Oskar Skog
email: oskar.skog@klinimm.uu.se

Olle Korsgren

Gun Frisk

Modulation of innate immunity in human pancreatic islets infected 1 with enterovirus in vitro 2 3

Keywords: type 1 diabetes, IP-10/CXCL10, CVB, estradiol, gender difference type 1 diabetes, CVB, IP-10/CXCL10, estradiol, gender difference, 12 13

Modulation of innate immunity in human pancreatic islets infected with enterovirus in vitro

Oskar Skog, Olle Korsgren, Gun

. 17β-estradiol reduces enterovirus-but not poly(I:C)-induced IP-10/CXCL10 secretion from human islets. Fifty hand-picked human islets were cultured for 24 h, with or without the addition of 17β-estradiol (1 µM) to the culture medium, before infection with enterovirus (Adr) (A) or exposure to poly(I:C) (B). Accumulation of IP-10/CXCL10 in the culture media was analyzed by ELISA at 3 days post exposure to 1 µg/mL poly(I:C) or at 5-6 days post infection with enterovirus. Exposure of islets to 17β-estradiol did not affect their ability to respond to high glucose, as tested in a dynamic perifusion system (C). Isolated islets were cultured for five days either untreated (■), or in the presence of 1 (○) or 10 µM (▲) 17β-estradiol. The figure shows insulin release after perifusion with glucose (1.67, 16.7 and 1.67 mmol/L). 76x104mm (300 x 300 DPI)

4

understanding the innate immune response triggered by EV in human pancreatic islets is essential 64 since it provides an immediate response to a viral challenge, while also controlling the later 65 antigen-specific adaptive immune response, possibly contributing to the β-cell destruction in type 66

diabetes. 67 68

There is an excess prevalence of males in the type 1 diabetes, beginning at the time of puberty 69 [START_REF] Nyström | Risk of Developing Insulin-Dependent Diabetes Mellitus 421 21 (IDDM) before 35 Years of Age: Indications of Climatological Determinants for Age at 422 Onset[END_REF]. The female sex hormone 17β-estradiol has been demonstrated to protect 70 mice from streptozotocin-induced diabetes [Le [START_REF] May | Estrogens protect pancreatic beta-cells from apoptosis and prevent insulin-deficient 396 diabetes mellitus in mice[END_REF], and to protect human pancreatic 71 islets from apoptosis induced by proinflammatory cytokines in vitro [START_REF] Contreras | 17beta-339 Estradiol protects isolated human pancreatic islets against proinflammatory cytokine-340 induced cell death: molecular mechanisms and islet functionality[END_REF]. 17β-72 estradiol has also been shown to modulate inflammation by suppressing chemokines induced by 73 synthetic dsRNA in human endometrial epithelial cells [Lesmeister et al., 2005], and to protect 74 mice from CVB3-induced myocarditis [START_REF] Huber | Coxsackievirus B3-induced myocarditis: infection of females during the estrus 382 phase of the ovarian cycle leads to activation of T regulatory cells[END_REF]. In this study, the ability of 17β-estradiol to 75 modulate the innate immune response induced in human islets by infection with EV or exposure 76 to synthetic dsRNA was evaluated. Islet secretion of the chemokine interferon inducible peptide-77 10/CXCL10 (IP-10/CXCL10) was measured as a sign of innate immune activation. IP-78 10/CXCL10 has been shown previously to be induced by CVB infection of human islets [Berg et 79 al., 2006;[START_REF] Moell | Antiviral effect of nicotinamide on 408 enterovirus-infected human islets in vitro: effect on virus replication and chemokine 409 secretion[END_REF], induce β-cell death [START_REF] Schulthess | CXCL10 impairs beta cell function and viability in diabetes 442 through TLR4 signaling[END_REF], and to be elevated in 80 serum [START_REF] Nicoletti | Serum concentrations of the interferon-gamma-inducible chemokine IP-417 10/CXCL10 are augmented in both newly diagnosed Type I diabetes mellitus patients and 418 subjects at risk of developing the disease[END_REF][START_REF] Shimada | Elevated serum IP-10 levels observed in type 1 diabetes[END_REF] and in pancreatic islets [START_REF] Tanaka | Enterovirus infection, CXC chemokine ligand 10 449 (CXCL10), and CXCR3 circuit: a mechanism of accelerated beta-cell failure in fulminant 450 type 1 diabetes[END_REF]] 81 from patients with type 1 diabetes at onset of the disease. Synthetic dsRNA, 82 polyinosinic:polycytidylic acid (poly(I:C)), is used often to mimic viral infection. When poly(I:C) 83 is administered in vitro to β-cells extracellularly to the culture medium, it induces innate 84 immunity in a TLR3-dependent fashion [START_REF] Dogusan | Double-stranded RNA induces pancreatic beta-cell 347 apoptosis by activation of the toll-like receptor 3 and interferon regulatory factor 3 348 pathways[END_REF][START_REF] Garcia | Regulation 375 and function of the cytosolic viral RNA sensor RIG-I in pancreatic beta cells[END_REF], while MDA5 85 is essential for detection of poly(I:C) in mice [START_REF] Kato | Differential roles of MDA5 and RIG-I helicases in the 390 recognition of RNA viruses[END_REF]

5

of type 1 interferon in mice, protecting against EV infectcion [START_REF] Liu | Type I interferons protect mice against enterovirus 71 infection[END_REF]. In this study, the 87 ability of poly(I:C) to induce an antiviral state in human islets was evaluated. 88

Most present knowledge of virus-host interactions relies on studies in mice or in immortalized 90 cell lines, although innate immune signalling varies heavily between different human primary cell 91 types [START_REF] Lundberg | Key differences in TLR3/poly I:C signaling and cytokine induction by human 406 primary cells: a phenomenon absent from murine cell systems[END_REF]. In this study, primary human islets were used, enabling the study of 92 innate immune signalling in a relevant system; i.e. a model for the possible induction of type 1 93 diabetes by EV. The aims were to evaluate the ability of 17β-estradiol to modulate the innate 94 immune response induced in human islets by infection with EV or exposure to synthetic dsRNA, 95 as well as to evaluate the ability of poly(I:C) to induce an antiviral state in human islets by the 96 induction of genes involved in antiviral innate immunity. 97 (range 19.7-30.9). Islets of Langerhans were isolated in Uppsala, Sweden, using a protocol 103 approved by the Local Ethics Committee, as described previously [START_REF] Goto | Refinement of the automated method for human islet 379 isolation and presentation of a closed system for in vitro islet culture[END_REF]. Islet 104 preparations were of good quality and available for research only because the total islet volume 105 was too low for clinical transplantation. The islets were kept in culture bags (Baxter Medical AB, 106 Sweden) with 200 mL CMRL-1066 (ICN Biomedicals, Costa Mesa, CA) supplemented with 10 107 mM HEPES, 2 mM L-glutamine, 50 µg/mL Gentamycin, 0.25 µg/mL Fungizone (GIBCO BRL), 108 20 µg/mL Ciproxfloxacin (Bayer healthcare AG, Leverkusen, Germany), 10 mM nicotinamide, 109 and 10% heat-inactivated human serum at 37°C in 5% CO 2 and humidified air for 1 to 7 days. 110

The culture medium was changed on day 1 and then every other day until the islet experiments 111 were performed. 112 113 Virus 114 A 1000 TCID 50 /200 µL stock of Adr, an EV strain isolated from a boy at onset of type 1 diabetes 115 [START_REF] Elshebani | Effects on isolated 360 human pancreatic islet cells after infection with strains of enterovirus isolated at clinical 361 presentation of type 1 diabetes[END_REF] and genotyped as Coxsackie virus B5 (GenBank accession number 116 FJ374273), was used. Studies have shown that this strain replicates well in islet cells resulting in 117 a pronounced destruction of the islets, impaired islet function, [START_REF] Elshebani | Effects on isolated 360 human pancreatic islet cells after infection with strains of enterovirus isolated at clinical 361 presentation of type 1 diabetes[END_REF] and 118 induction of chemokines [START_REF] Moell | Antiviral effect of nicotinamide on 408 enterovirus-infected human islets in vitro: effect on virus replication and chemokine 409 secretion[END_REF]. All virus infections were performed by addition of 119 200 µL of the 1000 TCID 50 CVB5/Adr stock, yielding a virus concentration of 100 TCID 50 . 120 9

perifused with two glucose concentrations (1.67 mmol/l and 16.7 mmol/l and then 1.67 mmol/l 169 again). Fractions were collected at 6-min intervals for 120 min and the insulin concentrations 170 were determined by ELISA (Mercodia, Uppsala, Sweden). 171 172

RNA extraction and real time RT-PCR 173

Total RNA from islets stored in RNAlater (Qiagen) was extracted with RNeasy Mini kit with on-174 column DNaseI digestion (Qiagen). RNA quantity and quality were determined using 175 spectrophotometry and gel electrophoresis. 50 ng total RNA per sample were primed with 176 oligo(dT) 12-18 and reverse transcribed to cDNA with SuperScript II™ RT (Invitrogen) according 177 to the manufacturer's instructions. Control reactions containing all reagents except the reverese 178 transcriptase were included to confirm the absence of interfering genomic DNA. RNaseOUT™ 179 (Invitrogen) was added to the reaction to avoid RNA degradation. Real Time PCRs were run with 180

Power SYBR Green master mix (Applied Biosystems, Sweden) on a StepOnePlus™ Real Time 181 PCR system (Applied Biosystems, Sweden). Pre-designed gene-specific primer sets 182 (QuantiTect® Primer Assays, Qiagen) were used for detection of TLR3 and MDA5 cDNA. 183

Custom primers for IFNβ (sense 5'-TAGGCGACACTGTTCGTGTTG-3', antisense 5'-184 CCAAGCAAGTTGTAGCTCATGG-3'), and β-actin [START_REF] Welsh | Is there a 454 role for locally produced interleukin-1 in the deleterious effects of high glucose or the 455 type 2 diabetes milieu to human pancreatic islets?[END_REF], were ordered from 185 Cybergene AB (Stockholm, Sweden). The expression level of each gene was normalized against 186 expression of the housekeeping gene β-actin, and is presented as 2 -(Ct gene of interest)-(Ct beta-actin) (2 - 187 ∆Ct)

). PCR specificity was verified by melt curve analysis of the final PCR products. separate pathways [START_REF] Kato | Differential roles of MDA5 and RIG-I helicases in the 390 recognition of RNA viruses[END_REF]. To test if poly(I:C) could induce IP-10/CXCL10 in human 213 islets, and whether such an induction could be reduced by 17β-estradiol like that induced by 214 CVB5/Adr infection, human islets were cultured for 24 h with or without the addition of 17β- The present study highlights important pathways of induction of innate immunity in primary 255 human pancreatic islets. It confirms the previous finding that proinflammatory chemokine, IP-256 10/CXCL10, is induced by infection of human islets with EV in vitro [START_REF] Berg | Induction of the chemokine interferon-gamma-inducible 336 protein-10 in human pancreatic islets during enterovirus infection[END_REF]Moell et 257 al., 2009], which indicates viral recognition and activation of innate immunity. The female sex 258 hormone 17β-estradiol, was shown previously to modulate inflammation induced by poly(I:C) in 259 an endometrial epithelial cell line in vitro [Lesmeister et al., 2005], and by infection of mice with 260 EV in vivo [START_REF] Huber | Coxsackievirus B3-induced myocarditis: infection of females during the estrus 382 phase of the ovarian cycle leads to activation of T regulatory cells[END_REF]. In the present study performed on isolated human pancreatic islets, 261 17β-estradiol reduced IP-10/CXCL10 secretion induced by infection with EV, whereas it had no 262 effect on IP-10/CXCL10 secretion induced by exposure to poly(I:C), suggesting that in primary 263 human islets of Langerhans, poly(I:C) and EV induce secretion of IP-10/CXCL10 by two 264 separate pathways. These divergent results further emphasize the importance of being careful 265 when comparing data from studies performed in separate systems. Since poly(I:C) introduced to 266 cells extracellularly is likely taken up by endocytosis and recognized by TLR3 in endsomes, 267 while dsRNA produced intracellularly as an intermediate during EV infection is more likely 268 recognized by intracellular helicases [START_REF] Dogusan | Double-stranded RNA induces pancreatic beta-cell 347 apoptosis by activation of the toll-like receptor 3 and interferon regulatory factor 3 348 pathways[END_REF], it is tempting to speculate that 17β-269 estradiol interferes with a step in the pathway leading from MDA5 activation to IP-10/CXCL10 270 transcription that is not shared by the TLR3 pathway. However, untreated islets in this study 271 contained TLR3 mRNA levels close to, or below, the RT-PCR detection limit, and it is therefore 272 questionable whether the basal TLR3 expression can be solely responsible for sensing poly 15

This individual variation could not be explained by donor differences in the ability of islets to 301 transcribe IFNβ, MDA5 or TLR3 in response to poly(I:C) exposure, since, even though induction 302 levels of these genes varied, they did not correlate with the level of antiviral activity. Possibilities 303 that need further studying are that other genes could be involved and induced differently, or there 304 may be genotype-specific differences in the genes degraded by 2A pro . The finding that poly(I:C) 305 reduced replication of EV, also when added 24 hpi, could suggest that EV infection cannot 306 counteract poly(I:C)-induced production of type I IFN. However, intact human islets contain 307 between 1000 and 2000 cells, all of which may not be permissive to infection. Thus, it is highly 308 plausible that uninfected cells, in which poly(I:C) could induce IFN expression, were available 309 even 24 hpi when poly(I:C) was added. The antiviral effect of poly(I:C) in isolated human 310 pancreatic islets may suggest that local administration of poly(I:C) could be exploited as an 311 antiviral strategy. However, when virus-induced auto-destructive immune responses are 312 suspected to contribute to the pathology of a disease, such as in type 1 diabetes [Richardson et al., 313 2010], additional stimulation of innate immunity is unlikely to be beneficial. 314

315

To conclude, this study shows for the first time that the female sex hormone, 17β-estradiol 316 modulates induction of the antiviral chemokine IP-10/CXCL10 in human islets infected with a 317 CVB5 strain isolated from a patient at onset of type 1 diabetes, introducing one possible 318 contributor to the high male to female ratio in the incidence of this disease after puberty. 319 Interestingly, 17β-estradiol does not affect secretion of IP-10/CXCL10 induced by exposure of 320 islets to poly(I:C), suggesting that, in human islets, different signalling cascades are involved in 321 induction of chemokines by EV and synthetic dsRNA respectively. This highlights further the 322 importance of being careful when interpreting results of studies performed in different cell 323 systems. Finally, this study shows that poly(I:C) can induce a form of antiviral state in human 324 secretion from human islets. Fifty hand-picked human islets were cultured for 24 h, with or 476 without the addition of 17β-estradiol (1 µM) to the culture medium, before infection with 477 enterovirus (Adr) (A) or exposure to poly(I:C) (B). Accumulation of IP-10/CXCL10 in the 478 culture media was analyzed by ELISA at 3 days post exposure to 1 µg/mL poly(I:C) or at 5-6 479 days post infection with enterovirus. Exposure of islets to 17β-estradiol did not affect their ability 480 to respond to high glucose, as tested in a dynamic perifusion system (C). Isolated islets were 481 cultured for five days either untreated (■), or in the presence of 1 (○) or 10 µM (▲) 17β-estradiol. 482

F

The figure shows insulin release after perifusion with glucose (1.67, 16.7 and 1.67 mmol/L). 483 484

  Figure1. 17β-estradiol reduces enterovirus-but not poly(I:C)-induced IP-10/CXCL10 secretion from human islets. Fifty hand-picked human islets were cultured for 24 h, with or without the addition of 17β-estradiol (1 µM) to the culture medium, before infection with enterovirus (Adr) (A) or exposure to poly(I:C) (B). Accumulation of IP-10/CXCL10 in the culture media was analyzed by ELISA at 3 days post exposure to 1 µg/mL poly(I:C) or at 5-6 days post infection with enterovirus. Exposure of islets to 17β-estradiol did not affect their ability to respond to high glucose, as tested in a dynamic perifusion system (C). Isolated islets were cultured for five days either untreated (■), or in the presence of 1 (○) or 10 µM (▲) 17β-estradiol. The figure shows insulin release after perifusion with glucose (1.67, 16.7 and 1.67 mmol/L). 76x104mm (300 x 300 DPI)

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. Antiviral genes are upregulated in human islets upon infection with enterovirus or exposure to poly(I:C). Hand-picked human islets were stored in RNAlater on the indicated timepoints post infection with enterovirus (Adr) (A) or exposure to poly(I:C) (B). Expression levels of interferon-β (IFNβ), melanoma differentiation-associated gene 5 (MDA5), and Toll-like receptor 3 (TLR3) are presented as their mRNA expression relative to expression of the house-keeping gene beta-actin (2-∆Ct). The dotted line (A) shows the extracellular enterovirus titer as a measure of viral replication. 82x101mm (300 x 300 DPI)

  obtained from 19 organ donors (8 females and 11 males) free of any 100 known pancreatic disease, from transplantation units in Sweden, Norway and Finland. The 101 donors had an average age of 56±9.2 years (range 34-77) and an average BMI of 25.8±2.5 kg/m 2 102

  (Sigma-Aldrich, St. Louis, US) was dissolved in DMSO and added to 50 hand-123 picked islets per well at a final 17β-estradiol concentration of 1 µM and a final DMSO 124 concentration of 0.001%. Islets were exposed to synthetic dsRNA, by addition of poly(I:C) 125 (Sigma-Aldrich, St. Louis, US) at a final concentration of 1 µg/mL. 126 127Islet Experiments 128Isolated human islets had a purity ranging from 40 to 95%, and were purified further for islet 129 experiments by handpicking with a micropipette under an inverted light microscope. All 130 experiments were performed on fifty handpicked islets per well, cultured in 6-well plates 131 (Sarstedt, NC, USA) in 2 mL RPMI-1640 containing 5.5 mM glucose (SVA, Uppsala, Sweden), 132 supplemented with 5% foetal bovine serum and 2 mM L-glutamine. 133 134 Islets from 9 donors were cultured with or without addition of 17β-estradiol for 24 hours before 135 infection with CVB5/Adr or exposure to poly(I:C) (Sigma-Aldrich, St. Louis, US). Mock 136 infection with 200 µL vehicle (medium only) was used as negative control. Samples of culture 137 medium were collected from all wells, directly after infection/exposure and on the indicated days 138 post infection/exposure, and stored at -20 ○ C for subsequent virus titration and chemokine 139 measurement. Islets from three of these experiments were lysed at 5 days post infection (dpi) for 140 analysis of the intracellular chemokine content. 141 were exposed to poly(I:C) 24 h before or 24 h post infection with CVB5/Adr 147 or mock infection, and samples of culture medium from all wells were collected at the time of 148 infection and at 1, 2, 4, and 6 dpi, for virus titrations. Islets from three of these donors were 149 washed with PBS at 24 h post poly(I:C) exposure and stored in RNAlater (Qiagen) at -80 ○ C for 150 subsequent RNA extraction and real-time RT-PCR analysis. Islets from one donor were exposed 151 to poly(I:C) in six separate wells (50 islets/well), washed with PBS, and stored in RNAlater 152 (Qiagen) at -80 ○ C at 0, 0.125, 1, 3, 4, and 6 dpi. 153 154Analysis of Virus Replication 155Virus replication was determined by tissue culture infectious dose-50 (TCID 50 ) titrations of 156 samples of culture medium as described previously[START_REF] Moell | Antiviral effect of nicotinamide on 408 enterovirus-infected human islets in vitro: effect on virus replication and chemokine 409 secretion[END_REF]. The samples were 157 collected from the infected islet cultures on the indicated days pi. 158 159Cytokine Measurement 160Culture media, sampled on the indicated time points, from CVB5/Adr-infected, poly(I:C)-161 exposed, or mock-infected human islets, cultured with or without 17β-estradiol, were analyzed 162 for IP-10/CXCL10 content using Human IP-10 Cytoscreen ELISA (Biosource, Nivelles, 163 Belgium) with a detection limit of 2 pg/mL. 164 165Islet Function Test 166Insulin secretion in response to glucose stimulation was assessed in a dynamic perifusion system 167 on day 6 of culture with or without 17β-estradiol in the culture medium (n = 2). Islets were 168Innate immunity in EV-infected human islets Skog et al.

  in virus titre and chemokine secretion were analyzed with Wilcoxon rank-sum test. 191 P-values <0.05 were considered statistically significant. Data are presented as mean+SEM. 192 effect of the female sex hormone, 17β-estradiol, on EV infection of pancreatic islets, 196 isolated human islets were cultured with or without the addition of 17β-estradiol to the culture 197 medium prior to infection with CVB5/Adr. CVB5/Adr replicated well in the islets and induced 198 the secretion of IP-10/CXCL10, measured as accumulation of IP-10/CXCL10 in the culture 199 medium during the first 5 or 6 dpi (n=6) (Fig 1a). When islets were cultured in the presence of 200 17β-estradiol prior to infection, the accumulation of IP-10/CXCL10 in the culture medium (Fig 201 1a) was reduced significantly (p=0.037, (n=6)). The intracellular IP-10/CXCL10 content was 202 reduced correspondingly (n=3, data not shown). The reduced secretion of IP-10/CXCL10 was not 203 due to any antiviral activity, since 17β-estradiol did not affect viral replication (data not shown). 204 To ensure that the used dose (1 µM) of 17β-estradiol did not affect islet function, the ability of 205 islets, untreated or treated with 10 or 1 µM of 17β-estradiol, to respond to glucose stimulation 206 was compared. Insulin release in response to glucose was not affected by any of the tested 207 concentrations of 17β-estradiol (n=2) (Fig 1c)suggested that picornaviral dsRNA and poly(I:C) trigger antiviral responses partly by 212

  (I:C) 273 in human islets. Other pathogen recognition receptors, such as RIG-I or MDA5, may contribute 274 to the initial islet response to poly(I:C). Signalling pathways initiated by dsRNA binding to 275 MDA5, RIG-I, or TLR3 are mediated by NFκB, MAPK and IRF-3, but the response pattern 276 Innate immunity in EV-infected human islets Skog et al.

  Figure legends

Figure 2 .Figure 3 .

 23 Figure 2. Antiviral genes are upregulated in human islets upon infection with enterovirus or 485

John Wiley & SonsJournal of Medical Virology

estradiol and exposed to poly(I:C). Analysis of IP-10/CXCL10 that accumulated in the culture

Innate immunity in EV-infected human islets Skog et al.

Innate immunity in EV-infected human islets Skog et al.12 exposure to poly(I:C) either blocked (5 donors), reduced (3 donors), or did not affect (1 donor)

Acknowledgements 328 We thank the Nordic Network and the islet isolation team for islet preparations. 329 Page 20 of 29 John Wiley & Sons Journal of Medical Virology

 [START_REF] Garcia | Regulation 375 and function of the cytosolic viral RNA sensor RIG-I in pancreatic beta cells[END_REF]This study was financially supported by the EU 7 th Framework Program (DIAPREPP) grant 20 202013, the Swedish Medical Research Council grant K2006-32X-14035-06-3, the Family 21 Ernfors Fund, and the Gillbergska Foundation.

John Wiley & Sons

Journal of Medical Virology


Kartikawati Dewi

Christien Paramita

Hedy Fatmawati

Frank G Juwana

Jajah Van Schaijk

Sophia Mubarika Fachiroh

Jaap M Haryana

Prof. dr J M Middeldorp

Dewi ; Paramita

Dewi K Paramita

Christien Fatmawati

Hedy Juwana

Frank G Van Schaijk

Jajah Fachiroh

Sofia M Haryana

Jaap M Middeldorp
email: j.middeldorp@vumc.nl

Extracellular Domains in Nasopharyngeal Carcinoma Patients and Regional Controls

Keywords: Epstein-Barr virus, tumor antigens, humoral immunity, immunisation, extracellular domains Epstein-Barr Virus, nasopharyngeal carcinoma, IgG, IgA, LMP1, LMP2, BARF1

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Epstein-Barr virus (EBV) is a human γ-herpesvirus, that infects more than 90% of the world population, and is associated with a spectrum of diseases, including infectious mononucleosis (IM) [START_REF] Henle | Epstein-Barr virus specific diagnostic tests in infectious mononucleosis[END_REF]], Burkitt's lymphoma (BL) [START_REF] Epstein | Virus Particles In Cultured Lymphoblasts From Burkitt's Lymphoma[END_REF]], Hodgkin's disease [START_REF] Kapatai | Contribution of the Epstein Barr virus to the molecular pathogenesis of Hodgkin lymphoma[END_REF][START_REF] Wu | Detection of EBV gene expression in Reed-Sternberg cells of Hodgkin's disease[END_REF]], extranodal T/NK cell lymphoma [START_REF] Bruin | Detection of Epstein-Barr virus nucleic acid sequences and protein in nodal T-cell lymphomas: relation between latent membrane protein-1 positivity and clinical course[END_REF][START_REF] Van Gorp | Expression of Epstein-Barr virus encoded latent genes in nasal T cell lymphomas[END_REF], immunoblastic B-cell lymphomas in immunocompromised individuals [START_REF] Snow | Epstein-Barr virus: evasive maneuvers in the development of PTLD[END_REF], gastric carcinoma [START_REF] Van Beek | EBV-positive gastric adenocarcinomas: a distinct clinicopathologic entity with a low frequency of lymph node involvement[END_REF] and nasopharyngeal carcinoma [zur Hausen et al., 1970].

EBV persists for life in its human host after the primary infection and is well controlled by the host's immune system. Life-long immunosurveillance is reflected by the persistence of antiviral antibodies and virus reactive (cytotoxic) T cells [START_REF] Rickinson | Epstein-Barr virus[END_REF]. Different sets of proteins expressed during EBV's lytic and latent life cycle induce qualitatively and quantitatively different immune responses [START_REF] Fachiroh | Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects[END_REF][START_REF] Fachiroh | Singleassay combination of Epstein-Barr Virus (EBV) EBNA1-and viral capsid antigen-p18-derived synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA antibody levels in sera from nasopharyngeal carcinoma patients: options for field screening[END_REF][START_REF] Hislop | Cellular responses to viral infection in humans: lessons from Epstein-Barr virus[END_REF]. Similar to other herpes viruses, EBV reactivation can occur in patients with immune defects or immune suppression reflected by aberrant IgG/M/A antibody responses [START_REF] Meij | Restricted low-level human antibody responses against Epstein-Barr virus (EBV)-encoded latent membrane protein 1 in a subgroup of patients with EBV-associated diseases[END_REF]. Importantly, EBV may cause a number of malignancies of lymphoid and epithelial origin in both immunosuppressed and immunocompetent individuals, which are also reflected by aberrant antibody responses to EBV.

In the neoplastic cells of these malignancies, several EBV latent gene products are expressed corresponding to the latency type. Nasopharyngeal carcinoma is one of the latency type II tumors and is characterized by expression of EBNA1, LMP1, LMP-2A/ -2B proteins [START_REF] Brooks | Epstein-Barr virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1, LMP1, and LMP2 transcripts[END_REF][START_REF] Heussinger | Expression of the Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) in EBV-associated nasopharyngeal carcinoma[END_REF][START_REF] Khabir | EBV latent membrane protein 1 abundance correlates with patient age but not with metastatic behavior in north African nasopharyngeal carcinomas[END_REF] with co-expression of the epithelial oncogene BARF1 [START_REF] Brink | Nucleic acid sequence-based amplification, a new method for analysis of spliced and unspliced Epstein-Barr virus latent transcripts, and its comparison with reverse transcriptase PCR[END_REF][START_REF] Decaussin | Expression of BARF1 gene encoded by Epstein-Barr virus in nasopharyngeal carcinoma biopsies[END_REF][START_REF] Seto | Epstein-Barr virus (EBV)-encoded BARF1 gene is expressed in nasopharyngeal carcinoma and EBV-associated gastric carcinoma tissues in the absence of lytic gene expression[END_REF]. In view of potential immunogenicity of virus-encoded "nonself" proteins, it is surprising that LMP1, LMP2 expressing tumors occur in immunocompetent individuals, who are considered to have the capacity of mounting an effective immune response to these "non-self" proteins. CD8 + T cell responses to EBV latent antigens are skewed towards immunodominant epitopes derived from the EBNA3A, 3B, and 3C protein family.

Accompanying subdominant responses map to additional epitopes from the same EBNA3 family or from LMP2, and much less often to epitopes from EBNA2, EBNA-LP, or LMP1 [START_REF] Hislop | Cellular responses to viral infection in humans: lessons from Epstein-Barr virus[END_REF][START_REF] Khanna | Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia: implications for vaccine development[END_REF][START_REF] Murray | Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBVpositive malignancies[END_REF]. Only limited data are available for T cell responses to BARF1 [START_REF] Martorelli | Spontaneous T cell responses to Epstein-Barr virusencoded BARF1 protein and derived peptides in patients with nasopharyngeal carcinoma: bases for improved immunotherapy[END_REF].

Early work on EBNA1 as CD8 + T cell target showed that the internal 250 amino acid glycine-alanine repeat (GAr) protects the endogenously expressed EBNA1 from CD8 + T recognition [START_REF] Levitskaya | Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1[END_REF], as consequence from GAr-mediated interference with proteasomal degradation [START_REF] Dantuma | Avoiding proteasomal processing: the case of EBNA1[END_REF]. EBV has multiple evasion strategies in establishing and maintaining latency in the face of a CD8 + T cell response by switching-off antigen expression in those cells constituting the latent reservoir [START_REF] Thorley-Lawson | Epstein-Barr virus: exploiting the immune system[END_REF], by inducing T-cell anergy [START_REF] Dukers | Direct immunosuppressive effects of EBV-encoded latent membrane protein 1[END_REF] or Treg's [START_REF] Marshall | Regulatory T cells secreting IL-10 dominate the immune response to EBV latent membrane protein 1[END_REF] or by active interference with antigen processing and presentation during lytic replication [START_REF] Wiertz | Herpesvirus interference with major histocompatibility complex class II-restricted T-cell activation[END_REF]Zuo et al., 2008]. In addition to the EBV-driven immune evasion, nasopharyngeal carcinoma cells can release HLA-class II positive exosomes containing galectin 9, which can trigger apoptosis of mature Th1 cells [START_REF] Keryer-Bibens | Exosomes released by EBV-infected nasopharyngeal carcinoma cells convey the viral latent membrane protein 1 and the immunomodulatory protein galectin 9[END_REF][START_REF] Klibi | Blood diffusion and Th1-suppressive effects of galectin-9-containing exosomes released by Epstein-Barr virus-infected nasopharyngeal carcinoma cells[END_REF].

EBNA1 is well recognized as a major target for humoral immune responses.

However, only few studies addressed the role of LMP1 and LMP2 proteins as targets for humoral immune responses in detail. Antibody reactivity to LMP1 has been described in different EBV-related patient populations, including nasopharyngeal carcinoma, Hodgkin Disease, mononucleosis, and Burkitt Lymphoma patients, using different techniques, such as ELISA, immunoblot, and migration inhibition assays [START_REF] Chen | Expression of the Epstein-Barr virus latent membrane protein (LMP) in insect cells and detection of antibodies in human sera against this protein[END_REF][START_REF] Lennette | Antibodies to LMP2A/2B in EBV-carrying malignancies[END_REF][START_REF] Meij | Restricted low-level human antibody responses against Epstein-Barr virus (EBV)-encoded latent membrane protein 1 in a subgroup of patients with EBV-associated diseases[END_REF][START_REF] Meij | Antibody responses to Epstein-Barr virus-encoded latent membrane protein-1 (LMP1) and expression of LMP1 in juvenile Hodgkin's disease[END_REF][START_REF] Modrow | Characterization of two related Epstein-Barr virus-encoded membrane proteins that are differentially expressed in Burkitt lymphoma and in vitro-transformed cell lines[END_REF][START_REF] Rowe | Characterization of the serological response in man to the latent membrane protein and the six nuclear antigens encoded by Epstein-Barr virus[END_REF][START_REF] Sulitzeanu | Antibodies in human sera against the Epstein-Barr virus encoded latent membrane protein (LMP)[END_REF]. Previous studies indicated that LMP1 is a protein with a low immunogenicity for the humoral immune response in humans. In nasopharyngeal carcinoma only 7.5% (3/40) patients had low serum levels of antibodies directed to LMP1, whereas antibodies to LMP2A/2B were detected at low titer in about 40-60% of nasopharyngeal carcinoma sera from different ethnicity [START_REF] Lennette | Antibodies to LMP2A/2B in EBV-carrying malignancies[END_REF][START_REF] Meij | Restricted low-level human antibody responses against Epstein-Barr virus (EBV)-encoded latent membrane protein 1 in a subgroup of patients with EBV-associated diseases[END_REF]. Structurally, LMP1 and LMP2A/B are suggested to protrude from the cell surface via several conserved small loop domains connecting the transmembrane helices [START_REF] Modrow | Characterization of two related Epstein-Barr virus-encoded membrane proteins that are differentially expressed in Burkitt lymphoma and in vitro-transformed cell lines[END_REF]. However these loop-domains have not been studied as target for humoral immune response to date. Importantly, such anti-loop antibodies may have potentially important function in targeting complement and/or FcR-bearing killer cells to LMP1, 2 expressing tumor cells. A prior study of antibody to BARF1 in sera with EBVassociated diseases including nasopharyngeal carcinoma suggested that the BARF1 protein may serve as target on EBV-infected cells for antibody dependent cytotoxicity [START_REF] Tanner | 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[END_REF]. However, this study has not been confirmed and recent data indicate that BARF1 is rapidly and completely secreted from the EBV positive cells, et al., 2005;[START_REF] Seto | Epstein-Barr virus (EBV)-encoded BARF1 gene is expressed in nasopharyngeal carcinoma and EBV-associated gastric carcinoma tissues in the absence of lytic gene expression[END_REF].

In this study, antibody responses to EBV-tumor associated antigens LMP1, LMP2 and BARF1 in nasopharyngeal carcinoma patients were evaluated in details compared to healthy EBV carriers. Specific antibodies to the putative LMP1 and LMP2 extracellular loop domains were further developed and evaluated whether such antibodies can mediate complement killing of the LMP1 and LMP2 expressed cell lines, e.g. RAJI and X50/7. The results may provide a basis for understanding EBV tumor immune escape and indicate options for a novel approach to target extracellular domains of LMP1 and LMP2 expressing tumor cells.

MATERIAL AND METHODS

Sera from nasopharyngeal carcinoma patients and Healthy EBV Carriers.

Serum panels from histologically confirmed nasopharyngeal carcinoma patients (overall n=125) were collected from department of Ear, Nose and Throat (ENT), Dr. Sardjito General Hospital, Yogyakarta. Nasopharyngeal carcinoma sera were taken on the first visit of patients to the clinic, prior to treatment. Nasopharyngeal carcinoma staging was done by ENT examination and CT-scan and classified according to the 1996 criteria established by UICC (Union International Cancer Control). Sera from healthy EBV carriers (overall n=100) were obtained from the local red-cross blood bank. All sera were extensively analysed for reactivity to multiple EBV-encoded lytic cycle proteins in prior studies [START_REF] Fachiroh | Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects[END_REF][START_REF] Fachiroh | Singleassay combination of Epstein-Barr Virus (EBV) EBNA1-and viral capsid antigen-p18-derived synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA antibody levels in sera from nasopharyngeal carcinoma patients: options for field screening[END_REF][START_REF] Paramita | Native early antigen of Epstein-Barr virus, a promising antigen for diagnosis of nasopharyngeal carcinoma[END_REF][START_REF] Paramita | Evaluation of commercial EBV RecombLine assay for diagnosis of nasopharyngeal carcinoma[END_REF]. Nasopharyngeal carcinoma tissues from available formalin fixed paraffin embedded nasopharyngeal carcinoma tumor biopsies were examined the EBV status by EBER in situ staining (DAKO, PNA) and analysed the expression of LMP1 using S12 or OT21C moAbs base immunohistochemistry [START_REF] Meij | Antibody responses to Epstein-Barr virus-encoded latent membrane protein-1 (LMP1) and expression of LMP1 in juvenile Hodgkin's disease[END_REF].

Cell culture. The EBV positive RAJI Burkitt Lymphoma cell line, the in vitro EBV transformed B cell line X50/7, BJAB-LMP1 (kind gift of M. Rowe) and Daudi-LMP1 (kind gift of P Busson) were cultured in RPMI-1640 medium comprising 25mM Hepes and glutamin (Sigma, St.Louis, USA), 10% fetal calf serum (FCS, Hyclone, Etten-Leur, The Netherlands), 100 IU/ml penicillin and 50 µg/ml streptomycin (p/s) at 37°C in a humidified 5% CO2 atmosphere. Both cell lines express relatively high levels of LMP1 and LMP2 [START_REF] Bernasconi | Quantitative profiling of housekeeping and Epstein-Barr virus gene transcription in Burkitt lymphoma cell lines using an oligonucleotide microarray[END_REF]Meij et al., 2000b]. Insect cells were cultured as described below.

The BJAB-LMP1 cell is originally from EBV negative cell line BJAB transfected with LMP1 expression vectors. The LMP1-transfected clones of BJAB were established using a tetracycline-regulated vector system and were maintained in culture medium containing 1.5 mg/ml G418, 0.5 mg/ml hygromycin B, and 1 µg/ml tetracycline.

Tetracycline withdrawal induced LMP1 expression as previously described [START_REF] Floettmann | Cytostatic effect of Epstein-Barr virus latent membrane protein-1 analyzed using tetracycline-regulated expression in B cell lines[END_REF].

Recombinant proteins. The Baculovirus constructs expressing full-length LMP1, LMP2A, BARF1 and EBNA1 without the GAr domain were made under control of the polyhedrin promoter [Meij et al., 2000a;Meij et al., 2000b]. Sf9 cells were cultured to the log phase (1 x 10 6 cells/mL) and infected with one of the Baculovirus constructs. A high dose of 1-5 PFU/cell was used for recombinant protein production and cells were harvested at 48 hours post infection (pi). For immunofluorescence experiments infection at 1 PFU/cell for 48 hours was used leaving about 50% uninfected cells in the preparation, which were used as specificity control. Insect cells were cultured in serum-free SF900-II medium at 28ºC. EBV synthetic peptides. Immunodominant epitopes on EBV proteins were derived by computer prediction techniques, as described by [START_REF] Modrow | Characterization of two related Epstein-Barr virus-encoded membrane proteins that are differentially expressed in Burkitt lymphoma and in vitro-transformed cell lines[END_REF], using high scores for hydrophilicity, flexibility, and β-turn probability.

Peptides mimicking different domains of LMP1, LMP2 and BARF1 proteins were synthesized with a peptide synthesizer (433A; Applied Biosystem, Foster City, CA).

Peptides representing putative extracellular loop domains of LMP1 and LMP2 were also synthesized as circular peptides by inserting two cysteine residues at the ends forming a S-S bridges upon oxidation [START_REF] Timmerman | Rapid and quantitative cyclization of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces[END_REF]. Most peptides were extended at the N-terminus with additional lysine residues for improving solubility and coupling options. All peptides were purified in reverse phase high performance liquid chromatography (Beckman System Gold, Mijdrecht, The Netherlands). Peptide coupling to carrier proteins KLH or TTd was performed by standard techniques using commercial reagents (Sigma, St.Louis, USA). Peptide denomination and amino acids sequences are listed in table 1.

Monoclonal and polyclonal antibodies. Monoclonal (MoAb) and polyclonal (PoAb) antibodies were obtained by immunization of mice and rabbits with synthetic peptides or purified recombinant EBNA1, LMP1, LMP2 and BARF1 proteins expressed in insect cells. Female Chinchilla rabbits were immunized with either keyhole limpet hemocyanine (KLH) or tetanus toxoid (TTd) conjugated synthetic peptides or isotachophoresis isolated recombinant proteins [START_REF] Meij | Restricted low-level human antibody responses against Epstein-Barr virus (EBV)-encoded latent membrane protein 1 in a subgroup of patients with EBV-associated diseases[END_REF]. Before immunization pre-serum of each rabbit was drained from the ear. For primary immunization 1 mg antigen was mixed well with 1 ml Freunds Complete Adjuvant (FCA) and injected subcutaneously and intramuscularly. Each rabbit was coded as k followed with numbers (xx). Approximately 30 days (+/-1 day) after primary immunization 5 ml blood was drawn and coded as kxx/-1. First, second and third immunizations with Freunds incomplete adjuvant (FIA) were given with an interval of approximately 1 month. Booster blood samples (kxx/ -2, -3, 4, or -5) were taken 10 days after booster injection [Aarbiou & Middeldorp, unpublished]. Production of monoclonal antibodies to various intracellular domains of LMP1 and LMP2 was described before [START_REF] Fruehling | Identification of latent membrane protein 2A (LMP2A) domains essential for the LMP2A dominant-negative effect on B-lymphocyte surface immunoglobulin signal transduction[END_REF][START_REF] Meij | Restricted low-level human antibody responses against Epstein-Barr virus (EBV)-encoded latent membrane protein 1 in a subgroup of patients with EBV-associated diseases[END_REF]Meij et al., 2000b], MoAbs

Immunofluorescent staining on fixed recombinant antigen-expressing cells.

Cytospins were made with Sf9 cells either infected with wild type (wt) baculovirus or recombinant baculovirus. Slides were fixed in cold (-20ºC) acetone and preincubated in PBS containing 2% fetal calf serum (PBS/2%FCS) for 10 min. All washings were done three times in PBS/0.05% Tween-20 (PBSt). Antibody dilutions were made in PBS/2% FCS and incubated at RT. MoAbs were diluted in 100 -1000 times and human sera were used in a 1:25, 1:50, 1:100 and 1:200 and incubated for 1 h unless stated otherwise. After washing, the slides were incubated for 30 min with FITC-labeled rabbit anti-mouse Ig or anti human IgG secondary antibodies (DAKO, Glostrup, Denmark). Finally slides were counterstained for 5 min. with a 1:1 mix of DAPI and Evans blue or 1:500 ToPro3 (Partec, Heerhugowaard, The Netherlands) washed, dipped with mounting fluid Vectashield, sealed with a coverslip and evaluated with a Leica DMRB fluorescence microscope (Leica, Cambridge, England).

SDS-PAGE and western blot analysis.

Recombinant proteins were solubilized in standard Laemmli sample and boiled for 5 min. and separated in 10% acrylamide gels using the Mini Protean II system (BioRad, Hercules, USA) under reducing condition. Polypeptides were transferred from the gel onto 0.2 µm nitrocellulose (Schleicher & Schuell, s'Hertogenbosch, the Netherlands) by Western blotting (Mini-Trans blot cells, BioRad). After transfer, nitrocellulose sheets were washed with H 2 O and dried between filter paper and stored at 4°C until use. Marker proteins (Bio-Rad Low MW marker) were run on the side to indicate the molecular weight of polypeptides. Non-specific binding sites were saturated with blocking buffer (5% horse serum and 5% non-fat dry milk (Campina, Eindhoven, the Netherlands) in PBS pH 7.2) followed by incubation with Moab or PoAb at appropriate dilutions or sera at different dilutions made in blocking buffer. After washing with PBSt, specific bound IgG and IgA were detected with horseradish-peroxidase (HRP)-conjugated secondary antibody (Dako) in blocking buffer and HRP-activity was visualized by using 4-chloro-1-naphtol [START_REF] Fachiroh | Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects[END_REF]. Synthetic Peptide ELISA. Standard microtiter plates (Biobasic, Toronto, Canada) were coated overnight at 4°C with 135 µl of one of the peptides in a concentration of 1 µg/ml in 0.05M carbonate buffer, pH 9.6. Excess coating fluid was removed and non-specific binding sites were blocked subsequently for 1 h with 200ul/well of PBS/3% BSA at 37°C. Further incubations were performed for 1 h at 37°C followed by four washes with PBSt. Human sera were diluted 1:50 in ELISA sample buffer (PBSt; 0.1% Triton-X100, 1% BSA), followed by washing and incubation with HRPlabeled rabbit anti-human IgG (1:3000) andIgA (1:2000) (DAKO) diluted in conjugate buffer (PBSt; 0.1% (v/v) Triton-X100, 1% BSA and 2% normal rabbit serum).

Peptide-specific Moab or PoAb were diluted in ELISA sample buffer and detected with rabbit anti-mouse or swine anti-rabbit HRP conjugates (Dako) (both at 1:1000), respectively. HRP activity was detected using 3,3',5,5'-tetramethylbenzidine (TMB) (BioMerieux, Boxtel, The Netherlands) and the reaction was stopped by adding 1M H2SO4. The optical density was determined at 450 nm (Anthos 2001 reader, Anthos Labtec, Wals, Austria).

Membrane immunofluorescence on viable cells.

Log-phase grown RAJI, X50/7, Daudi-LMP1, BJAB-LMP1 and BJAB cell suspensions were used and all incubations were performed on ice with pre-cooled solution unless mentioned otherwise. Prior to immunofluorescence, lymphoprep purification was performed to remove dead cells from the suspension. Cells were transferred to FACS tubes at 0.5 x 10 6 cells/ 100 µL staining buffer [Hank balanced salt solution (HBSS); 0.1% (w/v) NaN3; 1.0% (w/v) BSA, fraction V)]. Subsequently, appropriate dilutions of PoAb anti-LMP1 loop-1 and -3 and LMP2 loop-2, and -5 were added and incubated for 20 minutes. Following two washes with staining buffer, fluorescein isothiocyanate-labeled swine anti-rabbit Ig (1:100) in FACS buffer was added and incubated for 20 minutes. For confocal microscopy cells were washed in HBSS, cyto-centrifuged onto glass slides and counterstained for 5 min. Microscopic analysis was done using a Leica TCS confocal microscope (Leica, Cambridge, England) and results were digitally stored. For FACS analysis, the cells were washed three times with staining buffer and resuspended in 100 µL propidium iodide solution. Data acquisition was performed on FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). Cell staining with anti-β 2 M antibody (Dako) served as positive control.

MTT assay. To evaluate the cytolytic capacity of anti-LMP1 and -LMP2 loop-specific antibodies, complement cytotoxicity studies were performed with MTT read-out (Cell Proliferation Kit I, Roche, Mannheim, Germany) using EBV, LMP1, 2 positive RAJI and X50/7 cell lines and appropriate controls. All incubations were performed at 37ºC and 5% CO2. Prior to the experiment, lymphoprep purification was performed to remove dead cells. Cells were placed on a 96 well plate at 10 4 cells /25 µl per well. Antibody anti-loop-1 and -3 LMP1 and anti-loop-2 and -5 LMP2 (1:3, 1:10, 1:50 and 1:250) were added, followed by the addition of 50 µl 30 times diluted rabbit complement (Innovative Research, Novi, Michigan, USA) and incubated for 2 hrs. As controls, cells were incubated with rabbit preserum or beta-2 microglobuline.

Subsequently 5 µl MTT labeling reagent was added. After 4 hrs, 50 µL solubilization reagent was added and after overnight incubation, the optical density was determined at 550-600 nm. Percentage of dead cell was calculated by using the formula below.

Percentage (%) cell death:

OD of untreated cells (blank) -OD of treated cells X 100%

OD of untreated cells (blank)

Declaration on human and animal studies. From all nasopharyngeal carcinoma patients in this study informed consent was obtained on the use of their serum/plasma and tumor samples for research purposes and all procedures were approved by the medical ethical committee of the Sardjito University Hospital, Gadjah Mada University, Indonesia. Sera from healthy controls were obtained from the archives and used with permission as detailed in previous studies [START_REF] Fachiroh | Singleassay combination of Epstein-Barr Virus (EBV) EBNA1-and viral capsid antigen-p18-derived synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA antibody levels in sera from nasopharyngeal carcinoma patients: options for field screening[END_REF]. In this study, we explore the antibody responses of nasopharyngeal carcinoma patients to individual recombinant proteins LMP1, LMP2 and BARF1. Antibody responses to the individual proteins were analyzed by indirect immunofluorescence and immunoblot techniques. Sf9 insect cells infected with recombinant Baculovirus expressing full-length LMP1, LMP2A and BARF1 were used as antigen (rLMP1, rLMP2A, rBARF1, respectively), mainly as described previously [START_REF] Meij | Restricted low-level human antibody responses against Epstein-Barr virus (EBV)-encoded latent membrane protein 1 in a subgroup of patients with EBV-associated diseases[END_REF]Meij et al., 2000a;Meij et al., 2000b]. A low MOI was chosen to leave 40-60% Sf9 cells uninfected, serving as internal specificity control in each experiment.

Recombinant EBNA1 deleted of the GAr (rEBNA1) was used as positive control and all sera and MoAbs were analysed in parallel on Sf9-cells infected with wild type Baculovirus (wtBac). Expression of LMP1, LMP2A, BARF1 and EBNA1 in the infected Sf9 cells was confirmed by staining with specific MoAbs to the individual EBV proteins (Figure 1). Human antibody staining was interpreted with the MoAb staining pattern as reference.

Overall, immunofluorescence results with nasopharyngeal carcinoma sera showed rather low (most 1:25-1:100) IgG reactivity to acetone-fixed rLMP1, rLMP2A and rBARF1 being detectable in 81.2%, 95.6% and 84.8% of tested sera, respectively, whereas IgG to rEBNA1 was present at higher titers (> 1:200) in 100% of the sera (n=32) (Figure 1J). In general, observed background reactivity with uninfected Sf9 cells and Sf9-wtBac was minimal and, when present, wt-Bac staining pattern could be discriminated from EBV antigen-specific staining. In simultaneous immunofluorescence analysis, IgA reactivity to rLMP1, LMP2A and BARF1 was observed at even lower titer (< 1:25) and at lower frequency in 40.9%, 54.5%, and 59.0% of nasopharyngeal carcinoma sera, respectively. IgA to rEBNA1 was observed at slightly higher titer (1:100) in 81.8% of the sera (n=22) (data not shown).

Subsequently, to reveal potential immune responses to possible linear epitopes in fully denatured EBV tumor proteins, a set of nasopharyngeal carcinoma sera (n=123) was tested for IgG and IgA reactivity by immunoblot analysis at dilutions of 1:50 using lysates of Sf9 cells expressing either rLMP1, rLMP2A, rBARF1 or rEBNA1. Figure 2A-C show that control MoAbs OT21C, 14B7 and 4A6 recognize clear bands at 63kD (LMP1), 54kD (LMP2a) and 30kD (BARF1). In contrast to immunofluorescence, immunoblot analysis revealed very low IgG responses to LMP1, LMP2A and BARF1 indicated by weak intensity of the specific protein band in 24.2%, 12.5% and 12.5% nasopharyngeal carcinoma patients, respectively. In general EBV-protein specific staining by immunoblot was only detectable using the lowest dilution (1:50), if detectable at all. IgG reactivity to rEBNA1 was observed at 94.9% of nasopharyngeal carcinoma patients (Figure 2D), and showed similar clear band at 55kD as revealed by MoAb OT1X (figure not shown).

None of nasopharyngeal carcinoma patients had detectable IgA response to the LMP1, LMP2A and BARF1 by immunoblot analysis, but a weak IgA response to EBNA1 was observed at 56.5% nasopharyngeal carcinoma patients. These data indicated that nasopharyngeal carcinoma patients, who have high-level antibody reactivity to multiple lytic cycle antigens and EBNA1 [START_REF] Fachiroh | Singleassay combination of Epstein-Barr Virus (EBV) EBNA1-and viral capsid antigen-p18-derived synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA antibody levels in sera from nasopharyngeal carcinoma patients: options for field screening[END_REF][START_REF] Fachiroh | Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects[END_REF],

are largely lacking potent antibody responses to tumor associated membrane antigens LMP1 and LMP2A, as well as BARF1, as examined with intact full length recombinant proteins.

LMP1 expression and antibody reactivity in nasopharyngeal carcinoma cases.

No relation was found between LMP1, LMP2 and BARF1 responses (when present)

with TNM stage of the tumor. In cases analyzed for serological responses to LMP1 by immunofluorescence assay (n=32) or immunoblot (n=125), the presence of LMP1 was detected at the tumor level using MoAb base immunohistochemistry. Results are shown in table 3A and 3B. Overall 80% of the nasopharyngeal carcinoma were found to LMP1 expression using immunohistochemistry. In cases having antibody reactive with LMP1 by immunofluorescence assay has positive correlation with LMP expression on the tumor (68.8% concordance), but by IB has negative correlation (33.6% concordance) (table 3).

Immunofluorescence and immunoblot may detect different epitopes, which is related to the level of denaturation of the antigen used, being minimal in immunofluorescence assay using aceton fixation, and maximal in immunoblot using SDS boiling. Therefore, it was decided to analyse this option in more detail. The functional importance of detecting antibody responses to LMP1 and LMP2 conformational domain will be of particular interest when expressed on the tumor cell surface. and β-turn probability as described before [START_REF] Meij | Restricted low-level human antibody responses against Epstein-Barr virus (EBV)-encoded latent membrane protein 1 in a subgroup of patients with EBV-associated diseases[END_REF][START_REF] Middeldorp | Epitope-mapping on the Epstein-Barr virus major capsid protein using systematic synthesis of overlapping oligopeptides[END_REF][START_REF] Modrow | Characterization of two related Epstein-Barr virus-encoded membrane proteins that are differentially expressed in Burkitt lymphoma and in vitro-transformed cell lines[END_REF]]. In addition, for LMP1 and LMP2 synthetic peptides were also created representing the extracellular loop 1 and 3 (connecting the 1 st to 2 nd and 5 th to 6 th transmembrane helix, respectively) and loop 2 and 5 (connecting the 3 rd to 4 th and 9 th to 10 th helix respectively), respectively (Figure 3A & 3B). Synthesis of cytoplasmic peptide domains of LMP1 have been described previously [START_REF] Meij | Restricted low-level human antibody responses against Epstein-Barr virus (EBV)-encoded latent membrane protein 1 in a subgroup of patients with EBV-associated diseases[END_REF]. For LMP1, peptide domain in circular conformation to more closely mimick the in vivo structure was used. Circular peptides were created by oxidation of the sulfide bridge in peptides OTP 405 and OTP 407 (Table 1) [START_REF] Timmerman | Rapid and quantitative cyclization of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces[END_REF].

Antibody

These peptides were used as antigens in indirect ELISA. Epitope-specific antibodies were generated by rabbit immunization using carrier proteins conjugated to the peptides. These newly developed antibodies were used as positive control in the ELISA (Figure 3). All human sera used were strongly responsive to VCA-p18 and EBNA1 synthetic peptides as described before [START_REF] Fachiroh | Singleassay combination of Epstein-Barr Virus (EBV) EBNA1-and viral capsid antigen-p18-derived synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA antibody levels in sera from nasopharyngeal carcinoma patients: options for field screening[END_REF].

Analysis of LMP1, 2 and BARF1 peptide-epitope specific antibody response by ELISA did not show major differences between nasopharyngeal carcinoma patients and healthy EBV carriers. When detectable, positive responses were marginal in most cases and the most significant response (62.9% positive) in nasopharyngeal carcinoma patients is confined to IgG against the intracellular C-terminus of LMP1

(Figure 3C). Overall analysis is depicted in table 2. and LMP2A revealed a heterogeneous intracellular staining pattern between individual cells of a cell population as described before [START_REF] Lennette | Antibodies to LMP2A/2B in EBV-carrying malignancies[END_REF][START_REF] Rowe | Characterization of the serological response in man to the latent membrane protein and the six nuclear antigens encoded by Epstein-Barr virus[END_REF]. The presence of LMP1 and LMP2A loop domains on the surface of those cell lines were detected by indirect fluorescence and FACS analysis using antiloop specific antibodies for LMP1 loop 1 and 3 and LMP2 loop2 and 5 (Figure 4 A-G). LMP1 clearly expressed on RAJI and X50/7 (5-15% of the cells), but clearly negative with Namalwa and BJAB. Figure 4 shows a representative fine patch-like staining observed on 15-20% of RAJI cells for loop1 and 3 LMP1 (Figure 4C,D) and BJAB as negative control (figure 4A). FACS analysis of RAJI cells using similar antibodies confirmed the low-plevel staining in a restricted number of Raji cells (Figure 4G). On cells artificially expressing LMP1 (Daudi-LMP1 and BJAB-LMP1) by vector transfection much higher staining was seen (20-50%; Figure 4E). Best LMP2 expression was seen for loop2 on X50/7 cells (Figure 4F). Rabbit antibody against β2M served as positive control and strongly reacted with >88% of all cell lines (Figure 4B This study demonstrated that immunization of rabbits using synthetic peptides mimicking the extracellular loop domains of LMP1 and LMP2 could generate specific anti-loop antibodies. This approach might be applicable to humans as well, aiming for therapeutic vaccination. Considering this option, the functional activity of the anti-loop antibodies was evaluated and complement mediated lysis was analysed using RAJI and X50/7 cell lines. Figure 5 [START_REF] Cheng | Assessing the risk of nasopharyngeal carcinoma on the basis of EBV antibody spectrum[END_REF][START_REF] Fachiroh | Singleassay combination of Epstein-Barr Virus (EBV) EBNA1-and viral capsid antigen-p18-derived synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA antibody levels in sera from nasopharyngeal carcinoma patients: options for field screening[END_REF][START_REF] Fachiroh | Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects[END_REF][START_REF] Karray | Comparison of three different serological techniques for primary diagnosis and monitoring of nasopharyngeal carcinoma in two age groups from Tunisia[END_REF][START_REF] Ng | Screening for family members of patients with nasopharyngeal carcinoma[END_REF][START_REF] Paramita | Native early antigen of Epstein-Barr virus, a promising antigen for diagnosis of nasopharyngeal carcinoma[END_REF].

However most of the EBV antigens used for diagnosis are not expressed in tumor cells, but are derived from sporadic cells entering the lytic stages infection accompanying the malignant process.

Besides EBNA1, which is expressed universally in all EBV tumor cells, latent EBV proteins such as LMP1 and LMP2 are regularly detected in nasopharyngeal carcinoma [START_REF] Heussinger | Expression of the Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) in EBV-associated nasopharyngeal carcinoma[END_REF][START_REF] Khabir | EBV latent membrane protein 1 abundance correlates with patient age but not with metastatic behavior in north African nasopharyngeal carcinomas[END_REF]. This study could detect LMP1 expression about 80% of the cases analysed (n=125), but we did not have access to the LMP2-reactive antibodies used by Heussinger. In addition, recent studies revealed the expression and secretion of BARF1 protein in nasopharyngeal carcinoma and gastric cancer in absence of lytic gene expression [START_REF] Seto | Epstein-Barr virus (EBV)-encoded BARF1 gene is expressed in nasopharyngeal carcinoma and EBV-associated gastric carcinoma tissues in the absence of lytic gene expression[END_REF].

Due to the expression of non-self viral proteins in the nasopharyngeal carcinoma tumor cells, the possibility appears that these proteins might become targets of immune response, aiding in protection. Previous studies demonstrated that EBNA1, LMP2A and to a lesser extend LMP1 can elicit virus-specific cellular immunity and are proposed as antigen for immunotherapy [START_REF] Comoli | Adoptive transfer of allogeneic Epstein-Barr virus (EBV)-specific cytotoxic T cells with in vitro antitumor activity boosts LMP2-specific immune response in a patient with EBV-related nasopharyngeal carcinoma[END_REF][START_REF] Comoli | Cell therapy of stage IV nasopharyngeal carcinoma with autologous Epstein-Barr virus-targeted cytotoxic T lymphocytes[END_REF][START_REF] Hislop | Cellular responses to viral infection in humans: lessons from Epstein-Barr virus[END_REF][START_REF] Swanson-Mungerson | Identification of latent membrane protein 2A (LMP2A) specific targets for treatment and eradication of Epstein-Barr virus (EBV)-associated diseases[END_REF]]. However, information on humoral immune responses to LMP1, LMP2A and BARF1 antibodies is rather limited [START_REF] Frech | Characterization of the antibody response to the latent infection terminal proteins of Epstein-Barr virus in patients with nasopharyngeal carcinoma[END_REF][START_REF] Lennette | Antibodies to LMP2A/2B in EBV-carrying malignancies[END_REF][START_REF] Meij | Restricted low-level human antibody responses against Epstein-Barr virus (EBV)-encoded latent membrane protein 1 in a subgroup of patients with EBV-associated diseases[END_REF][START_REF] Tanner | 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[END_REF]]. In the present study using immunofluorescence-analysis on acetone-fixed recombinant proteins expressed in insect cells, IgG antibodies to LMP1 and LMP2 were found in a significant number of nasopharyngeal carcinoma patients (81.2 % and 95.6%, respectively), albeit in low titers, but hardly in controls. This confirms and extends previous studies that used smaller numbers of patients and controls [START_REF] Frech | Characterization of the antibody response to the latent infection terminal proteins of Epstein-Barr virus in patients with nasopharyngeal carcinoma[END_REF][START_REF] Lennette | Antibodies to LMP2A/2B in EBV-carrying malignancies[END_REF][START_REF] Meij | Restricted low-level human antibody responses against Epstein-Barr virus (EBV)-encoded latent membrane protein 1 in a subgroup of patients with EBV-associated diseases[END_REF]. In all samples tested, the LMP1,2 responses were much lower compared to IgG-EBNA1. By using a similar method this study found that 84.8% nasopharyngeal carcinoma patients have a detectable but have been studied before using sera from chronic and acute infectious mononucleosis and nasopharyngeal carcinoma patients [START_REF] Tanner | 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[END_REF]. Using transduced RAJI cells they demonstrated significant antibody dependent cytotoxicity reactivity to BARF1-expressing RAJI cells in sera from nasopharyngeal carcinoma patients. However no study has yet confirmed antibody responses to BARF1 to strengthen these findings. In fact, BARF1 seems to be rapidly and completely secreted by BARF1 expressing cells, leaving little protein in or on the cells for detection [de [START_REF] De Turenne-Tessier | Expression and analysis of the Epstein-Barr virus BARF1-encoded protein from a tetracycline-regulatable adenovirus system[END_REF][START_REF] Seto | Epstein-Barr virus (EBV)-encoded BARF1 gene is expressed in nasopharyngeal carcinoma and EBV-associated gastric carcinoma tissues in the absence of lytic gene expression[END_REF]. The role of anti-BARF1

immune responses remains to be further established.

Immunoblotting confirmed the low-level antibody responses, being detectable at 24.2%, 12.5%, and 12.5% of nasopharyngeal carcinoma patients for LMP1, LMP2

and BARF1, respectively. The lower response rates compared to immunofluorescence-analysis may be due to the fact that antigens prepared by SDS-PAGE may have lost certain conformational epitopes. Again anti-EBNA1 antibodies were clearly detected, confirming the immunodominance of EBNA1. IgA-specific analysis showed similar low responses to LMP1, LMP2A and BARF1, but again clearly detectable responses to EBNA1. This demonstrates a lack of local mucosaspecific responses to the tumor-associated latent EBV membrane antigens, hinting at specific defects in their presentation to the immune system. These observations are clearly in contrast to the responses to the marginally expressed but highly immunogenic lytic antigens, to which abundant IgG and IgA antibody responses are detectable in the same nasopharyngeal carcinoma patients [START_REF] Fachiroh | Singleassay combination of Epstein-Barr Virus (EBV) EBNA1-and viral capsid antigen-p18-derived synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA antibody levels in sera from nasopharyngeal carcinoma patients: options for field screening[END_REF][START_REF] Fachiroh | Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects[END_REF][START_REF] Paramita | Native early antigen of Epstein-Barr virus, a promising antigen for diagnosis of nasopharyngeal carcinoma[END_REF]. Importantly, most (80%) nasopharyngeal carcinoma cases analysed showed LMP1 expression. This study found a positive correlation between LMP1 expression and Ab-resposes using immunoflyorescence analysis, but a negative correlation when using immunoblot (table 3). This may suggest that conformational epitopes, which are more reactive by immunofluorescence assay may be triggered in LMP1 positive tumor cases, whereas antibodies to linear (denatured) LMP1 are triggered differently (i.e. by cross presentation). The finding in nasopharyngeal carcinoma differs from previous observation in Hodgkin Disease, where LMP1 antibodies were most prevalent in EBV seropositive but tumor negative cases [START_REF] Meij | Antibody responses to Epstein-Barr virus-encoded latent membrane protein-1 (LMP1) and expression of LMP1 in juvenile Hodgkin's disease[END_REF].

The data from this study using EBV-recombinant proteins showed that nasopharyngeal carcinoma patients only have weak humoral immune responses to extended our previous studies and explored responses to defined peptide epitopes mimicking these domain [START_REF] Meij | Restricted low-level human antibody responses against Epstein-Barr virus (EBV)-encoded latent membrane protein 1 in a subgroup of patients with EBV-associated diseases[END_REF]. In rabbits, polyclonal epitope-specific antibodies were developed directed against distinct domains of LMP1, LMP2 and BARF1. These antibodies, having a high affinity for their epitopes in denaturated as well as in the native conformation on viable cells, were used as positive controls.

Using these anti-loop antibody reagents, demonstration of the presence and functional accessibility of extracellular loop domains of LMP1 and LMP2 was done, opening option as targets for therapeutic applications [START_REF] Middeldorp | Method for the identification of extracellular domains of Epstein-Barr virus (EBV) tumor-associated latent membrane proteins and selection of antibody reagents reactive therewith[END_REF]. However, in naturally EBV infected nasopharyngeal carcinoma patients and healthy EBV carriers these LMP1 and LMP2 loop domains seem to evade from immune recognition, as anti-loop antibody responses are mostly negative (Figure 3 I,F,I,J;

Table 2). The results of peptide-specific analysis confirm the presence of some antibody responses to the intracellular C-and N-terminal domains of LMP1 and LMP2, although only at a low levels (Figure 3C,D). Intrinsic properties of LMP1 and LMP2 and their limited expression in the plasma membrane may be responsible for the low immunogenicity. On the other hand, this study shows that LMP1 and LMP2

antibodies specifically directed against the extracellular loop domains can be generated by immunization of rabbits using related peptides and these antibodies can activate the complement system to kill LMP1 and LMP2 expressing cells. X50/7 cells can be killed by complement (50.4% and 59.4%) in higher percentage compared to RAJI cells (35% and 35.9%) most likely reflecting different level of LMP1 and LMP2 expression or differences in loop-accessibility. This requires further analysis but is in line with known LMP1 expression levels in different cell lines [Meij et al., 2000b]. Detection of extracellular domains requires viable cells and low temperature incubation to inhibit aggregation and internalization activity. A heterogeneous staining pattern of small patches of FITC-labeled anti-loop antibodies was demonstrated in the cell membrane. Also individual cells among the cell population showed a clear distribution (Figure 5). This corresponds with the known heterogeneous intracellular expression of LMP1, being abundant in some cells and barely detectable in others in the same culture [START_REF] Rowe | Characterization of the serological response in man to the latent membrane protein and the six nuclear antigens encoded by Epstein-Barr virus[END_REF] active or passive immunization, may be important to the prevention and treatment of nasopharyngeal carcinoma as a member of latency type II tumors. The finding that immunization of rabbits using these peptides can generate highly reactive epitopespecific antibodies opens new prospects for immunotherapy and vaccination of patients suffering from EBV associated tumors [START_REF] Middeldorp | Method for the identification of extracellular domains of Epstein-Barr virus (EBV) tumor-associated latent membrane proteins and selection of antibody reagents reactive therewith[END_REF]. LMP1 loop-3 (blue line) and using rabbit anti-b2M as positive control (green line).

F
Background staining with rabbit pre-serum shows low signal as indicated by the red line, which is similar to anti-loop antiserum on control BJAB cells (data not shown). LMP1 and 35% by anti-loop-2 and loop-5 LMP2A. Similar results or even higher killed-cells were obtained in other EBV carrying LCL lines but not in Namalwa, an EBV positive cell lines with LMP1 and LMP2 negative. Anti-B2M antibody was taken as a positive control, and used at larger dilution (1:5000) and therefore is not reaching >80% lysis. Preserum was used as negative control and to distinguish with specific binding of specific antibodies.

F

  Figure 2 part 1

  disputable target for antibody dependent cytotoxicity [de Turenne-Tessier

  and C-terminal domains of BARF1 were made in-house by standard procedures[Klarenbeek and Middeldorp, unpublished].

  All animal experiments were performed under approval of specific animal handling and immunization protocols at Organon Teknika, Boxtel, and VU University medical center, Amsterdam, The Netherlands.All experiments were conducted in compliance with local laws and institutional guidelines, and are in concordance with ethical standards of the Declaration of Helsinki.

  in nasopharyngeal carcinoma patients and healthy EBV carriers to recombinant EBV-encoded Tumor Associated Protein.

  Responses to defined extracellular peptide-epitopes of and LMP1, LMP2 and BARF1. To more precisely study the epitope specificity in the sera of nasopharyngeal carcinoma patients, defined synthetic peptides representing putative LMP1, LMP2 and BARF1 were created and used as antigen in ELISA. Cytoplasmic peptide epitopes of LMP1 and LMP2 and extracellular domain of BARF1 were selected for having high scores for hydrophilicity, flexibility

  and 4G). Staining pattern of individual viable cells was determined by confocal microscopy, revealing a heterogenous staining pattern similar to the cytoplasmic staining patterns, with some cells being negative, and others being positive and showing a patch-wise distribution of LMP1 and LMP2 related epitopes. This is the first demonstration that extracellular LMP1 and LMP2 related loop domains, can potentially function as targets for antibody-based therapy. Complement lysis by anti-LMP1 and -LMP2 loop-specific antibodies. Since LMP1 and LMP2 are expressed in multiple EBV tumors, including nasopharyngeal of the extracellular domains may have therapeutic potential.

  response to BARF1. Antibody responses against BARF1 protein

  and BARF1. However the potential importance of LMP1 and LMP2 as targets for immunotherapy, prompted us to further analyze the presence of antibodies in nasopharyngeal carcinoma patients directed to defined extracellular epitopes of LMP1, LMP2 and BARF1 in the form of synthetic peptides. No such information was available yet, and, in fact, the extracellular accessibility of domains of LMP1 and LMP2 has not been clearly demonstrated before. Therefore we

  Figure 1. Antibody detection by indirect immunofluorescence of acetone-fixed Sf9 cells infected with Baculo-EBNA1 (A, B), Baculo-LMP1 (C, D), Baculo-LMP2A (E, F), Baculo-BARF1 (G, H) and WT-baculovirus (I). Cells were incubated with OT1X (1:200, mouse anti-EBNA1) (A), OT21C (1:100, mouse anti-LMP1) (C), 14B7 (1:100, rat anti-LMP2A) (E), K150-3 (1:100, rabbit anti-BARF1) (G), and serum of NPC patients containing antibodies anti-EBNA1, LMP1, LMP2A and BARF1 (B, D, F, H respectively). Sf9-WT-Bac served as negative control incubated with either Mo/PoAbs or NPC serum (I). Similar pattern was observed as Baculo-BARF1 incubated using K150-3, when the cells were incubated with mouse MoAb 4A6 (mouse anti-BARF1). (J) Bar chart shows the summary of antibody responses of NPC sera to Sf9 cells infected with Baculo-EBNA1, -LMP1, -LMP2A and -BARF1 respectively, as detected by indirect immunoflourescence. For an indication of the level of antibody response, NPC sera were examined at various dilutions, as Indicated.

Figure 2 .

 2 Figure 2. Immunoblot analysis of SF9-baculo expressed recombinant proteins stained with monoclonal, mono-reactive polyclonal antibodies and NPC sera. (A) Baculo-LMP1 strips, (B) Baculo-LMP2A strips, (C) Baculo-BARF1strips, stained with Mo/PoAbs specific to the protein (strip 1 for each immunoblot series) and stained with NPC sera (line 2-end for each series). (A-OT21C) Baculo-LMP1 strip stained with OT21C MoAb showing band on 63kD, (B-14B7) Baculo-LMP2A strip stained with 14B7 PoAb showing band on 54kD, (C-K150-3) Baculo-BARF1 strip stained with K150-3 PoAb showing band on 30kD. (A1 -A23) Baculo-LMP1 strip stained with NPC serum (1:50), (B1-B23) Baculo-LMP2A strip stained with NPC serum (1:50), (C1-C19) Baculo-BARF1 strip stained with NPC serum (1:50). (D) Bar charts summary of antibody responses of NPC sera to Sf9 cells infected with Baculo-EBNA1, -LMP1, -LMP2A and -BARF1 using IB. For an indication of the level of antibody response, NPC sera were examined at 1:50 dilutions.

Figure 4 .

 4 Figure 4. Accessibility of extracellular loops of LMP1 on viable RAJI and BJAB cells as determined by specific anti-loop antibodies. (A) In all experiments EBV negative BJAB cells produced a complete negative membrane staining with the LMP1 and LMP2 loop-specific sera. (B) As positive control, RAJI and BJAB cells showed more than 88% positive staining using anti-β-2microglobulin. (C, D) A fine patch-like staining was observed on the surface of RAJI cells with anti-LMP1 loop-1 and loop-3 specific antisera (C, D respectively). Note that some cells in the culture were negative for Loop-1-3 expression, which were generally having small nuclei, representing non-cycling cells. (E) Anti-LMP1 loop-1, -3 specific antisera produced a similar, but more abundant patch-like staining on stably transfected BJAB cells induced for LMP1 expression from a teracyclin regulated promoter during 24 hours, similar as observed with LMP1 transfected Daudi cells (data not shown). (F) Antisera to LMP2 loop-2 and -5, produced a similar patched staining pattern on X50-7 cells, with somewhat larger patches than observed for LMP1. (G) Flow cytometry histogram comparing the levels of accessibility of anti loops LMP1 on Raji cells. Cells were gated for viability by 7-AAD exclusion. Staining was obtained with the indicated rabbit anti-LMP1 loop-1 (purple line), rabbit anti-

Figure 5 .

 5 Figure 5.Complement mediated cell lysis. RAJI cells were incubated with a dilution series of anti-loop antibodies (3x, 10x, 50x and 250x) and 30 times diluted complement solution. Percentage cell death is plotted against antibody dose. In 3x dilution of antiloop antibodies, approximately 49% RAJI cells were killed by anti-loop-1 and -3

Table 2A

 2A

	Accessibility of LMP1 and LMP2 loop domains on viable EBV transformed
	cells. LMP1 and LMP2 are transmembrane proteins, with six or twelve membrane-
	spanning domains, respectively, connected by intracellular and extracellular loops.
	The extracellular loop domains are potential targets for functional immune
	responses, and may mediate killing of EBV transformed cells via complement
	dependent cytotoxicity or killer cell dependent cytotoxic pathways known as antibody
	dependent cytotoxicity. To study the accessibility of the extracellular loops of LMP1
	and LMP2 on viable cells, evaluation of specific antibody recognition of these
	proteins expressed on viable RAJI, X50/7, Daudi-LMP1, BJAB-LMP1 and BJAB cell
	lines was done by FACS analysis and confocal microscopy. All cell lines except
	BJAB cells were positive for LMP1 and LMP2A mRNA as determined by reverse
	transcription PCR and by intracellular protein staining. For the latter, permeabilized F cells were treated with monoclonal antibodies OT21C and 14B7, recognizing the o r intracellular epitopes of LMP1 and LMP2A respectively (data not shown). Both LMP1
	P
	e
	e r
	R
	e
	shows the number of donors and patients having IgG responses to the individual peptides of tumor-v i e associated EBV proteins. IgG responses to LMP2 in healthy EBV carriers were lower compared to responses to LMP1 and BARF1. There was no difference in w
	LMP1 loop-peptide responses when using circular (created by S-S bridge oxidation)
	or linear peptides (data not shown). None of healthy EBV carriers had IgG responses
	to LMP2 loop peptides. Responses to C-terminus and N-terminus LMP2 are found
	only in 2.0% and 1.8% of healthy EBV carriers, respectively. About 5% of healthy
	EBV carriers had IgG response to subfragments of LMP1 and BARF1. Table 2B
	shows the number of nasopharyngeal carcinoma patients and healthy EBV carriers
	with IgA responses to peptides of LMP1, LMP2 and BARF1. IgA responses are
	lower as compared to IgG reponses, and most of the IgA responses in
	nasopharyngeal carcinoma patients can also be addressed the C-terminus of LMP1
	(27.4%).

  Zuo J, Thomas W, van Leeuwen D,Middeldorp JM, Wiertz EJ, Ressing ME, Rowe M. 2008. The DNase of gammaherpesviruses impairs recognition by virusspecific CD8+ T cells through an additional host shutoff function. J Virol 82(5):2385-2393. zur Hausen H, Schulte-Holthausen H, Klein G, Henle W, Henle G, Clifford P, Santesson L. 1970. EBV DNA in biopsies of Burkitt tumours and anaplastic carcinomas of the nasopharynx. Nature 228(5276):1056-1058.

	o r F o r F o r F o r F o r F o r
	P P P P P P
	e e e e e e
	e r e r e r e r e r e r
	R R R R R R
	e e e e e e
	v i e v i e v i e v i e v i e v i e
	w w w w w w

Table 1 . Amino acid sequence of LMP1, LMP2 and BARF1-derived synthetic peptides

 1

	Peptide	Amino acid sequence					AA
											position
	LMP1:
	OTP 415 (Loop1)	H-KKKCYIVMSDWTGGALLVLYC-NH2				41-56
	OTP 417 (Loop3)	H-KKKCALYLQQNWWTLLVDLLC-NH2				157-172
	OTP 81 (N-	H-MEHDLERGPPGPRRPPRGPPLSS-OH				1-23
	term/LMP1-A)
	OTP 75 (C-	H-GSSGSGGDDDDPHGPVQLSYYD-OH				365-386
	term/LMP1-D)
	LMP2:
	OTP 307 (Loop2) H-AICLTWRIEDPPFNSLLFAL-OH				183-202
	OTP 308 (Loop5) H-GSILQTNFKSLSSTEFIPNLFGM-OH				363-385
	OTP 309 (C-term) H-RCCRYCCYYCLTLESEERPPTPYRNTV-OH			461-497
	OTP 310 (N-term) H-SGSSGNTPTPPNDEERESNEEPPPPYEDPY-OH		35-64
	BARF1:
	OTP 539 (C term) H-LGPEIEVSWFKLGPGEEQVLIGRMHHDVIFIEWP-		40-80
			FRGFFD-OH
	OTP 541 (N term) H-DLSLPKPWHLPVTCVGKNDKEEAHGVYVSGYL-SQ-	187-231
			OH
	Table 2A.
	Positive IgG responses (%) in NPC and healthy EBV carriers to LMP1, LMP2
	and BARF1 peptides.
			LMP1 (%)			LMP2A (%)		BARF1 (%)
	Loop1 Loop3	C-	N-	Loop2 Loop5	C-	N-		C-	N-
				term	term		term	term	term	term
	HC	5.0	5.0	5.0	6.3	0	0	2.0	1.8		5.0	5.0
	NPC 14.5	38.7	62.9 11.3	21.2	24.2	8.9	21.9		3.2	21
	Table 2B.
	Positive IgA responses (%) of NPC and healthy EBV carriers to LMP1, LMP2
	and BARF1 peptides.
			LMP1 (%)			LMP2A (%)		BARF1 (%)
	Loop1 Loop3	C-	N-	Loop2 Loop5	C-	N-		C-	N-
				term	term		term	term	term	term
	HC	5.0	5.0	10.0	3.0	0	0	1.0	5.5		5.0	5.0
	NPC	1.6	0	27.4	3.2	18.2	3.2	20.0 17.8		6.5	11.3

Table 3A . Correlation between LMP1 expression using IHC and IgG reactivity to LMP1 recombinant proteins by IFA in NPC patients

 3A

		+	IHC -		Concordance
	IFA	+ -	21 5	5 1	68.8%
	Table 3B.
	Correlation between LMP1 expression using IHC and IgG
	reactivity to LMP1 recombinant proteins by IB in NPC patients
		+	IHC -		Concordance
	IB	+ -	27 78	5 15	33.6%

Acknowledgement. We thank the nasopharyngeal carcinoma team of Dr. Sardjito Hospital, Faculty of Medicine, Gadjah Mada University, Indonesia for support in collecting patient samples and dr. Bambang Hariwiyanto, (ENT specialist) and dr.

Harijadi (pathologist) for providing clinical and pathological data, and Rurry T Oktariza, Ika Dian Fitria, Beni Sulistyono (students) for doing some peptide ELISAs.

We also thank the EBV team in Dept. Pathology, VU University Medical Centre, Amsterdam, the Netherlands, for providing facilities and assistance. We thank to P. Busson and M. Rowe for the kind gift of Daudi-LMP1 and BJAB-LMP1 cell lines. This research was funded by the Netherlands Cancer Foundation (grant KWF-IN 2000-02, and 2004-17), the European Union (grant Asia-link, Contract no: ASI/B7-301/98/679-034), Ministry of Health Republic of Indonesia (Risbin Iptekdok 2006), and LPPM UGM 2007.