Pergamon zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA E~ ~ nJarrmJofCrmcaVol.31A,No. 11,~ ~ . 1875-1878,1995 Elswier S&me Ltd hinted in GreatBritain 095%8049/95 $9.50+0.00 0959~8049(95)00354-l Original Paper Antibodies to LMP2A/2B in EBV-carrying Malignancies E.T. Lennette, G. Winberg, M. Yadav, G. Enblad and G. Klein Antibodies to the Epstein-Barr virus (EBV)-encoded membrane proteins, LMP2A and LMP2B, were assayed in 540 individuals, including 154 patients with nasopharyngeal carcinoma, 16 with African Burltitt’s lymphoma, 113 with Hodgkin’s disease, 14 with EBV-carrying gastric carcinoma, 14 with oral hairy leucoplakia (HIV+ patients), 37 with non-Hodgkin’s lymphoma, 49 with tumours of the head/neck, 19 with infectious mononucleosis, 62 with chronic illnesses with EBV titres consistent with re-activations, and 62 healthy controls. A novel assay, mouse monoclonal enhanced indirect ~~ofluo~~ence assay (MIPA) was designed and used to test the sera for antibodies to the LMP2A and 2B proteins, expressed in human keratinocytes. Antibody to both LMP2A and LMP2B was strikingly s:peci6c to NPC. Virtually all (99 of 101) of the LIMP2antibody positive individuals were NPC patients, 95% of whom had antibodies that reacted both with the LMP2A- and LMP2B-transfected indicator ceils, while the remaining 5% reacted only with the LMP2B expressing ceils. Key words: anti-LMPt, NPC, EBV malignancies EurJ Cancer, Vol. 31A, No. 11, pp. 1875-1878,1995 INTRODUCTION THREE DIFFERENT types of Epstein-Barr virus (EBV) latency havebeen identified,depencling on the expression pattern of the growth~~sfo~a~on-ass~iated EBNA (EBV-nuclear antigen) and LMP (latent membrane protein) complexes. EBV-carrying Burkitt’s lymphoma (BL) c~ells express only EBNA 1, and the same is true for BL-derived cell lines that have maintained a representative BL cell phenotype. EBV transformed B-lympho- cytes of non-neoplastic ori&n (l~phoblastoid cell lines, LCLs) and BL lines that have drifted to a more immunoblastic pheno- type during serial in vitro propagation express six nuclear antigens (EBNAl-6), together with the three membrane pro- teins LMPl, LMP2A and LMPZB. Un~erentiated NPC (nasopharyngeal carcinoma) cells express EBNAl but not EBNA2-6, and approximately 67% also express LMPl. In addition, LMPZA and LMP2B transcripts are regularly detected in the carcinoma cells, but there is no consensus as to their frequencies. Using nested PCR primers to increase assay sensi- tivity, we have recently found [l] that more than 90% of NPC biopsies express LMPl and LMP2B, while LMPZA and LMPZB Correspondence to E.T. Lennette. E.T. Lennette is at Virolab, Inc., 1204Tenth Street, Berkeley, Califor- nia 94710, U.S.A.; G. Winberg and G. Klein are at the Microbiology and Tumor Biology Center, Karolinska Institute, 517177 Stockholm, Sweden; M. Yadav is at the Department of Genetics and Cellular Biology, University of Malaya, Kuala Lumpur 22-11, Malaysia; and G. Enblad is at the University of Uppsala, Department of Oncology, S-751 85 Uppsala, Sweden. Revised 27 Feb. 1995; accepted 1 Mar. 19%. were co-expressed in only approximately 67% of the biopsies. Our findings were at variance to earlier reports [2-4] in which LMP2A transcripts were much more frequently detected than those of LMP2B or LMPl. There is even less information on the expression of the various LMPs in turnours. In NPC, LMPl protein is present in only 67% of the tumours detected by immunoblotting [5,6]. There is no information on the in situ expressions of LMP2A and LMP2B proteins. In 1993, Frech and colleagues, using LMP2A antigen (TPI) overexpressed in insect cells with baculovirus vectors, were able to detect LMPZA antibodies in approximately 40% of NPC patients of Mediterranean, Chinese and German ethnic origin. In contrast, all the HD (Hodgkin’s disease), BL, IM (Infectious mononucleosis) and control patients they examined were LMPZA antibody negative [7]. In this study, we attempted to confirm those fWings using LMP2A- and ZB-transfected human target cells to test 540 sera for LMPZ-specific antibodies in patients representing a variety of EBV-associated diseases and controls. Cells MATBRIALg AND METHODS The human keratinocyte cell line FEPlLI- 11 [8] was estab- lished by cotransfection of primary human foreskin kera- tinocytes with a plasmid clone of human papilloma virus 18. The coding sequences for the EBV LMP2A and LMP2B genes were derived from a LMPZA cDNA cloned in lambda gtl0 [9]. The genes were cloned into the retroviral vector pLNPOX [IO], which confers resistance to the drug G418. Recombinant retro- 1875