INTRODUCTION

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1. General IntroductionEpstein-Barr virus (EBV) was discovered in 1964 (111), initiated by the

finding of an unusual lymphoma in African children by Dennis Burkitt during his work in Mulago Hospital in Kampala Uganda around 1950s, which later become known as Burkitt's lymphoma (BL) (47, 302). In collaboration with Epstein, who already worked on the Rous sarcoma virus, research focused on the search of a virus causing BL. After a long failed investigation, they found a virus that differed from the known human herpesviruses in being unable to replicate in other cultured cells and in being non-reactive with antibodies to other human herpesvirus (205). In the laboratory in Phliladelphia, the virus inside BL cells was confirmed serologically as a new human herpesvirus (111).

EBV is classified into the gamma subfamily of potentially oncogenic herpesviruses and is also named human herpesvirus 4 (HHV-4). The gamma herpesvirus subfamily consists of the gamma 1 or Lymphocryptovirus (LCV) and gamma 2 or Rhadinovirus (RDV). EBV is the only human LCV, and is characterized by its capacity to drive B cell growth-transformation (205). Kaposi's sarcoma-associated herpesvirus (KHSV), also known as HHV-8, is the only human RDV (61).

EBV infects more than 90% of the world population via salivary contact. In developing countries, primary infection occurs in early infancy and is largely asymptomatic. In the West however, primary infection can be delayed until adolescence and in up to 25% cases can present as infectious mononucleosis (IM) (82, 112). Besides being the etiological agent of infectious mononucleosis, EBV is also associated with a variety of lymphoid and epithelial malignancies, including Burrkit's lymphoma, B-cell non Hodgkin lymphomas in immunocompromised patients, 40-90% of Hodgkin's disease cases, T/natural killer (NK)-cell lymphomas, 10% of gastric adenocarcinomas and nasopharyngeal carcinoma. In the tumor cells of these malignancies, different EBV latent genes are expressed (338).

2. The Virus and EBV genome structureAs other herpesviruses, EBV has toroid-shaped protein core which is

wrapped with DNA, a nucleocapsid with 162 capsomeres, a protein tegument between the nucleocapsid and envelope, and an outer envelope with external glycoprotein spikes (106, 107, 111, 183). The most abundant EBV envelope protein is gp350/220 (205). The schematic structure of herpesvirus can be seen in figure 1a.

EBV virions contain a linear double stranded 172 kb genome encoding approximately 85 putative open reading frames (ORF's) (94, 405). During latent infection EBV DNA is maintained in the nucleus of the host cells as an extra chromosomal circular episome. Twelve viral coding and non-coding genes can be expressed in different combinations during latent viral infection, while the remaining 70 major open reading frames are expressed during the replicative lytic cycle (8). The EBV latent genes on the double stranded viral DNA episome are shown in figure 1b.

The EBV genomic structure is characterized by short and long unique sequence domains (US and UL) that contain almost all the genome coding capacity, as well as internal (IR1-4) and terminal (TR) tandem and reiterated repeats. The

virus contains a linear genome, and upon EBV infection of a cell (typically by a single virion), the linear termini are joined via TR intracellularly to form circular, episomal DNA (338). The architecture of EBV genome can be seen in figure 1c.

The BamHI restriction fragments of B95.8 strain have been sequenced and constitute the first large pathogenic genome of which the full sequence was determined (20). The ORFs are named after BamHI restriction fragment containing the RNA start site and their leftward or rightward transcriptional orientation. BamHI-A is the largest fragment, BamHI-B the second largest, with BALF2 being the second leftward reading frame on the BamHI-A fragment and so on. The ORFs of latent EBV genes can be seen in Figure 1d.

Based on the isolates, EBV can be classified into EBV1 (type A) and EBV2 (type B). Prototypic type A and B viruses differ in the nucleotide sequence for EBNA2, 3A, 3B and 3C by 36%, 10%, 12% and 19% respectively (205). Divergence in the region around EBER genes has also been reported (15), but appears to be less consistence (354). Sequence variation was also found in LMP1 (392), LMP2 (48), EBNA1 (444), and BZLF1 (310). Other genes appear rather conserved, for example vIL-10 (186), EBER (15, 139, 344), and BARF1 [Hutajulu et al., Submitted.].

Figure 1a. The schematic structure of herpesvirus

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3. EBV infection & Latency

3.a. B cell infectionEBV mainly infects B lymphocytes through the binding of the major viral

envelope glycoprotein gp350 to the CD21 receptor on the surface of B cells. Penetration of virions into B cells requires a complex of three viral glycoproteins, gH, gL and gp42 (283, 445), with gp42 binding to human leucocyte antigen (HLA) class II molecules as a co-receptor (37, 146, 242, 446, 465). The binding of CD21 is followed by increased mRNA synthesis, blast transformation, homotypic cell adhesion, surface CD23 expression and IL6 production (140, 402, 403).

When the viral genome has been uncoated and delivered to the nucleus, Wp promoter expression initiates a cascade of events that lead to the expression of all of the EBNA proteins and the two membrane proteins. The EBNA-LP and EBNA2 proteins are the first proteins detected (5, 6). EBNA-LP has a role in enhancing EBNA-2 mediated transcriptional activation (205). EBNA2 is the major transcriptional regulator of viral gene expression, including EBV encoded LMP1 and LMP2, and auto-regulates the promoter for a long mRNA transcript that is alternatively spliced to encode EBNA2, LP, or the EBNA3 proteins (330, 351). EBNA2 also transcriptionally activates cellular genes by upregulating the expression of certain B cell antigen, CD21 and CD23, as well as viral genes (393, 441). EBNA2 plays an important role in immortalization by activating the Notch signaling pathway. EBNA2 may be a homologue of activated Notch and interacts with the RBP-Jk protein that provides sequence-specific DNA binding and transcriptional activation to the complex (156). EBNA2 also transactivates the viral C promoter (Cp), by inducing the switch from Wp to Cp observed early in B cell infection (205, 393, 441), driving the expression of EBNA3A, 3B and 3C (393, 395, 453). Studies using EBV recombinants have shown that EBNA3A and EBNA3C are essential for B-cell transformation in vitro, whereas EBNA3B is dispensable (341). EBNA3C induces the upregulation of both cellular (CD21) and viral (LMP1) gene expression (7), represses the Cp promoter (331) and might interact with pRb to promote transformation (318). Expression of EBNA3B correlates with the upregulation of vimentin and CD40 and downregulation of CD77 (365). The gene expression pattern, which is characterized by the expression nine viral latent genes under the control of EBNA2, is called growth program (latency type III) (Table 1) (411, 414). Together with EBNA-LP, EBNA2 is involved in G0-G1 transition (366), allowing the growth program activated B cells to become proliferating blasts and differentiate into resting memory B cell through the process of germinal centre reaction (248). Expression of EBNA3A and B will downregulate EBNA2 through competition for the binding of RBP-Jk protein (341). EBNA3C will activate G1 progression within the cell cycle. This will activate B cells to become growth-activated B cell blast not requiring further external signals. EBNA1 is co-expressed via Cp-transcription and provides essential signals for EBV genome maintenance in dividing cells (410). The only type of EBV-infected cells that are express the growth program is the infected naïve B cell in the Waldeyer's ring in healthy carriers (18, 168).

Figure 1b. EBV episome (464)

1d. Open reading frames (ORF) for EBV latent genes on the BamHI restriction endonuclease map of the prototype B95.8 genome. The Bam HI fragments are named according to size, with A being the largest and Z is the smallest. Lowercase letters indicate the smallest fragments. Note that the LMP2 proteins are produced from mRNAs that splice across the terminal repeats (TRs) in the circularized EBV genome. This region is referred to as N , to het

denote the heterogeneity in this region due to the variable number of TRs in different virus isolates and in different clones of EBV-infected cells (464)

Figure 1c. The architecture of EBV genome. EBV genome distinguished by short terminal repeat (TR) at both ends, and tandem array of internal repeats (IR). EBV clonality showed by its identical TR length. During infection, EBV enters host as linear DNA (uper figure), and forms episome in the host nucleus via the joining of TR (down figure). Lytic replication started from one or two origin of replication (oriLyt) and proceed repeatedly around the circular viral episome, producing long liner concotamer of viral genome (116, 205).

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Freshly EBV transformed B cells will migrate to the follicle, and subsequently downregulate Cp-driven EBNA2 and 3A-C, and switch to Qp-driven EBNA1, and EBNA2 independent expression of LMP1 and LMP2A gene expression, known as default program (latency type II) (Table 1) (19, 410, 411). The default program involves the expression of three latent membrane proteins (LMP), LMP1, LMP2A and LMP2B. These proteins are able to produce signals of the germinal center by mimicking T cell help and antigen-specific B cell receptor triggering to ensure the infected B cell to undergo germinal center reaction, proliferation, and become memory B cells without external signals (51, 207, 410).

The latently infected memory cells shut down the expression of viral protein by promoter methylation, except Qp-driven EBNA1 and the non-coding transcripts EBER1,2 and BARTs. Thus, the viral genome will be maintained in normal memory B cells (168) under the true latency program (latency I, EBNA1 only) (Table 1), and circulate primarily between peripheral blood and Waldeyer's ring (222).

Finally, latent EBV in memory B cells can be reactivated when B-cells become antibody producing plasma cells via antigen-stimulation and replicates at mucosal sites of the Waldeyer's ring in the nasopharynx yielding infectious virus that spreads in saliva to new hosts (414).

Table 1. Five Transcription Program Used by EBV to Establish and Maintain Infection

*Except where indicated, the types of cell are primarily restricted to the lymphoid tissue of Waldeyer's ring. Modified from Thorley-Lawson and Gross (414)

3.b. Epithelial infectionEBV is frequently found in certain epithelial pathologies, such as

nasopharygeal carcinoma (NPC), gastric adenocarinomas and oral hairy leukoplakia, indicating that the virus can infect epithelial cells in vivo (323). Most of the EBV+ epithelial cell lines have negative or extremely low CD21 expression, suggesting that the mechanism is CD21 independent (176).

The study in polarized human tongue and oropharyngeal epithelial cells shows that EBV enters these cells through multiple mechanisms, depending on the membrane domain that are distinct from those of lymphocyte infection. The apical membrane of polarized cells is the site of highly efficient cell-to-cell transmission from co-cultivated EBV infected lymphoblastoid cells. Cell-to cell contact of EBV-infected salivary cells with epithelial apical membranes suggest as the primary mode of EBV entry into oropharyngeal epithelium, since the apical site is resistant to cell-free EBV attachment and entry (425). Cell-free virions also infect oropharyngeal cells at the basolateral membrane, mediated in part through an interaction between b1 or a5b1 integrins and the EBV BMRF2 protein, which contain multi-span transmembrane domains and an arginine - glycine - aspartate (RGD) domain. This mechanism may occur when the epithelium is disrupted or injured. After initial infection, virus may spread directly across lateral membrane to adjacent epithelial cells. Virions are released from epithelial cells at both the apical and basolateral membranes (425). BMRF2 is an EBV glycoprotein with highly hydrophylic RGD motif in its major extracellular domain (425, 457). The RGD motif is present in extracellular matrix proteins, which bind to integrins and play a vital role in cell adhesion and cell-to-cell communication (123, 134, 135, 348). Viral proteins containing the RGD motif may serve as ligands for integrins leading to attachment of virions to host cells (419). BMRF2 is highly expressed in the upper differentiated layer of oral hairy leukoplakia (OHL) lesions, where the virus actively replicates (221, 311, 457). The promoter of BMRF-2 is up-regulated by mechanisms known to induce differentiation of epithelial cells (221). BMRF2 is associated with the viral envelope and binds to a3, a5, av and b1 integrin (425, 457). Antibodies to b1 family integrin and to the BMRF2 protein significantly block infection of EBV in polarized oral epithelial cells, which express high levels of integrins on the basolateral membranes. Treatment with the same antibodies does not affect EBV infection in B lymphocytes, which express lower levels of integrins (425). These findings indicate that interactions between EBV BMRF2 and EBV infection in oral epithelial cells but not B lymphocytes (456).

Another mechanism was suggested where the epithelial polymeric immunoglobulin receptor (pIgR) can mediate internalization of infectious virus-linked immunoglobulin A (IgA-EBV) complexes (367). Anti-EBV-gp350 IgA were used to prove the ability of EBV to infect epithelial cells (129, 426). The phenomena suggest that antibody-enhanced infection by pIgR-mediated transcytosis of pIgA-EBV through epitelium facilitates endogenous spread of EBV in long term virus carriers (129).

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3.c. Latent ReplicationDuring latent infection, the host cell is induced to proliferate and the EBV

genomes are maintained in the cells nucleus at a stable copy number as double stranded (ds), circular DNA episomes (460). These episomes undergo DNA replication semi-conservatively once per cell cycle only during S phase and are efficiently partitioned to the daughter cells during mitosis, alike to cellular chromosomes (2, 462, 463). Two viral components are required for the replication of the EBV genome, the oriP region on EBV DNA, which act in cis, and EBNA1 protein, which acts in trans (228, 463). EBNA1 is the only viral protein required for the replication of EBV latently-infected cells and accordingly it is found in all EBV associated malignancies (233). EBNA1 is essential for the maintenance and replication of the episomal EBV genome, through sequence-specific binding to the plasmid origin of viral replication, oriP (254, 463). OriP contains two functional elements, the dyad symmetry element (DS) and the family of repeats element (FR) (20, 334). The DS contains four EBNA1 binding sites and appears to be the initiation site of DNA replication (127, 332, 455). The FR contains 20 EBNA1 binding sites (332) and is involved in three viral processes. It activates DNA replication from the DS, enhances transcription from several promoters, and mediates the segregation of EBV episomes and oriP plasmids in dividing cells (128, 254, 334). EBNA1 has no helicase activity (122), which strongly indicates that the viral episomal replication depends on cellular replication machinery. It has been demonstrated that replication of oriP-containing plasmids is dependent on cellular initiation factors (104). The carboxy-terminal DNA-binding domain (DBD; aa451 to 640) of EBNA1 bind to two clusters of binding sites within oriP (FR and DS) (461). The recruitment of cellular replication system to DS requires the amino terminus of EBNA1 to contain glycine-arginine rich domains A (aa 33 to 89) and B (aa 328 to 378) (261, 356), which have similar function as AT hooks to associate with mitotic chromosomes and ensure stable replication and segregation of the oriP (357).

The EBNA1 AT-hook domains link the episome to mitotic chromosomes via heterotypic interaction with cellular protein p32/TAP with the A domain (65, 447), and EBP2 to B domain (364). The oriP plasmids can thus “piggyback” on the chromosomes, using the chromosome's centromere for its own partitioning into daughter cells (389).

Table 2. Function of EBV latent genes

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3.d. EBV-encoded non-translated RNA

3.d.1. EBV encoded RNAs (EBER1 and 2)EBER1 and 2 are small noncoding RNAs (167 and 172 nucleotide) that are

6produced in large numbers in EBV infected cells, at greater than 5 x 10 copies per cell (235, 396). EBER RNAs are expressed from their own promoter at 36 hr after infection of primary B lymphocytes in vitro, following other latent gene products (5, 396). The roles of EBERs in viral infection were unknown for long time, but recently several reports demonstrated that EBERs play a role in oncogenesis (398). A speculation about the role of EBERs in the normal EBV life cycle was proposed by Swaminathan (396).

EBERs are similar in size and organization to equally abundant small adenovirus, VAI and VAII, and are also transcribed by polymerase III and bind to cellular La protein. These similarities may indicate analogous function in infected cells. VA RNAs play an important role for adenovirus replication by rescuing cells from the shutdown of protein translation mediated by cellular kinase PKR, which induced by interferon and activated dsRNAs produced during replication of many viruses (133, 171). Mutant adenovirus with EBERs replacing VA RNAs, showed that EBER can functionally substitute the VA RNAs (30, 31). This evidence showed that EBER play a role in counteracting the antiviral effects of interferon and PKR activation in infected cells. In vitro studies demonstrated that EBER could directly bind PKR and inhibits its activity (74). PKR activation induces apoptosis by multiple mechanism including eIF2-phosphorylation and therefore PKR inhibition by EBERs gives protective effects against apoptosis (292).

Due to its expression in large amount in latently infected cells and all EBV-associated tumors, EBERs had been considered to have an important role in maintenance of transformation. Although in 1990s it was demonstrated that EBERs are not essential for transformation of B lymphocytes in vitro (397), recent findings indicated that EBERs do provide an advantage in viral transforming efficiency (459).

3.d.2. BamHI-A-rightward transcripts (BARTs) The EBV BARTs were first identified in NPC as multispliced transcripts

(370) and were later found in all EBV latency programs, including EBV infected B cells in healthy carriers and a wide range of EBV associated cancers. BART expression is low in B lymphocytes and high in epithelial tissues (50, 64, 137, 189, 371), suggesting that BARTs may be particularly important in epithelial malignancies. The finding of BARTs encoded miRNAs revealed the function of these transcript in modulating gene expression by forming imperfect or perfect complementary duplexes with their target mRNAs (22, 84). BARTs furthermore contain 3 ORF domains A73, RPMS1 and RK-BARF0 that may encode putative proteins (371). Although interesting functions have been described for these ORFs in artificial expression systems (415), the current consensus is that these ORFs are not translated into proteins in EBV infected cells (94, 415, 432). Therefore the main function of BARTs is considered being the generation of viral miRNAs.

3.d.3. EBV MicroRNAMicroRNAs (miRNAs) are small noncoding RNAs, generally 21-24 nt in

length, that can posttranscriptionally downregulate the expression of mRNAs bearing complementary target sequences (22). miRNAs are related to small interfering RNAs, approximately 22-nt-long noncoding RNAs that are generated by cleavage of double-stranded RNAs by the RNase III enzyme Dicer (22).

Viral miRNAs were first discovered in EBV (325), and now have been described for Kaposi's sarcoma-associated herpesviruses (KSHV), human cytomegalovirus (HCMV), mouse gammaherpesvirus 68 (MHV 68), herpes simplex virus I (HSVI) and simian virus 40 (SV40) (49, 145, 324, 350, 391). Five miRNAs were initially found in EBV, clustered into two genomic regions. One cluster of three miRNAs, miR-BHRF1-1, -2, -3 is proximal to the BHRF1 (Bam HI H Rightward Frame 1) ORF, and one cluster of miRNAs, miR-BART-1 and -2 is located within the BART (Bam HI A rightward transcript) gene (325). Recent study has identified an additional set of 18 EBV miRNAs, 13 of which map to a ~ 12 kb intronic region within the BART gene, which is deleted in the B95.8 strain (50).

There are some possibilities that viral miRNAs could directly act in oncogenesis. For instance, predicted targets of the EBV-encoded miR-BHRF1-1 including the tumor suppressor P53, in which mutations are found in 50% of all cancers (374). On the other hand, miR-BART1, another EBV-encoded miRNA that is coexpressed with miR-BHRF1-1 in latently infected B cell lymphomas, is predicted to target Bcl-2, an oncogene with function to block apoptotic cell death (80).

The EBV BARTs produce two clusters of miRNAs, 12 in cluster 1 and 15 in cluster 2. miR-BART2 is an individual miRNA. BART1 Cluster 1 miRNAs target the LMP1 3' UTR and negatively regulate LMP1 protein expression in NPC at posttranscriptional levels (250). The miR-BART2 serves as inhibitor for viral DNA replication through degradation of the mRNA for the viral DNA polymerase BALF5. BALF5 transcripts decrease in abundance when miR-BART2 is overexpressed. miR-BART2 is transcribed antisense to the BALF5 transcript and perfectly complementary to the BALF5 3' UTR (23). A quantitative PCR was recently described to measure miRNA levels in different EBV linked diseases (81).

4. Lytic replication

Reactivation from latency to lytic replication is essential for transmission of the virus from host to host (280). Lytic replication differs from the latent amplification state and is initiated within oriLyt (150). In contrast to latent replication, the viral lytic replication process is dependent on EBV-encoded proteins (119). EBV latent B cells can be activated to enter lytic replication by surface immunoglobulin cross-linking, cytokines or chemicals activating the transcription and function of two immediate early transactivators, Zta (BZLF1, Z, ZEBRA) and Rta (BRLF1, R) (253).

During the induction, like other herpesviruses, EBV gene expression follows a temporal and sequential order that is divided into three phases,

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immediate early (IE), early (E) and late (L) (Figure 2). Most of the early genes encode functions that are required for host cell survival and viral DNA replication, whereas late gene products are mainly structural proteins, which are expressed after viral DNA synthesis. The IE genes Zta and Rta are transactivators that are activated directly by cellular transcription factors induced by triggering the BCR, independent of the expression of other proteins. BZLF1 appears to be the major IE protein in EBV (33, 280).

The product of BZLF1, the ZEBRA protein, plays a role in both viral replication and transcription as an oriLyt binding protein and as transactivator. Therefore it can mediate the switch between latent and lytic forms of EBV infection and alone is sufficient to activate the EBV lytic cascade . Induction of the ZEBRA

promoter (Zp) appears to be the primary event in the ractivation of EBV. Zp promoter contain binding sites for multiple transcription factors . These factors coordinately initiate Zp activation, even from a methylated promoter, in response to lytic inducers and mediate ZEBRA transcription. ZEBRA binds to its own promoter and activates Rta promoter. ZEBRA and Rta then function synergistically to activate E and L promoters .

EBV genomic replication is initiated by sequence specific binding of ZEBRA to oriLyt followed by the recruitment of the cellular replication machinery to this site . There are seven viral encoded replication proteins, which essential for

oriLyt-dependent DNA replication, the BZLF1 (oriLyt binding protein), BALF5 (DNA polymerase), BALF2 (single-stranded DNA binding protein), BMRF1 (polymerase accessory factor), BBLF2/3 (primase associated factor), BBLF4 (helicase) and BSLF1 (primase) . During the induction, these replication proteins are expressed and clustered to newly replicated EBV DNA at discrete sites in the nuclei, termed replication compartment . The EBV core replication proteins are linked to each other through a series of contacts and with the viral oriLyt binding protein ZEBRA, which in turn binds to multiple sites within oriLyt . These proteins together have potential to open up the duplex DNA in the origin region and and synthesise RNA primers. The BALF5 Pol catalytic and BMRF1 Pol accessory subunits form a heterodimer to function as the Pol holoenzyme, which interact with BBLF4/BSLF1/BBLF2/3 complex . This interaction may play an important role in bringing the polymerase into the prepriming complex to initiate DNA synthesis by reducing its affinity for BZLF1, allowing the polymerase-helicase-primase complex to migrate away from oriLyt to the replication forks. The six viral replication proteins appear to all work at the replication fork in forming the replication machinery .

The final step in lytic cycle is initiated by the production of 100 to 1000 copies of viral DNA, which is followed by the transcription and translation of RNAs encoding the late viral protein including structural genes as BNRF1, BFRF2/3, BLRF1/2/3 and BcLF1 (major capsid protein), BFRF3 (VCA-p18, small capsid protein). BdRF1 (VCA-p40, scaffold protein) and virion envelopment i.e. BLLF1 (encoding gp 350/220), BXLF2 (encoding gp85), BZLF2 (encoding gp42), and BKRF2 (encoding gp25), anti-apoptotic genes as BHRF1 and BLRF1, and also immunomodulation factors such as BCRF1 (vIL-10) . This finally results in the formation of progeny virus. The expression of structural proteins (VCA, MA) depend on EA expression, in particular involving BALF5 the virus encoded DNA polymerase, which is a key enzyme during EBV replication. Inhibition of this enzyme by chemical (antiviral) agents such as phosphonoacetic acid (PAA), phosphonoformic acid (PFA), as well as some tannins from Eugenia uniflora will lead to block viral DNA synthesis, production of structural proteins and virus assembly . Another (theurapeutic) way to interfere with virus replication in vitro and in vivo is the use of acyclovir or (val) gancyclovir. Both are nucleoside analogous that, after specific phosphorylation by virus encoded thymidine kinase (TK) enzymes, are incorporated inti viral (and host) DNA genomes, leading to DNA-chain termination, and thus blocking replication .

5. EBV associated diseases

5. a. Infectious mononucleosis (IM)IM is the clinical manifestation forms of a primary EBV infection . IM is

benign disorder with expansion of the paracortex of lymphoid tissues. The proliferating cell populations are EBV infected polyclonal B blast , accompanied by the growth of activated T-cells . IM shows type III EBV latency pattern , even though the expression in a single cell is more heterogeneous. Most cells are EBER positive but do not express EBNA2 or LMP1 (type I latency), whereas some large

Figure 2. Overview of EBV lytic cycle. The lytic cycle can be induced with anti-IgG, which cross link the BCR to mimic antigen binding. Immediate early, early and late genes are expressed in sequential order. Late gene expression is prevented by inhibitor of lytic DNA replication such as acyclovir (10)

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immunoblasts express LMP1 in the absence of EBNA2 (type II latency) and small lymphocytes express EBNA2 but not LMP1 .

Primary EBV infection elicits strong cellular immune responses that bring the infection under control. The lymphocytosis in acute IM reflects the

+ hyperexpansion of cytotoxic CD8 T-cells that are reactive to lytic- and latent-cycle viral antigens. The limited clinical effect of antiviral drugs indicate that not EBV replication, but rather the strong T-cell reactivity during primary infection is causative for the clnical symptoms of IM . However these drugs do limit the amount of virus secretion via saliva, but have no effect on the number of circulating B-cells carrying latent EBV.

The diagnosis of IM relies on the detection of heterophile antibodies, and EBV seroconversion . Sera from IM patients have detectable IgM, IgG and IgA antibodies to one or more EBV marker protein(s). IgM antibody against EAd-p138 and –p47/54 marker to VCA-p18 and p23 , without any response against EBNA1. During acute IM IgG and IgA antibodies also can be detected against EAd-p138 and –p47/54 markers, dominant over reponses to VCA-p18 and -p23, without responses to EBNA1 .

5.b. Lymphoma in immunocompromised patients

5.b.1. Post transplant lymphoproliferative diseases (PTLDs)The T-cell immunosuppressive therapy given to patients after

transplantation is associated with an increased risk for EBV-driven lymphoproliferative diseases, which progress to malignant lymphoma if not treated properly at early stage . These post-transplantation lymphoproliferative diseases (PTLDs) are almost always of B-cell origin and EBV positive. The suppression of T cell responses allows uncontrolled proliferation of EBV-transformed B cells. PTLDs can present as polyclonal expansion or monoclonality . Some EBV-transformed B cell can acquire additional transforming events, which lead to the outgrowth of a malignant B-cell clone from an initially poly-or oligiclonal B cell expansion . The EBV-positive B cells in PTLDs overall have a latency III profile, but a more diverse and restricted gene-expression pattern is observed at the single cell level .

The expression of the full spectrum of latent EBV genes in PTLD indicates an important role for EBV in driving proliferation of infected B cells. In many cases of PTLD, the reduction of immunosuppression leads to the elimination of the EBV-positive B cells, at least during early stage of post transplant lymphoma development . Diagnostic monitoring of increased EBV-DNA loads is crucial for early and successful intervention and prevention of PTLD . In recent years the use of Rituximab (anti-CD20) has provided the new treatment option for PTLD, but is not without danger 5.b.2. AIDS-related lymphomas (ARLs)

A high incidence of malignant lymphoma is seen in highly immunocompromised AIDS patients. AIDS related lymphomas (ARLs) are mostly of B cell origin and occured in EBV positive patients. Like PTLDs, most ARLs

contain monoclonal EBV genomes . It is considered that chronic inflammation and antigen triggering is a causative factor in the outgrowth of ARLs .

There are two different types of ARL, which are differ amongst others in their EBV association and EBV gene expression pattern. Most of the cases of diffuse large B-cell lymphoma, particularly those arising in the central nervous system (CNS), are associated with EBV and have an EBV gene expression pattern comparable to that of PTLD . By contrast, only 30-40% of AIDS related Burkitt's Lymphoma (BL) is associated with EBV . These lymphomas are EBNA2 negative and rarely LMP1 positive . In addition AIDS related BL, like endemic and sporadic BL carry a translocation involving the c myc gene .

5.b.3. Oral hairy leukoplakia (OHL)Oral hairy leukoplakia (OHL) is a benign epithelial disorder with a white

epithelial lesion of the tongue, which can be found in immunocompromized individuals. EBV-DNA was first detected in this lesion in 1985, with localization restricted to the superficial epithelial cells (142). Expression of viral lytic cycle antigens has been shown in OHL and abundant production of virus particles has been demonstrated using electronmicroscopy, indicating that epithelial cells can support viral replication (224, 464). The detection of linear EBV genomes in the absence of episomal DNA (136, 299), the exclusive localization of the virus in upper epithelial cells, and the sensitivity of the lesion to acyclovir treatment (335), support this notion. Expression of latent proteins in OHL has also been reported but EBER RNAs are expressed at very low levels in OHL (136). A study by Webster-Cyraque indicates that LMP1 may contribute to the proliferation and apoptosis resistance of OHL epithelial cells (449).

5.c. Lymphomas in immunocompetent patients

5.c.1. Burkitt's Lymphoma (BL)Burkitt lymphoma (BL) was first described in children in equatorial Africa

and New Guinea. Most of the endemic BL are EBV positive. The tumours also occurs as a rare, sporadic lymphoma outside of the endemic areas, with an EBV association in 20-30% of cases (338). The first indication for a viral etiology was the climate-dependent distribution of the disease. BL had a high incidence in regions where malaria prevailed and EBV infection was present in approximately 95% of the childhood population. However in Europe and in the USA, the EBV association is only 10-20% and this sporadic form of BL mainly occurs in adults. This difference has been ascribed to the early age of infection in Africa compared with industrialized nations (257). Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) was recently shown to trigger growth activation of B-cells, and providing immunosuppression, thus setting the stage for unusual EBV B cells activation (66, 85, 108, 342, 412). In the endemic BL the EBV latent gene expression is restricted to the latency type I. EBNA1 are expressed from the Qp promoters (352). In contrast, a study from Malawi showed that BL expressed high proportion of immediate early (BZLF1) and early (IR2 and IR4) genes, a putative viral oncogene (BARF1), CST (BART) antisense transcripts and viral bcl-2

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homologue. This finding suggests that BL may not always be true latency type I (458). An important characteristic of both endemic and sporadic BL is the presence of the t(8;14)(q24;32) translocation (212, 217), which juxtaposes c-myc gene of chromosome 8 to the Ig heavy chain locus on chromosomes 14, resulting in overexpression of c-myc (466). Other translocations of c - myc to the promoter regions of Ig light chains is also seen. The pathogenesis of BL is further characterized by the epigenetic silencing of Bim, a proapoptotic gene, preventing apoptosis and exposing the cells to the growth inducing effects of c - myc. A direct role of EBV in Bim promotor silencing is indicated (14, 320).

5.c.2. Hodgkin lymphoma/ Hodgkin's Disease (HD)Hodgkin's disease (HD) is characterised by the disruption of normal lymph

node architecture and the presence of a minority (1%) of malignant Hodgkin and ReedSternberg (HRS) cells in a background of non-neoplastic cell populations comprising T- and B-lymphocytes and other cell types (154, 155). Based on differences in the histology and immunophenotype of HRS cells, the classical nodular sclerosing form of HD, accounting from 95% of cases, is distinguished from a lymphocyte-predominant form (154). HRS cells of lymphocyte-predominant HD are always EBV negative, whereas EBV is found in ~40% of cases of classical HD in the Western world. The association with EBV is higher in children with HD from Latin America, and in HIV-infected individuals, where most of cases are EBV positive (178). In EBV-positive cases, three EBV proteins are expressed, EBNA1, LMP1 and LMP2A (latency type II) (188, 216).

The expression of EBV genes in HRS cells indicates a role for EBV in rescue and transformation of pre-apoptotic germinal centre-B cells. The main survival signals for germinal centre - B cells are mediated through the BCR and through the stimulation of CD40 (249). These signals can be replaced by LMP2A and LMP1 respectively, both of which are expressed in EBV-positive HRS cells (161, 298, 312, 439), allowing cells with disadvantageous mutations to survive as germinal-centre B cells (216). EBV+ HRS cells also secrete multiple cytokines and other factors contributing to autocrine growth and immunomodulation (163, 188, 320, 326).

5.c.3. Extranodal T-cell and natural killer (NK) cell lymphomasEBV is also associated with rare T-cell and natural killer (NK)-cell

lymphomas (151, 185). How the virus accesses these cell lineages in vivo is still unclear, since T cell normally express EBV receptor CD21 at a rate 10-fold lower than B cells do (118). Interesting EBV+ T-/NK cell lymphomas always have a functionally activated cytotoxic phenotype, expressing granzyme B and perforin (35). These can arise as consequence of chronic active EBV infection and are highly aggressive (151, 185, 208). An example is extranodal lymphoma that usually present as an erosive lesion (lethal midline granuloma), which is most

+ -common in South-East Asia (151). Some of these tumours are CD3 CD56 T-cell + +lymphomas, but most are CD3 CD56 tumours of NK-cell origin. The viral gene

expression is restricted to the latency type I/II, with expression of EBNA1 and LMP2, with variable levels of LMP1 (187).

5.c.4. Nodal T cell lymphomasThe angioimmunoblastic lymphadenopathy (AILD) is a common human

T-cell lymphoma, in which EBV infected cells are often observed (12, 451). EBV can be detected in up to 30% of the nodal AILD-like lymphomas. These AILD-like lymphomas were shown to contain a clonal T-cell population (451), but EBV in these lymphomas is not present in the neoplastic T-cells, but predominantly in B-cells which are usually polyclonal or oligoclonal (369) and rarely become clonal in a later stage (268). This indicates that the proliferation of EBV-infected B-cells may be a secondary event and that EBV is not causally related to the pathogenesis of these lymphomas. The presence of EBV in B-cells in these lymphomas suggests that the expanded meshworks of follicular dendritic cells (FDCs), which are a hallmark of these lymphomas, stimulate proliferation of (EBV-infected) B blasts (276).

5.d. Epithelial and Other malignancies

5.d.1. Gastric Carcinoma (GC)EBV infection in gastric carcinoma (GC) was first reported by Burke et al

in 1990 using PCR (46). More than 90,000 patients worldwide are estimated to developed GC annually in association with EBV (approximately 10% of total gastric cancer) (362, 418, 430). In terms of histological feature, EBV associated GC occurs in two forms, the first one is lymphoepithelioma-like GC, which has a typical histology of poorly differentiated carcinoma with dense of infiltration of lymphocytes, and the second one is the ordinary type of GC (448). More than 80% of lymphoepithelioma-like GC is associated with EBV (361). The clinical feature of EBV-associated GC are located in the Gardia (upper part of stomach), less lymphnode metastasis, male predominance and relatively younger age compared to EBV-negative GC (60, 433). EBV-associated GC belongs to latency type I, with additional expression of LMP2A and BARF1, but no LMP1 (175, 390, 472). EBV positive GC have a better prognosis than EBV negative ones, possibly due to anti-tumor immune responses triggered by EBV genes in the tumor cells, not having immune escape function imposed by LMP1 (278, 434).

As in NPC, the precise role of EBV in the pathogenesis of gastric carcinoma remains to be determined, but the absence of EBV infection in pre-malignant gastric lesions supports the suggestion that viral infection is a relatively late event in gastric carcinoma (473). EBV-associated GC shows resistance to apoptosis and the production of immunomodulator molecules. Recently, global and non-random CpG island methylation of the promoter region of many cancer-related genes has been demonstrated in GC, correlating with their decreased expression such as p16, INK4A, p73 and E-chaderin. This abnormality is accompanied by methylation of EBV-genome, suggesting a process of virus driven hypermethylation in the development of neoplastic cells (428).

5.d.2. Nasopharyngeal carcinoma (NPC) NPC is a malignant disease that is classified into three types. The

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keratinized squamous cell carcinoma (WHO type I) is highly differentiated and characterized by epithelial growth patterns and keratin filaments. The non-keratinizing carcinoma (WHO type II) characterized with retaining epithelial shape and growth pattern. The undifferentiated carcinoma (WHO type III) does not produce keratin and lack of distinctively organized growth (329). Type I is seen in 25% of NPC cases. Approximately 20% of NPCs are of the type II, while type III constitutes 55% of the tumours seen in patients with NPC. In endemic areas, undifferentiated type III comprises the vast majority (>80%) of NPC, while keratinized squamous cell carcinoma is less common (269). The type III NPC is virtually 100% associated with clonal Epstein-Barr virus infection of all tumor cells that have a characteristic viral gene expression profile, known as Latency-II (default program). WHO type II NPC also is frequently (60-80%) EBV associated. WHO type I shows lower (0-20%) EBV linkage and is more associated with alcohol abuse and beetle nut chewing.

NPC is uncommon disease in most countries, with incidence for both sexes is less than one per 100,000 population (319). However, this disease occurs with much greater frequency in southern China, northern Africa, and Alaska (300). The Inuits Alaska and ethnic Chinese people living in Guangdong province are especially prone to the disease. The reported incidence of NPC among men and women in Hongkong is 20-30 per 100,000 and 15-20 per 100,000, respectively (319). The incidence of NPC remains high among Chinese people who have immigrated to Asia or North America, but is lower among Chinese people born in North America than in those born in southern China (43, 105). This finding suggests that genetic, ethnic and environmental factors may have a role in the cause of the disease (450). Recent data suggest that EBV positive NPC is a commont tumor also in non-chinese populations in SE-Asia (103, 372). However poor quality of local cancer registry data collections precludes more thorough analysis.

Patients with NPC can present with one or more symptoms of four categories, which consist of (1) presence of tumour mass in the nasopharynx (epistasis, nasal obstruction, and discharge); (2) dysfunction of the Eustachian tube, associated with the lateroposterior extension of the tumour to paranasopharyngeal space (tinnitus and deafness); (3) skull-base erosion palsy of the fifth and sixth cranial nerves, associated with the superior extension of the tumour (headache, diplopia, facial pain, and numbness); and (4) neck masses, usually appearing first in the upper neck. Other symptoms such as anorexia and weight loss are uncommon, when such symptoms are present, distant metastasis should be suspected. Unfortunately, due to the non-specific nature of the nasal and aural symptoms and the difficulty of making a clinical examination, most of the NPC patients are diagnosed when the tumour has reached an advance stage (stages III and IV) (450).

A retrospective study to 4768 patients showed the symptoms as neck mass (76%), nasal dysfunction (73%), aural dysfunction (62%), headache (35%), diplopia (11%), facial numbness (8%), weight loss (7%), and trismus (3%). The physical sign present at diagnosis were enlarged neck (75%) and cranial nerve palsy (20%) (226, 309). The symptoms in children are generally similar to those reported in adults (359).

Patients with the symptoms should be clinically assessed for the physical signs. An EBV serological test will give further justification and would justify an endoscopic examination and biopsy from the nasopharynx. If the clinical suspicion is high, even though the tumour is not visualized with endoscopic examination, cross-sectional imaging by CT and MRI should be undertaken. A definitive diagnosis of NPC needs a positive biopsy taken from the tumour in the nasopharynx (450).

The standard treatment for NPC is radiotherapy, but it can cause adverse complication because the location of the tumour at the base of skull, closely surrounded by and in close proximity to radiation dose-limiting organs, including the brain stem, spinal cord, pituitary-hypothalamic axis, temporal lobes, eyes, middle and inner ears, and parotid glands. Since NPC tends to infiltrate and spread towards these dose-limiting organs, they are even more difficult to protect. (450).

In radiotherapy a dose of 65-75 Gy is normally given to the primary tumour and 65-70 Gy to the involved neck nodes, whereas the dose for prophylactic treatment for node negative neck is 50-60 Gy. This treatment has successfully controlled T1 and T2 tumors in 75-90%of cases and T3 and T4 tumors in 50-75% of cases (72, 227, 450). Nodal control is achieved in 90% of N0 and N1 cases, but drops to 70% for N2 and N3 cases (72). Treatment schedule should be complied for success of the therapy. Interrupted or prolonged treatment reduces the benefits of radiotherapy (219). In combination with radiotherapy, the use of chemotherapy for the management of locoregional advanced cases have been reported for neoadjuvant, concurrent and adjuvant therapy or for combinations of these approaches (450). A study in 1997 (4) was the first study to show that the use of chemotherapy alongside radiotherapy improved overall survival compared with radiotherapy alone.

5.d.2.a. NPC and EBV NPC is the epithelial tumour most consistently associated with EBV

especially the WHO type III which is reflected by abnormal antibody profiles, increased circulating EBV DNA levels and by distinct EBV gene expression in the tumor cells (39, 40, 58, 114). Early pre-invasive hyperplastic nasopharyngeal lesions already carry clonal EBV, suggesting EBV infection is an early event in carcinogenesis, which contrast to EBV and GC (321, 473). The EBV association was initially suggested by the serological studies characterizing the antibody responses to EBV, which showed that patients with NPC had elevated IgG and IgA antibody titers to the EBV viral capsid antigen (VCA) and early antigen (EA) (157). The detection of EBV DNA and EBNA complex in NPC tumor cells was initially done by zur Hausen (1970) using in situ hybridization and the anticomplement immunofluorescence (ACIF) assay, respectively (474). NPC shows EBV latency type II, by showing several EBV genes consistently expressed in NPC biopsies, including EBERs, EBNA1, LMP1, LMP2A, and BARF1 (48, 98, 115, 137, 167, 193, 276). The early involvement of EBV infection in NPC development was recently further supported by observation that serological abnormalities precede NPC diagnosis by at least 2 years (180)

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5.d.2.b. EBV based diagnosis and therapy for NPCThe strong etiological link between NPC and EBV allows laboratory tests

for NPC to be based on EBV parameters, which improve the diagnosis, prognosis, prediction and prevention of the disease. Undifferentiated NPC (WHO type III) is nearly 100% EBV related as demonstrated by EBER in situ hybridization, which remain the gold standard for identifying latent infection (144).

Sero-epidemiological studies have indicated a close relation between EBV infection and NPC as revealed by elevated IgG and especially IgA to VCA, EA and EBNA complexes (157, 159). NPC patients particularly have elevated titers of IgA anti EBV-antibodies, reflecting the tumor's origin in the mucosa of the nasopharinx (100). Abnormal EBV serology is commonly used to support NPC diagnosis and provides an affordable approach for population screening to identify individuals with high NPC risk (294). Immunofluorescence assay (IFA), which allows the separate analysis of antibody responses to VCA, EA and EBNA complexes, is still used as golden standard for NPC diagnosis (130, 157), but this method is time consuming, laborious, and not suitable for mass screening (44, 131, 436). Enzyme-linked immunsorbent assay (ELISA) techniques are increasingly used and show better sensitivity and specificity compared to IFA (87, 113, 130, 190, 294, 295, 316).

Quantitative EBV DNA measurement is essential for discriminating the low-level infection of healthy carriers from the high levels characteristic of EBV related disease (379, 381). Patients with active infection or EBV related cancer tend to have high levels of EBV DNA in the circulation. In some types of cancer EBV DNA may resides in the cell-free fraction of blood (plasma or serum), whereas in healthy carriers the virus is restricted to cellular compartment of the blood (144, 379, 381). Circulating free EBV DNA (in serum or plasma) has been reported in patients with NPC (290), but the levels are relatively low (100-2000 copies/ml plasma or blood) in significant subset of patients, therefore making accurate quantification and definite diagnosis difficult (381). Recent studies have shown that plasma EBV DNA is highly fragmented and derives from apoptotic DNA released from dying tumor cells in both NPC and HD (59). This is in agreement with the absence of BARF1 mRNA in the circulation of patients, and excludes presence of circulating intact NPC tumor cells (381). The increased number of copies of EBV DNA in the blood during the initial phase of radiotherapy suggest that the viral DNA was released into circulation after tumor cell death by apoptosis (59, 251, 381). The quantity of free plasma EBV DNA is related to the stage of the disease in some studies, but not in all. The quantity of EBV DNA copies before and after treatment is significantly related to the rates of overall and disease-free survival (245). A study has reported that the (lower) levels of post treatment EBV DNA compared with pretreatment can be a good predictor of progression-free survival (57).

NPC cells are capable of immunologic processing for cytotoxic T-lymphocyte (CTL) recognition (200, 230), and on the basis of the success of adoptive EBV-targeted CTL therapy for EBV-related PTLD (387), the use of anti-EBV immunotherapy strategies has been receiving increasing attention as a possible additional treatment to improve prognosis of advanced NPC (71, 76, 203, 243).

Further details of EBV based therapy for latency type II malignancy, especially NPC, is mentioned in part 8 of introduction (strategy for immunotherapy).

5.d.2.c. NPC in IndonesiathIn Indonesia, NPC is the most prevalent head and neck cancer and the 4

thand 6 the most prevalent cancer overall in male and female respectively, with estimated incidence of 6.2/100.000/year in males and 4.6 in females (218, 372). The incidence peaks at the age of 45-54, but juvenile NPC is also quite common (372).

Histopathologically about 70-90% of the cases are WHO type III [Kurniawan, unpublished](372). Most of the patients come to the hospital at stage III and IV (>80%) when they already have metastasis in the cervical lymph node. Whereas, late stage disease has poor prognosis and requires combined chemo-radiotherapy, early stage NPC may reach complete remission by radiotherapy only (244). Therefore, screening for early stage NPC among the population is important and clinically relevant.

6. EBV Immunology

Primary EBV infection is followed by establish of viral latency in memory B lymphocytes. Infection in humans triggers both humoral and cellular immune responses to virus-encoded structural and non-structural proteins (antigens). In healthy individuals, EBV is effectively controlled by the immune system especially by antigen specific T lymphocytes (166, 339). Although the detection of antibodies against viral structural proteins and the EBV nuclear antigen is important for

+diagnosis, the cellular immune response, especially involving CD8 cytotoxic T lymphocytes (CTL) is more important in controlling primary (53) and life-long latent infection (337). The major targets for controling EBV are reactivating infected memory B cells. By killing these cells and preventing the spread of infectious virus by neutralizing antibody, the immune system may reduce the infection. However, the immune system is unable to eliminate the virus completely, and as consequence, viral shedding and virus infected cells persist at a low level, approximately 1 in 10,000 to 100,000 memory B cells (222, 414). New evidence is

+emerging for a critical role played by natural killer (NK) cells and CD4 effector T cells to control virus infected cells that is complementary to the CTL (314, 452).

6.a. Innate immune responseNatural killer cells are a key component of early innate immune response

to many microbial infections. An important role of NK cells in controlling EBV infection is revealed by the increasing numbers of activated NK cells in the peripheral lymphocyte inversely correlating with viral load (452). This finding suggests an active role for NK in controlling acute virus replication. In vitro study showed that NK cells can inhibit the EBV-induced transformation of resting B cells through the release of interferon (IFN)- g(303). However, the role of NK cells in controlling B-cell transformation in vivo is still in debate. Direct evidence against a

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major role of NK cells in controlling B-cell transformation in vivo, is provided by experiment in T-depleted stem cell transplant recipient. In these patients EBV-driven lymphoproliferative disease (LPD) is most common in the first 3-6 months post transplant, by which time NK cell numbers have recovered but the patients remain profoundly T cell-deficient (303). Furthermore, NK cells could also play role in control in lytic infection, since the reduction of HLA I expression on the surface of lytically infected cells, which increases sensitivity to NK cells (315).

6.b. Adaptive immune response

+ 6.b.1. Cellular responses: CD8 T cells+ CD8 T cells (CTLs) are known to recognize short peptide epitopes derived

from antigenic determinant in association with MHC class I molecules on the surface of appropriate antigen-presenting cells. Although synthetic peptides can directly prime target cells for lysis by CTLs, recognition of a native antigen requires intracellular processing which involves cleavage of the antigen into short peptides and their transport into the endoplasmic reticulum (ER), where they associate with MHC class I (256).

+ + The role of of CD3 CD8 CTL in controlling primary (53) and latent (337) +EBV-infection is well established. CD8 T cell responses to EBV latent antigent are

mainly directed to immunodominant epitopes derived from EBNA3A, 3B and 3C, and sometimes to other latent protein, but hardly ever to EBNA1 (36, 166, 197,

+289). Endogenously expressed EBNA1 cannot be presented to CD8 T cells because an internal glycine-alanine repeat (GAr) domain protects the protein from proteasomal degradation to peptides suitable for presentation in MHC I (236, 237). In addition the same GAr domain slows translation on ribosomes preventing DRiP formation and limiting the number of misfolded protein fragments available for degradation and CTL epitope formation (353).

+As observed in IM patients, 1-40% of all CD8 T cells reponses to individual + +epitopes are directed against EBV IE and E antigen. Cd4 and Cd8 Tcell responses

to latent cycle protein, with individual specificities, are 0.1-5% and mainly directed +to epitopes of the EBNA3A, 3B and 3C family (166). The CD8 T cells in IM blood are

perforin-positive, and rapidly die by apoptosis without antigen stimulation (54, 376, 377). They express low levels of anti-apoptotic proteins (Bcl-2 and Bcl-x) and high levels of pro-apoptotic proteins (Bax) (52, 400), together with activation marker (CD38) and cell cycle marker (Ki-67) (54, 55, 164, 165).

+CD8 T cell memory was studied in asymptomatic EBV carriers with no IM history, showing lackness of activation marker CD38 or CD69 and express higher

+ +levels of Bcl-2 than seen in CD8 T cell during acute infection. From the CD8 T cell memory population, 0.2-2% are specific to lytic epitopes and 0.05-1% to latent

+epitopes (34). EBV lytic antigen-specific CD8 T cells were identified to be mainly responsive to IE proteins (BZLF1 and BRLF1) or E protein (BMLF1) (355, 376). In

+EBV infected individuals the CD8 T cell responses particularly to EBNA1 and LMP1 seems to be marginal, which may be an evolutionary achievement beneficial for

+EBV persistence (271). In NPC patients normal EBV-specific circulating CD4 and

+CD8 T cell responses are detected in blood (246), but higher levels of T cells are reg

also apparent (223). Importantly, specific functional defects are detected in T cells obtained from fresh tumor biopsies indicative of local immune evasion

+mechanisms operated by EBV tumor cells in vivo (240).

+6.b.2. Cellular reponses: CD4 T cells+CD4 T cells recognize antigens via MHC II presentation. Extracellular

pathogens and viral protein comlexes that invade or are taken up into the cell are degraded into peptide in intracellular vesicle, which will fuse with vesicle containing MHC class II. The bound peptide is transported by MHC class II to the

+ cell surface and presented to CD4 T cells (288). CTLs seem to play major role in +eradicating virus-infected cells, but CD4 T-cell responses to viral infections are

critical role for the maintanance of virus-specific memory CTLs (267). +Furthermore, CD4 T cells can exhibit a direct effector function against virus-

infected or malignant cells that express MHC class II molecules (147, 305).+During the early stage of EBV infection the number of CD4 T cells is low

+compared to CD8 T cells (258). During primary infection, up to 2.7% of +circulating effector/memory CD4 T cells are EBV specific (327). Even though the

+ +direct effector role of CD4 T still in discussion, many lytic antigen-specific CD4 T cell are perforin-positive and cytotoxic, indicating their direct role in control of

+virus replicative lesions in vivo, especially HLA II positive target cells. CD4 T cells act by reducing EBV transformation efficiency through IFN- release, after being triggered by infected B-cells presenting antigen via MHC II upon processing of incoming virus (3).

+ +In IM blood, few CD4 T cells reactive to EBV compared with CD8 T cells (11). Their specificity as detected using individual lytic and latent cycle antigens (166, 327, 454), mainly composing IE ZEBRA (BZLF1) followed by EA protein (BMLF1) or to latent protein EBNA3A (327). In a recent study, the nuclear antigen

+(EBNA1) was found to be a main EBV latency antigen for CD4 T cell in healthy carriers (232, 286, 313). It seems that the hierarchy of immunodominance exists

+among the lytic cycle proteins for the CD4 T cell response, which is different from + the CD8 hierarchy (figure 3) (166).

+CD4 T cell responses against latent cycle antigens appear to be largely of +IFN- g producing Th1 type, but they are tenfold lower amount than CD8 T cells. The

+relevance of studying latent antigen-specific CD4 T cell responses is that most EBV-associated tumors are HLA class II-positive and express latent antigen (199).

+The use of HLA class II-recognizing CD4 T cells may be therapeutically useful against endogenously expressing the relevant latent protein (286, 421).

+More importantly, potentially, CD4 T helper cells can facilitate the expansion of CTL by direct costimulation through CD70/CD27, 4-1BBL/4-1BB interaction and by preventing CTL activation-induced cell death (138, 191).

g

+However, additional subsets of CD4 T lymphocytes, known as regulatory T cells (T ) can inhibit CTL responses, by producing significant amounts of IL-10 (263, reg

360).

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+Finally, CD4 T helper cells are important for induction and maturation of antibody reponses, providing CD40 ligand, IL-4 other cytokine to antigen triggered B-cells.

The quantitative changes of CD4+ and CD8+ T cell responses to EBV as from acute phase of IM through convalescence toward long-term asymptomatic virus carriership can be seen in figure 4. These responses parallel quantitative changes in the level of virus replication in the throat and the numbers of latently infected B cells seen in the blood (166).

Figure 4. Diagrammatic representation of changes over time in virus replication in the throat and in the load of latently infected B cells in the blood during acute and convalescent IM, and in the long-term carrier state. Changes in the magnitude of T cell responses are shown over the same time scale. The effect of T cell suppression on these parameters of the virus-host balance, as seen in post-transplant patients are also shown (166).

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Figure 3. Diagrammatic representation of the relative immunodominance in healthy virus carriers of representative +immediate early (IE), early (E) and late (L) proteins of the lytic cycle and of the eight latent cycle proteins for CD8 and

+ +CD4 T cell responses. Dotted arrows denote that CD4 T cell reponses have been observed to these proteins but that their relative immunodominance is not yet detemined. Proteins not testd are denoted n.t. (166)

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6.b.3. Humoral Immune ResponseAs mentioned previously, host immune responses are important both in

limiting the primary infection and controlling the life long latency. Combination of EBV specific antibodies and cytotoxic T lymphocytes (CTL) responses against latent and lytic EBV gene product will achieve the control of primary EBV infection. Serological studies have shown that primary and latent infections are associated with different combination of antibody reactivities to lytic and latent antigen (338). Antibodies to major virus envelope glycoprotein gp350 have capacity both to neutalize viral infectivity (413), and to mediate antibody-dependent cellular cytotoxicity against cells in lytic replication (322).

Most infected individuals develop EBV-specific antibodies against viral capsid antigens (VCAs), early (pre DNA replication) antigens (EAs), and EB nuclear antigens (EBNAs). In primary infection such as infectious mononucleosis (IM), serological responses can be classified into heterophile antibody responses and responses against specific EBV antigens (29). Antibody responses during acute IM are directed primarily against lytic cycle antigen, particularly membrane antigen complex, EA and VCA (158, 160, 277, 423, 435), while antibody responses to EBNAs are delayed by several months (Figure 5).

Antibody responses in EBV related cancer patients, is typically directed against EA and IgG VCA with variable anti-EBNA titer, although most have high EBNA titers as well, as long as cellular immunity is not affected (56, 378, 380, 437). However, the results should be interpreted with care since similar patterns are possible in autoimmune disease and other reactive condition. Moreover, serology is not reliable when the immune system is dysfunction, e.g. acquired immunodeficiency syndrome (AIDS) or allogeneic transplant patients including solid organ and marrow recipients. Unexpectedly, certain abnormal EBV serological patterns are a risk factor for developing EBV negative lymphoma, such as chronic lymphocytic leukemia, suggesting that atypical antibody response to EBV reflects a more general phenomenon and may underly development of other malignancies as well (95, 144).

EBV serology is important in NPC. Patients often have high IgA titers against lytic EBV proteins due to its tumor origin on the mucosal surface of nasopharynx (100, 144). Enhanced EBV lytic replication in nasopharyngeal epithelia is considered to preceed or at least parallel NPC development, leading to aberrant infection of epithelial cells, thus predisposing to carcinoma development. The contribution of humoral immunity to protect from tumor growth is poorly studied and will be addressed in this thesis (Chapter 7).

6.c. Immune responses to latent EBV proteins

6.c.1. Immune responses to EBNA1 EBNA1 is the single EBV antigen expressed in all EBV associated

malignancies, and has long been thought to go undetected by cell-mediated +immune system. EBNA1 is invisible to CD8 CTL, since its GAr domain prevents

proteasome-dependent processing for presentation on MHC I. However, recent + +studies show that EBNA1 can be presented to both CD4 and CD8 T cells, making it

a potential new target for immunotherapy of EBV related cancers (285). The EBNA1 protein can be processed and presented normally through the MHC class II

+processing pathway and elicits consistent CD4 T cell immune response (32, 125, 199, 286, 301, 375). It was shown that immunization with dendritic cells (DC) loaded with EBNA1-P peptide could elicit antigen-specific immunity, leading to 607-619

significant inhibition of the growth EBNA1-expressing tumor cells (125).+Several mechanisms of CD4 T cell mediated immunity have been

+proposed. Studies of EBNA1 specific CD4 T cell lines established from healthy +human donors have shown that some CD4 Th1 cells can directly kill Burkitt's

51lymphoma (BL) cells in an 18-hour Cr release assay (313), which suggest that +CD4 T cells might inhibit EBV-infected cells through cytotoxicity mediated by

+perforin or Fas ligand expressed by CD4 effector cells (125, 429). However, the experiment using EBNA1-P peptide activated T cell did not show any cytotoxic 607-619

+activity. Therefore, CD4 T cells might indirectly kill target cells through the production of cytokines, such as IFN-g, which have inhibitory activity in EBV-induced cell growth (13).

+A new hypothesis in EBNA1 specific CD8 T cell recognition suggest that

Figure 5. Schematic presentation of antibody responses against EBV antigen. Acute/ primary infection such as in acute IM is characterized by IgM-VCA and -EA, while in the late acute infection, beside IgM-VCA, the IgG-VCA and -EBNA1 also can be seen. In the healthy carrier individual the antibody responses generally is IgG-VCA and IgG-EBNA1.

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+EBNA1-derived CD8 T cell epitopes are not derived from full-length EBNA1 but from proteins that are prematurely truncated during translation or mal-folded after translation (defective ribosomal products or DRiPs), and these DRiPs are degraded by proteasomes for MHC class I presentation (285, 353).

EBNA proteins including EBNA1 are the first EBV-induced proteins expressed in an infected cell. However, anti-EBNA1 antibodies are not present during primary infection, but appear few months after infection and persist for life (9, 408). This early absence of anti-EBNA1 antibodies is thought to be due to late emergence or defective T-cell function (28, 56, 265, 438). Most of EBV healthy carriers show high IgG reactivity to EBNA1 (114).

6.c.2. Immune Response to LMP1LMP1 can be a target for CTL recognition and LMP1-specific MHC class I-

restricted epitopes have been reported, but, in fact, LMP1 is a subdominant target for CTL responses compred to other EBV latent and lytic antigen (166, 194, 198, 271). A previous study found that first transmembrane domain of LMP1 contains the sequence LALLFWL, which is highly homologous to immunosuppressive domain of retrovirus encoded transmembrane protein p15E (LDLLFL) (73, 306). This short sequence proved to have strong inhibitory effects on T cell proliferation and NK cytotoxicity in vitro (110), and modulates expression of immunosuppressive cytokine (IL-10 induction and IL-2 & IL-12 inhibition) (153, 263). In addition LMP1 is secreted in association with exosomes. These small MHC-II rich vesicles are directly and strongly immunosuppressive when incorporating LMP1 (121). This effect is directly mediated by full length LMP1 transferred to the T-cell, but not by LMP1 deleted LALLFWL (192). In the case of latency type II malignancies, when LMP1 is expressed, such as in EBV positive Hodgkin's Diseases tumor cells, LMP1 has direct role in local immunosuppression. It was demonstrate that EBV positive HRS cells expressing LMP1 abundantly

+produce IL-10, which can suppress CD8 T cell responses (109). Several studies reported than LMP1 derived peptides were recognized by

+human CD4 T cell (101, 213, 232). Kobayashi show that LMP1 antigen is +processed and presented to CD4 T cells in the context of HLA class II molecules

through the endogenous (direct presentation by transformed cells) or the exogenous (antigen captured and processed by conventional APC) pathways (213). Antigen presenting cells (APC) presenting LMP1 protein or synthetic

+peptides from LMP1 stimulated CD4 T cells from EBV-seropositive donors to produce significant amounts of IL-10 and to inhibit a variety of T cell responses (263). However, Kobayasi showed that one of LMP1 peptide (LMP1 )-specific 159-175

+CD4 T cells, although not tested for IL-10 production, show to secrete abundant of IFN- g in response to antigen stimulation, indicating that hese cells are not typical T lymphocytes (213). These findings show that much research is still needed to reg

understand the role of LMP1 in escape of host immune responses.Little is known about the humoral immune responses to LMP1, but it has

been detected in subgroups of patients with EBV associated diseases, such as NPC, HD, IM, BL patients using several approaches and different antigen protein

preparations. The result of those studies indicated that LMP1 is a protein with a low immunogenicity for the humoral immune response in humans (63, 272, 273, 282, 345).

6.c.3. Immune response to LMP2LMP2 is one of the few EBV-latent cycle proteins expressed in

+malignancies such as HD and NPC. LMP2 can serve as a CD8 T cell target (166, 225, 231). One of major EBV specific CTL response in healthy virus carriers is directed towards LMP2A (196). It has been demonstrated that LMP2A can be processed independently of TAP, furthermore tumor cells with down regulated TAP expression can be efficiently recognized by virus-specific T cell following infection with recombinant vaccinia virus encoding this protein. This indicates that LMP2A is a potential target in the immune control of EBV-associated malignancies such as HD and NPC (48, 97), and forms an obvious target for vaccination and immunotherapy.

+CD4 T cells reactivities to LMP2 have been considered to have additional therapeutic benefits, either through their helper or direct effector function (148,

+232, 386). A recent study demonstrated the CD4 T cell clones reactive to LMP2 display Th1-like reactivity secreting IFN-g, TNF-a and IL-2 in absence of typical Th2-like cytokines such as IL-10 and IL-4 (148).

Few studies addressed in detail the role of LMP2 protein as targets for humoral immune responses. Antibodies to LMP2A/2B were detected at low titer in about 40-60% of NPC sera from different ethnicity (234, 272).

6.c.4. Immune response to BARF1BARF1 protein is frequently expressed in undifferentiated NPC (98, 358).

It may be also secreted after signal sequence (residue 1-20) cleavage (349, 388). Moreover, BARF1 protein may function as soluble receptor (scavanger) for human colony-stimulating factor-1 (388), and regulate immune response by inhibiting a -

+interferon secretion by mononuclear cells (96). Recent study showed strong CD4 T

+and CD8 T cell reponses to BARF1 in healthy donors and NPC patients, by using autologous monocyte pulsed with recombinant BARF1p29 protein, in healthy

+donors and NPC patients (264). In contrast to the low CD8 T cell responses +observed for latent EBV protein LMP1 and LMP2, CD8 T cell responses to whole

BARF1 protein and derived peptides was significantly higher in NPC patients than in healthy donors. This discrepancy may relate to the biological property of the proteins. BARF1 protein can be actively secreted by undifferentiated NPC cells, and in the tumor microenvironment BARF1 protein may be captured and processed by local dendritic cells or other APC, which may provide a sustained presentation of antigenic BARF1 peptides. This may lead to the increased numbers of BARF1 specific T cells circulating in the blood of NPC patients, although it remains to be elucidated whether these effectors are detectable and functional within NPC lesions (264).

BARF1 is also able to induce humoral immune responses in EBV-seropositive individuals and may serve as a target for antibody-dependent cellular

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cytotoxicity in NPC patients (404). Like LMP1 and LMP2, BARF1 appear to be a protein of low immunogenicity for humoral immune responses (431). However, this remains to be confirmed in further studies.

6.d. Immune responses to lytic EBV proteinsEBV can establish both latent and productive (lytic) infection. The

+CD8 cytotoxic T lymphocytes (CTL) reponse to latently infected cells is well characterized, but very little is known about T cells control over lytic infection. Studies by Scotet (1996) and Steven (1997) shows that EBV lytic antigen specific

+CD8 T cells are present in the inflamed joint of rheumatoid arthritis patients (355) +and IM blood (376). As mentioned previously, the EBV lytic antigen-specific CD8

T cells were identified mainly to IE proteins (BZLF1 (ZEBRA) and BRLF1 (Rta)) or E protein (BMLF1, BMRF1 (EAd-p47/54), BALF2 (EAd-p138) and BALF5) (355,

+ 376). (see Adaptive immune response-Cellular responses-CD8 T cells section).+The CD4 T cel responses have been detected not only to ZEBRA and

BMLF1, but also to late proteins such as BCRF1 and the gp350 and gp110 envelope components (3, 440). We here limit discussion to immune responses against IE and E protein.

6.d.1. Immune response to IE proteins+Primary CD8 T cell response to EBV in IM patients as well as rheumatoid

arthritis contains multiple lytic antigen-specific reactivities. Clonality analysis revealed individual responses to two immediate early proteins BZLF1 and BRLF1 (355, 376).

In the case of autoimmunity, the finding of reactivity to IE antigen provide new understanding into the fine specificity of the anti-EBV T cell response and the immune control of EBV reactivation, because they provide the first clear-cut

+evidence that the ZEBRA EBV-transactivating proteins can be targets for CD8 T cells (355). T cell recognition to ZEBRA has been suspected in several EBV-associated diseases (e.g. acute IM, NPC, EBV reactivation in immunodepressed patients) during which antibody titers against ZEBRA and other early antigens were significantly increased (338). The physiological significant of ZEBRA recognition by T cells can be understood from its function during virus replication. ZEBRA is the first gene expressed during the immediate ealy stage of the lytic cycle and is thought to turn on expression of other early genes (205). It is therefore likely that T cell responses against this protein play a central role in controlling virus reactivation and spread under physiological and pathological situations (220).

Antibody responses against ZEBRA are marker of EBV reactivation and regularly found in undifferentiated nasopharyngeal carcinoma (NPC) patients (65-87%), but rarely among healthy people (181, 182, 266, 406, 407). Furthermore, studies done by Dardari (87, 89) found that IgG-ZEBRA in combination with IgA-EA-p47/54+138 improve the sensitivity of NPC diagnosis, and the IgG-ZEBRA can be used as diagnostic and post-therapeutic prognostic marker in juvenile NPC with low titers of IgA-VCA and EA.

6.d.2. Immune reponse to EA proteins+The primary CD8 T cell response to EA proteins was evaluated in IM

patients and rheumatoid arthritis as well as against ZEBRA and Rta (355, 376). The analysis revealed dominant responses to three early proteins BMLF1, BMRF1 (EAd-p47/54) and BALF2 (EAd-p138) (376). Clones reactive to other E and L proteins are detected but only in small number of patients and are only a minor component of total CD8 response (164, 328, 377).

Antibody responses to EA proteins were detected as well as to VCA and EBNAs (29). In acute IM, IgG reactivity to EAd-p47/54 and -p138 was detected. IgM and IgA reactivity in IM patients to EAd proteins was also detected but in a lesser extend compare to the IgG reactivity (277, 317).

Antibodies to EAd, especially IgA are important diagnostic and prognostic indicators in NPC as well (157, 158). Previous studies using recombinant proteins as well as synthetic peptide showed that IgA EA has high sensitivity and acceptable specificity for the diagnosis of NPC, either being used as single marker or in combination with other EBV antigen such as VCA or EBNA1 (69, 173, 252, 401, 422).

Antibody reponses to other EBV proteins are detected against DNAse and TK. EBV DNAse neutralizing antibodies have been found in 83%-94% of NPC patients (68). Stolzenberg et al. (384) detected IgA antibody against recombinant DNAse in NPC patients but rarely in patients with other EBV-realted malignancies. The use of thymidine kinase (TK) as antigen for diagnosis and screening of NPC was also suggested (78). However, present data indicate that a single EA marker protein as antigen might not be adequate to comprise the the diverse anti-EBV antibody responses for NPC diagnosis (114). Therefore multiple EA protein species may be required (67, 88, 114, 247). This thesis proposes the use of native EA proteins extracted from natural EBV infected cell lines for NPC diagnosis.

7. EBV Immune Escape

7.a. Immune escape in Latent infectionSuccessful viral persistence in immunocompetent individual is facilitated

by immune evasion strategies. In purpose of doing this, EBV has developed several mechanisms to escape from immune recognition. Latently infected cells express a restricted set of viral genes, which are usually non-immunogenic, making them invisible to the immune system. EBV is found in circulating memory B-cells that express low levels of MHC class I and co-stimulatory molecules (17, 281), which negatively affects the presentation of antigenic peptides (124). The true latency programme where EBV gene expression is completely shutdown or limited to the expression of LMP2A, may not be recognized by immune system (16, 17, 62, 281).

Another escape mechanism in latent infection is inhibition of antigen processing by the Gly-Ala repeat (GAr) present in EBNA1 protein. GAr generates a cis-acting inhibitory signal that interferes with antigen presentation, possibly by altering the kinetics of interaction with the proteasome, or sequesters the

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substrate to a cellular compartment that is inaccessible to the proteasome activity (238). More recently an important role for LMP1 in immune evasion is emerging (278).

Mutation in the CTL epitopes, as consequence of adaptation to the prevailing CTL responses in the infected host, provides an additional strategy of immune escape. These may occurs spontaneously at a very low frequency during EBV replication but they will prevail in the population when giving advantage for spreading to new hosts. Mutations in HLA A11 restricted epitopes in 23 EBV isolates from South East Asia, where HLA A11 is expressed in over 50% of the population, affect anchor residues in the peptides that are important for binding to HLA A11. The corresponding synthetic peptides are unable to form stable complexes with HLA A11 and LCLs carrying mutated viruses are not recognized by CTLs specific for the wild type sequence (92, 93, 239).

NK cells are considered to have an important role in controlling EBV infection, but are made inactive by the action of EBV induced gene 3 (EBI3). EBV transformed B-lymphocytes express high levels of EBV induced gene 3 (EBI3), and in a matter of immunosuppressive activity, a peptide derived from EBI3 bind to HLA-G, which is the ligand of inhibitory receptors expressed on NK cells and CTLs (102).

Another escape mechanism was demonstrated by Kitigawa (209) revealing the ingenious strategy of EBV to survive in immune hosts. This study demonstrated that EBERs were responsible for IL-10 induction. IL-10 allows it to escape the immune response by suppressing T-helper1 (Th1) cytotoxic T lymphocytes (117). Several studies have indicated that IL-10 is induced by EBV infection, in particular via LMP1 (109). The majority of EBV-transfomed lymphoblastoid cell lines and EBV carrying cell lines derived from BL and AIDS related lymphoma produce detectable amount of IL-10 (45). Most of these cells express LMP1, and Nakagomi (291) demonstrated that LMP1 is responsible for IL-10 induction, which was also shown for Hodgkin Lymphoma by Dukers et al (109).

+This may be a universal immune evasion effect in EBV cells, combining EBER and LMP1 effects to ensure persistent IL10 production since EBERs are expressed ubiquitously in all latently EBV infected-cells.

EBV associated malignancies are characterized by three different types of latency phenotypes (latency type I, II and III), which showing different escape mechanism. In latency type I tumor, such as BL, the tumor cells express only one viral antigen, EBNA1. EBNA1 can evade from immune recognition through its GAr, which acts as a cis-inhibitory signal for proteasomal degradation and blocks endogenous presentation of MHC-I epitopes (236). Furthermore, BL cells express very low levels of antigen processing genes (TAP-1 and TAP-2), which play a crucial role in the endogenous processing of CTL epitopes (195, 347). Therefore, BL cells resist CTL control by not only downregulating the expression of highly immunogenic antigens (EBNA3A, 3B and 3C), but also blocking the endogenous presentation of other potential tumor-associated epitopes (204).

Hodgkin disease and NPC represent the type II malignancies, which characterized by expression of EBNA1, BARF0, LMP1 and LMP2. LMP1 and LMP2 is considered to be strong targets for virus-specific CTLs. Immunological and

biochemical analysis of LMP1 and LMP2 sequences associated with HD and NPC have shown that these proteins are poorly immunogenic in murine models when compared with LMP1 sequences derived from normal EBV-infected B cells (270, 420). Moreover, Reed-Sternberg cell in HD secrete anti inflammatory cytokines (IL-10 and TGF-b), which can block tumour-infiltrating virus specific T cell responses (110, 162, 172).

Several mechanisms are proposed for immune escape in NPC. LMP1 and +LMP2 specific CD8 T cells are abundant in tumor infiltrating lymphocyte, but their

+ + +function is impaired (240), due to the present of CD4 CD25 FoxP3 natural regulatory T cell in the tumor tissue, which could suppress EBV specific immune reponses against NPC even after correct homing of effector T cells (223, 240). The majority of NPC down regulate MHC-I antigen processing machinery (304, 308, 467). High levels of granzyme B-positive tumor-infiltrating lymphocytes (TILs) in NPC associated with poor treatment outcome (308). It is assumed that under the pressure of a strong CTL-mediated immune response, selection for apoptosis-resistant tumour cells occurs, resulting in resistance to both CTL and radio therapy-induced apoptosis. This is substantiated by decreased activated Caspase-3 levels in these tumors (307). Another strategy of immune escape by LMP1 in NPC is directly mediated by immunosuppressive sequence of LMP1 LALLFWL (see part 6.c.2 immune response to LMP1) (278). It is suggested that NPC tumor cells impair EBV specific immune control locally, while systemic immune response against EBV remain intact (287). TILs, freshly obtained from NPC biopsies showed non-responsiveness and failed to produce IFN on specific stimulation (240). The general failure of CTL therapy in NPC also reflects the presence of local immune evasion by this tumor microenvironment (386).

7.b. Immune escape in Lytic infectionIn EBV lytic replication, more than 80 viral genes are expressed (183,

405). The escape strategies of the virus in this phase are different from those affecting the recognition of latently infected cells (336).

EBV infection inhibits the development of DCs from monocytes precursors (241), may temporarily halt the onset of immune responses during primary infection, creating time window for efficient viral replication. This could permit the accumulation of a sufficiently large pool of virus infected B-lymphocytes and allow their access to the memory B-cell compartment. Lytic EBV infection is associated with production of other factors that may affect the function of monocytes in trans. BARF1 gene that is transcribed early after EBV infection of B cells, encodes the soluble colony-stimulating factor-1 (CSF-1) receptor, which neutralizes the proliferative effect of CSF-1 on monocytes and inhibit the poly induced secretion of IFN- g by this cells (75). Therefore BARF1 may interfere with the IFN-g dependent antiviral effects of innate immunity and with delayed responses that depend on monocyte proliferation and differentiation (238).

During the lytic phase EBV has several strategies to escape antigen presentation and immune activation. Several immediate early and early product that can interfere directly or indirectly with T cell mediated immunity (238). EBV

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encodes BCRF1 protein, which close structural and functional similarity to human IL-10 (172), which affects the development of effector functions by suppressing IL-2 and IFN - g production by T-cells and enhancing the proliferation of EBV-infected B-cells (26). IL-10 also suppresses the migration of DCs to regional lymphnodes by inhibiting the expression of chemokine receptor CCR7 (399).

The early protein encoded by the EBV BHRF1 and BALF1 genes are homologues of the cellular bcl-2 gene and confer resistance to various apoptosis inducing signals including granzyme-B mediated cell killing by CTLs (27, 70, 91, 260).

One of the well described features of herpesvirus is host shutoff, and results in down regulation of surface expression of MHC class I and II molecules, which normally presenting peptide fragment of viral antigen for activation of specific T cells (214, 368). A recent study by Rowe (346) revealed a mechanism

+affecting MHC class I and II restricted antigen presentation by lytic cycle EBV cells (productively EBV-infected B cells). Expression of early-lytic gene product BGLF5 results in the mRNA degradation and causes the impairment of protein synthesis. One of the consequences of host shutoff is a block in the synthesis of HLA class I and II molecules, indicated by reduced levels of these antigen presenting complexes at the surface of lytic-cycle EBV infected cells (471). More recently, BILF1 was shown to down regulate MHCI expression by increasing the degradation (Rowe et al., 2008). Finally, like other herpesviruses EBV encodes an inhibitor of TAP function in the BNLF2a gene (170). These effects combined can lead to escape from T cell recognition and elimination of EBV-producing cells, and allowing generation of viral progeny in the face of memory T cell responses. Mechanism of imune escape in EBV latency & replication can be seen in figure 6.

8. Strategy for ImmunotherapyUnderstanding of the role of cytotoxic T lymphocytes (CTLs) in controlling

EBV-associated malignancies and overall biology of these diseases lead to the development of novel therapeutic strategies designed to specifically target viral antigens expressed in these malignancies. The success of these strategies is limited by the latency phenotypes in different diseases. Adoptive transfer of polyclonal virus specific CTLs has been used successfully to reverse the outgrowth of post-transplant lymphoproliferative disease (PTLD), which express full spectrum of EBV antigen (latency type III). However, limited viral gene expression in other EBV-associated malignancies such as BL, HD, and NPC limits the efficacy of this kind of immunotherapeutic strategies (204).

Phenotypic differences in EBV-associated malignancies provide an important basis for the development of immunotherapeutic strategies. In the type I malignancies, low expression of immunogenic antigens (eg. EBNA3A, 3B and 3C) and a consistent loss of antigen-processing function through the MHC calss I pathways result in the limitation of using antigen-specific immunotherapeutic strategies. On the other hand, a study by Herbst et al (162) showed that BL cell lines are efficiently recognized by EBNA2 reactive T cells. More recently similar results were shown for EBNA1-specific CD4+ CTLs (285, 287, 301).

Figure 6. Mechanism of immunoescape in EBV latency and replication. The essential immune escape feature in latent EBV infection is characterized by the limited expression of latent EBV proteins, while EBV expresses more than 80 antigens during lytic replication. During latent infection, the copy number of the expressed EBV antigen and of antigenic peptides processingfor MHC from these is kept very low. EBNA1 prevents efficient translation by glycine-alanin (Gly-Ala) repeat. The same GA repeat domain inhibits proteasomal processing for efficient MHC I presentation. LMP1 is considered as the main viral oncogenic gene driving cell growth, but also promotes metastasis, apoptotic resistance and immunomodulation. LMP1 expression provides EBV infected cells with an artificial T cell “help” (CD40), which is normaly only activated via specific ligand (CD40-CD40L) interaction present on the T cells. C terminus activating regions (CTARs) of LMP1are functionally homologous to the cytoplasmic tail of human CD40 and associate with the tumor necrosis factor (TNF) receptor associatedfactors (TRAFFs) and TRADDs (TNF-receptor 1-associated death domain proteins). LMP1 contains awell conserved immunosuppressive domain (LALLFWL) sequence, which actively suppresses antigen-, mitogen- and CD3+CD28- induced T cell activation. LMP1 is also secreted by EBV infected cells in the form of exosome, suggest a function in immunomodulation. Box: LMP1+ MHC-II/-I exosome may directly contact T cell receptors via MHC and co-receptor interaction, allowing exosome fusion into the T cell and LMP1 (LALLFWL) to physically disturb T cell signaling. Dyring lytic infection several mechanisms in imuune escape are proposed. BZLF1 inhibits the LMP1-induced upregulation of MHC class I mRNA levels. BNLF2a prevents the import of peptides into the ER by TAP by blocking the binding of both peptides and ATP to the transporter complex. vIL-10, the product of the lytic phase BCRF-1 gene, decreases the mRNA and, consequently, the protein levels of TAP1 and b1i/LMP2, a subunit of the immunoproteasome that shares a bidirectional promoter with the TAP1 gene. BILF1 downregulates MHC class I expression at the cell surface by targeting MHC class I molecules for lysosomal degradation. BGLF5 inhibits the synthesis of host cell proteins, including MHC class I molecules, through its virus host-shutoff function. BGLF5 also blocks MHC class II synthesis. Adopted from (83, 278, 287, 336, 469).

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Another strategy for treatment of the BL cells is using soluble CD40 ligand (CD40L). This treatment can upregulate the TAP-1 and TAP-2 gene expression involved in MHC class I-restricted presentation (201), therefore CD40L-treated BL cells regain susceptibility to EBV-specific CTL mediated lysis.

EBV associated malignancies expressing latency type II genes provide an excellent opportunity to adopt a CTL-based immunotherapy for the treatment of HD and NPC. LMP1 and LMP2 proteins are expressed in these malignancies, and have been proposed to be used as targets for immunotherapy. The first challenge to treat NPC using adoptive T cell therapy was reported by Chua et al (71). LCL-reactivated T cell lines were generated in vitro by stimulating the peripheral blood lymphocytes with EBV-transformed lymphoblastoid cell lines (LCLs) and used to treat advance cases of NPC. However, there was no evidence of tumour regression. The major limitation of this study is that LCL reactivation of PBMCs preferentially stimulate T cells that are specific for EBNA3 antigens rather than to viral protein expressed in NPC (196, 230, 289). Another approach involves stimulation of T cells with autologous dendritic cells transduced with a viral vector that expresses individual LMP antigen. This approach can be used to selectively expand LMP2-specific CTLs with minimal reactivity to other EBV antigens (343). Khanna et al (204) developed two therapeutic strategies for HD and NPC, based on the T cell activation with viral or DNA expression vectors encoding multiple LMP1 and LMP2 CTL epitopes known as polyepitope. The first approach is adoptive transfer of in vitro activated LMP-specific CTLs. These CTL were stimulated by autologous DCs loaded with recombinant vaccinia virus containing full length LMP2A or LMP polyepitope. The second approach is based on the active immunization with DNA vaccine or a recombinant viral delivery system containing LMP polyepitope construct (202, 255).

A later study by Comoli proposed a therapeutic approach for NPC using adoptive transfer of EBV-specific CTLs reactivated ex vivo from human leukocyte antigen identical siblings, showing an increasing of endogenous tumor-infiltrating

+CD8 T lymphocytes and a long-term increase of latent membrane protein 2-specific immunity (76). Another study by Comoli used the same approach on ten patients with EBV related NPC stage IV with progression after radio and chemotherapy (77). Additional study by Straathoff et al also supported this idea (385). Because of CTL resistance and immunosuppressive phenotype of NPC, CTL therapy may not be efficient and required improved targeting (LMP2A) and resistance to local immunosuppressive effect(s) (141).

Totally different approach by vaccination inducing antibody against EBV were also developed. The first prophylactic vaccination with vaccinia gp350 result in increasing EBV MA-neutralizing antibodies in juvenile (143). Another phase I/II studies using a recombinant gp350 in healthy adults were also done shows that the gp350 vaccine induce gp350 specific antibody reponses, including neutralizing antibodies. The adjuvanted gp350 vaccine display significant higher than non-adjuvanted gp350 vaccine (284, 373). In the same study by Sokal (373), which suggest that 3 doses of vaccine are necessary before full protection showed that no cases of infectious mononucleosis was declared in the vaccinated healthy

individual group after completion the vaccination schedule, whereas cases still occurred regularly in the placebo group. This is the first demonstration of considerable efficacy of vaccine to prevent infectious mononucleosis. A recent phase-I trial failed to induce protective immunity in young transplant recipients by using a gp350 based vaccine (333). The use of potent adjuvant seems crucial.

Surprisingly, in contrast to T cell based and gp-350 based vaccination or immunotherapy, very little attention has been done for Ab based for immunotherapy directed to tumor. A combination of early stage detection of NPC and specific vaccination or immunotherapy targeting tumor-associated antigen may provide an effective approach in prophylactic treatment of NPC.

9. Aims and outline of this thesisNasopharyngeal carcinoma (NPC) is one of the EBV associated malignant

diseases, which shows latency type II by expression of several EBV gene products including EBER-1, EBER-2 and BART transcripts encoding miRNAs, as well as EBNA-1, LMP-1, LMP-2A, -2B and BARF1. This etiological link between EBV and NPC is also indicated by the elevated IgG and especially IgA antibodies against EBV antigen complexes prior to clinical manifestation. Aberrant EBV serology is commonly used to support NPC diagnosis and provides an affordable approach for population screening to identify individuals with high NPC risk and to monitor treatment outcome. For the development of affordable diagnostic tools we developed in this study a protocol to provide a reliable and reproducible EA antigen and analysed each component in the EA complex for its diagnostic value.

The LMP1 and LMP2 “non-self” proteins can be an effective target for the immune response, despite the fact that tumor cells expressing LMP1 and LMP2 are not eradicated by natural immune responses. Many studies developed T cell therapy specific to these proteins, but their effectiveness is still under debate. Importantly, LMP1 and LMP2A/B are suggested to stick out from the cell surface via several conserved small loop domains connecting the transmembrane helices. These loop-domains have not been studied as target for humoral immune response. Such anti-loop antibodies may have potentially important function in targeting complement and/or FcR-bearing killer cells to LMP1, 2 expressing tumor cells. In this study we evaluate the humoral immune responses to the protein expressed on the tumor i.e. LMP1, LMP2 and BARF1 and analysed the possibility to use specific antibody to extracellular loop domains of these proteins for therapeutic approach for NPC.

Chapter 2. What is the predominant EBV marker for NPC diagnosis? In this chapter we analysed the IgG and IgA responses against individual

EBV antigens in NPC patients and healthy EBV carriers using serum panels of Indonesian, Caucasian, and Chinese ethnical background. By employing immunoblot-strips containing EBNA1 and a large spectrum of EBV lytic antigens separated by molecular weight we further identify IgG and IgA responses to individual EBV antigens and defined their diagnostic value.

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Chapter 3. Can we use individual EBV markers alone or in combination for NPC diagnosis?

A number of EpsteinBarr virus (EBV) proteins were defined as being immunodominant markers for diagnostic serology. Specific reactivity patterns to these proteins have been described for infectious mononucleosis (IM), nasopharyngeal carcinoma (NPC), various types of lymphoma, and healthy EBV carriers.

In this study we compare the NPC-related diagnostic value of EBV RecombLine test containing EBNA1, VCA-p18, VCA-p23, EAd-p47/54 and EAd-p138 with a standardized immunoblot assay (chapter 2). Furthermore, we define the diagnostic value of individual EBV marker proteins in a population with high incidence of NPC.

Chapter 4. What is the diagnostic value of individual EA components versus the complex “native” EA proteins for NPC?

The EA protein complex contains multiple proteins, which are relevant markers for diagnosis of acute, chronic and malignant EBV related diseases, including nasopharyngeal carcinoma (NPC). Multiple studies have addressed the value of individual EA marker proteins for NPC diagnosis, but no consensus is obtained thus far. Considering the diagnostic relevance of EA-specific antibody responses in NPC patients, we evaluate in chapter 4, the antibody responses of patients with NPC and healthy EBV carriers against individual recombinant EA proteins expressed in Sf9 insect cells (TK, DNAse, ZEBRA) and E. coli (EAd-p47/54 and EAd-p138), by using immunofluorescence assay (IFA) and immunoblotting (IB). Evaluation of the responses was also done to the synthetic peptides of immunodominant epitopes EAd-p47/54 and -p138 using enzyme-linked immunosorbent assay (ELISA).

For comparison with individual recombinant EA proteins and synthetic peptides, an EA-specific extract was prepared by differential salt extraction from induced HH514.c16 cells, and used as antigen for ELISA to evaluate the antibody responses of patients with NPC in compare with healthy EBV carriers and other EBV associated diseases. Furthermore we defined the diagnostic value of native EA extract for NPC.

Chapter 5. Can we use IgA-EA ELISA as confirmation test in the screening of NPC. Therapy failure in NPC cases is high, since most patients come to the

hospital at advanced stage of disease. Therefore screening for early-stage NPC is needed. In this chapter we propose to use two-step EBV ELISA for screening and confirmation of NPC. The first step consist of a cheap peptide based test for IgA-[EBNA1+VCA-p18] with high sensitivity and second step consist of IgA-EA ELISA with high specificity described in chapter 4 as confirmation test.

Chapter 6. Can we use the Epstein-Barr Virus (EBV) DNA Load in Blood of NPC Patients as confirmation test for screening?

NPC patients generally show strong IgG and especially IgA responses to

EA, VCA, and EBNA1. Serodiagnostic assays based on defined EBV-derived epitopes may facilitate population-based screening to identify NPC at early stage. However, an independent marker might be useful as confirmation for serology testing. In this chapter we use a real time PCR method targeting a conserved region in the single copy EBNA1 gene of EBV-DNA to quantify EBV-DNA levels in whole blood of NPC patiens and controls.

Chapter 7. Is there any antibody response in patients with NPC against EBV protein expressed on the tumor i.e. LMP1, LMP2, EBNA1 and BARF1 and what is the potential of this response?

NPC is one of the latency type II tumors and is characterized by expression of EBNA1, LMP1, LMP-2A/-2B and BARF1. 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.

+ Many studies addressing CD8 T cell responses against EBV latent antigens, identified immunodominant epitopes derived from the EBNA3A, 3B, and 3C protein family, and subdominant epitopes from the same EBNA3 family or from LMP2, and much less often to epitopes from EBNA2, EBNA-LP, or LMP1. Only limited data are available for T cell responses to BARF1.

Using IFA and IB methods, in this chapter we evaluate the humoral immune reponses (IgG and IgA) of NPC to the individual recombinant EBV tumor associated protein (EBNA1, LMP1, LMP2A and BARF1) expressed on Sf9 cells. PEPSCAN analysis of LMP1 and LMP2 was used to define immunogenic parts of the proteins, followed by the generation of synthetic peptides of each immunogenic epitopes. IgG and IgA responses to epitope-specific synthetic peptides were evaluated using ELISA. Immunization of rabbits with synthetic peptides representing extracellular domain yielded specific antibodies serving as positive control. Furthermore, we show that antibodies against putative LMP1 and LMP2 extracellular domain can mediate complement-driven cytolysis of LMP1 and LMP2 expressing cell lines. This opens possibilities for inducing therapeutic antibodies in NPC patients targeting tumor associated antigen expressed on the NPC tumor cells, using either using active or passive immunization.

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Reference: 1. Abbot, S. D., M. Rowe, K. Cadwallader, A. Ricksten, J. Gordon, F. Wang, L. Rymo,

and A. B. Rickinson. 1990. Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein. J Virol 64:2126-34.

2. Adams, A. 1987. Replication of latent Epstein-Barr virus genomes in Raji cells. J Virol 61:1743-6.

3. Adhikary, D., U. Behrends, A. Moosmann, K. Witter, G. W. Bornkamm, and J. Mautner. 2006. Control of Epstein-Barr virus infection in vitro by T helper cells specific for virion glycoproteins. J Exp Med 203:995-1006.

4. Al-Sarraf, M., M. LeBlanc, P. G. Giri, K. K. Fu, J. Cooper, T. Vuong, A. A. Forastiere, G. Adams, W. A. Sakr, D. E. Schuller, and J. F. Ensley. 1998. Chemoradiotherapy versus radiotherapy in patients with advanced nasopharyngeal cancer: phase III randomized Intergroup study 0099. J Clin Oncol 16:1310-7.

5. Alfieri, C., M. Birkenbach, and E. Kieff. 1991. Early events in Epstein-Barr virus infection of human B lymphocytes. Virology 181:595-608.

6. Allday, M. J., D. H. Crawford, and B. E. Griffin. 1989. Epstein-Barr virus latent gene expression during the initiation of B cell immortalization. J Gen Virol 70 ( Pt 7):1755-64.

7. Allday, M. J., and P. J. Farrell. 1994. Epstein-Barr virus nuclear antigen EBNA3C/6 expression maintains the level of latent membrane protein 1 in G1-arrested cells. J Virol 68:3491-8.

8. Almqvist, J. 2005. Epstein_Barr virus nuclear antigen 1, Oct and Groucho/TLE in control of promoter regulation. Karolinska Institute.

9. Ambinder, R. F., and R. B. Mann. 1994. Detection and characterization of Epstein-Barr virus in clinical specimens. Am J Pathol 145:239-52.

10. Amon, W., and P. J. Farrell. 2005. Reactivation of Epstein-Barr virus from latency. Rev Med Virol 15:149-56.

11. Amyes, E., C. Hatton, D. Montamat-Sicotte, N. Gudgeon, A. B. Rickinson, A. J. McMichael, and M. F. Callan. 2003. Characterization of the CD4+ T cell response to Epstein-Barr virus during primary and persistent infection. J Exp Med 198:903-11.

12. Anagnostopoulos, I., M. Hummel, T. Finn, M. Tiemann, P. Korbjuhn, C. Dimmler, K. Gatter, F. Dallenbach, M. R. Parwaresch, and H. Stein. 1992. Heterogeneous Epstein-Barr virus infection patterns in peripheral T-cell lymphoma of angioimmunoblastic lymphadenopathy type. Blood 80:1804-12.

13. Andersson, J., B. Isberg, B. Christensson, B. Veress, A. Linde, and T. Bratel. 1999. Interferon gamma (IFN-gamma) deficiency in generalized Epstein-Barr virus infection with interstitial lymphoid and granulomatous pneumonia, focal cerebral lesions, and genital ulcers: remission following IFN-gamma substitution therapy. Clin Infect Dis 28:1036-42.

14. Anderton, E., J. Yee, P. Smith, T. Crook, R. E. White, and M. J. Allday. 2008. Two Epstein-Barr virus (EBV) oncoproteins cooperate to repress expression of the proapoptotic tumour-suppressor Bim: clues to the pathogenesis of Burkitt's lymphoma. Oncogene 27:421-33.

15. Arrand, J. R., L. S. Young, and J. D. Tugwood. 1989. Two families of sequences in the small RNA-encoding region of Epstein-Barr virus (EBV) correlate with EBV types A and B. J Virol 63:983-6.

16. Babcock, G. J., L. L. Decker, R. B. Freeman, and D. A. Thorley-Lawson. 1999. Epstein-barr virus-infected resting memory B cells, not proliferating lymphoblasts,

accumulate in the peripheral blood of immunosuppressed patients. J Exp Med 190:567-76.

17. Babcock, G. J., L. L. Decker, M. Volk, and D. A. Thorley-Lawson. 1998. EBV persistence in memory B cells in vivo. Immunity 9:395-404.

18. Babcock, G. J., D. Hochberg, and A. D. Thorley-Lawson. 2000. The expression pattern of Epstein-Barr virus latent genes in vivo is dependent upon the differentiation stage of the infected B cell. Immunity 13:497-506.

19. Babcock, G. J., and D. A. Thorley-Lawson. 2000. Tonsillar memory B cells, latently infected with Epstein-Barr virus, express the restricted pattern of latent genes previously found only in Epstein-Barr virus-associated tumors. Proc Natl Acad Sci U S A 97:12250-5.

20. Baer, R., A. T. Bankier, M. D. Biggin, P. L. Deininger, P. J. Farrell, T. J. Gibson, G. Hatfull, G. S. Hudson, S. C. Satchwell, C. Seguin, and et al. 1984. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature 310:207-11.

21. Balfour, H. H., Jr., K. M. Hokanson, R. M. Schacherer, C. M. Fietzer, D. O. Schmeling, C. J. Holman, H. E. Vezina, and R. C. Brundage. 2007. A virologic pilot study of valacyclovir in infectious mononucleosis. J Clin Virol 39:16-21.

22. Bartel, D. P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281-97.

23. Barth, S., T. Pfuhl, A. Mamiani, C. Ehses, K. Roemer, E. Kremmer, C. Jaker, J. Hock, G. Meister, and F. A. Grasser. 2008. Epstein-Barr virus-encoded microRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. Nucleic Acids Res 36:666-75.

24. Bauer, G. 2001. Simplicity through complexity: immunoblot with recombinant antigens as the new gold standard in Epstein-Barr virus serology. Clin Lab 47:223-30.

25. Baumann, M., R. Feederle, E. Kremmer, and W. Hammerschmidt. 1999. Cellular transcription factors recruit viral replication proteins to activate the Epstein-Barr virus origin of lytic DNA replication, oriLyt. Embo J 18:6095-105.

26. Bejarano, M. T., and M. G. Masucci. 1998. Interleukin-10 abrogates the inhibition of Epstein-Barr virus-induced B-cell transformation by memory T-cell responses. Blood 92:4256-62.

27. Bellows, D. S., M. Howell, C. Pearson, S. A. Hazlewood, and J. M. Hardwick. 2002. Epstein-Barr virus BALF1 is a BCL-2-like antagonist of the herpesvirus antiapoptotic BCL-2 proteins. J Virol 76:2469-79.

28. Berkel, A. I., W. Henle, G. Henle, G. Klein, F. Ersoy, and O. Sanal. 1979. Epstein-Barr virus-related antibody patterns in ataxia-telangiectasia. Clin Exp Immunol 35:196-201.

29. Bhaduri-McIntosh, S., M. L. Landry, S. Nikiforow, M. Rotenberg, A. El-Guindy, and G. Miller. 2007. Serum IgA antibodies to Epstein-Barr virus (EBV) early lytic antigens are present in primary EBV infection. J Infect Dis 195:483-92.

30. Bhat, R. A., and B. Thimmappaya. 1985. Construction and analysis of additional adenovirus substitution mutants confirm the complementation of VAI RNA function by two small RNAs encoded by Epstein-Barr virus. J Virol 56:750-6.

31. Bhat, R. A., and B. Thimmappaya. 1983. Two small RNAs encoded by Epstein-Barr virus can functionally substitute for the virus-associated RNAs in the lytic growth of adenovirus 5. Proc Natl Acad Sci U S A 80:4789-93.

32. Bickham, K., C. Munz, M. L. Tsang, M. Larsson, J. F. Fonteneau, N. Bhardwaj, and R. Steinman. 2001. EBNA1-specific CD4+ T cells in healthy carriers of Epstein-Barr virus are primarily Th1 in function. J Clin Invest 107:121-30.

Introdction

4140

Chapter 1

33. Biggin, M., M. Bodescot, M. Perricaudet, and P. Farrell. 1987. Epstein-Barr virus gene expression in P3HR1-superinfected Raji cells. J Virol 61:3120-32.

34. Bihl, F., N. Frahm, L. Di Giammarino, J. Sidney, M. John, K. Yusim, T. Woodberry, K. Sango, H. S. Hewitt, L. Henry, C. H. Linde, J. V. Chisholm, 3rd, T. M. Zaman, E. Pae, S. Mallal, B. D. Walker, A. Sette, B. T. Korber, D. Heckerman, and C. Brander. 2006. Impact of HLA-B alleles, epitope binding affinity, functional avidity, and viral coinfection on the immunodominance of virus-specific CTL responses. J Immunol 176:4094-101.

35. Bladergroen, B. A., C. J. Meijer, R. L. ten Berge, C. E. Hack, J. J. Muris, D. F. Dukers, A. Chott, Y. Kazama, J. J. Oudejans, O. van Berkum, and J. A. Kummer. 2002. Expression of the granzyme B inhibitor, protease inhibitor 9, by tumor cells in patients with non-Hodgkin and Hodgkin lymphoma: a novel protective mechanism for tumor cells to circumvent the immune system? Blood 99:232-7.

36. Blake, N., S. Lee, I. Redchenko, W. Thomas, N. Steven, A. Leese, P. Steigerwald-Mullen, M. G. Kurilla, L. Frappier, and A. Rickinson. 1997. Human CD8+ T cell responses to EBV EBNA1: HLA class I presentation of the (Gly-Ala)-containing protein requires exogenous processing. Immunity 7:791-802.

37. Borza, C. M., and L. M. Hutt-Fletcher. 2002. Alternate replication in B cells and epithelial cells switches tropism of Epstein-Barr virus. Nat Med 8:594-9.

38. Brink, A. A., D. F. Dukers, A. J. van den Brule, J. J. Oudejans, J. M. Middeldorp, C. J. Meijer, and M. Jiwa. 1997. Presence of Epstein-Barr virus latency type III at the single cell level in post-transplantation lymphoproliferative disorders and AIDS related lymphomas. J Clin Pathol 50:911-8.

39. Brink, A. A., M. B. Vervoort, J. M. Middeldorp, C. J. Meijer, and A. J. van den Brule. 1998. 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. J Clin Microbiol 36:3164-9.

40. Brooks, L., Q. Y. Yao, A. B. Rickinson, and L. S. Young. 1992. Epstein-Barr virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1, LMP1, and LMP2 transcripts. J Virol 66:2689-97.

41. Brown, N. A., C. Liu, C. R. Garcia, Y. F. Wang, A. Griffith, R. S. Sparkes, and K. L. Calame. 1986. Clonal origins of lymphoproliferative disease induced by Epstein-Barr virus. J Virol 58:975-8.

42. Bryant, H., and P. J. Farrell. 2002. Signal Transduction and Transcription Factor Modification during Reactivation of Epstein-Barr Virus from Latency. J Virol 76:10290-8.

43. Buell, P. 1974. The effect of migration on the risk of nasopharyngeal cancer among Chinese. Cancer Res 34:1189-91.

44. Buisson, M., B. Fleurent, M. Mak, P. Morand, L. Chan, A. Ng, M. Guan, D. Chin, and J. M. Seigneurin. 1999. Novel immunoblot assay using four recombinant antigens for diagnosis of Epstein-Barr virus primary infection and reactivation. J Clin Microbiol 37:2709-14.

45. Burdin, N., C. Peronne, J. Banchereau, and F. Rousset. 1993. Epstein-Barr virus transformation induces B lymphocytes to produce human interleukin 10. J Exp Med 177:295-304.

46. Burke, A. P., T. S. Yen, K. M. Shekitka, and L. H. Sobin. 1990. Lymphoepithelial carcinoma of the stomach with Epstein-Barr virus demonstrated by polymerase chain reaction. Mod Pathol 3:377-80.

47. Burkitt, D., and G. T. O'Conor. 1961. Malignant lymphoma in African children. I. A

clinical syndrome. Cancer 14:258-69.48. Busson, P., R. McCoy, R. Sadler, K. Gilligan, T. Tursz, and N. Raab-Traub. 1992.

Consistent transcription of the Epstein-Barr virus LMP2 gene in nasopharyngeal carcinoma. J Virol 66:3257-62.

49. Cai, X., S. Lu, Z. Zhang, C. M. Gonzalez, B. Damania, and B. R. Cullen. 2005. Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells. Proc Natl Acad Sci U S A 102:5570-5.

50. Cai, X., A. Schafer, S. Lu, J. P. Bilello, R. C. Desrosiers, R. Edwards, N. Raab-Traub, and B. R. Cullen. 2006. Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed. PLoS Pathog 2:e23.

51. Caldwell, R. G., J. B. Wilson, S. J. Anderson, and R. Longnecker. 1998. Epstein-Barr virus LMP2A drives B cell development and survival in the absence of normal B cell receptor signals. Immunity 9:405-11.

52. Callan, M. F., C. Fazou, H. Yang, T. Rostron, K. Poon, C. Hatton, and A. J. McMichael. 2000. CD8(+) T-cell selection, function, and death in the primary immune response in vivo. J Clin Invest 106:1251-61.

53. Callan, M. F., N. Steven, P. Krausa, J. D. Wilson, P. A. Moss, G. M. Gillespie, J. I. Bell, A. B. Rickinson, and A. J. McMichael. 1996. Large clonal expansions of CD8+ T cells in acute infectious mononucleosis. Nat Med 2:906-11.

54. Callan, M. F., L. Tan, N. Annels, G. S. Ogg, J. D. Wilson, C. A. O'Callaghan, N. Steven, A. J. McMichael, and A. B. Rickinson. 1998. Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus In vivo. J Exp Med 187:1395-402.

55. Catalina, M. D., J. L. Sullivan, K. R. Bak, and K. Luzuriaga. 2001. Differential evolution and stability of epitope-specific CD8(+) T cell responses in EBV infection. J Immunol 167:4450-7.

56. Cen, H., P. A. Williams, H. P. McWilliams, M. C. Breinig, M. Ho, and J. L. McKnight. 1993. Evidence for restricted Epstein-Barr virus latent gene expression and anti-EBNA antibody response in solid organ transplant recipients with posttransplant lymphoproliferative disorders. Blood 81:1393-403.

57. Chan, A. T., Y. M. Lo, B. Zee, L. Y. Chan, B. B. Ma, S. F. Leung, F. Mo, M. Lai, S. Ho, D. P. Huang, and P. J. Johnson. 2002. Plasma Epstein-Barr virus DNA and residual disease after radiotherapy for undifferentiated nasopharyngeal carcinoma. J Natl Cancer Inst 94:1614-9.

58. Chan, K. C., and Y. M. Lo. 2002. Circulating EBV DNA as a tumor marker for nasopharyngeal carcinoma. Semin Cancer Biol 12:489-96.

59. Chan, K. C., J. Zhang, A. T. Chan, K. I. Lei, S. F. Leung, L. Y. Chan, K. C. Chow, and Y. M. Lo. 2003. Molecular characterization of circulating EBV DNA in the plasma of nasopharyngeal carcinoma and lymphoma patients. Cancer Res 63:2028-32.

60. Chang, M. S., H. S. Lee, H. S. Kim, S. H. Kim, S. I. Choi, B. L. Lee, C. W. Kim, Y. I. Kim, M. Yang, and W. H. Kim. 2003. Epstein-Barr virus and microsatellite instability in gastric carcinogenesis. J Pathol 199:447-52.

61. Chang, Y., E. Cesarman, M. S. Pessin, F. Lee, J. Culpepper, D. M. Knowles, and P. S. Moore. 1994. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science 266:1865-9.

62. Chen, F., J. Z. Zou, L. di Renzo, G. Winberg, L. F. Hu, E. Klein, G. Klein, and I. Ernberg. 1995. A subpopulation of normal B cells latently infected with Epstein-Barr virus resembles Burkitt lymphoma cells in expressing EBNA-1 but not EBNA-2 or LMP1. J Virol 69:3752-8.

Introdction

4342

Chapter 1

63. Chen, H. F., S. Kevan-Jah, K. O. Suentzenich, F. A. Grasser, and N. Mueller-Lantzsch. 1992. Expression of the Epstein-Barr virus latent membrane protein (LMP) in insect cells and detection of antibodies in human sera against this protein. Virology 190:106-15.

64. Chen, H. L., M. M. Lung, J. S. Sham, D. T. Choy, B. E. Griffin, and M. H. Ng. 1992. Transcription of BamHI-A region of the EBV genome in NPC tissues and B cells. Virology 191:193-201.

65. Chen, M. R., J. F. Yang, C. W. Wu, J. M. Middeldorp, and J. Y. Chen. 1998. Physical association between the EBV protein EBNA-1 and P32/TAP/hyaluronectin. J Biomed Sci 5:173-9.

66. Chene, A., D. Donati, A. O. Guerreiro-Cacais, V. Levitsky, Q. Chen, K. I. Falk, J. Orem, F. Kironde, M. Wahlgren, and M. T. Bejarano. 2007. A molecular link between malaria and Epstein-Barr virus reactivation. PLoS Pathog 3:e80.

67. Cheng, W. M., K. H. Chan, H. L. Chen, R. X. Luo, S. P. Ng, W. Luk, B. J. Zheng, M. F. Ji, J. S. Liang, J. S. Sham, D. K. Wang, Y. S. Zong, and M. H. Ng. 2002. Assessing the risk of nasopharyngeal carcinoma on the basis of EBV antibody spectrum. Int J Cancer 97:489-92.

68. Cheng, Y. C., J. Y. Chen, R. Glaser, and W. Henle. 1980. Frequency and levels of antibodies to Epstein-Barr virus-specific DNase are elevated in patients with nasopharyngeal carcinoma. Proc Natl Acad Sci U S A 77:6162-5.

69. Chow, K. C., J. Ma, L. S. Lin, K. H. Chi, S. H. Yen, S. M. Liu, W. T. Liu, W. K. Chen, T. H. Chang, and K. Y. Chen. 1997. Serum responses to the combination of Epstein-Barr virus antigens from both latent and acute phases in nasopharyngeal carcinoma: complementary test of EBNA-1 with EA-D. Cancer Epidemiol Biomarkers Prev 6:363-8.

70. Chu, P. G., K. L. Chang, W. G. Chen, Y. Y. Chen, D. Shibata, K. Hayashi, C. Bacchi, M. Bacchi, and L. M. Weiss. 1999. Epstein-Barr virus (EBV) nuclear antigen (EBNA)-4 mutation in EBV-associated malignancies in three different populations. Am J Pathol 155:941-7.

71. Chua, D., J. Huang, B. Zheng, S. Y. Lau, W. Luk, D. L. Kwong, J. S. Sham, D. Moss, K. Y. Yuen, S. W. Im, and M. H. Ng. 2001. Adoptive transfer of autologous Epstein-Barr virus-specific cytotoxic T cells for nasopharyngeal carcinoma. Int J Cancer 94:73-80.

72. Chua, D. T., J. S. Sham, W. I. Wei, W. K. Ho, and G. K. Au. 2001. The predictive value of the 1997 American Joint Committee on Cancer stage classification in determining failure patterns in nasopharyngeal carcinoma. Cancer 92:2845-55.

73. Cianciolo, G. J., T. D. Copeland, S. Oroszlan, and R. Snyderman. 1985. Inhibition of lymphocyte proliferation by a synthetic peptide homologous to retroviral envelope proteins. Science 230:453-5.

74. Clarke, P. A., N. A. Sharp, and M. J. Clemens. 1990. Translational control by the Epstein-Barr virus small RNA EBER-1. Reversal of the double-stranded RNA-induced inhibition of protein synthesis in reticulocyte lysates. Eur J Biochem 193:635-41.

75. Cohen, J. I., and K. Lekstrom. 1999. Epstein-Barr virus BARF1 protein is dispensable for B-cell transformation and inhibits alpha interferon secretion from mononuclear cells. J Virol 73:7627-32.

76. Comoli, P., R. De Palma, S. Siena, A. Nocera, S. Basso, F. Del Galdo, R. Schiavo, O. Carminati, A. Tagliamacco, G. F. Abbate, F. Locatelli, R. Maccario, and P. Pedrazzoli. 2004. 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. Ann Oncol 15:113-7.77. Comoli, P., P. Pedrazzoli, R. Maccario, S. Basso, O. Carminati, M. Labirio, R.

Schiavo, S. Secondino, C. Frasson, C. Perotti, M. Moroni, F. Locatelli, and S. Siena. 2005. Cell therapy of stage IV nasopharyngeal carcinoma with autologous Epstein-Barr virus-targeted cytotoxic T lymphocytes. J Clin Oncol 23:8942-9.

78. Connolly, Y., E. Littler, N. Sun, X. Chen, P. C. Huang, S. N. Stacey, and J. R. Arrand. 2001. Antibodies to Epstein-Barr virus thymidine kinase: a characteristic marker for the serological detection of nasopharyngeal carcinoma. Int J Cancer 91:692-7.

79. Cordier, M., A. Calender, M. Billaud, U. Zimber, G. Rousselet, O. Pavlish, J. Banchereau, T. Tursz, G. Bornkamm, and G. M. Lenoir. 1990. Stable transfection of Epstein-Barr virus (EBV) nuclear antigen 2 in lymphoma cells containing the EBV P3HR1 genome induces expression of B-cell activation molecules CD21 and CD23. J Virol 64:1002-13.

80. Cory, S., D. C. Huang, and J. M. Adams. 2003. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 22:8590-607.

81. Cosmopoulos, K., M. Pegtel, J. Hawkins, H. Moffett, C. Novina, J. Middeldorp, and D. A. Thorley-Lawson. 2009. Comprehensive profiling of Epstein-Barr virus microRNAs in nasopharyngeal carcinoma. J Virol 83:2357-67.

82. Crawford, D. H., K. F. Macsween, C. D. Higgins, R. Thomas, K. McAulay, H. Williams, N. Harrison, S. Reid, M. Conacher, J. Douglas, and A. J. Swerdlow. 2006. A cohort study among university students: identification of risk factors for Epstein-Barr virus seroconversion and infectious mononucleosis. Clin Infect Dis 43:276-82.

83. Croft, N. P., C. Shannon-Lowe, A. I. Bell, D. Horst, E. Kremmer, M. E. Ressing, E. J. Wiertz, J. M. Middeldorp, M. Rowe, A. B. Rickinson, and A. D. Hislop. 2009. Stage-specific inhibition of MHC class I presentation by the Epstein-Barr virus BNLF2a protein during virus lytic cycle. PLoS Pathog 5:e1000490.

84. Cullen, B. R. 2006. Viruses and microRNAs. Nat Genet 38 Suppl:S25-30.85. D'Ombrain, M. C., T. S. Voss, A. G. Maier, J. A. Pearce, D. S. Hansen, A. F. Cowman,

and L. Schofield. 2007. Plasmodium falciparum erythrocyte membrane protein-1 specifically suppresses early production of host interferon-gamma. Cell Host Microbe 2:130-8.

86. Daikoku, T., A. Kudoh, M. Fujita, Y. Sugaya, H. Isomura, N. Shirata, and T. Tsurumi. 2005. Architecture of replication compartments formed during Epstein-Barr virus lytic replication. J Virol 79:3409-18.

87. Dardari, R., W. Hinderer, D. Lang, A. Benider, B. El Gueddari, I. Joab, A. Benslimane, and M. Khyatti. 2001. Antibody responses to recombinant Epstein-Barr virus antigens in nasopharyngeal carcinoma patients: complementary test of ZEBRA protein and early antigens p54 and p138. J Clin Microbiol 39:3164-70.

88. Dardari, R., M. Khyatti, A. Benider, H. Jouhadi, A. Kahlain, C. Cochet, A. Mansouri, B. El Gueddari, A. Benslimane, and I. Joab. 2000. Antibodies to the Epstein-Barr virus transactivator protein (ZEBRA) as a valuable biomarker in young patients with nasopharyngeal carcinoma. Int J Cancer 86:71-5.

89. Dardari, R., J. Menezes, E. Drouet, I. Joab, A. Benider, H. Bakkali, L. Kanouni, H. Jouhadi, N. Benjaafar, B. El Gueddari, M. Hassar, and M. Khyatti. 2008. Analyses of the prognostic significance of the Epstein-Barr virus transactivator ZEBRA protein and diagnostic value of its two synthetic peptides in nasopharyngeal carcinoma. J Clin Virol 41:96-103.

90. Datta, A. K., and R. E. Hood. 1981. Mechanism of inhibition of Epstein-Barr virus replication by phosphonoformic acid. Virology 114:52-9.

Introdction

4544

Chapter 1

91. Davis, J. E., V. R. Sutton, M. J. Smyth, and J. A. Trapani. 2000. Dependence of granzyme B-mediated cell death on a pathway regulated by Bcl-2 or its viral homolog, BHRF1. Cell Death Differ 7:973-83.

92. de Campos-Lima, P. O., R. Gavioli, Q. J. Zhang, L. E. Wallace, R. Dolcetti, M. Rowe, A. B. Rickinson, and M. G. Masucci. 1993. HLA-A11 epitope loss isolates of Epstein-Barr virus from a highly A11+ population. Science 260:98-100.

93. de Campos-Lima, P. O., V. Levitsky, J. Brooks, S. P. Lee, L. F. Hu, A. B. Rickinson, and M. G. Masucci. 1994. T cell responses and virus evolution: loss of HLA A11-restricted CTL epitopes in Epstein-Barr virus isolates from highly A11-positive populations by selective mutation of anchor residues. J Exp Med 179:1297-305.

94. de Jesus, O., P. R. Smith, L. C. Spender, C. Elgueta Karstegl, H. H. Niller, D. Huang, and P. J. Farrell. 2003. Updated Epstein-Barr virus (EBV) DNA sequence and analysis of a promoter for the BART (CST, BARF0) RNAs of EBV. J Gen Virol 84:1443-50.

95. de Sanjose, S., R. Bosch, T. Schouten, S. Verkuijlen, A. Nieters, L. Foretova, M. Maynadie, P. L. Cocco, A. Staines, N. Becker, P. Brennan, Y. Benavente, P. Boffetta, C. J. Meijer, and J. M. Middeldorp. 2007. Epstein-Barr virus infection and risk of lymphoma: immunoblot analysis of antibody responses against EBV-related proteins in a large series of lymphoma subjects and matched controls. Int J Cancer 121:1806-12.

96. de Turenne-Tessier, M., P. Jolicoeur, J. M. Middeldorp, and T. Ooka. 2005. Expression and analysis of the Epstein-Barr virus BARF1-encoded protein from a tetracycline-regulatable adenovirus system. Virus Res 109:9-18.

97. Deacon, E. M., G. Pallesen, G. Niedobitek, J. Crocker, L. Brooks, A. B. Rickinson, and L. S. Young. 1993. Epstein-Barr virus and Hodgkin's disease: transcriptional analysis of virus latency in the malignant cells. J Exp Med 177:339-49.

98. Decaussin, G., F. Sbih-Lammali, M. de Turenne-Tessier, A. Bouguermouh, and T. Ooka. 2000. Expression of BARF1 gene encoded by Epstein-Barr virus in nasopharyngeal carcinoma biopsies. Cancer Res 60:5584-8.

99. DeMario, M. D., and D. N. Liebowitz. 1998. Lymphomas in the immunocompromised patient. Semin Oncol 25:492-502.

100. Deng, H., Y. Zeng, Y. Lei, Z. Zhao, P. Wang, B. Li, Z. Pi, B. Tan, Y. Zheng, W. Pan, and et al. 1995. Serological survey of nasopharyngeal carcinoma in 21 cities of south China. Chin Med J (Engl) 108:300-3.

101. Depil, S., O. Morales, F. A. Castelli, N. Delhem, V. Francois, B. Georges, F. Dufosse, F. Morschhauser, J. Hammer, B. Maillere, C. Auriault, and V. Pancre. 2007. Determination of a HLA II promiscuous peptide cocktail as potential vaccine against EBV latency II malignancies. J Immunother 30:215-26.

102. Devergne, O., A. Coulomb-L'Hermine, F. Capel, M. Moussa, and F. Capron. 2001. Expression of Epstein-Barr virus-induced gene 3, an interleukin-12 p40-related molecule, throughout human pregnancy: involvement of syncytiotrophoblasts and extravillous trophoblasts. Am J Pathol 159:1763-76.

103. Devi, B. C., P. Pisani, T. S. Tang, and D. M. Parkin. 2004. High incidence of nasopharyngeal carcinoma in native people of Sarawak, Borneo Island. Cancer Epidemiol Biomarkers Prev 13:482-6.

104. Dhar, S. K., K. Yoshida, Y. Machida, P. Khaira, B. Chaudhuri, J. A. Wohlschlegel, M. Leffak, J. Yates, and A. Dutta. 2001. Replication from oriP of Epstein-Barr virus requires human ORC and is inhibited by geminin. Cell 106:287-96.

105. Dickson, R. I., and A. D. Flores. 1985. Nasopharyngeal carcinoma: an evaluation of

134 patients treated between 1971-1980. Laryngoscope 95:276-83.106. Dolyniuk, M., R. Pritchett, and E. Kieff. 1976. Proteins of Epstein-Barr virus. I.

Analysis of the polypeptides of purified enveloped Epstein-Barr virus. J Virol 17:935-49.

107. Dolyniuk, M., E. Wolff, and E. Kieff. 1976. Proteins of Epstein-Barr Virus. II. Electrophoretic analysis of the polypeptides of the nucleocapsid and the glucosamine- and polysaccharide-containing components of enveloped virus. J Virol 18:289-97.

108. Donati, D., L. P. Zhang, A. Chene, Q. Chen, K. Flick, M. Nystrom, M. Wahlgren, and M. T. Bejarano. 2004. Identification of a polyclonal B-cell activator in Plasmodium falciparum. Infect Immun 72:5412-8.

109. Dukers, D. F., L. H. Jaspars, W. Vos, J. J. Oudejans, D. Hayes, S. Cillessen, J. M. Middeldorp, and C. J. Meijer. 2000. Quantitative immunohistochemical analysis of cytokine profiles in Epstein-Barr virus-positive and -negative cases of Hodgkin's disease. J Pathol 190:143-9.

110. Dukers, D. F., P. Meij, M. B. Vervoort, W. Vos, R. J. Scheper, C. J. Meijer, E. Bloemena, and J. M. Middeldorp. 2000. Direct immunosuppressive effects of EBV-encoded latent membrane protein 1. J Immunol 165:663-70.

111. Epstein, M. A., G. Henle, B. G. Achong, and Y. M. Barr. 1965. Morphological And Biological Studies On A Virus In Cultured Lymphoblasts From Burkitt's Lymphoma. J Exp Med 121:761-70.

112. Evans, A. S. 1972. Clinical syndromes associated with EB virus infection. Adv Intern Med 18:77-93.

113. Fachiroh, J., D. K. Paramita, B. Hariwiyanto, A. Harijadi, H. L. Dahlia, S. R. Indrasari, H. Kusumo, Y. S. Zeng, T. Schouten, S. Mubarika, and J. M. Middeldorp. 2006. Single-assay 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. J Clin Microbiol 44:1459-67.

114. Fachiroh, J., T. Schouten, B. Hariwiyanto, D. K. Paramita, A. Harijadi, S. M. Haryana, M. H. Ng, and J. M. Middeldorp. 2004. Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects. J Infect Dis 190:53-62.

115. Fahraeus, R., H. L. Fu, I. Ernberg, J. Finke, M. Rowe, G. Klein, K. Falk, E. Nilsson, M. Yadav, P. Busson, and et al. 1988. Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma. Int J Cancer 42:329-38.

116. Farrell, P. 2005. Epstein-Barr Virus Genome, p. 263-287. In E. S. Robertson (ed.), Epstein-Barr Virus. Caister Academic Press, Philadelphia.

117. Fiorentino, D. F., M. W. Bond, and T. R. Mosmann. 1989. Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J Exp Med 170:2081-95.

118. Fischer, E., C. Delibrias, and M. D. Kazatchkine. 1991. Expression of CR2 (the C3dg/EBV receptor, CD21) on normal human peripheral blood T lymphocytes. J Immunol 146:865-9.

119. Fixman, E. D., G. S. Hayward, and S. D. Hayward. 1995. Replication of Epstein-Barr virus oriLyt: lack of a dedicated virally encoded origin-binding protein and dependence on Zta in cotransfection assays. J Virol 69:2998-3006.

120. Fixman, E. D., G. S. Hayward, and S. D. Hayward. 1992. trans-acting requirements for replication of Epstein-Barr virus ori-Lyt. J Virol 66:5030-9.

Introdction

4746

Chapter 1

121. Flanagan, J., J. Middeldorp, and T. Sculley. 2003. Localization of the Epstein-Barr virus protein LMP 1 to exosomes. J Gen Virol 84:1871-9.

122. Frappier, L., and M. O'Donnell. 1991. Overproduction, purification, and characterization of EBNA1, the origin binding protein of Epstein-Barr virus. J Biol Chem 266:7819-26.

123. Friedl, P., E. B. Brocker, and K. S. Zanker. 1998. Integrins, cell matrix interactions and cell migration strategies: fundamental differences in leukocytes and tumor cells. Cell Adhes Commun 6:225-36.

124. Frisan, T., V. Levitsky, and M. G. Masucci. 2000. Variations in proteasome subunit composition and enzymatic activity in B-lymphoma lines and normal B cells. Int J Cancer 88:881-8.

125. Fu, T., K. S. Voo, and R. F. Wang. 2004. Critical role of EBNA1-specific CD4+ T cells in the control of mouse Burkitt lymphoma in vivo. J Clin Invest 114:542-50.

126. Fujii, K., N. Yokoyama, T. Kiyono, K. Kuzushima, M. Homma, Y. Nishiyama, M. Fujita, and T. Tsurumi. 2000. The Epstein-Barr virus pol catalytic subunit physically interacts with the BBLF4-BSLF1-BBLF2/3 complex. J Virol 74:2550-7.

127. Gahn, T. A., and C. L. Schildkraut. 1989. The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication. Cell 58:527-35.

128. Gahn, T. A., and B. Sugden. 1995. An EBNA-1-dependent enhancer acts from a distance of 10 kilobase pairs to increase expression of the Epstein-Barr virus LMP gene. J Virol 69:2633-6.

129. Gan, Y. J., J. Chodosh, A. Morgan, and J. W. Sixbey. 1997. Epithelial cell polarization is a determinant in the infectious outcome of immunoglobulin A-mediated entry by Epstein-Barr virus. J Virol 71:519-26.

130. Gartner, B. C., J. M. Fischinger, K. Roemer, M. Mak, B. Fleurent, and N. Mueller-Lantzsch. 2001. Evaluation of a recombinant line blot for diagnosis of Epstein-Barr Virus compared with ELISA, using immunofluorescence as reference method. J Virol Methods 93:89-96.

131. Gartner, B. C., R. D. Hess, D. Bandt, A. Kruse, A. Rethwilm, K. Roemer, and N. Mueller-Lantzsch. 2003. Evaluation of four commercially available Epstein-Barr virus enzyme immunoassays with an immunofluorescence assay as the reference method. Clin Diagn Lab Immunol 10:78-82.

132. Gergely, L., G. Klein, and I. Ernberg. 1971. The action of DNA antagonists on Epstein-Barr virus (EBV)-associated early antigen (EA) in Burkitt lymphoma lines. Int J Cancer 7:293-302.

133. Ghadge, G. D., P. Malhotra, M. R. Furtado, R. Dhar, and B. Thimmapaya. 1994. In vitro analysis of virus-associated RNA I (VAI RNA): inhibition of the double-stranded RNA-activated protein kinase PKR by VAI RNA mutants correlates with the in vivo phenotype and the structural integrity of the central domain. J Virol 68:4137-51.

134. Giancotti, F. G., and E. Ruoslahti. 1999. Integrin signaling. Science 285:1028-32.135. Gilcrease, M. Z. 2007. Integrin signaling in epithelial cells. Cancer Lett 247:1-25.136. Gilligan, K., P. Rajadurai, L. Resnick, and N. Raab-Traub. 1990. Epstein-Barr virus

small nuclear RNAs are not expressed in permissively infected cells in AIDS-associated leukoplakia. Proc Natl Acad Sci U S A 87:8790-4.

137. Gilligan, K. J., P. Rajadurai, J. C. Lin, P. Busson, M. Abdel-Hamid, U. Prasad, T. Tursz, and N. Raab-Traub. 1991. Expression of the Epstein-Barr virus BamHI A fragment in nasopharyngeal carcinoma: evidence for a viral protein expressed in vivo. J Virol

65:6252-9.138. Giuntoli, R. L., 2nd, J. Lu, H. Kobayashi, R. Kennedy, and E. Celis. 2002. Direct

costimulation of tumor-reactive CTL by helper T cells potentiate their proliferation, survival, and effector function. Clin Cancer Res 8:922-31.

139. Glickman, J. N., J. G. Howe, and J. A. Steitz. 1988. Structural analyses of EBER1 and EBER2 ribonucleoprotein particles present in Epstein-Barr virus-infected cells. J Virol 62:902-11.

140. Gordon, J., L. Walker, G. Guy, G. Brown, M. Rowe, and A. Rickinson. 1986. Control of human B-lymphocyte replication. II. Transforming Epstein-Barr virus exploits three distinct viral signals to undermine three separate control points in B-cell growth. Immunology 58:591-5.

141. Gottschalk, S., H. E. Heslop, and C. M. Rooney. 2005. Adoptive immunotherapy for EBV-associated malignancies. Leuk Lymphoma 46:1-10.

142. Greenspan, J. S., D. Greenspan, E. T. Lennette, D. I. Abrams, M. A. Conant, V. Petersen, and U. K. Freese. 1985. Replication of Epstein-Barr virus within the epithelial cells of oral "hairy" leukoplakia, an AIDS-associated lesion. N Engl J Med 313:1564-71.

143. Gu, S. Y., T. M. Huang, L. Ruan, Y. H. Miao, H. Lu, C. M. Chu, M. Motz, and H. Wolf. 1995. First EBV vaccine trial in humans using recombinant vaccinia virus expressing the major membrane antigen. Dev Biol Stand 84:171-7.

144. Gulley, M. L., and W. Tang. 2008. Laboratory assays for Epstein-Barr virus-related disease. J Mol Diagn 10:279-92.

145. Gupta, A., J. J. Gartner, P. Sethupathy, A. G. Hatzigeorgiou, and N. W. Fraser. 2006. Anti-apoptotic function of a microRNA encoded by the HSV-1 latency-associated transcript. Nature 442:82-5.

146. Haan, K. M., W. W. Kwok, R. Longnecker, and P. Speck. 2000. Epstein-Barr virus entry utilizing HLA-DP or HLA-DQ as a coreceptor. J Virol 74:2451-4.

147. Hahn, S., R. Gehri, and P. Erb. 1995. Mechanism and biological significance of CD4-mediated cytotoxicity. Immunol Rev 146:57-79.

148. Haigh, T. A., X. Lin, H. Jia, E. P. Hui, A. T. Chan, A. B. Rickinson, and G. S. Taylor. 2008. EBV latent membrane proteins (LMPs) 1 and 2 as immunotherapeutic targets: LMP-specific CD4+ cytotoxic T cell recognition of EBV-transformed B cell lines. J Immunol 180:1643-54.

149. Hamilton-Dutoit, S. J., D. Rea, M. Raphael, K. Sandvej, H. J. Delecluse, C. Gisselbrecht, L. Marelle, H. J. van Krieken, and G. Pallesen. 1993. Epstein-Barr virus-latent gene expression and tumor cell phenotype in acquired immunodeficiency syndrome-related non-Hodgkin's lymphoma. Correlation of lymphoma phenotype with three distinct patterns of viral latency. Am J Pathol 143:1072-85.

150. Hammerschmidt, W., and B. Sugden. 1988. Identification and characterization of oriLyt, a lytic origin of DNA replication of Epstein-Barr virus. Cell 55:427-33.

151. Harabuchi, Y., N. Yamanaka, A. Kataura, S. Imai, T. Kinoshita, F. Mizuno, and T. Osato. 1990. Epstein-Barr virus in nasal T-cell lymphomas in patients with lethal midline granuloma. Lancet 335:128-30.

152. Harada, S., and E. Kieff. 1997. Epstein-Barr virus nuclear protein LP stimulates EBNA-2 acidic domain-mediated transcriptional activation. J Virol 71:6611-8.

153. Haraguchi, S., R. A. Good, and N. K. Day. 1995. Immunosuppressive retroviral peptides: cAMP and cytokine patterns. Immunol Today 16:595-603.

154. Harris, N. L. 1999. Hodgkin's lymphomas: classification, diagnosis, and grading.

Introdction

4948

Chapter 1

Semin Hematol 36:220-32.155. Harris, N. L., E. S. Jaffe, H. Stein, P. M. Banks, J. K. Chan, M. L. Cleary, G. Delsol, C. De

Wolf-Peeters, B. Falini, K. C. Gatter, and et al. 1994. A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood 84:1361-92.

156. Henkel, T., P. D. Ling, S. D. Hayward, and M. G. Peterson. 1994. Mediation of Epstein-Barr virus EBNA2 transactivation by recombination signal-binding protein J kappa. Science 265:92-5.

157. Henle, G., and W. Henle. 1976. Epstein-Barr virus-specific IgA serum antibodies as an outstanding feature of nasopharyngeal carcinoma. Int J Cancer 17:1-7.

158. Henle, W., and G. Henle. 1976. The sero-epidemiology of Epstein-Barr virus. Adv Pathobiol:5-17.

159. Henle, W., G. Henle, H. C. Ho, P. Burtin, Y. Cachin, P. Clifford, A. de Schryver, G. de-The, V. Diehl, and G. Klein. 1970. Antibodies to Epstein-Barr virus in nasopharyngeal carcinoma, other head and neck neoplasms, and control groups. J Natl Cancer Inst 44:225-31.

160. Henle, W., G. Henle, B. A. Zajac, G. Pearson, R. Waubke, and M. Scriba. 1970. Differential reactivity of human serums with early antigens induced by Epstein-Barr virus. Science 169:188-90.

161. Herbst, H., F. Dallenbach, M. Hummel, G. Niedobitek, S. Pileri, N. Muller-Lantzsch, and H. Stein. 1991. Epstein-Barr virus latent membrane protein expression in Hodgkin and Reed-Sternberg cells. Proc Natl Acad Sci U S A 88:4766-70.

162. Herbst, H., H. D. Foss, J. Samol, I. Araujo, H. Klotzbach, H. Krause, A. Agathanggelou, G. Niedobitek, and H. Stein. 1996. Frequent expression of interleukin-10 by Epstein-Barr virus-harboring tumor cells of Hodgkin's disease. Blood 87:2918-29.

163. Herbst, H., J. Samol, H. D. Foss, T. Raff, and G. Niedobitek. 1997. Modulation of interleukin-6 expression in Hodgkin and Reed-Sternberg cells by Epstein-Barr virus. J Pathol 182:299-306.

164. Hislop, A. D., N. E. Annels, N. H. Gudgeon, A. M. Leese, and A. B. Rickinson. 2002. Epitope-specific evolution of human CD8(+) T cell responses from primary to persistent phases of Epstein-Barr virus infection. J Exp Med 195:893-905.

165. Hislop, A. D., M. Kuo, A. B. Drake-Lee, A. N. Akbar, W. Bergler, N. Hammerschmitt, N. Khan, U. Palendira, A. M. Leese, J. M. Timms, A. I. Bell, C. D. Buckley, and A. B. Rickinson. 2005. Tonsillar homing of Epstein-Barr virus-specific CD8+ T cells and the virus-host balance. J Clin Invest 115:2546-55.

166. Hislop, A. D., G. S. Taylor, D. Sauce, and A. B. Rickinson. 2007. Cellular responses to viral infection in humans: lessons from Epstein-Barr virus. Annu Rev Immunol 25:587-617.

167. Hitt, M. M., M. J. Allday, T. Hara, L. Karran, M. D. Jones, P. Busson, T. Tursz, I. Ernberg, and B. E. Griffin. 1989. EBV gene expression in an NPC-related tumour. Embo J 8:2639-51.

168. Hochberg, D., J. M. Middeldorp, M. Catalina, J. L. Sullivan, K. Luzuriaga, and D. A. Thorley-Lawson. 2004. Demonstration of the Burkitt's lymphoma Epstein-Barr virus phenotype in dividing latently infected memory cells in vivo. Proc Natl Acad Sci U S A 101:239-44.

169. Hopwood, P., and D. H. Crawford. 2000. The role of EBV in post-transplant malignancies: a review. J Clin Pathol 53:248-54.

170. Horst, D., D. van Leeuwen, N. P. Croft, M. A. Garstka, A. D. Hislop, E. Kremmer, A. B.

Rickinson, E. J. Wiertz, and M. E. Ressing. 2009. Specific targeting of the EBV lytic phase protein BNLF2a to the transporter associated with antigen processing results in impairment of HLA class I-restricted antigen presentation. J Immunol 182:2313-24.

171. Hovanessian, A. G. 1989. The double stranded RNA-activated protein kinase induced by interferon: dsRNA-PK. J Interferon Res 9:641-7.

172. Hsu, D. H., R. de Waal Malefyt, D. F. Fiorentino, M. N. Dang, P. Vieira, J. de Vries, H. Spits, T. R. Mosmann, and K. W. Moore. 1990. Expression of interleukin-10 activity by Epstein-Barr virus protein BCRF1. Science 250:830-2.

173. Hsu, M. M., W. C. Hsu, T. S. Sheen, and C. L. Kao. 2001. Specific IgA antibodies to recombinant early and nuclear antigens of Epstein-Barr virus in nasopharyngeal carcinoma. Clin Otolaryngol Allied Sci 26:334-8.

174. Huang, J., G. Liao, H. Chen, F. Y. Wu, L. Hutt-Fletcher, G. S. Hayward, and S. D. Hayward. 2006. Contribution of C/EBP proteins to Epstein-Barr virus lytic gene expression and replication in epithelial cells. J Virol 80:1098-109.

175. Imai, S., S. Koizumi, M. Sugiura, M. Tokunaga, Y. Uemura, N. Yamamoto, S. Tanaka, E. Sato, and T. Osato. 1994. Gastric carcinoma: monoclonal epithelial malignant cells expressing Epstein-Barr virus latent infection protein. Proc Natl Acad Sci U S A 91:9131-5.

176. Imai, S., J. Nishikawa, and K. Takada. 1998. Cell-to-cell contact as an efficient mode of Epstein-Barr virus infection of diverse human epithelial cells. J Virol 72:4371-8.

177. Israel, B. F., and S. C. Kenney. 2005. EBV Lytic Infection, p. 571-611. In E. S. Robertson (ed.), Epstein-Barr Virus. Caister Academic Press, Philadelphia.

178. Jarrett, R. F., and J. MacKenzie. 1999. Epstein-Barr virus and other candidate viruses in the pathogenesis of Hodgkin's disease. Semin Hematol 36:260-9.

179. Jenkins, P. J., U. K. Binne, and P. J. Farrell. 2000. Histone acetylation and reactivation of Epstein-Barr virus from latency. J Virol 74:710-20.

180. Ji, M. F., D. K. Wang, Y. L. Yu, Y. Q. Guo, J. S. Liang, W. M. Cheng, Y. S. Zong, K. H. Chan, S. P. Ng, W. I. Wei, D. T. Chua, J. S. Sham, and M. H. Ng. 2007. Sustained elevation of Epstein-Barr virus antibody levels preceding clinical onset of nasopharyngeal carcinoma. Br J Cancer 96:623-30.

181. Joab, I., J. C. Nicolas, G. Schwaab, G. de-The, B. Clausse, M. Perricaudet, and Y. Zeng. 1991. Detection of anti-Epstein-Barr-virus transactivator (ZEBRA) antibodies in sera from patients with nasopharyngeal carcinoma. Int J Cancer 48:647-9.

182. Joab, I., H. Triki, J. de Saint Martin, M. Perricaudet, and J. C. Nicolas. 1991. Detection of anti-Epstein-Barr virus trans-activator (ZEBRA) antibodies in sera from patients with human immunodeficiency virus. J Infect Dis 163:53-6.

183. Johannsen, E., M. Luftig, M. R. Chase, S. Weicksel, E. Cahir-McFarland, D. Illanes, D. Sarracino, and E. Kieff. 2004. Proteins of purified Epstein-Barr virus. Proc Natl Acad Sci U S A 101:16286-91.

184. Johannsen, E., C. L. Miller, S. R. Grossman, and E. Kieff. 1996. EBNA-2 and EBNA-3C extensively and mutually exclusively associate with RBPJkappa in Epstein-Barr virus-transformed B lymphocytes. J Virol 70:4179-83.

185. Jones, J. F., S. Shurin, C. Abramowsky, R. R. Tubbs, C. G. Sciotto, R. Wahl, J. Sands, D. Gottman, B. Z. Katz, and J. Sklar. 1988. T-cell lymphomas containing Epstein-Barr viral DNA in patients with chronic Epstein-Barr virus infections. N Engl J Med 318:733-41.

186. Kanai, K., Y. Satoh, H. Yamanaka, A. Kawaguchi, K. Horie, K. Sugata, Y. Hoshikawa, T. Sata, and T. Sairenji. 2007. The vIL-10 gene of the Epstein-Barr virus (EBV) is

Introdction

5150

Chapter 1

conserved in a stable manner except for a few point mutations in various EBV isolates. Virus Genes 35:563-9.

187. Kanavaros, P., M. C. Lescs, J. Briere, M. Divine, F. Galateau, I. Joab, J. Bosq, J. P. Farcet, F. Reyes, and P. Gaulard. 1993. Nasal T-cell lymphoma: a clinicopathologic entity associated with peculiar phenotype and with Epstein-Barr virus. Blood 81:2688-95.

188. Kapatai, G., and P. Murray. 2007. Contribution of the Epstein Barr virus to the molecular pathogenesis of Hodgkin lymphoma. J Clin Pathol 60:1342-9.

189. Karran, L., Y. Gao, P. R. Smith, and B. E. Griffin. 1992. Expression of a family of complementary-strand transcripts in Epstein-Barr virus-infected cells. Proc Natl Acad Sci U S A 89:8058-62.

190. Karray, H., W. Ayadi, L. Fki, A. Hammami, J. Daoud, M. M. Drira, M. Frikha, R. Jlidi, and J. M. Middeldorp. 2005. Comparison of three different serological techniques for primary diagnosis and monitoring of nasopharyngeal carcinoma in two age groups from Tunisia. J Med Virol 75:593-602.

191. Kennedy, R., and E. Celis. 2006. T helper lymphocytes rescue CTL from activation-induced cell death. J Immunol 177:2862-72.

192. Keryer-Bibens, C., C. Pioche-Durieu, C. Villemant, S. Souquere, N. Nishi, M. Hirashima, J. Middeldorp, and P. Busson. 2006. Exosomes released by EBV-infected nasopharyngeal carcinoma cells convey the viral latent membrane protein 1 and the immunomodulatory protein galectin 9. BMC Cancer 6:283.

193. Khabir, A., H. Karray, S. Rodriguez, M. Rose, J. Daoud, M. Frikha, T. Boudawara, J. Middeldorp, R. Jlidi, and P. Busson. 2005. EBV latent membrane protein 1 abundance correlates with patient age but not with metastatic behavior in north African nasopharyngeal carcinomas. Virol J 2:39.

194. Khanna, R., and S. R. Burrows. 2000. Role of cytotoxic T lymphocytes in Epstein-Barr virus-associated diseases. Annu Rev Microbiol 54:19-48.

195. Khanna, R., S. R. Burrows, V. Argaet, and D. J. Moss. 1994. Endoplasmic reticulum signal sequence facilitated transport of peptide epitopes restores immunogenicity of an antigen processing defective tumour cell line. Int Immunol 6:639-45.

196. Khanna, R., S. R. Burrows, M. G. Kurilla, C. A. Jacob, I. S. Misko, T. B. Sculley, E. Kieff, and D. J. Moss. 1992. Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia: implications for vaccine development. J Exp Med 176:169-76.

197. Khanna, R., S. R. Burrows, and D. J. Moss. 1995. Immune regulation in Epstein-Barr virus-associated diseases. Microbiol Rev 59:387-405.

198. Khanna, R., S. R. Burrows, J. Nicholls, and L. M. Poulsen. 1998. Identification of cytotoxic T cell epitopes within Epstein-Barr virus (EBV) oncogene latent membrane protein 1 (LMP1): evidence for HLA A2 supertype-restricted immune recognition of EBV-infected cells by LMP1-specific cytotoxic T lymphocytes. Eur J Immunol 28:451-8.

199. Khanna, R., S. R. Burrows, S. A. Thomson, D. J. Moss, P. Cresswell, L. M. Poulsen, and L. Cooper. 1997. Class I processing-defective Burkitt's lymphoma cells are recognized efficiently by CD4+ EBV-specific CTLs. J Immunol 158:3619-25.

200. Khanna, R., P. Busson, S. R. Burrows, C. Raffoux, D. J. Moss, J. M. Nicholls, and L. Cooper. 1998. Molecular characterization of antigen-processing function in nasopharyngeal carcinoma (NPC): evidence for efficient presentation of Epstein-Barr virus cytotoxic T-cell epitopes by NPC cells. Cancer Res 58:310-4.

201. Khanna, R., L. Cooper, N. Kienzle, D. J. Moss, S. R. Burrows, and K. K. Khanna. 1997. Engagement of CD40 antigen with soluble CD40 ligand up-regulates peptide transporter expression and restores endogenous processing function in Burkitt's lymphoma cells. J Immunol 159:5782-5.

202. Khanna, R., D. Moss, and M. Gandhi. 2005. Technology insight: Applications of emerging immunotherapeutic strategies for Epstein-Barr virus-associated malignancies. Nat Clin Pract Oncol 2:138-49.

203. Khanna, R., D. J. Moss, and S. R. Burrows. 1999. Vaccine strategies against Epstein-Barr virus-associated diseases: lessons from studies on cytotoxic T-cell-mediated immune regulation. Immunol Rev 170:49-64.

204. Khanna, R., J. Tellam, J. Duraiswamy, and L. Cooper. 2001. Immunotherapeutic strategies for EBV-associated malignancies. Trends Mol Med 7:270-6.

205. Kieff, E., and A. B. Rickinson. 2006. Epstein-Barr Virus and Its Replication, p. 2603-2654. In D. Knipe and P. Howley (ed.), Fields Virology, 5th ed. Lippincott Williams & Wilkins, Philadelphia.

206. Kieser, A., C. Kaiser, and W. Hammerschmidt. 1999. LMP1 signal transduction differs substantially from TNF receptor 1 signaling in the molecular functions of TRADD and TRAF2. Embo J 18:2511-21.

207. Kilger, E., A. Kieser, M. Baumann, and W. Hammerschmidt. 1998. Epstein-Barr virus-mediated B-cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor. Embo J 17:1700-9.

208. Kimura, H. 2006. Pathogenesis of chronic active Epstein-Barr virus infection: is this an infectious disease, lymphoproliferative disorder, or immunodeficiency? Rev Med Virol 16:251-61.

209. Kitagawa, N., M. Goto, K. Kurozumi, S. Maruo, M. Fukayama, T. Naoe, M. Yasukawa, K. Hino, T. Suzuki, S. Todo, and K. Takada. 2000. Epstein-Barr virus-encoded poly(A)(-) RNA supports Burkitt's lymphoma growth through interleukin-10 induction. Embo J 19:6742-50.

210. Knowles, D. M. 1999. Immunodeficiency-associated lymphoproliferative disorders. Mod Pathol 12:200-17.

211. Knowles, D. M. 1998. The molecular genetics of post-transplantation lymphoproliferative disorders. Springer Semin Immunopathol 20:357-73.

212. Knowles, D. M., and E. C. Pirog. 2001. Pathology of AIDS-related lymphomas and other AIDS-defining neoplasms. Eur J Cancer 37:1236-50.

213. Kobayashi, H., T. Nagato, M. Takahara, K. Sato, S. Kimura, N. Aoki, M. Azumi, M. Tateno, Y. Harabuchi, and E. Celis. 2008. Induction of EBV-latent membrane protein 1-specific MHC class II-restricted T-cell responses against natural killer lymphoma cells. Cancer Res 68:901-8.

214. Koppers-Lalic, D., F. A. Rijsewijk, S. B. Verschuren, J. A. van Gaans-Van den Brink, A. Neisig, M. E. Ressing, J. Neefjes, and E. J. Wiertz. 2001. The UL41-encoded virion host shutoff (vhs) protein and vhs-independent mechanisms are responsible for down-regulation of MHC class I molecules by bovine herpesvirus 1. J Gen Virol 82:2071-81.

215. Kulwichit, W., R. H. Edwards, E. M. Davenport, J. F. Baskar, V. Godfrey, and N. Raab-Traub. 1998. Expression of the Epstein-Barr virus latent membrane protein 1 induces B cell lymphoma in transgenic mice. Proc Natl Acad Sci U S A 95:11963-8.

216. Kuppers, R. 2003. B cells under influence: transformation of B cells by Epstein-Barr virus. Nat Rev Immunol 3:801-12.

217. Kuppers, R., and R. Dalla-Favera. 2001. Mechanisms of chromosomal

Introdction

5352

Chapter 1

translocations in B cell lymphomas. Oncogene 20:5580-94.218. Kurniawan, A., R. Kodariah, M. Elisabeth, A. Roezin, and S. Gondhowihardjo. 2002.

Evaluation of EBV-LMP1 as prognostic indicator of nasopharyngeal carcinoma. Medical Indonesian Journal 11:81-86.

219. Kwong, D. L., J. S. Sham, D. T. Chua, D. T. Choy, G. K. Au, and P. M. Wu. 1997. The effect of interruptions and prolonged treatment time in radiotherapy for nasopharyngeal carcinoma. Int J Radiat Oncol Biol Phys 39:703-10.

220. Lacerda, J. F., M. Ladanyi, D. C. Louie, J. M. Fernandez, E. B. Papadopoulos, and R. J. O'Reilly. 1996. Human Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes home preferentially to and induce selective regressions of autologous EBV-induced B cell lymphoproliferations in xenografted C.B-17 scid/scid mice. J Exp Med 183:1215-28.

221. Lagenaur, L. A., and J. M. Palefsky. 1999. Regulation of Epstein-Barr virus promoters in oral epithelial cells and lymphocytes. J Virol 73:6566-72.

222. Laichalk, L. L., D. Hochberg, G. J. Babcock, R. B. Freeman, and D. A. Thorley-Lawson. 2002. The dispersal of mucosal memory B cells: evidence from persistent EBV infection. Immunity 16:745-54.

223. Lau, K. M., S. H. Cheng, K. W. Lo, S. A. Lee, J. K. Woo, C. A. van Hasselt, S. P. Lee, A. B. Rickinson, and M. H. Ng. 2007. Increase in circulating Foxp3+CD4+CD25(high) regulatory T cells in nasopharyngeal carcinoma patients. Br J Cancer 96:617-22.

224. Lau, R., J. Middeldorp, and P. J. Farrell. 1993. Epstein-Barr virus gene expression in oral hairy leukoplakia. Virology 195:463-74.

225. Lautscham, G., S. Mayrhofer, G. Taylor, T. Haigh, A. Leese, A. Rickinson, and N. Blake. 2001. Processing of a multiple membrane spanning Epstein-Barr virus protein for CD8(+) T cell recognition reveals a proteasome-dependent, transporter associated with antigen processing-independent pathway. J Exp Med 194:1053-68.

226. Lee, A. W., W. Foo, S. C. Law, Y. F. Poon, W. M. Sze, S. K. O, S. Y. Tung, and W. H. Lau. 1997. Nasopharyngeal carcinoma: presenting symptoms and duration before diagnosis. Hong Kong Med J 3:355-361.

227. Lee, A. W., S. C. Law, S. H. Ng, D. K. Chan, Y. F. Poon, W. Foo, S. Y. Tung, F. K. Cheung, and J. H. Ho. 1992. Retrospective analysis of nasopharyngeal carcinoma treated during 1976-1985: late complications following megavoltage irradiation. Br J Radiol 65:918-28.

228. Lee, M. A., M. E. Diamond, and J. L. Yates. 1999. Genetic evidence that EBNA-1 is needed for efficient, stable latent infection by Epstein-Barr virus. J Virol 73:2974-82.

229. Lee, M. H., J. F. Chiou, K. Y. Yen, and L. L. Yang. 2000. EBV DNA polymerase inhibition of tannins from Eugenia uniflora. Cancer Lett 154:131-6.

230. Lee, S. P., A. T. Chan, S. T. Cheung, W. A. Thomas, D. CroomCarter, C. W. Dawson, C. H. Tsai, S. F. Leung, P. J. Johnson, and D. P. Huang. 2000. CTL control of EBV in nasopharyngeal carcinoma (NPC): EBV-specific CTL responses in the blood and tumors of NPC patients and the antigen-processing function of the tumor cells. J Immunol 165:573-82.

231. Lee, S. P., R. J. Tierney, W. A. Thomas, J. M. Brooks, and A. B. Rickinson. 1997. Conserved CTL epitopes within EBV latent membrane protein 2: a potential target for CTL-based tumor therapy. J Immunol 158:3325-34.

232. Leen, A., P. Meij, I. Redchenko, J. Middeldorp, E. Bloemena, A. Rickinson, and N. Blake. 2001. Differential immunogenicity of Epstein-Barr virus latent-cycle proteins for human CD4(+) T-helper 1 responses. J Virol 75:8649-59.

233. Leight, E. R., and B. Sugden. 2000. EBNA-1: a protein pivotal to latent infection by Epstein-Barr virus. Rev Med Virol 10:83-100.

234. Lennette, E. T., G. Winberg, M. Yadav, G. Enblad, and G. Klein. 1995. Antibodies to LMP2A/2B in EBV-carrying malignancies. Eur J Cancer 31A:1875-8.

235. Lerner, M. R., N. C. Andrews, G. Miller, and J. A. Steitz. 1981. Two small RNAs encoded by Epstein-Barr virus and complexed with protein are precipitated by antibodies from patients with systemic lupus erythematosus. Proc Natl Acad Sci U S A 78:805-9.

236. Levitskaya, J., M. Coram, V. Levitsky, S. Imreh, P. M. Steigerwald-Mullen, G. Klein, M. G. Kurilla, and M. G. Masucci. 1995. Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1. Nature 375:685-8.

237. Levitskaya, J., A. Sharipo, A. Leonchiks, A. Ciechanover, and M. G. Masucci. 1997. Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein-Barr virus nuclear antigen 1. Proc Natl Acad Sci U S A 94:12616-21.

238. Levitsky, V., and M. G. Masucci. 2002. Manipulation of immune responses by Epstein-Barr virus. Virus Res 88:71-86.

239. Levitsky, V., Q. J. Zhang, J. Levitskaya, M. G. Kurilla, and M. G. Masucci. 1997. Natural variants of the immunodominant HLA A11-restricted CTL epitope of the EBV nuclear antigen-4 are nonimmunogenic due to intracellular dissociation from MHC class I:peptide complexes. J Immunol 159:5383-90.

240. Li, J., X. H. Zeng, H. Y. Mo, U. Rolen, Y. F. Gao, X. S. Zhang, Q. Y. Chen, L. Zhang, M. S. Zeng, M. Z. Li, W. L. Huang, X. N. Wang, Y. X. Zeng, and M. G. Masucci. 2007. Functional inactivation of EBV-specific T-lymphocytes in nasopharyngeal carcinoma: implications for tumor immunotherapy. PLoS ONE 2:e1122.

241. Li, L., D. Liu, L. Hutt-Fletcher, A. Morgan, M. G. Masucci, and V. Levitsky. 2002. Epstein-Barr virus inhibits the development of dendritic cells by promoting apoptosis of their monocyte precursors in the presence of granulocyte macrophage-colony-stimulating factor and interleukin-4. Blood 99:3725-34.

242. Li, Q., M. K. Spriggs, S. Kovats, S. M. Turk, M. R. Comeau, B. Nepom, and L. M. Hutt-Fletcher. 1997. Epstein-Barr virus uses HLA class II as a cofactor for infection of B lymphocytes. J Virol 71:4657-62.

243. Lin, C. L., W. F. Lo, T. H. Lee, Y. Ren, S. L. Hwang, Y. F. Cheng, C. L. Chen, Y. S. Chang, S. P. Lee, A. B. Rickinson, and P. K. Tam. 2002. Immunization with Epstein-Barr Virus (EBV) peptide-pulsed dendritic cells induces functional CD8+ T-cell immunity and may lead to tumor regression in patients with EBV-positive nasopharyngeal carcinoma. Cancer Res 62:6952-8.

244. Lin, J. C., J. S. Jan, C. Y. Hsu, W. M. Liang, R. S. Jiang, and W. Y. Wang. 2003. Phase III study of concurrent chemoradiotherapy versus radiotherapy alone for advanced nasopharyngeal carcinoma: positive effect on overall and progression-free survival. J Clin Oncol 21:631-7.

245. Lin, J. C., W. Y. Wang, K. Y. Chen, Y. H. Wei, W. M. Liang, J. S. Jan, and R. S. Jiang. 2004. Quantification of plasma Epstein-Barr virus DNA in patients with advanced nasopharyngeal carcinoma. N Engl J Med 350:2461-70.

246. Lin, X., N. H. Gudgeon, E. P. Hui, H. Jia, X. Qun, G. S. Taylor, M. C. Barnardo, C. K. Lin, A. B. Rickinson, and A. T. Chan. 2008. CD4 and CD8 T cell responses to tumour-associated Epstein-Barr virus antigens in nasopharyngeal carcinoma patients. Cancer Immunol Immunother 57:963-75.

247. Liu, M. Y., Y. T. Huang, T. S. Sheen, J. Y. Chen, and C. H. Tsai. 2004. Immune

Introdction

5554

Chapter 1

responses to Epstein-Barr virus lytic proteins in patients with nasopharyngeal carcinoma. J Med Virol 73:574-82.

248. Liu, Y. J., and C. Arpin. 1997. Germinal center development. Immunol Rev 156:111-26.249. Liu, Y. J., D. E. Joshua, G. T. Williams, C. A. Smith, J. Gordon, and I. C. MacLennan.

1989. Mechanism of antigen-driven selection in germinal centres. Nature 342:929-31.

250. Lo, A. K., K. F. To, K. W. Lo, R. W. Lung, J. W. Hui, G. Liao, and S. D. Hayward. 2007. Modulation of LMP1 protein expression by EBV-encoded microRNAs. Proc Natl Acad Sci U S A 104:16164-9.

251. Lo, Y. M., S. F. Leung, L. Y. Chan, A. T. Chan, K. W. Lo, P. J. Johnson, and D. P. Huang. 2000. Kinetics of plasma Epstein-Barr virus DNA during radiation therapy for nasopharyngeal carcinoma. Cancer Res 60:2351-5.

252. Low, W. K., J. L. Leong, Y. H. Goh, and K. W. Fong. 2000. Diagnostic value of Epstein-Barr viral serology in nasopharyngeal carcinoma. Otolaryngol Head Neck Surg 123:505-7.

253. Lu, C., and M. Chen. 2006. Lytic replication of Epstein-Barr virus. Future Virology 1:435-446.

254. Lupton, S., and A. J. Levine. 1985. Mapping genetic elements of Epstein-Barr virus that facilitate extrachromosomal persistence of Epstein-Barr virus-derived plasmids in human cells. Mol Cell Biol 5:2533-42.

255. Lutzky, V. P., M. Corban, L. Heslop, L. E. Morrison, P. Crooks, D. F. Hall, W. B. Coman, S. A. Thomson, and D. J. Moss. 2009. A novel approach to the formulation of an EBV antigen-based nasopharyngeal carcinoma vaccine. J Virol.

256. Maffei, A., and P. E. Harris. 1998. Peptides bound to major histocompatibility complex molecules. Peptides 19:179-98.

257. Magrath, I., V. Jain, and K. Bhatia. 1992. Epstein-Barr virus and Burkitt's lymphoma. Semin Cancer Biol 3:285-95.

258. Maini, M. K., N. Gudgeon, L. R. Wedderburn, A. B. Rickinson, and P. C. Beverley. 2000. Clonal expansions in acute EBV infection are detectable in the CD8 and not the CD4 subset and persist with a variable CD45 phenotype. J Immunol 165:5729-37.

259. Mannick, J. B., J. I. Cohen, M. Birkenbach, A. Marchini, and E. Kieff. 1991. The Epstein-Barr virus nuclear protein encoded by the leader of the EBNA RNAs is important in B-lymphocyte transformation. J Virol 65:6826-37.

260. Marchini, A., B. Tomkinson, J. I. Cohen, and E. Kieff. 1991. BHRF1, the Epstein-Barr virus gene with homology to Bc12, is dispensable for B-lymphocyte transformation and virus replication. J Virol 65:5991-6000.

261. Marechal, V., A. Dehee, R. Chikhi-Brachet, T. Piolot, M. Coppey-Moisan, and J. C. Nicolas. 1999. Mapping EBNA-1 domains involved in binding to metaphase chromosomes. J Virol 73:4385-92.

262. Marshall, D., and C. Sample. 1995. Epstein-Barr virus nuclear antigen 3C is a transcriptional regulator. J Virol 69:3624-30.

263. Marshall, N. A., M. A. Vickers, and R. N. Barker. 2003. Regulatory T cells secreting IL-10 dominate the immune response to EBV latent membrane protein 1. J Immunol 170:6183-9.

264. Martorelli, D., K. Houali, L. Caggiari, E. Vaccher, L. Barzan, G. Franchin, A. Gloghini, A. Pavan, A. Da Ponte, R. M. Tedeschi, V. De Re, A. Carbone, T. Ooka, P. De Paoli, and R. Dolcetti. 2008. Spontaneous T cell responses to Epstein-Barr virus-encoded BARF1 protein and derived peptides in patients with nasopharyngeal carcinoma: bases for improved immunotherapy. Int J Cancer 123:1100-7.

265. Masucci, M. G., R. Szigeti, I. Ernberg, G. Masucci, G. Klein, J. Chessels, C. Sieff, S. Lie, A. Glomstein, L. Businco, W. Henle, G. Henle, G. Pearson, K. Sakamoto, and D. T. Purtilo. 1981. Cellular immune defects to Epstein-Barr virus-determined antigens in young males. Cancer Res 41:4284-91.

266. Mathew, A., H. M. Cheng, C. K. Sam, I. Joab, U. Prasad, and C. Cochet. 1994. A high incidence of serum IgG antibodies to the Epstein-Barr virus replication activator protein in nasopharyngeal carcinoma. Cancer Immunol Immunother 38:68-70.

267. Matloubian, M., R. J. Concepcion, and R. Ahmed. 1994. CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection. J Virol 68:8056-63.

268. Matsue, K., M. Itoh, K. Tsukuda, T. Kokubo, and Y. Hirose. 1998. Development of Epstein-Barr virus-associated B cell lymphoma after intensive treatment of patients with angioimmunoblastic lymphadenopathy with dysproteinemia. Int J Hematol 67:319-29.

269. McGuire, L. J., and J. C. Lee. 1990. The histopathologic diagnosis of nasopharyngeal carcinoma. Ear Nose Throat J 69:229-36.

270. Mehl, A. M., N. Fischer, M. Rowe, F. Hartmann, H. Daus, L. Trumper, M. Pfreundschuh, N. Muller-Lantzsch, and F. A. Grasser. 1998. Isolation and analysis of two strongly transforming isoforms of the Epstein-Barr-Virus(EBV)-encoded latent membrane protein-1 (LMP1) from a single Hodgkin's lymphoma. Int J Cancer 76:194-200.

271. Meij, P., A. Leen, A. B. Rickinson, S. Verkoeijen, M. B. Vervoort, E. Bloemena, and J. M. Middeldorp. 2002. Identification and prevalence of CD8(+) T-cell responses directed against Epstein-Barr virus-encoded latent membrane protein 1 and latent membrane protein 2. Int J Cancer 99:93-9.

272. Meij, P., M. B. Vervoort, J. Aarbiou, P. van Dissel, A. Brink, E. Bloemena, C. J. Meijer, and J. M. Middeldorp. 1999. 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. J Infect Dis 179:1108-15.

273. Meij, P., M. B. Vervoort, E. Bloemena, T. E. Schouten, C. Schwartz, S. Grufferman, R. F. Ambinder, and J. M. Middeldorp. 2002. Antibody responses to Epstein-Barr virus-encoded latent membrane protein-1 (LMP1) and expression of LMP1 in juvenile Hodgkin's disease. J Med Virol 68:370-7.

274. Merchant, M., R. Swart, R. B. Katzman, M. Ikeda, A. Ikeda, R. Longnecker, M. L. Dykstra, and S. K. Pierce. 2001. The effects of the Epstein-Barr virus latent membrane protein 2A on B cell function. Int Rev Immunol 20:805-35.

275. Middeldorp, J. M. 2002. Molecular diagnosis of viral infections in renal transplant recipients. Curr Opin Nephrol Hypertens 11:665-72.

276. Middeldorp, J. M., A. A. Brink, A. J. van den Brule, and C. J. Meijer. 2003. Pathogenic roles for Epstein-Barr virus (EBV) gene products in EBV-associated proliferative disorders. Crit Rev Oncol Hematol 45:1-36.

277. Middeldorp, J. M., and P. Herbrink. 1988. Epstein-Barr virus specific marker molecules for early diagnosis of infectious mononucleosis. J Virol Methods 21:133-46.

278. Middeldorp, J. M., and D. M. Pegtel. 2008. Multiple roles of LMP1 in Epstein-Barr virus induced immune escape. Semin Cancer Biol 18:388-96.

279. Miller, C. L., A. L. Burkhardt, J. H. Lee, B. Stealey, R. Longnecker, J. B. Bolen, and E. Kieff. 1995. Integral membrane protein 2 of Epstein-Barr virus regulates reactivation from latency through dominant negative effects on protein-tyrosine

Introdction

5756

Chapter 1

kinases. Immunity 2:155-66.280. Miller, G., A. El-Guindy, J. Countryman, J. Ye, and L. Gradoville. 2007. Lytic cycle

switches of oncogenic human gammaherpesviruses. Adv Cancer Res 97:81-109.281. Miyashita, E. M., B. Yang, G. J. Babcock, and D. A. Thorley-Lawson. 1997.

Identification of the site of Epstein-Barr virus persistence in vivo as a resting B cell. J Virol 71:4882-91.

282. Modrow, S., and H. Wolf. 1986. Characterization of two related Epstein-Barr virus-encoded membrane proteins that are differentially expressed in Burkitt lymphoma and in vitro-transformed cell lines. Proc Natl Acad Sci U S A 83:5703-7.

283. Molesworth, S. J., C. M. Lake, C. M. Borza, S. M. Turk, and L. M. Hutt-Fletcher. 2000. Epstein-Barr virus gH is essential for penetration of B cells but also plays a role in attachment of virus to epithelial cells. J Virol 74:6324-32.

284. Moutschen, M., P. Leonard, E. M. Sokal, F. Smets, M. Haumont, P. Mazzu, A. Bollen, F. Denamur, P. Peeters, G. Dubin, and M. Denis. 2007. Phase I/II studies to evaluate safety and immunogenicity of a recombinant gp350 Epstein-Barr virus vaccine in healthy adults. Vaccine 25:4697-705.

285. Munz, C. 2004. Epstein-barr virus nuclear antigen 1: from immunologically invisible to a promising T cell target. J Exp Med 199:1301-4.

286. Munz, C., K. L. Bickham, M. Subklewe, M. L. Tsang, A. Chahroudi, M. G. Kurilla, D. Zhang, M. O'Donnell, and R. M. Steinman. 2000. Human CD4(+) T lymphocytes consistently respond to the latent Epstein-Barr virus nuclear antigen EBNA1. J Exp Med 191:1649-60.

287. Munz, C., and A. Moormann. 2008. Immune escape by Epstein-Barr virus associated malignancies. Semin Cancer Biol 18:381-7.

288. Murphy, K., P. Travers, and M. Walport. 2008. Jeneway's Immunobiology, 7 ed. Garland Science, Taylor & Francis Group, LLC, New York.

289. Murray, R. J., M. G. Kurilla, J. M. Brooks, W. A. Thomas, M. Rowe, E. Kieff, and A. B. Rickinson. 1992. Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies. J Exp Med 176:157-68.

290. Mutirangura, A., W. Pornthanakasem, A. Theamboonlers, V. Sriuranpong, P. Lertsanguansinchi, S. Yenrudi, N. Voravud, P. Supiyaphun, and Y. Poovorawan. 1998. Epstein-Barr viral DNA in serum of patients with nasopharyngeal carcinoma. Clin Cancer Res 4:665-9.

291. Nakagomi, H., R. Dolcetti, M. T. Bejarano, P. Pisa, R. Kiessling, and M. G. Masucci. 1994. The Epstein-Barr virus latent membrane protein-1 (LMP1) induces interleukin-10 production in Burkitt lymphoma lines. Int J Cancer 57:240-4.

292. Nanbo, A., K. Inoue, K. Adachi-Takasawa, and K. Takada. 2002. Epstein-Barr virus RNA confers resistance to interferon-alpha-induced apoptosis in Burkitt's lymphoma. Embo J 21:954-65.

293. Neri, A., F. Barriga, G. Inghirami, D. M. Knowles, J. Neequaye, I. T. Magrath, and R. Dalla-Favera. 1991. Epstein-Barr virus infection precedes clonal expansion in Burkitt's and acquired immunodeficiency syndrome-associated lymphoma. Blood 77:1092-5.

294. Ng, M. H., K. H. Chan, S. P. Ng, and Y. S. Zong. 2006. Epstein-Barr virus serology in early detection and screening of nasopharyngeal carcinoma. Ai Zheng 25:250-6.

295. Ng, W. T., T. K. Yau, R. W. Yung, W. M. Sze, A. H. Tsang, A. L. Law, and A. W. Lee. 2005. Screening for family members of patients with nasopharyngeal carcinoma. Int J Cancer 113:998-1001.

296. Niedobitek, G., A. Agathanggelou, H. Herbst, L. Whitehead, D. H. Wright, and L. S. Young. 1997. Epstein-Barr virus (EBV) infection in infectious mononucleosis: virus latency, replication and phenotype of EBV-infected cells. J Pathol 182:151-9.

297. Niedobitek, G., A. Agathanggelou, N. Steven, and L. S. Young. 2000. Epstein-Barr virus (EBV) in infectious mononucleosis: detection of the virus in tonsillar B lymphocytes but not in desquamated oropharyngeal epithelial cells. Mol Pathol 53:37-42.

298. Niedobitek, G., E. Kremmer, H. Herbst, L. Whitehead, C. W. Dawson, E. Niedobitek, C. von Ostau, N. Rooney, F. A. Grasser, and L. S. Young. 1997. Immunohistochemical detection of the Epstein-Barr virus-encoded latent membrane protein 2A in Hodgkin's disease and infectious mononucleosis. Blood 90:1664-72.

299. Niedobitek, G., L. S. Young, R. Lau, L. Brooks, D. Greenspan, J. S. Greenspan, and A. B. Rickinson. 1991. Epstein-Barr virus infection in oral hairy leukoplakia: virus replication in the absence of a detectable latent phase. J Gen Virol 72 ( Pt 12):3035-46.

300. Nielsen, N. H., F. Mikkelsen, and J. P. Hansen. 1977. Nasopharyngeal cancer in Greenland. The incidence in an Arctic Eskimo population. Acta Pathol Microbiol Scand [A] 85:850-8.

301. Nikiforow, S., K. Bottomly, G. Miller, and C. Munz. 2003. Cytolytic CD4(+)-T-cell clones reactive to EBNA1 inhibit Epstein-Barr virus-induced B-cell proliferation. J Virol 77:12088-104.

302. O'Conor, G. T. 1961. Malignant lymphoma in African children. II. A pathological entity. Cancer 14:270-83.

303. O'Reilly, R. J., T. N. Small, E. Papadopoulos, K. Lucas, J. Lacerda, and L. Koulova. 1997. Biology and adoptive cell therapy of Epstein-Barr virus-associated lymphoproliferative disorders in recipients of marrow allografts. Immunol Rev 157:195-216.

304. Ogino, T., S. Moriai, Y. Ishida, H. Ishii, A. Katayama, N. Miyokawa, Y. Harabuchi, and S. Ferrone. 2007. Association of immunoescape mechanisms with Epstein-Barr virus infection in nasopharyngeal carcinoma. Int J Cancer 120:2401-10.

305. Omiya, R., C. Buteau, H. Kobayashi, C. V. Paya, and E. Celis. 2002. Inhibition of EBV-induced lymphoproliferation by CD4(+) T cells specific for an MHC class II promiscuous epitope. J Immunol 169:2172-9.

306. Oostendorp, R. A., W. M. Schaaper, J. Post, B. M. Von Blomberg, R. H. Meloen, and R. J. Scheper. 1992. Suppression of lymphocyte proliferation by a retroviral p15E-derived hexapeptide. Eur J Immunol 22:1505-11.

307. Oudejans, J. J., A. Harijadi, S. A. Cillessen, P. Busson, I. B. Tan, D. F. Dukers, W. Vos, B. Hariwiyanto, J. Middeldorp, and C. J. Meijer. 2005. Absence of caspase 3 activation in neoplastic cells of nasopharyngeal carcinoma biopsies predicts rapid fatal outcome. Mod Pathol 18:877-85.

308. Oudejans, J. J., H. Harijadi, J. A. Kummer, I. B. Tan, E. Bloemena, J. M. Middeldorp, B. Bladergroen, D. F. Dukers, W. Vos, and C. J. Meijer. 2002. High numbers of granzyme B/CD8-positive tumour-infiltrating lymphocytes in nasopharyngeal carcinoma biopsies predict rapid fatal outcome in patients treated with curative intent. J Pathol 198:468-75.

309. Ozyar, E., I. L. Atahan, F. H. Akyol, M. Gurkaynak, and A. F. Zorlu. 1994. Cranial nerve involvement in nasopharyngeal carcinoma: its prognostic role and response to radiotherapy. Radiat Med 12:65-8.

Introdction

5958

Chapter 1

310. Packham, G., M. Brimmell, D. Cook, A. J. Sinclair, and P. J. Farrell. 1993. Strain variation in Epstein-Barr virus immediate early genes. Virology 192:541-50.

311. Palefsky, J. M., M. E. Penaranda, L. T. Pierik, L. A. Lagenaur, L. A. MacPhail, D. Greenspan, and J. S. Greenspan. 1997. Epstein-Barr virus BMRF-2 and BDLF-3 expression in hairy leukoplakia. Oral Dis 3 Suppl 1:S171-6.

312. Pallesen, G., S. J. Hamilton-Dutoit, M. Rowe, and L. S. Young. 1991. Expression of Epstein-Barr virus latent gene products in tumour cells of Hodgkin's disease. Lancet 337:320-2.

313. Paludan, C., K. Bickham, S. Nikiforow, M. L. Tsang, K. Goodman, W. A. Hanekom, J. F. Fonteneau, S. Stevanovic, and C. Munz. 2002. Epstein-Barr nuclear antigen 1-specific CD4(+) Th1 cells kill Burkitt's lymphoma cells. J Immunol 169:1593-603.

314. Paludan, C., and C. Munz. 2003. CD4+ T cell responses in the immune control against latent infection by Epstein-Barr virus. Curr Mol Med 3:341-7.

315. Pappworth, I. Y., E. C. Wang, and M. Rowe. 2007. The switch from latent to productive infection in epstein-barr virus-infected B cells is associated with sensitization to NK cell killing. J Virol 81:474-82.

316. Paramita, D. K., J. Fachiroh, W. T. Artama, E. van Benthem, S. M. Haryana, and J. M. Middeldorp. 2007. Native early antigen of Epstein-Barr virus, a promising antigen for diagnosis of nasopharyngeal carcinoma. J Med Virol 79:1710-21.

317. Paramita, D. K., J. Fachiroh, S. M. Haryana, and J. M. Middeldorp. 2008. Evaluation of commercial EBV RecombLine assay for diagnosis of nasopharyngeal carcinoma. J Clin Virol 42:343-52.

318. Parker, G. A., T. Crook, M. Bain, E. A. Sara, P. J. Farrell, and M. J. Allday. 1996. Epstein-Barr virus nuclear antigen (EBNA)3C is an immortalizing oncoprotein with similar properties to adenovirus E1A and papillomavirus E7. Oncogene 13:2541-9.

319. Parkin, D., S. Whelan, J. Ferlay, L. Raymond, and J. Young (ed.). 1997. Cancer incidence in five continents. IARC.

320. Paschos, K., P. Smith, E. Anderton, J. M. Middeldorp, R. E. White, and M. J. Allday. 2009. Epstein-barr virus latency in B cells leads to epigenetic repression and CpG methylation of the tumour suppressor gene Bim. PLoS Pathog 5:e1000492.

321. Pathmanathan, R., U. Prasad, R. Sadler, K. Flynn, and N. Raab-Traub. 1995. Clonal proliferations of cells infected with Epstein-Barr virus in preinvasive lesions related to nasopharyngeal carcinoma. N Engl J Med 333:693-8.

322. Pearson, G. R., L. F. Qualtiere, G. Klein, T. Norin, and I. S. Bal. 1979. Epstein-Barr virus-specific antibody-dependent cellular cytotoxicity in patients with Burkitt's lymphoma. Int J Cancer 24:402-6.

323. Pegtel, D. M., J. Middeldorp, and D. A. Thorley-Lawson. 2004. Epstein-Barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers. J Virol 78:12613-24.

324. Pfeffer, S., A. Sewer, M. Lagos-Quintana, R. Sheridan, C. Sander, F. A. Grasser, L. F. van Dyk, C. K. Ho, S. Shuman, M. Chien, J. J. Russo, J. Ju, G. Randall, B. D. Lindenbach, C. M. Rice, V. Simon, D. D. Ho, M. Zavolan, and T. Tuschl. 2005. Identification of microRNAs of the herpesvirus family. Nat Methods 2:269-76.

325. Pfeffer, S., M. Zavolan, F. A. Grasser, M. Chien, J. J. Russo, J. Ju, B. John, A. J. Enright, D. Marks, C. Sander, and T. Tuschl. 2004. Identification of virus-encoded microRNAs. Science 304:734-6.

326. Poppema, S. 2005. Immunobiology and pathophysiology of hodgkin lymphomas. Hematology Am Soc Hematol Educ Program:231-8.

327. Precopio, M. L., J. L. Sullivan, C. Willard, M. Somasundaran, and K. Luzuriaga. 2003.

Differential kinetics and specificity of EBV-specific CD4+ and CD8+ T cells during primary infection. J Immunol 170:2590-8.

328. Pudney, V. A., A. M. Leese, A. B. Rickinson, and A. D. Hislop. 2005. CD8+ immunodominance among Epstein-Barr virus lytic cycle antigens directly reflects the efficiency of antigen presentation in lytically infected cells. J Exp Med 201:349-60.

329. Raab-Traub, N. 2005. Epstein-Barr virus in the pathogenesis of NPC. P 71-92 In E.S. Robertson (ed), Epstein-Barr virus Caister Academic Press, Philadelphi.

330. Raab-Traub, N. 2002. Epstein-Barr virus in the pathogenesis of NPC. Semin Cancer Biol 12:431-41.

331. Radkov, S. A., M. Bain, P. J. Farrell, M. West, M. Rowe, and M. J. Allday. 1997. Epstein-Barr virus EBNA3C represses Cp, the major promoter for EBNA expression, but has no effect on the promoter of the cell gene CD21. J Virol 71:8552-62.

332. Rawlins, D. R., G. Milman, S. D. Hayward, and G. S. Hayward. 1985. Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region. Cell 42:859-68.

333. Rees, L., E. J. Tizard, A. J. Morgan, W. D. Cubitt, S. Finerty, T. A. Oyewole-Eletu, K. Owen, C. Royed, S. J. Stevens, R. C. Shroff, M. K. Tanday, A. D. Wilson, J. M. Middeldorp, P. L. Amlot, and N. M. Steven. 2009. A phase I trial of epstein-barr virus gp350 vaccine for children with chronic kidney disease awaiting transplantation. Transplantation 88:1025-9.

334. Reisman, D., J. Yates, and B. Sugden. 1985. A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components. Mol Cell Biol 5:1822-32.

335. Resnick, L., J. S. Herbst, D. V. Ablashi, S. Atherton, B. Frank, L. Rosen, and S. N. Horwitz. 1988. Regression of oral hairy leukoplakia after orally administered acyclovir therapy. Jama 259:384-8.

336. Ressing, M. E., D. Horst, B. D. Griffin, J. Tellam, J. Zuo, R. Khanna, M. Rowe, and E. J. Wiertz. 2008. Epstein-Barr virus evasion of CD8 and CD4 T cell immunity via concerted actions of multiple gene products. Semin Cancer Biol.

337. Rickinson, A. B., M. F. Callan, and N. E. Annels. 2000. T-cell memory: lessons from Epstein-Barr virus infection in man. Philos Trans R Soc Lond B Biol Sci 355:391-400.

338. Rickinson, A. B., and E. Kieff. 2006. Epstein-Barr Virus, p. 2655-2700. In D. Knipe and P. Howley (ed.), Fields Virology, 5th ed. Lippincott Williams & Wilkins, Philadelphia.

339. Rickinson, A. B., and D. J. Moss. 1997. Human cytotoxic T lymphocyte responses to Epstein-Barr virus infection. Annu Rev Immunol 15:405-31.

340. Robertson, E. S., S. Grossman, E. Johannsen, C. Miller, J. Lin, B. Tomkinson, and E. Kieff. 1995. Epstein-Barr virus nuclear protein 3C modulates transcription through interaction with the sequence-specific DNA-binding protein J kappa. J Virol 69:3108-16.

341. Robertson, E. S., J. Lin, and E. Kieff. 1996. The amino-terminal domains of Epstein-Barr virus nuclear proteins 3A, 3B, and 3C interact with RBPJ(kappa). J Virol 70:3068-74.

342. Rochford, R., M. J. Cannon, and A. M. Moormann. 2005. Endemic Burkitt's lymphoma: a polymicrobial disease? Nat Rev Microbiol 3:182-7.

343. Rooney, C. M., M. A. Roskrow, C. A. Smith, M. K. Brenner, and H. E. Heslop. 1998. Immunotherapy for Epstein-Barr virus-associated cancers. J Natl Cancer Inst Monogr:89-93.

344. Rosa, M. D., E. Gottlieb, M. R. Lerner, and J. A. Steitz. 1981. Striking similarities are

Introdction

6160

Chapter 1

exhibited by two small Epstein-Barr virus-encoded ribonucleic acids and the adenovirus-associated ribonucleic acids VAI and VAII. Mol Cell Biol 1:785-96.

345. Rowe, M., J. Finke, R. Szigeti, and G. Klein. 1988. Characterization of the serological response in man to the latent membrane protein and the six nuclear antigens encoded by Epstein-Barr virus. J Gen Virol 69 ( Pt 6):1217-28.

346. Rowe, M., B. Glaunsinger, D. van Leeuwen, J. Zuo, D. Sweetman, D. Ganem, J. Middeldorp, E. J. Wiertz, and M. E. Ressing. 2007. Host shutoff during productive Epstein-Barr virus infection is mediated by BGLF5 and may contribute to immune evasion. Proc Natl Acad Sci U S A 104:3366-71.

347. Rowe, M., R. Khanna, C. A. Jacob, V. Argaet, A. Kelly, S. Powis, M. Belich, D. Croom-Carter, S. Lee, S. R. Burrows, and et al. 1995. Restoration of endogenous antigen processing in Burkitt's lymphoma cells by Epstein-Barr virus latent membrane protein-1: coordinate up-regulation of peptide transporters and HLA-class I antigen expression. Eur J Immunol 25:1374-84.

348. Ruoslahti, E. 1996. RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 12:697-715.

349. Sall, A., S. Caserta, P. Jolicoeur, L. Franqueville, M. de Turenne-Tessier, and T. Ooka. 2004. Mitogenic activity of Epstein-Barr virus-encoded BARF1 protein. Oncogene 23:4938-44.

350. Samols, M. A., J. Hu, R. L. Skalsky, and R. Renne. 2005. Cloning and identification of a microRNA cluster within the latency-associated region of Kaposi's sarcoma-associated herpesvirus. J Virol 79:9301-5.

351. Sample, J., M. Hummel, D. Braun, M. Birkenbach, and E. Kieff. 1986. Nucleotide sequences of mRNAs encoding Epstein-Barr virus nuclear proteins: a probable transcriptional initiation site. Proc Natl Acad Sci U S A 83:5096-100.

352. Schaefer, B. C., M. Woisetschlaeger, J. L. Strominger, and S. H. Speck. 1991. Exclusive expression of Epstein-Barr virus nuclear antigen 1 in Burkitt lymphoma arises from a third promoter, distinct from the promoters used in latently infected lymphocytes. Proc Natl Acad Sci U S A 88:6550-4.

353. Schubert, U., L. C. Anton, J. Gibbs, C. C. Norbury, J. W. Yewdell, and J. R. Bennink. 2000. Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 404:770-4.

354. Schuster, V., S. Seidenspinner, and H. W. Kreth. 1996. Detection of a nuclear antigen 2 (EBNA2)-variant Epstein-Barr virus strain in two siblings with fatal lymphoproliferative disease. J Med Virol 48:114-20.

355. Scotet, E., J. David-Ameline, M. A. Peyrat, A. Moreau-Aubry, D. Pinczon, A. Lim, J. Even, G. Semana, J. M. Berthelot, R. Breathnach, M. Bonneville, and E. Houssaint. 1996. T cell response to Epstein-Barr virus transactivators in chronic rheumatoid arthritis. J Exp Med 184:1791-800.

356. Sears, J., J. Kolman, G. M. Wahl, and A. Aiyar. 2003. Metaphase chromosome tethering is necessary for the DNA synthesis and maintenance of oriP plasmids but is insufficient for transcription activation by Epstein-Barr nuclear antigen 1. J Virol 77:11767-80.

357. Sears, J., M. Ujihara, S. Wong, C. Ott, J. Middeldorp, and A. Aiyar. 2004. The amino terminus of Epstein-Barr Virus (EBV) nuclear antigen 1 contains AT hooks that facilitate the replication and partitioning of latent EBV genomes by tethering them to cellular chromosomes. J Virol 78:11487-505.

358. Seto, E., L. Yang, J. Middeldorp, T. S. Sheen, J. Y. Chen, M. Fukayama, Y. Eizuru, T. Ooka, and K. Takada. 2005. 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. J Med Virol 76:82-8.

359. Sham, J. S., Y. F. Poon, W. I. Wei, and D. Choy. 1990. Nasopharyngeal carcinoma in young patients. Cancer 65:2606-10.

360. Shevach, E. M., R. S. McHugh, C. A. Piccirillo, and A. M. Thornton. 2001. Control of T-cell activation by CD4+ CD25+ suppressor T cells. Immunol Rev 182:58-67.

361. Shibata, D., M. Tokunaga, Y. Uemura, E. Sato, S. Tanaka, and L. M. Weiss. 1991. Association of Epstein-Barr virus with undifferentiated gastric carcinomas with intense lymphoid infiltration. Lymphoepithelioma-like carcinoma. Am J Pathol 139:469-74.

362. Shibata, D., and L. M. Weiss. 1992. Epstein-Barr virus-associated gastric adenocarcinoma. Am J Pathol 140:769-74.

363. Shibata, D., L. M. Weiss, A. M. Hernandez, B. N. Nathwani, L. Bernstein, and A. M. Levine. 1993. Epstein-Barr virus-associated non-Hodgkin's lymphoma in patients infected with the human immunodeficiency virus. Blood 81:2102-9.

364. Shire, K., D. F. Ceccarelli, T. M. Avolio-Hunter, and L. Frappier. 1999. EBP2, a human protein that interacts with sequences of the Epstein-Barr virus nuclear antigen 1 important for plasmid maintenance. J Virol 73:2587-95.

365. Silins, S. L., and T. B. Sculley. 1994. Modulation of vimentin, the CD40 activation antigen and Burkitt's lymphoma antigen (CD77) by the Epstein-Barr virus nuclear antigen EBNA-4. Virology 202:16-24.

366. Sinclair, A. J., I. Palmero, G. Peters, and P. J. Farrell. 1994. EBNA-2 and EBNA-LP cooperate to cause G0 to G1 transition during immortalization of resting human B lymphocytes by Epstein-Barr virus. Embo J 13:3321-8.

367. Sixbey, J. W., and Q. Y. Yao. 1992. Immunoglobulin A-induced shift of Epstein-Barr virus tissue tropism. Science 255:1578-80.

368. Smiley, J. R. 2004. Herpes simplex virus virion host shutoff protein: immune evasion mediated by a viral RNase? J Virol 78:1063-8.

369. Smith, J. L., E. Hodges, C. T. Quin, K. P. McCarthy, and D. H. Wright. 2000. Frequent T and B cell oligoclones in histologically and immunophenotypically characterized angioimmunoblastic lymphadenopathy. Am J Pathol 156:661-9.

370. Smith, P. 2001. Epstein-Barr virus complementary strand transcripts (CSTs/BARTs) and cancer. Semin Cancer Biol 11:469-76.

371. Smith, P. R., O. de Jesus, D. Turner, M. Hollyoake, C. E. Karstegl, B. E. Griffin, L. Karran, Y. Wang, S. D. Hayward, and P. J. Farrell. 2000. Structure and coding content of CST (BART) family RNAs of Epstein-Barr virus. J Virol 74:3082-92.

372. Soeripto. 1998. Epidemiology of Nasopharyngeal Carcinoma. Berita Kedokteran Masyarakat XIV:207-211.

373. Sokal, E. M., K. Hoppenbrouwers, C. Vandermeulen, M. Moutschen, P. Leonard, A. Moreels, M. Haumont, A. Bollen, F. Smets, and M. Denis. 2007. Recombinant gp350 vaccine for infectious mononucleosis: a phase 2, randomized, double-blind, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of an Epstein-Barr virus vaccine in healthy young adults. J Infect Dis 196:1749-53.

374. Soussi, T., and G. Lozano. 2005. p53 mutation heterogeneity in cancer. Biochem Biophys Res Commun 331:834-42.

375. Steigerwald-Mullen, P., M. G. Kurilla, and T. J. Braciale. 2000. Type 2 cytokines predominate in the human CD4(+) T-lymphocyte response to Epstein-Barr virus nuclear antigen 1. J Virol 74:6748-59.

376. Steven, N. M., N. E. Annels, A. Kumar, A. M. Leese, M. G. Kurilla, and A. B. Rickinson.

Introdction

6362

Chapter 1

1997. Immediate early and early lytic cycle proteins are frequent targets of the Epstein-Barr virus-induced cytotoxic T cell response. J Exp Med 185:1605-17.

377. Steven, N. M., A. M. Leese, N. E. Annels, S. P. Lee, and A. B. Rickinson. 1996. Epitope focusing in the primary cytotoxic T cell response to Epstein-Barr virus and its relationship to T cell memory. J Exp Med 184:1801-13.

378. Stevens, S. J., B. S. Blank, P. H. Smits, P. L. Meenhorst, and J. M. Middeldorp. 2002. High Epstein-Barr virus (EBV) DNA loads in HIV-infected patients: correlation with antiretroviral therapy and quantitative EBV serology. Aids 16:993-1001.

379. Stevens, S. J., I. Pronk, and J. M. Middeldorp. 2001. Toward standardization of Epstein-Barr virus DNA load monitoring: unfractionated whole blood as preferred clinical specimen. J Clin Microbiol 39:1211-6.

380. Stevens, S. J., P. H. Smits, S. A. Verkuijlen, D. A. Rockx, E. C. van Gorp, J. W. Mulder, and J. M. Middeldorp. 2007. Aberrant Epstein-Barr virus persistence in HIV carriers is characterized by anti-Epstein-Barr virus IgA and high cellular viral loads with restricted transcription. Aids 21:2141-9.

381. Stevens, S. J., S. A. Verkuijlen, B. Hariwiyanto, Harijadi, J. Fachiroh, D. K. Paramita, I. B. Tan, S. M. Haryana, and J. M. Middeldorp. 2005. Diagnostic value of measuring Epstein-Barr virus (EBV) DNA load and carcinoma-specific viral mRNA in relation to anti-EBV immunoglobulin A (IgA) and IgG antibody levels in blood of nasopharyngeal carcinoma patients from Indonesia. J Clin Microbiol 43:3066-73.

382. Stevens, S. J., E. A. Verschuuren, I. Pronk, W. van Der Bij, M. C. Harmsen, T. H. The, C. J. Meijer, A. J. van Den Brule, and J. M. Middeldorp. 2001. Frequent monitoring of Epstein-Barr virus DNA load in unfractionated whole blood is essential for early detection of posttransplant lymphoproliferative disease in high-risk patients. Blood 97:1165-71.

383. Stevens, S. J., E. A. Verschuuren, S. A. Verkuujlen, A. J. Van Den Brule, C. J. Meijer, and J. M. Middeldorp. 2002. Role of Epstein-Barr virus DNA load monitoring in prevention and early detection of post-transplant lymphoproliferative disease. Leuk Lymphoma 43:831-40.

384. Stolzenberg, M. C., S. Debouze, M. Ng, J. Sham, D. Choy, A. Bouguermouh, K. H. Chan, and T. Ooka. 1996. Purified recombinant EBV desoxyribonuclease in serological diagnosis of nasopharyngeal carcinoma. Int J Cancer 66:337-41.

385. Straathof, K. C., C. M. Bollard, U. Popat, M. H. Huls, T. Lopez, M. C. Morriss, M. V. Gresik, A. P. Gee, H. V. Russell, M. K. Brenner, C. M. Rooney, and H. E. Heslop. 2005. Treatment of nasopharyngeal carcinoma with Epstein-Barr virus--specific T lymphocytes. Blood 105:1898-904.

386. Straathof, K. C., A. M. Leen, E. L. Buza, G. Taylor, M. H. Huls, H. E. Heslop, C. M. Rooney, and C. M. Bollard. 2005. Characterization of latent membrane protein 2 specificity in CTL lines from patients with EBV-positive nasopharyngeal carcinoma and lymphoma. J Immunol 175:4137-47.

387. Straathof, K. C., B. Savoldo, H. E. Heslop, and C. M. Rooney. 2002. Immunotherapy for post-transplant lymphoproliferative disease. Br J Haematol 118:728-40.

388. Strockbine, L. D., J. I. Cohen, T. Farrah, S. D. Lyman, F. Wagener, R. F. DuBose, R. J. Armitage, and M. K. Spriggs. 1998. The Epstein-Barr virus BARF1 gene encodes a novel, soluble colony-stimulating factor-1 receptor. J Virol 72:4015-21.

389. Sugden, B., and N. Warren. 1988. Plasmid origin of replication of Epstein-Barr virus, oriP, does not limit replication in cis. Mol Biol Med 5:85-94.

390. Sugiura, M., S. Imai, M. Tokunaga, S. Koizumi, M. Uchizawa, K. Okamoto, and T. Osato. 1996. Transcriptional analysis of Epstein-Barr virus gene expression in EBV-

positive gastric carcinoma: unique viral latency in the tumour cells. Br J Cancer 74:625-31.

391. Sullivan, C. S., A. T. Grundhoff, S. Tevethia, J. M. Pipas, and D. Ganem. 2005. SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells. Nature 435:682-6.

392. Sung, N. S., R. H. Edwards, F. Seillier-Moiseiwitsch, A. G. Perkins, Y. Zeng, and N. Raab-Traub. 1998. Epstein-Barr virus strain variation in nasopharyngeal carcinoma from the endemic and non-endemic regions of China. Int J Cancer 76:207-15.

393. Sung, N. S., S. Kenney, D. Gutsch, and J. S. Pagano. 1991. EBNA-2 transactivates a lymphoid-specific enhancer in the BamHI C promoter of Epstein-Barr virus. J Virol 65:2164-9.

394. Svedmyr, E., and M. Jondal. 1975. Cytotoxic effector cells specific for B Cell lines transformed by Epstein-Barr virus are present in patients with infectious mononucleosis. Proc Natl Acad Sci U S A 72:1622-6.

395. Swaminathan, S. 1996. Characterization of Epstein-Barr virus recombinants with deletions of the BamHI C promoter. Virology 217:532-41.

396. Swaminathan, S. 2008. Noncoding RNAs produced by oncogenic human herpesviruses. J Cell Physiol 216:321-6.

397. Swaminathan, S., B. Tomkinson, and E. Kieff. 1991. Recombinant Epstein-Barr virus with small RNA (EBER) genes deleted transforms lymphocytes and replicates in vitro. Proc Natl Acad Sci U S A 88:1546-50.

398. Takada, K. 2005. Role of EBERs, p. 429-438. In E. S. Robertson (ed.), Epstein-Barr Virus. Caister Academic Press, Norfolk.

399. Takayama, T., A. E. Morelli, N. Onai, M. Hirao, K. Matsushima, H. Tahara, and A. W. Thomson. 2001. Mammalian and viral IL-10 enhance C-C chemokine receptor 5 but down-regulate C-C chemokine receptor 7 expression by myeloid dendritic cells: impact on chemotactic responses and in vivo homing ability. J Immunol 166:7136-43.

400. Tamaru, Y., T. Miyawaki, K. Iwai, T. Tsuji, R. Nibu, A. Yachie, S. Koizumi, and N. Taniguchi. 1993. Absence of bcl-2 expression by activated CD45RO+ T lymphocytes in acute infectious mononucleosis supporting their susceptibility to programmed cell death. Blood 82:521-7.

401. Tang, J. W., E. Rohwader, I. M. Chu, R. K. Tsang, K. Steinhagen, A. C. Yeung, K. F. To, and P. K. Chan. 2007. Evaluation of Epstein-Barr virus antigen-based immunoassays for serological diagnosis of nasopharyngeal carcinoma. J Clin Virol 40:284-8.

402. Tanner, J., J. Weis, D. Fearon, Y. Whang, and E. Kieff. 1987. Epstein-Barr virus gp350/220 binding to the B lymphocyte C3d receptor mediates adsorption, capping, and endocytosis. Cell 50:203-13.

403. Tanner, J. E., C. Alfieri, T. A. Chatila, and F. Diaz-Mitoma. 1996. Induction of interleukin-6 after stimulation of human B-cell CD21 by Epstein-Barr virus glycoproteins gp350 and gp220. J Virol 70:570-5.

404. Tanner, J. E., M. X. Wei, C. Alfieri, A. Ahmad, P. Taylor, T. Ooka, and J. Menezes. 1997. 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. J Infect Dis 175:38-46.

405. Tarbouriech, N., M. Buisson, T. Geoui, S. Daenke, S. Cusack, and W. P. Burmeister. 2006. Structural genomics of the Epstein-Barr virus. Acta Crystallogr D Biol Crystallogr 62:1276-85.

Introdction

6564

Chapter 1

406. Tedeschi, R., Y. T. Foong, H. M. Cheng, P. dePaoli, T. Lehtinen, T. Elfborg, and J. Dillner. 1995. The disease associations of the antibody response against the Epstein-Barr virus transactivator protein ZEBRA can be separated into different epitopes. J Gen Virol 76 ( Pt 6):1393-400.

407. Tedeschi, R., E. Pin, D. Martorelli, E. Bidoli, A. Marus, C. Pratesi, M. T. Bortolin, S. Zanussi, E. Vaccher, R. Dolcetti, and P. De Paoli. 2007. Serum antibody response to lytic and latent Epstein-Barr virus antigens in undifferentiated nasopharyngeal carcinoma patients from an area of nonendemicity. Clin Vaccine Immunol 14:435-41.

408. Thiele, G. M., A. M. Johnson, M. Okano, G. L. Woods, and D. T. Purtilo. 1990. Comparison of the classical immunofluorescent antibody tests for Epstein-Barr virus with commercially available test kits. J Clin Lab Anal 4:256-60.

409. Thorley-Lawson, D., and J. L. Strominger. 1976. Transformation of human lymphocytes by Epstein-Barr virus is inhibited by phosphonoacetic acid. Nature 263:332-4.

410. Thorley-Lawson, D. A. 2005. EBV the prototypical human tumor virus--just how bad is it? J Allergy Clin Immunol 116:251-61; quiz 262.

411. Thorley-Lawson, D. A. 2001. Epstein-Barr virus: exploiting the immune system. Nat Rev Immunol 1:75-82.

412. Thorley-Lawson, D. A., and M. J. Allday. 2008. The curious case of the tumour virus: 50 years of Burkitt's lymphoma. Nat Rev Microbiol 6:913-24.

413. Thorley-Lawson, D. A., and K. Geilinger. 1980. Monoclonal antibodies against the major glycoprotein (gp350/220) of Epstein-Barr virus neutralize infectivity. Proc Natl Acad Sci U S A 77:5307-11.

414. Thorley-Lawson, D. A., and A. Gross. 2004. Persistence of the Epstein-Barr virus and the origins of associated lymphomas. N Engl J Med 350:1328-37.

415. Thornburg, N. J., S. Kusano, and N. Raab-Traub. 2004. Identification of Epstein-Barr virus RK-BARF0-interacting proteins and characterization of expression pattern. J Virol 78:12848-56.

416. Tierney, R. J., N. Steven, L. S. Young, and A. B. Rickinson. 1994. Epstein-Barr virus latency in blood mononuclear cells: analysis of viral gene transcription during primary infection and in the carrier state. J Virol 68:7374-85.

417. Timms, J. M., A. Bell, J. R. Flavell, P. G. Murray, A. B. Rickinson, A. Traverse-Glehen, F. Berger, and H. J. Delecluse. 2003. Target cells of Epstein-Barr-virus (EBV)-positive post-transplant lymphoproliferative disease: similarities to EBV-positive Hodgkin's lymphoma. Lancet 361:217-23.

418. Tokunaga, M., C. E. Land, Y. Uemura, T. Tokudome, S. Tanaka, and E. Sato. 1993. Epstein-Barr virus in gastric carcinoma. Am J Pathol 143:1250-4.

419. Triantafilou, K., and M. Triantafilou. 2001. A biochemical approach reveals cell-surface molecules utilised by Picornaviridae: Human Parechovirus 1 and Echovirus 1. J Cell Biochem 80:373-81.

420. Trivedi, P., L. F. Hu, F. Chen, B. Christensson, M. G. Masucci, G. Klein, and G. Winberg. 1994. Epstein-Barr virus (EBV)-encoded membrane protein LMP1 from a nasopharyngeal carcinoma is non-immunogenic in a murine model system, in contrast to a B cell-derived homologue. Eur J Cancer 30A:84-8.

421. Tsang, C. W., X. Lin, N. H. Gudgeon, G. S. Taylor, H. Jia, E. P. Hui, A. T. Chan, C. K. Lin, and A. B. Rickinson. 2006. CD4+ T-cell responses to Epstein-Barr virus nuclear antigen EBNA1 in Chinese populations are highly focused on novel C-terminal domain-derived epitopes. J Virol 80:8263-6.

422. Tsang, R. K., A. C. Vlantis, R. W. Ho, J. S. Tam, K. F. To, and C. A. van Hasselt. 2004. Sensitivity and specificity of Epstein-Barr virus IGA titer in the diagnosis of nasopharyngeal carcinoma: a three-year institutional review. Head Neck 26:598-602.

423. Tsuchiya, S. 2002. Diagnosis of Epstein-Barr virus-associated diseases. Crit Rev Oncol Hematol 44:227-38.

424. Tsurumi, T., M. Fujita, and A. Kudoh. 2005. Latent and lytic Epstein-Barr virus replication strategies. Rev Med Virol 15:3-15.

425. Tugizov, S. M., J. W. Berline, and J. M. Palefsky. 2003. Epstein-Barr virus infection of polarized tongue and nasopharyngeal epithelial cells. Nat Med 9:307-14.

426. Turk, S. M., R. Jiang, L. S. Chesnokova, and L. M. Hutt-Fletcher. 2006. Antibodies to gp350/220 enhance the ability of Epstein-Barr virus to infect epithelial cells. J Virol 80:9628-33.

427. Uchida, J., T. Yasui, Y. Takaoka-Shichijo, M. Muraoka, W. Kulwichit, N. Raab-Traub, and H. Kikutani. 1999. Mimicry of CD40 signals by Epstein-Barr virus LMP1 in B lymphocyte responses. Science 286:300-3.

428. Uozaki, H., and M. Fukayama. 2008. Epstein-Barr Virus and Gastric Carcinoma - Viral Carcinogenesis through Epigenetic Mechanisms. Int J Clin Exp Pathol 1:198-216.

429. Usherwood, E. J., J. W. Brooks, S. R. Sarawar, R. D. Cardin, W. D. Young, D. J. Allen, P. C. Doherty, and A. A. Nash. 1997. Immunological control of murine gammaherpesvirus infection is independent of perforin. J Gen Virol 78 ( Pt 8):2025-30.

430. Ushiku, T., J. M. Chong, H. Uozaki, R. Hino, M. S. Chang, M. Sudo, B. R. Rani, K. Sakuma, H. Nagai, and M. Fukayama. 2007. p73 gene promoter methylation in Epstein-Barr virus-associated gastric carcinoma. Int J Cancer 120:60-6.

431. van Beek, J. 2005. EBV-brearing gastric carcinomas: expression patterns, pathogenesis and clinical features Thesis. Vrije Universiteit, Amsterdam.

432. van Beek, J., A. A. Brink, M. B. Vervoort, M. J. van Zijp, C. J. Meijer, A. J. van den Brule, and J. M. Middeldorp. 2003. In vivo transcription of the Epstein-Barr virus (EBV) BamHI-A region without associated in vivo BARF0 protein expression in multiple EBV-associated disorders. J Gen Virol 84:2647-59.

433. van Beek, J., A. zur Hausen, E. Klein Kranenbarg, C. J. van de Velde, J. M. Middeldorp, A. J. van den Brule, C. J. Meijer, and E. Bloemena. 2004. EBV-positive gastric adenocarcinomas: a distinct clinicopathologic entity with a low frequency of lymph node involvement. J Clin Oncol 22:664-70.

434. van Beek, J., A. zur Hausen, S. N. Snel, J. Berkhof, E. K. Kranenbarg, C. J. van de Velde, A. J. van den Brule, J. M. Middeldorp, C. J. Meijer, and E. Bloemena. 2006. Morphological evidence of an activated cytotoxic T-cell infiltrate in EBV-positive gastric carcinoma preventing lymph node metastases. Am J Surg Pathol 30:59-65.

435. van Grunsven, W. M., A. Nabbe, and J. M. Middeldorp. 1993. Identification and molecular characterization of two diagnostically relevant marker proteins of the Epstein-Barr virus capsid antigen complex. J Med Virol 40:161-9.

436. van Grunsven, W. M., W. J. Spaan, and J. M. Middeldorp. 1994. Localization and diagnostic application of immunodominant domains of the BFRF3-encoded Epstein-Barr virus capsid protein. J Infect Dis 170:13-9.

437. Verschuuren, E., W. van der Bij, W. de Boer, W. Timens, J. Middeldorp, and T. H. The. 2003. Quantitative Epstein-Barr virus (EBV) serology in lung transplant recipients with primary EBV infection and/or post-transplant lymphoproliferative disease. J Med Virol 69:258-66.

Introdction

6766

Chapter 1

438. Vilmer, E., G. M. Lenoir, J. L. Virelizier, and C. Griscelli. 1984. Epstein-Barr serology in immunodeficiencies: an attempt to correlate with immune abnormalities in Wiskott-Aldrich and Chediak-Higashi syndromes and ataxia telangiectasia. Clin Exp Immunol 55:249-56.

439. Vockerodt, M., S. L. Morgan, M. Kuo, W. Wei, M. B. Chukwuma, J. R. Arrand, D. Kube, J. Gordon, L. S. Young, C. B. Woodman, and P. G. Murray. 2008. The Epstein-Barr virus oncoprotein, latent membrane protein-1, reprograms germinal centre B cells towards a Hodgkin's Reed-Sternberg-like phenotype. J Pathol 216:83-92.

440. Wallace, L. E., J. Wright, D. O. Ulaeto, A. J. Morgan, and A. B. Rickinson. 1991. Identification of two T-cell epitopes on the candidate Epstein-Barr virus vaccine glycoprotein gp340 recognized by CD4+ T-cell clones. J Virol 65:3821-8.

441. Wang, F., C. Gregory, C. Sample, M. Rowe, D. Liebowitz, R. Murray, A. Rickinson, and E. Kieff. 1990. Epstein-Barr virus latent membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23. J Virol 64:2309-18.

442. Wang, F., C. D. Gregory, M. Rowe, A. B. Rickinson, D. Wang, M. Birkenbach, H. Kikutani, T. Kishimoto, and E. Kieff. 1987. Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23. Proc Natl Acad Sci U S A 84:3452-6.

443. Wang, F., S. F. Tsang, M. G. Kurilla, J. I. Cohen, and E. Kieff. 1990. Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1. J Virol 64:3407-16.

444. Wang, W. Y., Y. C. Chien, J. S. Jan, C. M. Chueh, and J. C. Lin. 2002. Consistent sequence variation of Epstein-Barr virus nuclear antigen 1 in primary tumor and peripheral blood cells of patients with nasopharyngeal carcinoma. Clin Cancer Res 8:2586-90.

445. Wang, X., and L. M. Hutt-Fletcher. 1998. Epstein-Barr virus lacking glycoprotein gp42 can bind to B cells but is not able to infect. J Virol 72:158-63.

446. Wang, X., W. J. Kenyon, Q. Li, J. Mullberg, and L. M. Hutt-Fletcher. 1998. Epstein-Barr virus uses different complexes of glycoproteins gH and gL to infect B lymphocytes and epithelial cells. J Virol 72:5552-8.

447. Wang, Y., J. E. Finan, J. M. Middeldorp, and S. D. Hayward. 1997. P32/TAP, a cellular protein that interacts with EBNA-1 of Epstein-Barr virus. Virology 236:18-29.

448. Watanabe, H., M. Enjoji, and T. Imai. 1976. Gastric carcinoma with lymphoid stroma. Its morphologic characteristics and prognostic correlations. Cancer 38:232-43.

449. Webster-Cyriaque, J., J. Middeldorp, and N. Raab-Traub. 2000. Hairy leukoplakia: an unusual combination of transforming and permissive Epstein-Barr virus infections. J Virol 74:7610-8.

450. Wei, W. I., and J. S. Sham. 2005. Nasopharyngeal carcinoma. Lancet 365:2041-54.451. Weiss, L. M., E. S. Jaffe, X. F. Liu, Y. Y. Chen, D. Shibata, and L. J. Medeiros. 1992.

Detection and localization of Epstein-Barr viral genomes in angioimmunoblastic lymphadenopathy and angioimmunoblastic lymphadenopathy-like lymphoma. Blood 79:1789-95.

452. Williams, H., K. McAulay, K. F. Macsween, N. J. Gallacher, C. D. Higgins, N. Harrison, A. J. Swerdlow, and D. H. Crawford. 2005. The immune response to primary EBV infection: a role for natural killer cells. Br J Haematol 129:266-74.

453. Woisetschlaeger, M., X. W. Jin, C. N. Yandava, L. A. Furmanski, J. L. Strominger, and S. H. Speck. 1991. Role for the Epstein-Barr virus nuclear antigen 2 in viral promoter switching during initial stages of infection. Proc Natl Acad Sci U S A 88:3942-6.

454. Woodberry, T., T. J. Suscovich, L. M. Henry, J. K. Davis, N. Frahm, B. D. Walker, D. T. Scadden, F. Wang, and C. Brander. 2005. Differential targeting and shifts in the immunodominance of Epstein-Barr virus--specific CD8 and CD4 T cell responses during acute and persistent infection. J Infect Dis 192:1513-24.

455. Wysokenski, D. A., and J. L. Yates. 1989. Multiple EBNA1-binding sites are required to form an EBNA1-dependent enhancer and to activate a minimal replicative origin within oriP of Epstein-Barr virus. J Virol 63:2657-66.

456. Xiao, J., J. M. Palefsky, R. Herrera, J. Berline, and S. M. Tugizov. 2008. The Epstein-Barr virus BMRF-2 protein facilitates virus attachment to oral epithelial cells. Virology 370:430-42.

457. Xiao, J., J. M. Palefsky, R. Herrera, and S. M. Tugizov. 2007. Characterization of the Epstein-Barr virus glycoprotein BMRF-2. Virology 359:382-96.

458. Xue, S. A., L. G. Labrecque, Q. L. Lu, S. K. Ong, I. A. Lampert, P. Kazembe, E. Molyneux, R. L. Broadhead, E. Borgstein, and B. E. Griffin. 2002. Promiscuous expression of Epstein-Barr virus genes in Burkitt's lymphoma from the central African country Malawi. Int J Cancer 99:635-43.

459. Yajima, M., T. Kanda, and K. Takada. 2005. Critical role of Epstein-Barr Virus (EBV)-encoded RNA in efficient EBV-induced B-lymphocyte growth transformation. J Virol 79:4298-307.

460. Yates, J. 1996. Epstein-Barr virus replication, p. 751-773. In M. DePamphilis (ed.), DNA replication in eukaryotic cells. Cold Spring Laboratory Press, New York.

461. Yates, J., N. Warren, D. Reisman, and B. Sugden. 1984. A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc Natl Acad Sci U S A 81:3806-10.

462. Yates, J. L., and N. Guan. 1991. Epstein-Barr virus-derived plasmids replicate only once per cell cycle and are not amplified after entry into cells. J Virol 65:483-8.

463. Yates, J. L., N. Warren, and B. Sugden. 1985. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 313:812-5.

464. Young, L. S., R. Lau, M. Rowe, G. Niedobitek, G. Packham, F. Shanahan, D. T. Rowe, D. Greenspan, J. S. Greenspan, A. B. Rickinson, and et al. 1991. Differentiation-associated expression of the Epstein-Barr virus BZLF1 transactivator protein in oral hairy leukoplakia. J Virol 65:2868-74.

465. Young, L. S., and A. B. Rickinson. 2004. Epstein-Barr virus: 40 years on. Nat Rev Cancer 4:757-68.

466. Zech, L., U. Haglund, K. Nilsson, and G. Klein. 1976. Characteristic chromosomal abnormalities in biopsies and lymphoid-cell lines from patients with Burkitt and non-Burkitt lymphomas. Int J Cancer 17:47-56.

467. Zeidler, R., G. Eissner, P. Meissner, S. Uebel, R. Tampe, S. Lazis, and W. Hammerschmidt. 1997. Downregulation of TAP1 in B lymphocytes by cellular and Epstein-Barr virus-encoded interleukin-10. Blood 90:2390-7.

468. Zhao, B., and C. E. Sample. 2000. Epstein-barr virus nuclear antigen 3C activates the latent membrane protein 1 promoter in the presence of Epstein-Barr virus nuclear antigen 2 through sequences encompassing an spi-1/Spi-B binding site. J Virol 74:5151-60.

469. Zimber-Strobl, U., E. Kremmer, F. Grasser, G. Marschall, G. Laux, and G. W. Bornkamm. 1993. The Epstein-Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis-element of the terminal protein 1 gene promoter. Embo J 12:167-75.

470. Zuo, J., A. Currin, B. D. Griffin, C. Shannon-Lowe, W. A. Thomas, M. E. Ressing, E. J. Wiertz, and M. Rowe. 2009. The Epstein-Barr virus G-protein-coupled receptor

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contributes to immune evasion by targeting MHC class I molecules for degradation. PLoS Pathog 5:e1000255.

471. Zuo, J., W. Thomas, D. van Leeuwen, J. M. Middeldorp, E. J. Wiertz, M. E. Ressing, and M. Rowe. 2008. The DNase of gammaherpesviruses impairs recognition by virus-specific CD8+ T cells through an additional host shutoff function. J Virol 82:2385-93.

472. zur Hausen, A., A. A. Brink, M. E. Craanen, J. M. Middeldorp, C. J. Meijer, and A. J. van den Brule. 2000. Unique transcription pattern of Epstein-Barr virus (EBV) in EBV-carrying gastric adenocarcinomas: expression of the transforming BARF1 gene. Cancer Res 60:2745-8.

473. Zur Hausen, A., B. P. van Rees, J. van Beek, M. E. Craanen, E. Bloemena, G. J. Offerhaus, C. J. Meijer, and A. J. van den Brule. 2004. Epstein-Barr virus in gastric carcinomas and gastric stump carcinomas: a late event in gastric carcinogenesis. J Clin Pathol 57:487-91.

474. zur Hausen, H., H. Schulte-Holthausen, G. Klein, W. Henle, G. Henle, P. Clifford, and L. Santesson. 1970. EBV DNA in biopsies of Burkitt tumours and anaplastic carcinomas of the nasopharynx. Nature 228:1056-8.

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