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.

Neutralizing Antibodies Protect against Oral Transmission of Lymphocryptovirus.

Epstein-Barr virus (EBV) is a cancer-associated pathogen for which there is no vaccine. Successful anti-viral vaccines elicit antibodies that neutralize infectivity; however, it is unknown whether neutralizing antibodies prevent EBV acquisition. Here we assessed whether passively delivered AMMO1, a monoclonal antibody that neutralizes EBV in a cell-type-independent manner, could protect against experimental EBV challenge in two animal infection models. When present prior to a high-dose intravenous EBV challenge, AMMO1 prevented viremia and reduced viral loads to nearly undetectable levels in humanized mice. AMMO1 conferred sterilizing immunity to three of four macaques challenged orally with rhesus lymphocryptovirus, the EBV ortholog that infects rhesus macaques. The infected macaque had lower plasma neutralizing activity than the protected animals. These results indicate that a vaccine capable of eliciting adequate titers of neutralizing antibodies targeting the AMMO1 epitope may protect against EBV acquisition and are therefore highly relevant to the design of an effective EBV vaccine.

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.

Adenovirus-based vaccines against rhesus lymphocryptovirus EBNA-1 induce expansion of specific CD8+ and CD4+ T cells in persistently infected rhesus macaques.

UNLABELLED: The impact of Epstein-Barr virus (EBV) on human health is substantial, but vaccines that prevent primary EBV infections or treat EBV-associated diseases are not yet available. The Epstein-Barr nuclear antigen 1 (EBNA-1) is an important target for vaccination because it is the only protein expressed in all EBV-associated malignancies. We have designed and tested two therapeutic EBV vaccines that target the rhesus (rh) lymphocryptovirus (LCV) EBNA-1 to determine if ongoing T cell responses during persistent rhLCV infection in rhesus macaques can be expanded upon vaccination. Vaccines were based on two serotypes of E1-deleted simian adenovirus and were administered in a prime-boost regimen. To further modulate the response, rhEBNA-1 was fused to herpes simplex virus glycoprotein D (HSV-gD), which acts to block an inhibitory signaling pathway during T cell activation. We found that vaccines expressing rhEBNA-1 with or without functional HSV-gD led to expansion of rhEBNA-1-specific CD8(+) and CD4(+) T cells in 33% and 83% of the vaccinated animals, respectively. Additional animals developed significant changes within T cell subsets without changes in total numbers. Vaccination did not increase T cell responses to rhBZLF-1, an immediate early lytic phase antigen of rhLCV, thus indicating that increases of rhEBNA-1-specific responses were a direct result of vaccination. Vaccine-induced rhEBNA-1-specific T cells were highly functional and produced various combinations of cytokines as well as the cytolytic molecule granzyme B. These results serve as an important proof of principle that functional EBNA-1-specific T cells can be expanded by vaccination. IMPORTANCE: EBV is a common human pathogen that establishes a persistent infection through latency in B cells, where it occasionally reactivates. EBV infection is typically benign and is well controlled by the host adaptive immune system; however, it is considered carcinogenic due to its strong association with lymphoid and epithelial cell malignancies. Latent EBNA-1 is a promising target for a therapeutic vaccine, as it is the only antigen expressed in all EBV-associated malignancies. The goal was to determine if rhEBNA-1-specific T cells could be expanded upon vaccination of infected animals. Results were obtained with vaccines that target EBNA-1 of rhLCV, a virus closely related to EBV. We found that vaccination led to expansion of rhEBNA-1 immune cells that exhibited functions fit for controlling viral infection. This confirms that rhEBNA-1 is a suitable target for therapeutic vaccines. Future work should aim to generate more-robust T cell responses through modified vaccines.

Measuring T-cell responses against LCV and CMV in cynomolgus macaques using ELISPOT: potential application to non-clinical testing of immunomodulatory therapeutics.

A number of immunomodulatory therapeutics increase the risk of disease associated with latent herpesviruses such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV), a member of the lymphocryptovirus (LCV) family that infects humans. The diseases associated with loss of immunity to these viruses can have major impacts on patients as well as on the commercial viability of the immunomodulatory therapeutics. In an effort to develop non-clinical methods for measuring effects on anti-viral immunity, we have developed an interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISPOT) assay to quantify the number of CMV or LCV-reactive T-cells in peripheral blood of cynomolgus macaques. After optimization of various parameters, the IFN-γ ELISPOT assay was characterized for specificity, intra-assay, monkey-to-monkey, and longitudinal variability and sensitivity to immunosuppression. The results show that nearly all animals have detectable responses against both CMV and LCV and responses were derived from T-cells specific to the virus of interest. Analyses of variability show assay reproducibility (≤23% CV), and that variability over time in anti-viral responses in individual animals (larger for LCV than for CMV) was ∼2-fold in most animals over a 3-month time period, which is predicted to allow for detection of drug-induced changes when using group sizes typical of non-clinical studies. In addition, the IFN-γ ELISPOT assay was capable of detecting decreases in the numbers of CMV and LCV reactive T-cells induced by immunosuppressive drugs in vitro. This assay may allow for non-clinical assessment of the effects of immunomodulatory therapeutics on anti-viral T-cell immunity in monkeys, and may help determine if therapeutics increase the risk of reactivating latent viral infections.

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.

Induction of encephalitis in rhesus monkeys infused with lymphocryptovirus-infected B-cells presenting MOG(34-56) peptide.

The overlapping epidemiology of multiple sclerosis (MS) and Epstein-Barr virus (EBV), the increased risk to develop MS after infectious mononucleosis (IM) and the localization of EBV-infected B-cells within the MS brain suggest a causal link between EBV and MS. However, the underlying mechanism is unknown. We hypothesize that EBV-infected B-cells are capable of eliciting a central nervous system (CNS) targeting autoimmune reaction. To test this hypothesis we have developed a novel experimental model in rhesus monkeys of IM-like disease induced by infusing autologous B-lymphoblastoid cells (B-LCL). Herpesvirus papio (HVP) is a lymphocryptovirus related to EBV and was used to generate rhesus monkey B-LCL. Three groups of five animals were included; each group received three intravenous infusions of B-LCL that were either pulsed with the encephalitogenic self peptide MOG(34-56) (group A), a mimicry peptide (981-1003) of the major capsid protein of cytomegalovirus (CMVmcp(981-1003); group B) or the citrullinated MOG(34-56) (cMOG(34-56); group C). Groups A and B received on day 98 a single immunization with MOG(34-56) in incomplete Freund's adjuvant (IFA). Group C monkeys were euthanized just prior to day 98 without booster immunization. We observed self-peptide-specific proliferation of T-cells, superimposed on similar strong proliferation of CD3(+)CD8(+) T-cells against the B-LCL as observed in IM. The brains of several monkeys contained perivascular inflammatory lesions of variable size, comprising CD3(+) and CD68(+) cells. Moreover, clusters of CD3(+) and CD20(+) cells were detected in the meninges. The only evident clinical sign was substantial loss of bodyweight (>15%), a symptom observed both in early autoimmune encephalitis and IM. In conclusion, this model suggests that EBV-induced B-LCL can elicit a CNS targeting inflammatory (auto)immune reaction.

CD4+ and CD8+ T-cell responses to latent antigen EBNA-1 and lytic antigen BZLF-1 during persistent lymphocryptovirus infection of rhesus macaques.

Epstein-Barr virus (EBV) infection leads to lifelong viral persistence through its latency in B cells. EBV-specific T cells control reactivations and prevent the development of EBV-associated malignancies in most healthy carriers, but infection can sometimes cause chronic disease and malignant transformation. Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein consistently expressed during all forms of latency and in all EBV-associated malignancies and is a promising target for a therapeutic vaccine. Here, we studied the EBNA-1-specific immune response using the EBV-homologous rhesus lymphocryptovirus (rhLCV) infection in rhesus macaques. We assessed the frequency, phenotype, and cytokine production profiles of rhLCV EBNA-1 (rhEBNA-1)-specific T cells in 15 rhesus macaques and compared them to the lytic antigen of rhLCV BZLF-1 (rhBZLF-1). We were able to detect rhEBNA-1-specific CD4(+) and/or CD8(+) T cells in 14 of the 15 animals screened. In comparison, all 15 animals had detectable rhBZLF-1 responses. Most peptide-specific CD4(+) T cells exhibited a resting phenotype of central memory (TCM), while peptide-specific CD8(+) T cells showed a more activated phenotype, belonging mainly to the effector cell subset. By comparing our results to the human EBV immune response, we demonstrate that the rhLCV model is a valid system for studying chronic EBV infection and for the preclinical development of therapeutic vaccines.

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.

Soluble rhesus lymphocryptovirus gp350 protects against infection and reduces viral loads in animals that become infected with virus after challenge.

Epstein-Barr virus (EBV) is a human lymphocryptovirus that is associated with several malignancies. Elevated EBV DNA in the blood is observed in transplant recipients prior to, and at the time of post-transplant lymphoproliferative disease; thus, a vaccine that either prevents EBV infection or lowers the viral load might reduce certain EBV malignancies. Two major approaches have been suggested for an EBV vaccine- immunization with either EBV glycoprotein 350 (gp350) or EBV latency proteins (e.g. EBV nuclear antigens [EBNAs]). No comparative trials, however, have been performed. Rhesus lymphocryptovirus (LCV) encodes a homolog for each gene in EBV and infection of monkeys reproduces the clinical, immunologic, and virologic features of both acute and latent EBV infection. We vaccinated rhesus monkeys at 0, 4 and 12 weeks with (a) soluble rhesus LCV gp350, (b) virus-like replicon particles (VRPs) expressing rhesus LCV gp350, (c) VRPs expressing rhesus LCV gp350, EBNA-3A, and EBNA-3B, or (d) PBS. Animals vaccinated with soluble gp350 produced higher levels of antibody to the glycoprotein than those vaccinated with VRPs expressing gp350. Animals vaccinated with VRPs expressing EBNA-3A and EBNA-3B developed LCV-specific CD4 and CD8 T cell immunity to these proteins, while VRPs expressing gp350 did not induce detectable T cell immunity to gp350. After challenge with rhesus LCV, animals vaccinated with soluble rhesus LCV gp350 had the best level of protection against infection based on seroconversion, viral DNA, and viral RNA in the blood after challenge. Surprisingly, animals vaccinated with gp350 that became infected had the lowest LCV DNA loads in the blood at 23 months after challenge. These studies indicate that gp350 is critical for both protection against infection with rhesus LCV and for reducing the viral load in animals that become infected after challenge. Our results suggest that additional trials with soluble EBV gp350 alone, or in combination with other EBV proteins, should be considered to reduce EBV infection or virus-associated malignancies in humans.

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.

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.

The BZLF1 homolog of an Epstein-Barr-related gamma-herpesvirus is a frequent target of the CTL response in persistently infected rhesus macaques.

Although CD8(+) T lymphocytes targeting lytic infection proteins dominate the immune response to acute and persistent EBV infection, their role in immune control of EBV replication is not known. Rhesus lymphocryptovirus (rhLCV) is a gamma-herpesvirus closely related to EBV, which establishes persistent infection in rhesus macaques. In this study, we investigated cellular immune responses to the rhLCV BZLF1 (rhBZLF1) homolog in a cohort of rhLCV-seropositive rhesus macaques. rhBZLF1-specific IFN-gamma ELISPOT responses ranging between 56 and 3070 spot-forming cells/10(6) PBMC were detected in 36 of 57 (63%) rhesus macaques and were largely mediated by CD8(+) T lymphocytes. The prevalence and magnitude of ELISPOT responses were greater in adult (5-15 years of age) rather than juvenile macaques (<5 years of age), suggesting that rhBZLF1-specific CTL increase over time following early primary infection. A highly immunogenic region in the carboxyl terminus of the rhBZLF1 protein containing overlapping CTL epitopes restricted by Mamu-A*01 and other as yet unidentified MHC class I alleles was identified. The presence of a robust CD8(+) T lymphocyte response targeting this lytic infection protein in both rhesus macaques and humans suggests that these CTL may be important for immune control of EBV-related gamma-herpesvirus infection. These data underscore the utility of the rhLCV-macaque model for studies of EBV pathogenesis.

The CD8+ T-cell response to an Epstein-Barr virus-related gammaherpesvirus infecting rhesus macaques provides evidence for immune evasion by the EBNA-1 homologue.

Epstein-Barr virus (EBV) infection persists for life in humans, similar to other gammaherpesviruses in the same lymphocryptovirus (LCV) genus that naturally infect Old World nonhuman primates. The specific immune elements required for control of EBV infection and potential immune evasion strategies essential for persistent EBV infection are not well defined. We evaluated the cellular immune response to latent infection proteins in rhesus macaques with naturally and experimentally acquired rhesus LCV (rhLCV) infection. RhLCV EBNA-1 (rhEBNA-1) was the most frequently targeted latent infection protein and induced the most robust responses by peripheral blood mononuclear cells tested ex vivo using the gamma interferon ELISPOT assay. In contrast, although in vitro stimulation and expansion of rhLCV-specific T lymphocytes demonstrated cytotoxic T-lymphocyte (CTL) activity against autologous rhLCV-infected B cells, rhEBNA-1-specific CTL activity could not be detected. rhEBNA-1 CTL epitopes were identified and demonstrated that rhEBNA-1-specific CTL were stimulated and expanded in vitro but did not lyse targets expressing rhEBNA-1. Similarly, rhEBNA-1-specific CTL clones were able to lyse targets pulsed with rhEBNA-1 peptides or expressing rhEBNA-1 deleted for the glycine-alanine repeat (GAR) but not full-length rhEBNA-1 or rhLCV-infected B cells. These studies show that the rhLCV-specific immune response to latent infection proteins is similar to the EBV response in humans, and a potential immune evasion mechanism for EBNA-1 has been conserved in rhLCV. Thus, the rhLCV animal model can be used to analyze the immune responses important for control of persistent LCV infection and the role of the EBNA-1 GAR for immune evasion in vivo.

Reduced prevalence of Epstein-Barr virus-related lymphocryptovirus infection in sera from a new world primate.

The recent discovery of an Epstein-Barr virus (EBV)-related lymphocryptovirus (LCV) naturally infecting common marmosets demonstrated that gamma-1 herpesviruses are not limited to human and Old World nonhuman primate hosts. We developed serologic assays to detect serum antibodies against lytic- and latent-infection marmoset LCV antigens in order to perform the first seroepidemiologic study of LCV infection in New World primates. In three different domestic colonies and in animals recently captured from the wild, we found that the seroprevalence of marmoset LCV infection was not as ubiquitous as with EBV or Old World LCV. These biologic differences in LCV infection of New World versus human and Old World primate hosts correlate with the evolution of the LCV viral gene repertoire.

Cloning of the rhesus lymphocryptovirus viral capsid antigen and Epstein-Barr virus-encoded small RNA homologues and use in diagnosis of acute and persistent infections.

Epstein-Barr virus (EBV) is the most common cause of infectious mononucleosis and is associated with the development of several human malignancies. A closely related herpesvirus in the same lymphocryptovirus (LCV) genera as EBV naturally infects rhesus monkeys and provides an important animal model for studying EBV pathogenesis. We cloned the small viral capsid antigen (sVCA) homologue from the rhesus LCV and developed a peptide enzyme-linked immunosorbent assay (ELISA) to determine whether epitopes in the rhesus LCV sVCA are a reliable indicator of rhesus LCV infection. In order to define a "gold standard" for rhesus LCV infection, we also cloned the EBV-encoded small RNA 1 (EBER1) and EBER2 homologues from rhesus LCV and developed a reverse transcription (RT)-PCR assay to detect persistent LCV infection in rhesus monkey peripheral blood lymphocytes. Animals from a conventional and a hand-reared colony were studied to compare the prevalence of rhesus LCV infection in the two groups. There was a 100% correlation between the peptide ELISA and EBER RT-PCR results for rhesus LCV infection. In addition, specificity for LCV infection and exclusion of potential cross-reactivity to the rhesus rhadinovirus sVCA homologue could be demonstrated using sera from experimentally infected animals. These studies establish two novel assays for reliable diagnosis of acute and persistent rhesus LCV infections. The rhesus LCV sVCA peptide ELISA provides a sensitive and reliable assay for routine screening, and these studies of the hand-reared colony confirm the feasibility of raising rhesus LCV-naive animals.

Sequence analysis and variation of EBNA-1 in Epstein-Barr virus-related herpesvirus of cynomolgus monkey.

OBJECTIVES: The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is an important protein for immortalization and tumorigenesis of infected cells. EBNA-1 gene variants may play a role in tumorigenesis. We determined the nucleotide and amino acid (aa) sequences of EBNA-1 in EBV-related herpesviruses from cynomolgus monkeys (cynomolgus-EBV) which induced malignant lymphomas in its natural host and in rabbits, and compared them with sequences of EBV and other lymphocryptoviruses (LCVs). METHODS: Polymerase chain reaction and direct sequencing methods were performed using extracted DNA from cynomolgus-EBV-infected cell lines. RESULTS: The amino acid sequences of cynomolgus-EBV EBNA-1 from two cell lines (Si-IIA: 588 aa; Ts-B6: 619 aa) which are antigenically cross-reactive to human EBV EBNA-1 showed homology with human EBV (Si-IIA: 53%; Ts-B6: 58%) and other LCVs from baboons (54 and 52%) and rhesus monkeys (60 and 58%), especially in the C-terminal unique domain. Homology of the EBNA-1 sequence between Si-IIA and Ts-B6 was 92%. The sequence difference between EBV and the related LCVs was manifested mainly in the length of the internal repeat 3-corresponding region, which contains serine in the glycine/alanine repeat region of nonhuman LCVs. CONCLUSION: Sequence variation of cynomolgus-EBV EBNA-1 from different cell lines was observed. However, their sequences show a relatively high homology with human EBV and share the common features of EBNA-1 of EBV and other LCVs.

Inhibition of antigen presentation by the glycine/alanine repeat domain is not conserved in simian homologues of Epstein-Barr virus nuclear antigen 1.

Most humans and Old World nonhuman primates are infected for life with Epstein-Barr virus (EBV) or closely related gammaherpesviruses in the same lymphocryptovirus (LCV) subgroup. Several potential strategies for immune evasion and persistence have been proposed based on studies of EBV infection in humans, but it has been difficult to test their actual contribution experimentally. Interest has focused on the EBV nuclear antigen 1 (EBNA1) because of its essential role in the maintenance and replication of the episomal viral genome in latently infected cells and because EBNA1 endogenously expressed in these cells is protected from presentation to the major histocompatibility complex class-I restricted cytotoxic T-lymphocyte (CTL) response through the action of an internal glycine-alanine repeat (GAR). Given the high degree of biologic conservation among LCVs which infect humans and Old World primates, we hypothesized that strategies essential for viral persistence would be well conserved among viruses of this subgroup. We show that the rhesus LCV EBNA1 shares sequence homology with the EBV and baboon LCV EBNA1 and that the rhesus LCV EBNA1 is a functional homologue for EBV EBNA1-dependent plasmid maintenance and replication. Interestingly, all three LCVs possess a GAR domain, but the baboon and rhesus LCV EBNA1 GARs fail to inhibit antigen processing and presentation as determined by using three different in vitro CTL assays. These studies suggest that inhibition of antigen processing and presentation by the EBNA1 GAR may not be an essential mechanism for persistent infection by all LCV and that other mechanisms may be important for immune evasion during LCV infection.

An animal model for acute and persistent Epstein-Barr virus infection.

Epstein-Barr virus (EBV) is a human lymphocryptovirus that causes infectious mononucleosis, persists asymptomatically for life in nearly all adults, and is associated with the development of B cell lymphomas and nasopharyngeal carcinomas. A highly similar rhesus lymphocryptovirus naturally endemic in rhesus monkeys was used to orally infect naïve animals from a pathogen-free colony. This animal model reproduced key aspects of human EBV infection, including oral transmission, atypical lymphocytosis, lymphadenopathy, activation of CD23(+) peripheral blood B cells, sustained serologic responses to lytic and latent EBV antigens, latent infection in the peripheral blood, and virus persistence in oropharyngeal secretions. This system may be useful for studying the pathogenesis, prevention, and treatment of EBV infection and associated oncogenesis.