Journal of Medical Virology 68:370–377 (2002) Antibody Responses to Epstein-Barr Virus-Encoded Latent Membrane Protein-1 (LMP1) and Expression of LMP1 in Juvenile Hodgkin’s Disease Pauline Meij, 1 Marcel B.H.J. Vervoort, 1 Elisabeth Bloemena, 1 Tabitha E. Schouten, 1 Cindy Schwartz, 2 Seymour Grufferman, 4 Richard F. Ambinder, 3 and Jaap M. Middeldorp 1 * 1 Department of Pathology, VU Medical Center, Amsterdam, The Netherlands 2 Department of Oncology and Pediatrics, Johns Hopkins University, School of Medicine, Baltimore, Maryland 3 Department of Oncology, Johns Hopkins University, School of Medicine, Baltimore, Maryland 4 Department of Family Medicine and Clinical Epidemiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania A large group of juvenile Hodgkin’s disease patients (n ¼ 242, mean age 11.7 years, 75% [n ¼ 181] seropositive) was evaluated for anti- Epstein-Barr virus (EBV) antibody responses and the presence of EBV-encoded EBER-RNA and latent membrane protein-1 (LMP1)-protein expression in the tumor. The molecular diversity of anti-EBV antibody responses in Hodgkin’s disease patients with EBV-positive and-negative tumors was studied by enzyme-linked immuno- sorbent assay (ELISA) and immunoblot. Using purified recombinant LMP1 protein as antigen, the presence of antibodies to LMP1 was related to expression of LMP1 in the tumor cells and specific EBV-serological patterns. Antibodies to LMP1 were detected in 30% of the EBV-seropo- sitive Hodgkin’s disease patients. The presence of antibodies to LMP1 was not associated with a distinct anti-EBV antibody diversity profile (ELISA), but a significantly higher percentage of patients with antibodies to LMP1 had anti- bodies to ZEBRA and viral capsid antigen (VCA)- p18 (Immunoblot). Significantly more patients with an EBV-positive tumor had detectable anti- body responses to LMP1, but the presence of antibodies to LMP1 did not reflect the expres- sion of LMP1 protein in the tumor cells. Interest- ingly, all patients with the strongest antibody responses to LMP1 had EBV-negative tumors, suggesting immunological selection in vivo. J. Med. Virol. 68:370 – 377, 2002. ß 2002 Wiley-Liss, Inc. KEY WORDS: EBV; serology; immunologi- cal selection; immunity; onco- genesis INTRODUCTION Epstein-Barr virus (EBV) is a g-herpesvirus and the etiological agent of infectious mononucleosis. After primary infection, EBV establishes a lifelong persistent infection. More than 90% of the world population is infected with EBV. EBV is also associated with variety of lymphoid and epithelial malignancies, including Burkitt’s lymphoma, nasopharyngeal carcinoma, B- non-Hodgkin lymphomas in immunocompromised patients, 40 – 90% of Hodgkin’s disease cases, T/natural killer (NK)-cell lymphomas, and 10% of gastric carcino- mas. In the tumor cells of these malignancies, different EBV latent genes are expressed [Rickinson and Kieff, 1996]. In Hodgkin’s disease, a restricted set of latent genes is expressed, encoding the so-called latency type II antigens: Epstein-Barr nuclear antigen 1 (EBNA1), latent membrane proteins-1 and -2 (LMP1 and -2), two abundant small noncoding RNAs (EBER1 and -2), and BamHI-A rightward transcripts (BARTs) of unknown function [Herbst et al., 1991; Pallesen et al., 1991; Brooks et al., 1993; Niedobitek et al., 1997]. Hodgkin’s disease has a typical bimodal age distribution, with an early peak in children and young adults and in those >60 years. Individuals with a history of infectious mono- nucleosis and individuals with elevated antibody titers Grant sponsor: National Institutes of Health; Grant number: RO1 CA 47473; Grant sponsor: Pediatric Oncology Group; Grant sponsor: Children’s Cancer Group; Grant sponsor: National Cancer Institute; Grant numbers: CA30969, CA13539. *Correspondence to: Dr. Jaap M. Middeldorp, Department of Pathology, VU Medical Center, de Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. E-mail: j.middeldorp@vumc.nl Accepted 26 May 2002 DOI 10.1002/jmv.10213 Published online in Wiley InterScience (www.interscience.wiley.com) ß 2002 WILEY-LISS, INC.