCC) response could be detected in the presence of NPC sera on a BARF1-transfected NKresistant Raji cell line from our laboratory (Tanner et al, 1997), it would be interesting, in consideration of BARF1 expression in NPC biopsies, to know whether BARF1-expressing epithelial cells could also be targets for speci®c killer cells.…”

“…Concerning immuno¯uorescence, Figure 5 shows the results obtained on acetone ®xed slides from primary Patas cells and BARF1 transfectants treated either with the NPC serum Tu 115 (A and B), or with rabbit polyclonal antibodies raised to a synthetic peptide from the BARF1 ORF (C and D). Tu 115 has long been used for the detection of the BARF1 protein by immuno¯uorescence, as well as immunoprecipitation and immunoblot (Zhang et al, 1988;Wei and Ooka, 1989;and de Turenne-Tessier et al, submitted), and our anti-peptide rabbit serum has been shown to recognize the BARF1 protein at the surface of transfected lymphoblastoid cells (Tanner et al, 1997). While no immuno¯uorescence was observed with either human EBV-negative or rabbit preimmune sera (not shown), both the Tu 115 serum and the anit-peptide rabbit antibodies gave a cytoplasmic and membrane response with BARF1 transfectants (B and D) but not with primary Patas cells (A and C).…”

“…The BARF1 mRNA in transfectants showed a larger size than both major BARF1 transcripts in B95-8 cells. This might represent a fusion transcript resulting from either retroviral vector splicing (Cepko et al, 1984;Tanner et al, 1997) and/or retroviral integration. BARF1 transcription was still detected in transfected cells after the 52th passage (about 1 year in culture).…”

“…After cell culture, the slides were washed with PBS and ®xed with cold acetone for 15 min. They were then incubated for 45 min at 378C with one of the following primary antibodies, at 1 : 200 dilution : TU 115, a human serum from tunisian NPC patient containing a high titer of antibodies to both early (EA) and late (VCA) EBV antigens (EA = 1 : 2560, and VCA = 1 : 2560); III-5, a polyclonal rabbit antiserum prepared against a synthetic peptide (produced by Socie te Bioatlantic, Nantes, France) corresponding to a presumed epitope (NGGVMKEKD, aminoacids 172 to 180) of the BARF1 protein, and previously used in immunoblot and Facs analysis (Tanner et al, 1997); and the anti-keratin monoclonal antibodies: K18 (Sigma) directed to keratin 18, K8-60 (Sigma) speci®c for keratins 10 and 11, or AE1/AE3 (Boehringer Mannheim) raised to both acidic and basic simple keratins.…”

Mitogenic activity of Epstein–Barr virus-encoded BARF1 protein

“…On the other hand, BARF1 has been shown to serve as a target for antibody-dependent cellular cytotoxicity (ADCC) (Tanner et al, 1997). These observations suggest that the biological activity of BARF1 might concern immunomodulation besides oncogenicity.…”