Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein.

Infection of Epstein-Barr virus-negative human B-lymphoma cell lines with the fully transforming B95.8 Epstein-Barr virus strain was associated with complete virus latent gene expression and a change in the cell surface and growth phenotype toward that of in vitro-transformed lymphoblastoid cell lines. In contrast, the cells infected with the P3HR1 Epstein-Barr virus strain, a deletion mutant that cannot encode Epstein-Barr nuclear antigen 2 (EBNA2) or a full-length EBNA-LP, expressed EBNAs1, 3a, 3b, and 3c but were negative for the latent membrane protein (LMP) and showed no change in cellular phenotype. This suggests that EBNA2 and/or EBNA-LP may be required for subsequent expression of LMP in Epstein-Barr virus-infected B cells. Recombinant vectors capable of expressing the B95.8 EBNA2A protein were introduced by electroporation into two P3HR1-converted B-lymphoma cell lines, BL30/P3 and BL41/P3. In both cases, stable expression of EBNA2A was accompanied by activation of LMP expression from the resident P3HR1 genome; control transfectants that did not express the EBNA2A protein never showed induction of LMP. In further experiments, a recombinant vector capable of expressing the full-length B95.8 EBNA-LP was introduced into the same target lines. Strong EBNA-LP expression was consistently observed in the transfected clones but was never accompanied by induction of LMP. The EBNA2A gene transfectants expressing EBNA2A and LMP showed a dramatic change in cell surface and growth phenotype toward a pattern like that of lymphoblastoid cell lines; some but not all of these changes could be reproduced in the absence of EBNA2A by transfection of P3HR1-converted cell lines with a recombinant vector expressing LMP. These studies suggest that EBNA2 plays an important dual role in the process of B-cell activation to the lymphoblastoid phenotype; the protein can have a direct effect upon cellular gene expression and is also involved in activating the expression of a second virus-encoded effector protein, LMP.     

Epstein-Barr virus-specific cytotoxic T-cell recognition of transfectants expressing the virus-coded latent membrane protein LMP

Cytotoxic T cells from Epstein-Barr virus (EBV)-immune individuals specifically kill EBV-transformed B cells from HLA class I antigen-matched donors even though the latently infected cells express only a restricted set of virus genes. The virus-induced target antigens recognized by these immune T cells have not been identified. In our experiments, EBV DNA sequences encoding the virus latent gene products Epstein-Barr nuclear antigen (EBNA)1, EBNA 2, and EBNA-LP and the latent membrane protein (LMP) were individually expressed in a virus-negative human B-lymphoma cell line, Louckes. Transfected clones expressing LMP were killed by EBV-specific cytotoxic T-cell preparations from each of three virus-immune donors HLA matched with Louckes through HLA-A2, B44 antigens; control transfectants or clones expressing one of the EBNA proteins were not recognized. Expression of LMP in a second virus-negative B-cell line, BL41, sensitized these cells to EBV-specific cytolysis restricted through the HLA-A11 antigen. To distinguish between the viral protein and an induced human B-cell activation antigen as the target for T-cell recognition, LMP was then expressed in a murine mastocytoma cell line, P815-A11-restricted human T cells. The LMP-expressing P815-A11 transfectants were susceptible to lysis by EBV-specific cytotoxic T cells from three HLA-A11-positive individuals. Both Louckes and P815-A11 cells were also transfected with constructs capable of encoding a truncated form of LMP (Tr-LMP) which lacks the N-terminal 128 amino acids of the full-length protein. Tr-LMP-expressing transfectants were not recognized by the above T-cell preparations. The results suggest that LMP, and, in particular, epitopes derived from the N-terminal region of the protein, provides one of the target antigens for the EBV-induced human cytotoxic T-cell response.     

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

Undifferentiated nasopharyngeal carcinoma (NPC) is latently infected with EBV and expresses a restricted number of viral proteins. Studies in healthy virus carriers have demonstrated that at least some of these proteins can act as targets for HLA class I-restricted CTLs. Therefore we have explored the possibility of a CTL-based therapy for NPC by characterizing EBV-specific CTL responses in 10 newly diagnosed NPC cases and 21 healthy virus carriers from Southeast Asia. Using the autologous EBV-transformed lymphoblastoid cell line, virus-specific CTL were reactivated in vitro from PBMC, cloned, and screened for cytotoxicity against target cells expressing individual EBV proteins from recombinant vaccinia vectors. EBV-specific CTLs were identified in 6 of 10 patients and 14 of 21 controls and mainly targeted the EBV nuclear Ag 3 (EBNA3) family of viral latent proteins. However, in 3 of 10 patients and 11 of 21 controls, CTLs specific for the NPC-associated protein LMP2 were also detected, albeit at low frequency. EBV-specific CTLs were detected in tumor biopsy material obtained from 3 of 6 of the patients, indicating that functional CTL are present at the tumor site, but none was specific for tumor-associated viral proteins. To assess the Ag-presenting function in NPC we studied two NPC-derived cell lines (C15 and c666.1) and demonstrated that both were capable of processing and presenting endogenously synthesized protein to HLA class I-restricted CTL clones. Overall, our data provide a sound theoretical basis for therapeutic strategies that aim to boost or elicit LMP2-specific CTL responses in NPC patients.     

Dual stimulation of Epstein-Barr Virus (EBV)-specific CD4+- and CD8+-T-cell responses by a chimeric antigen construct: potential therapeutic vaccine for EBV-positive nasopharyngeal carcinoma

Virus-associated malignancies are potential targets for immunotherapeutic vaccines aiming to stimulate T-cell responses against viral antigens expressed in tumor cells. Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma, a high-incidence tumor in southern China, expresses a limited set of EBV proteins, including the nuclear antigen EBNA1, an abundant source of HLA class II-restricted CD4(+) T-cell epitopes, and the latent membrane protein LMP2, a source of subdominant CD8(+) T-cell epitopes presented by HLA class I alleles common in the Chinese population. We used appropriately modified gene sequences from a Chinese EBV strain to generate a modified vaccinia virus Ankara recombinant, MVA-EL, expressing the CD4 epitope-rich C-terminal domain of EBNA1 fused to full-length LMP2. The endogenously expressed fusion protein EL is efficiently processed via the HLA class I pathway, and MVA-EL-infected dendritic cells selectively reactivate LMP2-specific CD8(+) memory T-cell responses from immune donors in vitro. Surprisingly, endogenously expressed EL also directly accesses the HLA class II presentation pathway and, unlike endogenously expressed EBNA1 itself, efficiently reactivates CD4(+) memory T-cell responses in vitro. This unscheduled access to the HLA class II pathway is coincident with EL-mediated redirection of the EBNA1 domain from its native nuclear location to dense cytoplasmic patches. Given its immunogenicity to both CD4(+) and CD8(+) T cells, MVA-EL has potential as a therapeutic vaccine in the context of nasopharyngeal carcinoma.     

HLA A2.1-restricted cytotoxic T cells recognizing a range of Epstein-Barr virus isolates through a defined epitope in latent membrane protein LMP2.

Cytotoxic T-lymphocyte (CTL) responses induced by persistent Epstein-Barr virus (EBV) infection in normal B-lymphoid tissues could potentially be directed against EBV-positive malignancies if expression of the relevant viral target proteins is maintained in tumor cells. For malignancies such as nasopharyngeal carcinoma and Hodgkin's disease, this will require CTL targeting against the nuclear antigen EBNA1 or the latent membrane proteins LMP1 and LMP2. Here we analyze in detail a B95.8 EBV-reactivated CTL response which is specific for LMP2 and restricted through a common HLA allele, A2.1. We found that in vitro-reactivated CTL preparations from several A2.1-positive virus-immune donors contained detectable reactivity against A2.1-bearing target cells expressing either LMP2A or the smaller LMP2B protein from recombinant vaccinia virus vectors. Peptide sensitization experiments then mapped the A2.1-restricted response to a single epitope, the nonamer CLGGLLTMV (LMP2A residues 426 to 434), whose sequence accords well with the proposed peptide binding motif for A2.1. Most Caucasian and African virus isolates (whether of type 1 or type 2) were identical in sequence to B95.8 across this LMP2 epitope region, although 2 of 12 such isolates encoded a Leu-->Ile change at epitope position 6. In contrast, most Southeast Asian and New Guinean isolates (whether of type 1 or type 2) constituted a different virus group with a Cys-->Ser mutation at epitope position 1. CTLs raised against the B95.8-encoded epitope were nevertheless able to recognize these variant epitope sequences in the context of A2.1 whether they were provided exogenously as synthetic peptides or generated endogenously in B cells transformed with the variant viruses. A CTL response of this kind could have therapeutic potential in that it is directed against a protein expressed in many EBV-positive malignancies, is reactive across a range of virus isolates, and is restricted through a relatively common HLA allele.     

Epstein-Barr Virus (EBV)-derived BARF1 encodes CD4- and CD8-restricted epitopes as targets for T-cell immunotherapy

Background aims

EBV type II latency tumors, such as Hodgkin lymphoma (HL), Non-Hodgkin lymphoma (NHL) and nasopharyngeal carcinoma, express a limited array of EBV antigens including Epstein-Barr nuclear antigen (EBNA)1, latent membrane protein (LMP)1, LMP2, and BamH1-A right frame 1 (BARF1). Adoptive immunotherapy for these malignancies have focused on EBNA1, LMP1 and LMP2 because little is known about the cellular immune response to BARF1.

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.

A set of B-cell activation molecules, including the Epstein-Barr virus (EBV) receptor CR2 (CD21) and the B-cell activation antigen CD23 (Blast2/Fc epsilon RII), is turned on by infecting EBV-negative B-lymphoma cell lines with immortalizing strains of the viruslike B95-8 (BL/B95 cells). This up regulation may represent one of the mechanisms involved in EBV-mediated B-cell immortalization. The P3HR1 nonimmortalizing strain of the virus, which is deleted for the entire Epstein-Barr nuclear antigen 2 (EBNA2) protein open reading frame, is incapable of inducing the expression of CR2 and CD23, suggesting a crucial role for EBNA2 in the activation of these molecules. In addition, lymphoma cells containing the P3HR1 genome (BL/P3HR1 cells) do not express the viral latent membrane protein (LMP), which is regularly expressed in cells infected with immortalizing viral strains. Using electroporation, we have transfected the EBNA2 gene cloned in an episomal vector into BL/P3HR1 cells and have obtained cell clones that stably express the EBNA2 protein. In these clones, EBNA2 expression was associated with an increased amount of CR2 and CD23 steady-state RNAs. Of the three species of CD23 mRNAs described, the Fc epsilon RIIa species was preferentially expressed in these EBNA2-expressing clones. An increased cell surface expression of CR2 but not of CD23 was observed, and the soluble form of CD23 molecule (SCD23) was released. We were, however, not able to detect any expression of LMP in these cell clones. These data demonstrate that EBNA2 gene is able to complement P3HR1 virus latent functions to induce the activation of CR2 and CD23 expression, and they emphasize the role of EBNA2 protein in the modulation of cellular gene implicated in B-cell proliferation and hence in EBV-mediated B-cell immortalization. Nevertheless, EBNA2 expression in BL/P3HR1 cells is not able to restore the level of CR2 and CD23 expression observed in BL/B95 cells, suggesting that other cellular or viral proteins may also have an important role in the activation of these molecules: the viral LMP seems to be a good candidate.     

Three pathways of Epstein-Barr virus gene activation from EBNA1-positive latency in B lymphocytes.

Previous studies on Epstein-Barr virus (EBV)-positive B-cell lines have identified two distinct forms of virus latency. Lymphoblastoid cell lines generated by virus-induced transformation of normal B cells in vitro, express the full spectrum of six EBNAs and three latent membrane proteins (LMP1, LMP2A, and LMP2B); furthermore, these lines often contain a small fraction of cells spontaneously entering the lytic cycle. In contrast, Burkitt's lymphoma-derived cell lines retaining the tumor biopsy cell phenotype express only one of the latent proteins, the nuclear antigen EBNA1; such cells do not enter the lytic cycle spontaneously but may be induced to do so by treatment with such agents as tetradecanoyl phorbol acetate and anti-immunoglobulin. The present study set out to determine whether activation of full virus latent-gene expression was a necessary accompaniment to induction of the lytic cycle in Burkitt's lymphoma lines. Detailed analysis of Burkitt's lymphoma lines responding to anti-immunoglobulin treatment revealed three response pathways of EBV gene activation from EBNA1-positive latency. A first, rapid response pathway involves direct entry of cells into the lytic cycle without broadening of the pattern of latent gene expression; thereafter, the three "latent" LMPs are expressed as early lytic cycle antigens. A second, delayed response pathway in another cell subpopulation involves the activation of full latent gene expression and conversion to a lymphoblastoidlike cell phenotype. A third response pathway in yet another subpopulation involves the selective activation of LMPs, with no induction of the lytic cycle and with EBNA expression still restricted to EBNA1; this type of latent infection in B lymphocytes has hitherto not been described. Interestingly, the EBNA1+ LMP+ cells displayed some but not all of the phenotypic changes normally induced by LMP1 expression in a B-cell environment. These studies highlight the existence of four different types of EBV infection in B cells, including three distinct forms of latency, which we now term latency I, latency II, and latency III.     

Differential immunogenicity of Epstein-Barr virus latent-cycle proteins for human CD4(+) T-helper 1 responses

Human CD4(+) T-helper 1 cell responses to Epstein-Barr virus (EBV) infection are likely to be important in the maintenance of virus-specific CD8(+) memory and/or as antiviral effectors in their own right. The present work has used overlapping peptides as stimulators of gamma interferon release (i) to identify CD4(+) epitopes within four EBV latent-cycle proteins, i.e., the nuclear antigens EBNA1 and EBNA3C and the latent membrane proteins LMP1 and LMP2, and (ii) to determine the frequency and magnitude of memory responses to these proteins in healthy virus carriers. Responses to EBNA1 and EBNA3C epitopes were detected in the majority of donors, and in the case of EBNA1, their antigen specificity was confirmed by in vitro reactivation and cloning of CD4(+) T cells using protein-loaded dendritic cell stimulators. By contrast, responses to LMP1 and LMP2 epitopes were seen much less frequently. EBV latent-cycle proteins therefore display a marked hierarchy of immunodominance for CD4(+) T-helper 1 cells (EBNA1, EBNA3C > LMP1, LMP2) which is different from that identified for the same proteins with respect to CD8(+)-T-cell responses (EBNA3C > EBNA1 > LMP2 > LMP1). Furthermore, the range of CD4(+) memory T-cell frequencies in peripheral blood of healthy virus carriers was noticeably lower and narrower than the corresponding range of latent antigen-specific CD8(+)-T-cell frequencies.     

A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1.

CaM kinase-Gr is a multifunctional Ca2+/calmodulin-dependent protein kinase which is enriched in neurons and T lymphocytes. The kinase is absent from primary human B lymphocytes but is expressed in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines, suggesting that expression of the kinase can be upregulated by an EBV gene product(s). We investigated the basis of CaM kinase-Gr expression in EBV-transformed cells and the mechanisms that regulate its activity therein by using an EBV-negative Burkitt lymphoma cell line, BJAB, and BJAB cells converted to expression of individual EBV proteins by single-gene transfer. CaM kinase-Gr expression was upregulated in BJAB cells by EBV latent-infection membrane protein 1 (LMP1) but not by LMP2A or by nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C. In LMP1-converted BJAB cells, the kinase was functional and was dramatically activated upon cross-linking of surface immunoglobulin M. Overlapping cDNA clones that encode human CaM kinase-Gr were sequenced, revealing 81% amino acid identity between the rat and human proteins. Transfection of BJAB cells with an expression construct for the human enzyme resulted in a functional kinase which was shown by epitope tagging to localize primarily to cytoplasmic and perinuclear structures. Induction of CaM kinase-Gr expression by LMP1 provides the first example of a Ca2+/calmodulin-dependent protein kinase upregulated by a viral protein. In view of the key role played by LMP1 in B-lymphocyte immortalization by EBV, these findings implicate CaM kinase-Gr as a potential mediator of B-lymphocyte growth transformation.     

Epstein-Barr virus gene expression in malignant lymphomas induced by experimental virus infection of cottontop tamarins.

Inoculation of cottontop tamarins with a large dose of Epstein-Barr virus (EBV) leads to the induction of multiple EBV genome-positive lymphomas. These tumors have been characterized as oligoclonal or monoclonal large-cell malignant lymphomas that closely resemble the EBV genome-positive B-cell lymphomas that arise in human allograft recipients. The expression of latent and lytic EBV-encoded proteins was investigated in these virus-induced tamarin lymphomas and in derived cell lines. The tamarin tumors were found to express EBV nuclear antigen 1 (EBNA 1), EBNA 2, EBNA leader protein, and the latent membrane protein (LMP) as determined both by immunohistochemical staining and by immunoblotting. However, within the limits of the immunoblotting assays, no expression of the EBNA 3a protein family could be detected. Assays for lytic-cycle proteins by using both polyclonal human sera and monoclonal antibodies against viral capsid antigen, early antigen, and membrane antigen (gp340/220) showed minimal, if any, expression of these antigens in the lymphoma biopsies. In contrast, the cell lines derived from these lymphomas, even in early passage, expressed abundant levels of the lytic-cycle antigens and also expressed the EBNA 3a protein as well as EBNA 1, EBNA 2, EBNA leader protein, and LMP. This finding suggests that the virus-lymphoma cell interaction, in particular the switch to lytic cycle, is subject to some form of host control in vivo. The expression of EBNA 2 and LMP in these tamarin lymphomas strengthens their resemblance to posttransplant lymphomas in humans, since these human tumors are also EBNA 2 and LMP positive (L. S. Young, C. Alfieri, K. Hennessy, H. Evans, C. O'Hara, K. Anderson, A. Rickinson, E. Kieff, and J. I. Cohen, submitted for publication). Since both proteins are known to be important effector molecules of virus-induced B-cell growth transformation in vitro, their expression in these lymphomas constitutes the best evidence for a direct oncogenic role for EBV in vivo.     

Distinction between Epstein-Barr virus type A (EBNA 2A) and type B (EBNA 2B) isolates extends to the EBNA 3 family of nuclear proteins.

The Epstein-Barr virus (EBV) nuclear antigens EBNA 3a, 3b, and 3c have recently been mapped to adjacent reading frames in the BamHI L and E fragments of the B95.8 EBV genome. We studied by immunoblotting the expression of the family of EBNA 3 proteins in a panel of 20 EBV-transformed lymphoblastoid cell lines (LCLs) carrying either type A (EBNA 2A-encoding) or type B (EBNA 2B-encoding) virus isolates. Certain human sera from donors naturally infected with type A isolates detected the EBNA 3a, 3b, and 3c proteins in all type A virus-transformed LCLs (with a single exception in which EBNA 3b was not detected) but detected only EBNA 3a in LCLs carrying type B isolates. These results were confirmed with human and murine antibodies with specific reactivity against sequences of the type A EBNA 3a, 3b, or 3c expressed in bacterial fusion proteins. Conversely, selected human sera from donors naturally infected with type B strains of EBV identified the EBNA 3a encoded by both types of isolates plus two novel EBNAs present only in type B, and not in type A, virus-transformed LCLs; these novel proteins appear to be the type B homologs of EBNA 3b and 3c. The distinction between type A and type B EBV isolates therefore extends beyond the EBNA 2 gene to the EBNA 3 family of proteins. This has important implications with respect to the evolutionary origin of these two EBV types and also places in a new light recent studies which identified differences between type A and type B transformants in terms of growth phenotype (A. B. Rickinson, L. S. Young, and M. Rowe, J. Virol. 61:1310-1317, 1987) and of detection by EBV-specific cytotoxic T cells (D. J. Moss, I. S. Misko, S. R. Burrows, K. Burman, R. McCarthy, and T. B. Sculley, Nature [London] 331:719-721, 1988).     

Dendritic cells transduced with an adenovirus vector encoding Epstein-Barr virus latent membrane protein 2B: a new modality for vaccination

Epstein-Barr virus (EBV) is a herpesvirus commonly associated with several malignancies, particularly in immunocompromised hosts. As a strategy for stimulating immunity against EBV for the treatment of EBV-associated tumors, we have genetically engineered dendritic cells (DC) to express EBV antigens, such as latent membrane protein 2B (LMP2B), using recombinant adenovirus vectors. CD8(+) T lymphocytes from HLA-A2.1(+), EBV-seropositive healthy donors were cultured with autologous DC infected with recombinant adenovirus vector AdEGFP, encoding an enhanced green fluorescent protein (EGFP), or AdLMP2B at a multiplicity of infection of 250. After 48 h, >95% of the DC were positive for EGFP expression as assessed by fluorescence-activated cell sorting analysis, indicating efficient gene transfer. AdLMP2-transduced DC were used to stimulate CD8(+) T cells. Responder CD8(+) T cells were tested for gamma interferon (IFN-gamma) release by enzyme-linked spot (ELISPOT) assay and cytotoxic activity. Prior to in vitro stimulation, the frequencies of T-cells directed against two HLA-A2-presented LMP2 peptides (LMP2 329-337 and LMP2 426-434) were very low as assessed by IFN-gamma spot formation (T-cell frequency, <0.003%). IFN-gamma ELISPOT assays performed at day 14 showed a significant (2-log) increase of the day 0 frequency of T cells reactive against the LMP2 329-337 peptide, from 0.003 to 0.3 (P < 0.001). Moreover, specific cytolytic activity was observed against the autologous EBV B-lymphoblastoid cell lines after 21 days of stimulation of T-cell responders with AdLMP2-transduced DC (P < 0.01). In summary, autologous mature DC genetically modified with an adenovirus encoding EBV antigens stimulate the generation of EBV-specific CD8(+) effector T cells in vitro, supporting the potential application of EBV-based adenovirus vector vaccination for the immunotherapy of the EBV-associated malignancies.     

Holoendemic Malaria Exposure Is Associated with Altered Epstein-Barr Virus-Specific CD8+ T-Cell Differentiation

Coinfection with Plasmodium falciparum malaria and Epstein-Barr virus (EBV) is a major risk factor for endemic Burkitt lymphoma (eBL), still one of the most prevalent pediatric cancers in equatorial Africa. Although malaria infection has been associated with immunosuppression, the precise mechanisms that contribute to EBV-associated lymphomagenesis remain unclear. In this study, we used polychromatic flow cytometry to characterize CD8⁺ T-cell subsets specific for EBV-derived lytic (BMFL1 and BRLF1) and latent (LMP1, LMP2, and EBNA3C) antigens in individuals with divergent malaria exposure. No malaria-associated differences in EBV-specific CD8⁺ T-cell frequencies were observed. However, based on a multidimensional analysis of CD45RO, CD27, CCR7, CD127, CD57, and PD-1 expression, we found that individuals living in regions with intense and perennial (holoendemic) malaria transmission harbored more differentiated EBV-specific CD8⁺ T-cell populations that contained fewer central memory cells than individuals living in regions with little or no (hypoendemic) malaria. This profile shift was most marked for EBV-specific CD8⁺ T-cell populations that targeted latent antigens. Importantly, malaria exposure did not skew the phenotypic properties of either cytomegalovirus (CMV)-specific CD8⁺ T cells or the global CD8⁺ memory T-cell pool. These observations define a malaria-associated aberration localized to the EBV-specific CD8⁺ T-cell compartment that illuminates the etiology of eBL.     

CD4+ T-cell responses to Epstein-Barr virus (EBV) latent-cycle antigens and the recognition of EBV-transformed lymphoblastoid cell lines

There is considerable interest in the potential of Epstein-Barr virus (EBV) latent antigen-specific CD4+ T cells to act as direct effectors controlling EBV-induced B lymphoproliferations. Such activity would require direct CD4+ T-cell recognition of latently infected cells through epitopes derived from endogenously expressed viral proteins and presented on the target cell surface in association with HLA class II molecules. It is therefore important to know how often these conditions are met. Here we provide CD4+ epitope maps for four EBV nuclear antigens, EBNA1, -2, -3A, and -3C, and establish CD4+ T-cell clones against 12 representative epitopes. For each epitope we identify the relevant HLA class II restricting allele and determine the efficiency with which epitope-specific effectors recognize the autologous EBV-transformed B-lymphoblastoid cell line (LCL). The level of recognition measured by gamma interferon release was consistent among clones to the same epitope but varied between epitopes, with values ranging from 0 to 35% of the maximum seen against the epitope peptide-loaded LCL. These epitope-specific differences, also apparent in short-term cytotoxicity and longer-term outgrowth assays on LCL targets, did not relate to the identity of the source antigen and could not be explained by the different functional avidities of the CD4+ clones; rather, they appeared to reflect different levels of epitope display at the LCL surface. Thus, while CD4+ T-cell responses are detectable against many epitopes in EBV latent proteins, only a minority of these responses are likely to have therapeutic potential as effectors directly recognizing latently infected target cells.     

HLA-A11-restricted epitope polymorphism among Epstein-Barr virus strains in the highly HLA-A11-positive Chinese population: incidence and immunogenicity of variant epitope sequences

An individual's CD8(+)-cytotoxic-T-lymphocyte (CTL) response to Epstein-Barr virus (EBV) latent cycle antigens focuses on a small number of immunodominant epitopes often presented by just one of the available HLA class I alleles; for example, HLA-A11-positive Caucasians frequently respond to two immunodominant HLA A11 epitopes, IVTDFSVIK (IVT) and AVFDRKSDAK (AVF), within the nuclear antigen EBNA3B. Here, we reexamine the spectrum of EBV strains present in the highly HLA-A11-positive Chinese population for sequence changes in these epitopes relative to the Caucasian type 1 prototype strain B95.8. The IVT epitope was altered in 61 of 64 Chinese type 1 viruses, with four different sequence variants being observed, and the AVF epitope was altered in 46 cases with six different sequence variants; by contrast, all 10 Chinese type 2 viruses retained the prototype 2 epitope sequences. All but one of the type 1 epitope variants were poorly recognized by IVT- or AVF-specific CTLs in pulse-chase assays of peptide-mediated target cell lysis. More importantly, we screened HLA-A11-positive Chinese donors carrying viruses with known epitope mutations for evidence of epitope-specific CTL memory by enzyme-linked immunospot assays: none of the type 1 variants tested, nor the type 2 prototype, appeared to be immunogenic in vivo. The data remain consistent with the possibility that, during virus-host coevolution, pressure from the host CTL-mediated immune response has given A11 epitope-loss viruses a selective advantage.     

Interferon regulatory factor 7 regulates expression of Epstein-Barr virus latent membrane protein 1: a regulatory circuit

We have shown previously that interferon regulatory factor 7 (IRF7), a multifunctional protein intimately involved in latent Epstein-Barr virus (EBV) infection, is induced as well as activated by EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein. Since the LMP1 promoter (LMP1p) contains an interferon-stimulated response element (ISRE), we hypothesized that IRF7 might be able to regulate LMP1 expression and thus participate in a regulatory circuit between these two genes. In this study, IRF7 was shown first to activate LMP1p in transient transfection assays. Compared with EBV nuclear antigen 2 (EBNA2), the most potent viral transactivator of LMP1p, IRF7 has a lesser effect (approximately 10% that of EBNA2) on induction of LMP1p. Study with IRF7 deletion mutants showed that IRF7 functional domains have similar effects on both the beta interferon (IFN-beta) and LMP1 promoters in BJAB and 293 cells, and study with IRF7 phosphomimetic mutants showed that IRF7 phosphorylation may be involved in the activation of these two promoters. Further, the ISRE in LMP1p responds to IRF7 induction and IRF7 binds to this element. In the EBV-positive cell line P3HR1, which lacks the complete EBNA2 and EBV-encoded leader protein genes and hence expresses low-level LMP1, IRF7 alone can notably increase the endogenous LMP1 mRNA and protein levels. These results indicate that LMP1 is regulated by this host cell gene in addition to the viral factor, EBNA2, and may help to explain how LMP1 is expressed in type II latency in the absence of EBNA2. Moreover, IRF7 can regulate a viral gene in addition to a host cellular gene such as the IFN-beta gene. Together with the previous data that LMP1 can induce IRF7 expression and facilitate IRF7 phosphorylation and nuclear translocation, these results suggest a positive regulatory circuit between IRF7 and LMP1.