Neutralizing antibodies against Epstein-Barr virus infection of B cells can protect from oral viral challenge in the rhesus macaque animal model.

Epstein-Barr virus (EBV) and related lymphocryptoviruses (LCVs) from nonhuman primates are transmitted through oral secretions, penetrate the mucosal epithelium, and establish persistent infection in B cells. To determine whether neutralizing antibodies against epithelial or B cell infection could block oral transmission and persistent LCV infection, we use rhesus macaques, the most accurate animal model for EBV infection by faithfully reproducing acute and persistent infection in humans. Naive animals are infused with monoclonal antibodies neutralizing epithelial cell infection or B cell infection and then challenged orally with recombinant rhesus LCV. Our data show that high-titer B cell-neutralizing antibodies alone, but not epithelial cell-neutralizing antibodies, can provide complete protection of rhesus macaques from oral LCV challenge, but not in all hosts. Thus, neutralizing antibodies against B cell infection are important targets for EBV vaccine development, but they may not be sufficient.

Species-specific functions of Epstein-Barr virus nuclear antigen 2 (EBNA2) reveal dual roles for initiation and maintenance of B cell immortalization.

Epstein-Barr virus (EBV) and related lymphocryptoviruses (LCV) from non-human primates infect B cells, transform their growth to facilitate life-long viral persistence in the host, and contribute to B cell oncogenesis. Co-evolution of LCV with their primate hosts has led to species-specificity so that LCVs preferentially immortalize B cells from their natural host in vitro. We investigated whether the master regulator of transcription, EBV nuclear antigen 2 (EBNA2), is involved in LCV species-specificity. Using recombinant EBVs, we show that EBNA2 orthologues of LCV isolated from chimpanzees, baboons, cynomolgus or rhesus macaques cannot replace EBV EBNA2 for the immortalization of human B cells. Thus, LCV species-specificity is functionally linked to viral proteins expressed during latent, growth-transforming infection. In addition, we identified three independent domains within EBNA2 that act through species-specific mechanisms. Importantly, the EBNA2 orthologues and species-specific EBNA2 domains separate unique roles for EBNA2 in the initiation of B cell immortalization from those responsible for maintaining the immortalized state. Investigating LCV species-specificity provides a novel approach to identify critical steps underlying EBV-induced B cell growth transformation, persistent infection, and oncogenesis.

Epstein-Barr virus-specific adoptive immunotherapy for recurrent, metastatic nasopharyngeal carcinoma.

BACKGROUND: Early-stage and intermediate-stage nasopharyngeal cancer (NPC) generally carry a good prognosis, but for patients with recurrent, metastatic disease, options are limited. In the current study, the authors present a phase 1/2 study to evaluate the efficacy of Epstein-Barr virus (EBV)-stimulated cytotoxic T-lymphocyte (EBV-CTL) immunotherapy in this patient population. METHODS: Screening for patients with active, recurrent, metastatic EBV-associated NPC began in February 2007, and the study was closed to accrual in January 2012. After informed consent was obtained, patients had their blood drawn to initiate manufacturing of the EBV-CTL product. During product manufacturing, patients were placed on interim standard-of-care chemotherapy, and only after disease progression on the interim chemotherapy did patients receive investigational immunotherapy. Patients were restaged every 2 months until disease progression and then followed for survival. RESULTS: A total of 28 patients were enrolled, and 21 patients were treated. There was 1 complete response achieved, and at the time of last follow-up, the patient had been in remission for >8 years since treatment. The median progression-free survival was 2.2 months, and the median overall survival was 16.7 months. Two other patients, after failing EBV-CTL immunotherapy, unexpectedly demonstrated strong responses to the chemotherapy regimens they had previously failed. Patient EBV viral load and EBV-CTL specificity for tumor-associated viral antigens did not appear to correlate with clinical response. CONCLUSIONS: A durable response was observed with EBV-CTL immunotherapy, but the overall response rate for patients with recurrent, metastatic NPC was low. Further research is necessary to increase the efficacy of EBV-specific immunotherapy in patients with incurable NPC, and to characterize mechanisms for refacilitation to chemotherapy. Cancer 2017;123:2642-50. © 2017 American Cancer Society.

Epstein-Barr Virus gp350 Can Functionally Replace the Rhesus Lymphocryptovirus Major Membrane Glycoprotein and Does Not Restrict Infection of Rhesus Macaques.

UNLABELLED: Primary Epstein-Barr virus (EBV) infection is the most common cause of infectious mononucleosis, and persistent infection is associated with multiple cancers. EBV vaccine development has focused on the major membrane glycoprotein, gp350, since it is the major target for antibodies that neutralize infection of B cells. However, EBV has tropism for both B cells and epithelial cells, and it is unknown whether serum neutralizing antibodies against B cell infection will provide sufficient protection against virus infection initiated at the oral mucosa. This could be stringently tested by passive antibody transfer and oral virus challenge in the rhesus macaque model for EBV infection. However, only neutralizing monoclonal antibodies (MAbs) against EBV are available, and EBV is unable to infect rhesus macaques because of a host range restriction with an unknown mechanism. We cloned the prototypic EBV-neutralizing antibody, 72A1, and found that recombinant 72A1 did not neutralize rhesus lymphocryptovirus (rhLCV) infection of macaque B cells. Therefore, we constructed a chimeric rhLCV in which the native major membrane glycoprotein was replaced with EBV gp350. This chimeric rhLCV became sensitive to neutralization by the 72A1 MAb, efficiently immortalized macaque B cells in vitro, and successfully established acute and persistent infection after oral inoculation of rhesus macaques. Thus, EBV gp350 can functionally replace rhLCV gp350 and does not restrict rhLCV infection in vitro or in vivo. The chimeric rhLCV enables direct use of an EBV-specific MAb to investigate the effects of serum neutralizing antibodies against B cell infection on oral viral challenge in rhesus macaques. IMPORTANCE: This study asked whether the EBV major membrane glycoprotein could functionally replace the rhLCV major membrane glycoprotein. We found that an rhLCV humanized with EBV gp350 is capable of efficiently immortalizing monkey B cells in vitro and reproduces acute and persistent infection after oral inoculation of macaques. These results advance our understanding of why EBV cannot infect rhesus macaques by proving that viral attachment through gp350 is not the mechanism for EBV host range restriction. Humanization of rhLCV with EBV gp350 also confers susceptibility to a potent EBV-neutralizing MAb and provides a novel and significant enhancement to the rhesus macaque animal model where both the clinical utility and biological role of neutralizing MAbs against B cell or epithelial cell infection can now be directly tested in the most accurate animal model for EBV infection.

Non-human Primate Lymphocryptoviruses: Past, Present, and Future.

Epstein-Barr virus (EBV) orthologues from non-human primates (NHPs) have been studied for nearly as long as EBV itself. Cross-reactive sera and DNA hybridization studies provided the first glimpses of the closely related herpesviruses that belonged to the same gamma-1 herpesvirus, or lymphocryptovirus, genus, as EBV. Over the years, detailed molecular and sequence analyses of LCVs that infect humans and other NHPs revealed similar colinear genome structures and homologous viral proteins expressed during latent and lytic infection. Despite these similarities, experimental infection of NHPs with EBV did not result in acute symptoms or persistent infection as observed in humans, suggesting some degree of host species restriction. Genome sequencing and a molecular clone of an LCV isolate from naturally infected rhesus macaques combined with domestic colonies of LCV-naïve rhesus macaques have opened the door to a unique experimental animal model that accurately reproduces the normal transmission, acute viremia, lifelong persistence, and immune responses found in EBV-infected humans. This chapter will summarize the advances made over the last 50 years in our understanding of LCVs that naturally infect both Old and New World NHPs, the recent, groundbreaking developments in the use of rhesus macaques as an animal model for EBV infection, and how NHP LCVs and the rhLCV animal model can advance future EBV research and the development of an EBV vaccine.

Host Range Restriction of Epstein-Barr Virus and Related Lymphocryptoviruses.

Epstein-Barr-related herpesviruses, or lymphocryptoviruses (LCV), naturally infect humans and nonhuman primates (NHP), but their host range is not well characterized. Using LCV and B cells from multiple species of Hominidae and Cercopithecidae, we show that LCV can immortalize B cells from some nonnative species but that growth transformation is restricted to B cells from their own family of hominoids or Old World NHP, suggesting a high degree of LCV adaptation to their natural primate host.

Therapeutic vaccination against the rhesus lymphocryptovirus EBNA-1 homologue, rhEBNA-1, elicits T cell responses to novel epitopes in rhesus macaques.

Epstein-Barr virus (EBV) is a vaccine/immunotherapy target due to its association with several human malignancies. EBNA-1 is an EBV protein consistently expressed in all EBV-associated cancers. Herein, EBNA-1-specific T cell epitopes were evaluated after AdC-rhEBNA-1 immunizations in chronically lymphocryptovirus-infected rhesus macaques, an EBV infection model. Preexisting rhEBNA-1-specific responses were augmented in 4/12 animals, and new epitopes were recognized in 5/12 animals after vaccinations. This study demonstrated that EBNA-1-specific T cells can be expanded by vaccination.

Nonhuman primate models for Epstein-Barr virus infection.

Epstein-Barr virus (EBV) is a human herpesvirus that infects nearly all humans by adulthood and is associated with a spectrum of human diseases including Infectious Mononucleosis, Hodgkin Lymphoma, Nasopharyngeal Carcinoma, and lymphomas in immunosuppressed hosts. Nonhuman primate (NHP) animal models provide important experimental systems for studying EBV infection. There has been significant progress in studies of EBV-related herpesviruses, or lymphocryptoviruses (LCV), that naturally infect New and Old World NHPs. Prototypes for New and Old World LCV have been cloned and sequenced, humoral and cellular immune responses to LCV in NHP have been characterized, experimental LCV infections in naïve rhesus macaques have been successful, and a genetic system to manipulate specific viral genes in rhesus LCV (rhLCV) has been developed. These advances have led to new insights in the dynamic interactions with the host during acute and persistent EBV infection and can provide a novel platform for EBV vaccine development. Further development and utilization of the rhLCV animal model would be greatly enhanced by expansion of LCV-free breeding colonies as a reliable source of naïve animals for experimental studies. NHP animal models for EBV infection provide unique opportunities for understanding the biology of EBV infection in humans and translating that knowledge into effective vaccines against EBV-induced diseases.

Therapeutic targeting of regulatory T cells enhances tumor-specific CD8+ T cell responses in Epstein-Barr virus associated nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) is associated with multiple malignancies including nasopharyngeal carcinoma (NPC). In nasopharynx cancer, CD8+ T cells specific for EBV Nuclear Antigen-1 (EBNA-1) and Latent Membrane Protein 2 (LMP2) are important components of anti-tumor immunity since both are consistently expressed in NPC. We have previously shown that EBNA-1-specific CD8+ T cell responses were suppressed in NPC patients compared to healthy controls. We now find that CD8+ T cell responses specific for LMP2 are also abnormal in NPC patients, and both EBNA-1- and LMP2-specific responses are suppressed by regulatory T cells (Treg). EBNA-1 and LMP2-specific CD8+ T cell responses, as well as immune control of EBV-infected cells in vitro, could be restored by the depletion of Tregs and by use of a clinically approved drug targeting Tregs. Thus, in vivo modulation of Tregs may be an effective means of enhancing these anti-tumor immune responses in NPC patients.

EBV BILF1 evolved to downregulate cell surface display of a wide range of HLA class I molecules through their cytoplasmic tail.

Coevolution of herpesviruses and their hosts has driven the development of both host antiviral mechanisms to detect and eliminate infected cells and viral ploys to escape immune surveillance. Among the immune-evasion strategies used by the lymphocryptovirus (γ(1)-herpesvirus) EBV is the downregulation of surface HLA class I expression by the virally encoded G protein-coupled receptor BILF1, thereby impeding presentation of viral Ags and cytotoxic T cell recognition of the infected cell. In this study, we show EBV BILF1 to be expressed early in the viral lytic cycle. BILF1 targets a broad range of HLA class I molecules, including multiple HLA-A and -B types and HLA-E. In contrast, HLA-C was only marginally affected. We advance the mechanistic understanding of the process by showing that the cytoplasmic C-terminal tail of EBV BILF1 is required for reducing surface HLA class I expression. Susceptibility to BILF1-mediated downregulation, in turn, is conferred by specific residues in the intracellular tail of the HLA class I H chain. Finally, we explore the evolution of BILF1 within the lymphocryptovirus genus. Although the homolog of BILF1 encoded by the lymphocryptovirus infecting Old World rhesus primates shares the ability of EBV to downregulate cell surface HLA class I expression, this function is not possessed by New World marmoset lymphocryptovirus BILF1. Therefore, this study furthers our knowledge of the evolution of immunoevasive functions by the lymphocryptovirus genus of herpesviruses.

An Epstein-Barr virus encoded inhibitor of Colony Stimulating Factor-1 signaling is an important determinant for acute and persistent EBV infection.

Acute Epstein-Barr virus (EBV) infection is the most common cause of Infectious Mononucleosis. Nearly all adult humans harbor life-long, persistent EBV infection which can lead to development of cancers including Hodgkin Lymphoma, Burkitt Lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and lymphomas in immunosuppressed patients. BARF1 is an EBV replication-associated, secreted protein that blocks Colony Stimulating Factor 1 (CSF-1) signaling, an innate immunity pathway not targeted by any other virus species. To evaluate effects of BARF1 in acute and persistent infection, we mutated the BARF1 homologue in the EBV-related herpesvirus, or lymphocryptovirus (LCV), naturally infecting rhesus macaques to create a recombinant rhLCV incapable of blocking CSF-1 (ΔrhBARF1). Rhesus macaques orally challenged with ΔrhBARF1 had decreased viral load indicating that CSF-1 is important for acute virus infection. Surprisingly, ΔrhBARF1 was also associated with dramatically lower virus setpoints during persistent infection. Normal acute viral load and normal viral setpoints during persistent rhLCV infection could be restored by Simian/Human Immunodeficiency Virus-induced immunosuppression prior to oral inoculation with ΔrhBARF1 or infection of immunocompetent animals with a recombinant rhLCV where the rhBARF1 was repaired. These results indicate that BARF1 blockade of CSF-1 signaling is an important immune evasion strategy for efficient acute EBV infection and a significant determinant for virus setpoint during persistent EBV infection.

Summary of roundtable discussion meeting: non-human primates to assess risk for EBV-related lymphomas in humans.

Epstein-Barr virus (EBV)-associated lymphomas are a known risk for immunosuppressed individuals. Non-clinical methods to determine the potential of new immunomodulatory compounds to produce EBV-associated lymphomas (hazard identification) have not been developed. Since lymphocryptovirus (LCV) in non-human primates (NHP) has similar characteristics to EBV in humans, a Roundtable meeting was held in October 2010 to explore how the potential for EBV-related lymphomas in humans can be assessed by using surrogate biomarkers for lymphoma risk in NHP toxicity studies. Stakeholders from regulatory agencies, academia, and industry came together to determine the research gaps and potential benefits and considerations of such an approach given the current state-of-the-science. Key conclusions from the discussion included considerations raised about the potential usefulness of LCV-related biomarkers from NHP studies since there is significant controversy over the reliability of using EBV viral load or EBV-specific T-lymphocytes to predict for lymphoproliferative disorders in transplant patients. In addition, there are technical challenges that need to be further addressed in order to develop methods to measure LCV viral load and LCV-specific T-lymphocytes from cynomolgus monkeys.

Persistent infection drives the development of CD8+ T cells specific for late lytic infection antigens in lymphocryptovirus-infected macaques and Epstein-Barr virus-infected humans.

We examined the CD8(+) T cell repertoire against lytic infection antigens in rhesus macaques persistently infected with the Epstein-Barr virus (EBV)-related lymphocryptovirus (rhLCV). CD8(+) T cells specific for late (L) antigens were detected at rates comparable to those for early antigens and were associated with increasing duration of infection. L antigen-specific CD8(+) T cells were also readily detected in adult, EBV-positive humans. Thus, viral major histocompatibility complex class I (MHCI) immune evasion genes expressed during lytic LCV infection do not prevent L-specific CD8(+) T cell development over time during persistent infection.

Antibodies to lytic infection proteins in lymphocryptovirus-infected rhesus macaques: a model for humoral immune responses to epstein-barr virus infection.

Humoral immune responses to rhesus lymphocryptovirus (rhLCV) lytic infection proteins were evaluated in the rhesus macaque animal model for Epstein-Barr virus (EBV) infection. We found a hierarchy of humoral responses to 14 rhLCV lytic infection proteins in naturally infected rhesus macaques, with (i) widespread and robust responses to four glycoproteins expressed as late proteins, (ii) frequent but less robust responses to a subset of early proteins, and (iii) low-level responses to immediate-early proteins. This hierarchy of humoral responses was similar to that reported for EBV-infected humans, with the notable exception of the response to rhBARF1. Serum antibodies to rhBARF1 were frequently detected in healthy rhLCV-infected macaques, but in humans, anti-BARF1 antibodies have been reported primarily in patients with EBV-positive nasopharyngeal carcinoma (NPC). The macaque data accurately predicted that serum antibodies against BARF1 are a normal response to EBV infection when human serum samples are analyzed. The rhesus macaque animal provides a unique perspective on humoral responses to EBV infection in humans and can be a valuable model for EBV vaccine development.

Cloning of the Epstein-Barr virus-related rhesus lymphocryptovirus as a bacterial artificial chromosome: a loss-of-function mutation of the rhBARF1 immune evasion gene.

Rhesus macaques are naturally infected with a gammaherpesvirus which is in the same lymphocryptovirus (LCV) genus as and closely related to Epstein-Barr virus (EBV). The rhesus macaque LCV (rhLCV) contains a repertoire of genes identical to that of EBV, and experimental rhLCV infection of naive rhesus macaques accurately models acute and persistent EBV infection of humans. We cloned the LCL8664 rhLCV strain as a bacterial artificial chromosome to create recombinant rhLCV for investigation in this animal model system. A recombinant rhLCV (clone 16 rhLCV) carrying a mutation in the putative immune evasion gene rhBARF1 was created along with a rescued wild-type (rWT) rhLCV in which the rhBARF1 open reading frame (ORF) was repaired. The rWT rhLCV molecular clone demonstrated viral replication and B-cell immortalization properties comparable to those of the naturally derived LCL8664 rhLCV. Qualitatively, clone 16 rhLCV carrying a mutated rhBARF1 was competent for viral replication and B-cell immortalization, but quantitative assays showed that clone 16 rhLCV immortalized B cells less efficiently than LCL8664 and rWT rhLCV. Functional studies showed that rhBARF1 could block CSF-1 cytokine signaling as well as EBV BARF1, whereas the truncated rhBARF1 from clone 16 rhLCV was a loss-of-function mutant. These recombinant rhLCV can be used in the rhesus macaque animal model system to better understand how a putative viral immune evasion gene contributes to the pathogenesis of acute and persistent EBV infection. The development of a genetic system for making recombinant rhLCV constitutes a major advance in the study of EBV pathogenesis in the rhesus macaque animal model.

T cell detection of a B-cell tropic virus infection: newly-synthesised versus mature viral proteins as antigen sources for CD4 and CD8 epitope display.

Viruses that naturally infect cells expressing both MHC I and MHC II molecules render themselves potentially visible to both CD8+ and CD4+ T cells through the de novo expression of viral antigens. Here we use one such pathogen, the B-lymphotropic Epstein-Barr virus (EBV), to examine the kinetics of these processes in the virally-infected cell, comparing newly synthesised polypeptides versus the mature protein pool as viral antigen sources for MHC I- and MHC II-restricted presentation. EBV-transformed B cell lines were established in which the expression of two cognate EBV antigens, EBNA1 and EBNA3B, could be induced and then completely suppressed by doxycycline-regulation. These cells were used as targets for CD8+ and CD4+ T cell clones to a range of EBNA1 and EBNA3B epitopes. For both antigens, when synthesis was induced, CD8 epitope display rose quickly to near maximum within 24 h, well before steady state levels of mature protein had been reached, whereas CD4 epitope presentation was delayed by 36-48 h and rose only slowly thereafter. When antigen expression was suppressed, despite the persistence of mature protein, CD8 epitope display fell rapidly at rates similar to that seen for the MHC I/epitope half-life in peptide pulse-chase experiments. By contrast, CD4 epitope display persisted for many days and, following peptide stripping, recovered well on cells in the absence of new antigen synthesis. We infer that, in virally-infected MHC I/II-positive cells, newly-synthesised polypeptides are the dominant source of antigen feeding the MHC I pathway, whereas the MHC II pathway is fed by the mature protein pool. Hence, newly-infected cells are rapidly visible only to the CD8 response; by contrast, latent infections, in which viral gene expression has been extinguished yet viral proteins persist, will remain visible to CD4+ T cells.

Decreased EBNA-1-specific CD8+ T cells in patients with Epstein-Barr virus-associated nasopharyngeal carcinoma.

The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) is potentially a universal target for immune recognition of EBV-infected normal or malignant cells. EBNA-1-specific CD8+ T-cell responses have been assessed against a few epitopes presented on a limited number of HLA class I alleles. We now assess CD8+ T-cell responses to a complete panel of EBNA-1 peptides in an HLA-characterized population. We detected EBNA-1-specific CD8+ T cells in 10 of 14 healthy donors by analysis of peripheral blood mononuclear cells and EBV-specific T-cell lines. The frequent detection of CD8+ T-cell responses was confirmed by mapping EBNA-1 epitopes and demonstrating HLA class I presentation to CD8+ T cells in 6 of 6 donors, including 2 new EBNA-1 epitopes presented by HLA A0206 and A6802. Importantly, EBNA-1-specific CD8+ T cells were significantly less frequent in EBV-specific T-cell lines from patients with EBV-associated nasopharyngeal carcinoma (3 out of 22, P = 0.0003), whereas the frequency of LMP2-specific responses (14 out of 22) was not significantly different from healthy donors (11 out of 14). EBNA-1-specific CD8+ T-cell responses were rescued in approximately half of nasopharyngeal carcinoma patients by peptide and cytokine stimulation of peripheral blood mononuclear cells, suggesting these EBNA-1-specific CD8+ T cells were functionally defective in their response to EBV-infected cells. These results indicate that humans normally mount a significant EBNA-1-specific CD8+ T-cell response to EBV infection, but the immune response to this tumor antigen has been significantly altered in nasopharyngeal carcinoma patients. Overcoming this defect in EBV-specific immunity may prevent or enhance treatment of EBV-associated nasopharyngeal carcinoma.

Lytic and latent antigens of the human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus induce T-cell responses with similar functional properties and memory phenotypes.

The cellular immunity against Kaposi's sarcoma-associated herpesvirus (KSHV) is poorly characterized and has not been compared to T-cell responses against other human herpesviruses. Here, novel and dominant targets of KSHV-specific cellular immunity are identified and compared to T cells specific for lytic and latent antigens in a second human gammaherpesvirus, Epstein-Barr virus. The data identify a novel HLA-B57- and HLA-B58-restricted epitope in the Orf57 protein and show consistently close parallels in immune phenotypes and functional response patterns between cells targeting lytic or latent KSHV- and EBV-encoded antigens, suggesting common mechanisms in the induction of these responses.

Influence of translation efficiency of homologous viral proteins on the endogenous presentation of CD8+ T cell epitopes.

A significant proportion of endogenously processed CD8(+) T cell epitopes are derived from newly synthesized proteins and rapidly degrading polypeptides (RDPs). It has been hypothesized that the generation of rapidly degrading polypeptides and CD8(+) T cell epitopes from these RDP precursors may be influenced by the efficiency of protein translation. Here we address this hypothesis by using the Epstein-Barr virus-encoded nuclear antigen 1 protein (EBNA1), with or without its internal glycine-alanine repeat sequence (EBNA1 and EBNA1DeltaGA, respectively), which display distinct differences in translation efficiency. We demonstrate that RDPs constitute a significant proportion of newly synthesized EBNA1 and EBNA1DeltaGA and that the levels of RDPs produced by each of these proteins directly correlate with the translation efficiency of either EBNA1 or EBNA1DeltaGA. As a consequence, a higher number of major histocompatibility complex-peptide complexes can be detected on the surface of cells expressing EBNA1DeltaGA, and these cells are more efficiently recognized by virus-specific cytotoxic T lymphocytes compared to the full-length EBNA1. More importantly, we also demonstrate that the endogenous processing of these CD8(+) T cell epitopes is predominantly determined by the rate at which the RDPs are generated rather than the intracellular turnover of these proteins.

Translation efficiency of EBNA1 encoded by lymphocryptoviruses influences endogenous presentation of CD8+ T cell epitopes.

Lymphocryptoviruses (LCV) that infect humans and Old World primates display a significant degree of genetic identity. These viruses use B lymphocytes as primary host cells to establish a long-term latent infection and express highly homologous latent viral proteins. Of particular interest is the expression of the EBV-encoded nuclear antigen-1 (EBNA1), which plays a crucial role in maintaining the viral genome in B cells. Using human and Old World primate homologues of EBNA1, we show that the internal repeat sequences differentially influence their in vitro translation efficiency. Although the glycine-alanine repeat domain of human LCV (EBV) EBNA1 inhibits its self-synthesis, the repeat domains within the simian LCV homologues of EBNA1 do not inhibit self-synthesis. As a consequence, simian LCV EBNA1-expressing cells are more efficiently recognized by virus-specific CTL when compared to human EBV EBNA1, even though both proteins are highly stable in B cells. Interestingly, we also show that similar to human EBNA1, CD8+ T cell epitopes from simian LCV EBNA1 are predominantly derived from newly synthesized protein rather than the long-lived pool of stable protein. These observations provide additional evidence that supports the theory that immune recognition of EBNA1 can occur without compromising the biological maintenance function of this protein.