Epstein-Barr virus: induction and control of cell transformation

Epstein-Barr virus (EBV), a ubiquitous human herpes virus, is associated with an increasing number of lymphoid and epithelial malignancies. The ability of the virus to establish life-long persistent infections and induce growth transformation is related to the function of a set of viral proteins that are variously expressed in both normal and malignant cells. Recent evidence indicates that these viral proteins are able to usurp cellular pathways that promote the cell growth and survival, while impairing anti-viral immune responses. Elucidation of the mechanisms by which EBV induces cell transformation and escapes host immune control provides the rational background for the design of new strategies of intervention for EBV-related malignancies.     

Epstein-Barr病毒的免疫调控与逃逸机制北大核心CSCD

EB病毒(Epstein-Barr Virus,EBV)属于γ疱疹病毒科,是第一个被发现与人类肿瘤相关的DNA病毒。EB病毒通过激活Toll样受体(Toll like receptors,TLRs)信号通路,诱导I型干扰素的大量释放和功能性的自噬机制,从而引起机体的免疫应答。然而,相对于其他疱疹病毒,EB病毒已进化出更为精细且错综复杂的机制来破坏和逃逸宿主的免疫系统,如限制自身蛋白表达、活化宿主的泛素-蛋白酶体系统、干扰或逆转自噬与泛素化修饰等。这些机制会引发EB病毒在宿主体内的持续性感染,导致宿主免疫功能失调,引发EB病毒相关疾病(如鼻咽癌、传染性单核细胞增多症等)。因此,研究EB病毒特异性的免疫调控机制不仅对深入理解EB病毒的潜伏性感染和致癌性至关重要,而且还将为EB病毒诱发的相关疾病的免疫预防与治疗鉴定出新的潜在靶点。此文主要阐述了EB病毒调控宿主免疫应答和逃逸先天免疫应答的分子机制。     

CD4+ T cell responses in the immune control against latent infection by Epstein-Barr virus

The human gamma-herpesvirus Epstein-Barr virus establishes latent, life-long infection in more than 95% of the human adult population. Despite its growth transforming capacity, most carriers control EBV associated malignancies efficiently and remain free of EBV+ tumors. It is commonly accepted that lymphoblastoid cells, expressing all EBV latent antigens, are targeted by the immune system and cause tumors only in immune-suppressed individuals. However, immune control of EBV associated malignancies which express only three or one EBV latent antigen is less obvious. Recent studies have addressed the pattern of EBV latent infection in healthy EBV carriers and the identity of EBV derived target antigens for CD4+ T cells. The results suggest that immune surveillance also extends to tumors, which have down-regulated most EBV latent antigens and therefore escape EBV specific immune recognition at least in part. EBV specific immunity that targets these tumors in healthy EBV carriers seems to fail specifically during the development of Hodgkin's disease, nasopharyngeal carcinoma and Burkitt's lymphoma. These three EBV+ tumors appear to subdue EBV immunity against the remaining EBV latent antigens in different ways or profit from the effect of other pathogens on EBV specific immune responses, when they develop in otherwise immune competent individuals. While immune control and immune escape of these so-called spontaneously arising EBV associated malignancies is just beginning to be understood, immune control of persisting EBV infection can serve as a model for tumor immune surveillance in general.     

Differential evolution and stability of epitope-specific CD8(+) T cell responses in EBV infection

Murine models of lymphocytic choriomeningitis virus infection suggest that the memory CD8(+) T cell repertoire is reflective of the CD8(+) T cell repertoire generated during acute infection. Less is known regarding the evolution of CD8(+) T cell repertoires during human viral infections. We therefore examined epitope-specific CD8(+) T cell responses in a large cohort of individuals with acute through latent Epstein-Barr virus infection. Using 16 of 20 published EBV epitopes restricted by HLA-A2, HLA-A3 or HLA-B7, we showed that lytic cycle-specific CD8(+) T cell responses predominated during acute EBV infection. However, whereas HLA-A2(+)-restricted BMLF-1-specific CD8(+) T cell responses were maintained through latency, HLA-A2(+)- and HLA-B7(+)-restricted BZLF-1, as well as HLA-A3(+)-restricted BRLF-1 CD8(+) T cell responses, were generated but not readily maintained. Analyses of CD8(+) T cell responses to EBV latent cycle Ags showed delayed detection and lower frequencies of latent epitope-specific CD8(+) T cell responses during acute EBV infection, with maintenance of these responses 1 yr post-EBV infection. Early BMLF-1 and EBNA-3A epitope-specific CD8(+) T cell frequencies did not correlate with their frequencies at 1 yr postinfection. Interestingly, populations of EBV-specific CD8(+) T cells were stable during 20 mo in our long term EBV-seropositive populations, suggesting homeostasis between virus and the host immune system. This study demonstrates that CD8(+) T cell repertoires generated during persistent viral infections are not simply reflective of the initial pool of CD8(+) T cells and provides evidence that the generation of CD8(+) T cell responses to a persistent infection is a dynamic process.     

Differences among human immunodeficiency virus strains in their capacities to induce cytolysis or persistent infection of a lymphoblastoid cell line immortalized by Epstein-Barr virus.

Four strains of human immunodeficiency virus (HIV) manifest consistent differences in biologic behavior after infection of the X50-7 line of human umbilical cord lymphocytes immortalized by Epstein-Barr virus (EBV). Some dilutions of the first strain examined, human T-cell lymphotropic virus type III B, which is derived from a pool of patient isolates propagated in H9 cells, caused transient cytopathic effects (CPE) followed by recovery of a subpopulation of X50-7 cells which became virus carrier cultures. Other dilutions of the same virus stock completely lysed X50-7 cells. Two other strains, RF2 and YW, both from individual patients with acquired immune deficiency syndrome, always induced complete cytolysis of X50-7 cells at all dilutions which infected the cells. However, RF2 did establish persistent infection of H9 cells. A fourth strain, PH1-MN, from a child with acquired immune deficiency syndrome-related complex, induced only transient CPE in X50-7 and H9 cells, which thereafter always recovered to form carrier cultures. For all four strains, the dilutions of HIV stocks which caused CPE corresponded to dilutions which resulted in the detection of HIV polypeptides by immunoblot. Cytolysis in HIV-infected X50-7 cells was accompanied by a decrease in the amount of EBV nuclear antigen; however, HIV infection did not induce EBV replication. Thus CPE in X50-7 cells is due to replication of HIV per se and not to activation of EBV. The observations indicate that there are differences in the cytolytic properties of HIVs and that these differences are influenced by the target cell.     

The dynamics of the cellular immune response to HIV infection: implications for vaccination

Recent advances in measuring T-cell responses to viruses have led to new insights into how these T cells respond. In the acute infection there are massive CD8+ T-cell responses to both Epstein-Barr virus (EBV) and to human immunodeficiency virus (HIV). Many of these T cells are effector cells and only a minority appear to be capable of maintaining immunological memory. In persistent virus infections, high levels of antigen-specific effector cells persist. If virus does not persist, the effectors fade in number but memory is maintained and is primed to react rapidly to a new challenge. A vaccine that stimulates only T-cell responses may protect when these memory cells respond rapidly enough to generate high numbers of effectors before the infecting virus becomes established.     

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.     

Epstein-Barr virus induces MCP-1 secretion by human monocytes via TLR2

Epstein-Barr virus (EBV) is a gammaherpesvirus infecting the majority of the human adult population in the world. TLR2, a member of the Toll-like receptor (TLR) family, has been implicated in the immune responses to different viruses including members of the herpesvirus family, such as human cytomegalovirus, herpes simplex virus type 1, and varicella-zoster virus. In this report, we demonstrate that infectious and UV-inactivated EBV virions lead to the activation of NF-kappaB through TLR2 using HEK293 cells cotransfected with TLR2-expressing vector along with NF-kappaB-Luc reporter plasmid. NF-kappaB activation in HEK293-TLR2 cells (HEK293 cells transfected with TLR2) by EBV was not enhanced by the presence of CD14. The effect of EBV was abrogated by pretreating HEK293-TLR2 cells with blocking anti-TLR2 antibodies or by preincubating viral particles with neutralizing anti-EBV antibodies 72A1. In addition, EBV infection of primary human monocytes induced the release of MCP-1 (monocyte chemotactic protein 1), and the use of small interfering RNA targeting TLR2 significantly reduced such a chemokine response to EBV. Taken together, these results indicate that TLR2 may be an important pattern recognition receptor in the immune response directed against EBV infection.     

Herpesvirus-specific CD8 T cell immunity in old age: cytomegalovirus impairs the response to a coresident EBV infection

Aging in humans is associated with increased infections and the reduced proliferative capacity of T cells, part of the more global phenomenon termed immune senescence. The etiology of immune senescence is unknown but the accumulation of virus-specific memory T cells may be a contributory factor. We have examined CD8 T cell responses to two persistent herpesvirus infections, CMV and EBV, and to a recurrent virus infection, influenza, in different age cohorts of healthy donors using HLA-peptide tetramers and intracellular cytokine detection. Of these, CMV appears to be the most immunogenic, with the CD8 T cell response representing over 10% of the CD8 pool in many elderly donors. Interestingly, the effect of age upon EBV-specific responses depends upon donor CMV sero-status. In CMV seropositive donors, the magnitude of the EBV-specific immune response is stable with age, but in CMV seronegative donors, the response to EBV increases significantly with age. By contrast, the influenza-specific CD8 T cell immune response decreases with age, independent of CMV status. The functional activity of the herpesvirus-specific immune response decreases in elderly donors, although the characteristic phenotypes of CMV- and EBV-specific memory populations are retained. This demonstrates that aging is associated with a marked accumulation of CMV-specific CD8 T cells together with a decrease in immediate effector function. Moreover, infection with CMV can reduce prevailing levels of immunity to EBV, another persistent virus. These results suggest that carriage of CMV may be detrimental to the immunocompetent host by suppressing heterologous virus-specific immunity during aging.     

Tonsilar NK cells restrict B cell transformation by the Epstein-Barr virus via IFN-gamma

Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, including Epstein-Barr virus (EBV), activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56(bright)CD16(-) NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. DCs elicited 50-fold stronger interferon-gamma (IFN-gamma) secretion from tonsilar NK cells than from peripheral blood NK cells, reaching levels that inhibited B cell transformation by EBV. In fact, 100- to 1,000-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The high IFN-gamma concentrations, produced by tonsilar NK cells, delayed latent EBV antigen expression, resulting in decreased B cell proliferation during the first week after EBV infection in vitro. These results suggest that NK cell activation by DCs can limit primary EBV infection in tonsils until adaptive immunity establishes immune control of this persistent and oncogenic human pathogen.     

The EBV Immunoevasins vIL-10 and BNLF2a Protect Newly Infected B Cells from Immune Recognition and Elimination

Lifelong persistence of Epstein-Barr virus (EBV) in infected hosts is mainly owed to the virus'' pronounced abilities to evade immune responses of its human host. Active immune evasion mechanisms reduce the immunogenicity of infected cells and are known to be of major importance during lytic infection. The EBV genes BCRF1 and BNLF2a encode the viral homologue of IL-10 (vIL-10) and an inhibitor of the transporter associated with antigen processing (TAP), respectively. Both are known immunoevasins in EBV''s lytic phase. Here we describe that BCRF1 and BNLF2a are functionally expressed instantly upon infection of primary B cells. Using EBV mutants deficient in BCRF1 and BNLF2a, we show that both factors contribute to evading EBV-specific immune responses during the earliest phase of infection. vIL-10 impairs NK cell mediated killing of infected B cells, interferes with CD4+ T-cell activity, and modulates cytokine responses, while BNLF2a reduces antigen presentation and recognition of newly infected cells by EBV-specific CD8+ T cells. Together, both factors significantly diminish the immunogenicity of EBV-infected cells during the initial, pre-latent phase of infection and may improve the establishment of a latent EBV infection in vivo.     

EBV lytic-phase protein BGLF5 contributes to TLR9 downregulation during productive infection

Viruses use a wide range of strategies to modulate the host immune response. The human gammaherpesvirus EBV, causative agent of infectious mononucleosis and several malignant tumors, encodes proteins that subvert immune responses, notably those mediated by T cells. Less is known about EBV interference with innate immunity, more specifically at the level of TLR-mediated pathogen recognition. The viral dsDNA sensor TLR9 is expressed on B cells, a natural target of EBV infection. Here, we show that EBV particles trigger innate immune signaling pathways through TLR9. Furthermore, using an in vitro system for productive EBV infection, it has now been possible to compare the expression of TLRs by EBV(-) and EBV(+) human B cells during the latent and lytic phases of infection. Several TLRs were found to be differentially expressed either in latently EBV-infected cells or after induction of the lytic cycle. In particular, TLR9 expression was profoundly decreased at both the RNA and protein levels during productive EBV infection. We identified the EBV lytic-phase protein BGLF5 as a protein that contributes to downregulating TLR9 levels through RNA degradation. Reducing the levels of a pattern-recognition receptor capable of sensing the presence of EBV provides a mechanism by which the virus could obstruct host innate antiviral responses.     

Tonsilar NK Cells Restrict B Cell Transformation by the Epstein-Barr Virus via IFN-��

Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, including Epstein-Barr virus (EBV), activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56^brightCD16⁻ NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. DCs elicited 50-fold stronger interferon-γ (IFN-γ) secretion from tonsilar NK cells than from peripheral blood NK cells, reaching levels that inhibited B cell transformation by EBV. In fact, 100- to 1,000-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The high IFN-γ concentrations, produced by tonsilar NK cells, delayed latent EBV antigen expression, resulting in decreased B cell proliferation during the first week after EBV infection in vitro. These results suggest that NK cell activation by DCs can limit primary EBV infection in tonsils until adaptive immunity establishes immune control of this persistent and oncogenic human pathogen.     

Epstein-Barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers

Epstein-Barr virus (EBV) is found frequently in certain epithelial pathologies, such as nasopharyngeal carcinoma and oral hairy leukoplakia, indicating that the virus can infect epithelial cells in vivo. Recent studies of cell lines imply that epithelial cells may also play a role in persistent EBV infection in vivo. In this report, we show the establishment and characterization of an ex vivo culture model of tonsil epithelial cells, a likely site for EBV infection in vivo. Primary epithelial-cell cultures, generated from tonsil explants, contained a heterogeneous mixture of cells with an ongoing process of differentiation. Keratin expression profiles were consistent with the presence of cells from both surface and crypt epithelia. A small subset of cells could be latently infected by coculture with EBV-releasing cell lines, but not with cell-free virus. We also detected viral-DNA, -mRNA, and -protein expression in cultures from EBV-positive tonsil donors prior to in vitro infection. We conclude that these cells were either already infected at the time of explantation or soon after through cell-to-cell contact with B cells replicating EBV in the explant. Taken together, these findings suggest that the tonsil epithelium of asymptomatic virus carriers is able to sustain EBV infection in vivo. This provides an explanation for the presence of EBV in naso- and oropharyngeal pathologies and is consistent with epithelial cells playing a role in the egress of EBV during persistent infection.     

A new model of Epstein-Barr virus infection reveals an important role for early lytic viral protein expression in the development of lymphomas

Epstein-Barr virus (EBV) infects cells in latent or lytic forms, but the role of lytic infection in EBV-induced lymphomas is unclear. Here, we have used a new humanized mouse model, in which both human fetal CD34(+) hematopoietic stem cells and thymus/liver tissue are transplanted, to compare EBV pathogenesis and lymphoma formation following infection with a lytic replication-defective BZLF1-deleted (Z-KO) virus or a lytically active BZLF1(+) control. Both the control and Z-KO viruses established long-term viral latency in all infected animals. The infection appeared well controlled in some animals, but others eventually developed CD20(+) diffuse large B cell lymphomas (DLBCL). Animals infected with the control virus developed tumors more frequently than Z-KO virus-infected animals. Specific immune responses against EBV-infected B cells were generated in mice infected with either the control virus or the Z-KO virus. In both cases, forms of viral latency (type I and type IIB) were observed that are less immunogenic than the highly transforming form (type III) commonly found in tumors of immunocompromised hosts, suggesting that immune pressure contributed to the outcome of the infection. These results point to an important role for lytic EBV infection in the development of B cell lymphomas in the context of an active host immune response.     

Antibody-mediated control of persistent gamma-herpesvirus infection

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, establish life-long latency and can reactivate in immunocompromised individuals. T cells play an important role in controlling persistent EBV infection, whereas a role for humoral immunity is less clear. The murine gamma-herpesvirus-68 has biological and structural similarities to the human gamma-herpesviruses, and provides an important in vivo experimental model for dissecting mechanisms of immune control. In the current studies, CD28(-/-) mice were used to address the role of Abs in control of persistent murine gamma-herpesvirus-68 infection. Lytic infection was controlled in the lungs of CD28(-/-) mice, and latency was maintained in B cells at normal frequencies. Although class-switched virus-specific Abs were initially generated in the absence of germinal centers, titers and viral neutralizing activity rapidly waned. T cell depletion in CD28(-/-) mice with compromised Ab responses, but not in control mice with intact Ab responses, resulted in significant recrudescence from latency, both in the spleen and the lung. Recrudescence could be prevented by passive transfer of immune serum. These data directly demonstrate an important contribution of humoral immunity to control of gamma-herpesvirus latency, and have significant implications for clinical intervention.     

The human T cell immune response to Epstein-Barr virus

The Epstein-Barr virus (EBV) is a gamma-herpes virus which establishes latent, life-long infection in more than 95% of the human adult population. Despite its growth transforming capacity, most carriers control EBV associated malignacies efficiently and remain free of EBV+ tumors. Though EBV is controlled by a potent immune response, this virus uses latency to persist in vivo. This review summarizes work which has been done to characterize T cell responses to EBV. The CD8 T cell responses are rather well characterized and have been shown by several groups to be highly focused towards early lytic antigens. Much less is known about CD4 T cell epitopes, due to the small size of the CD4 compartment. However, recent data indicate a control of lytic and latent cycles of EBV by specific CD4+ T cells. A clear understanding of the T cell response to EBV is important with a view to developing immunotherapies for the virus and its related malignancies.