Epstein-Barr Virus Lytic Cycle Proteins Research Articles

Cotargeting EBV lytic as well as latent cycle antigens increases T-cell potency against lymphoma

AbstractThe remarkable efficacy of Epstein-Barr virus (EBV)-specific T cells for the treatment of posttransplant lymphomas has not been reproduced for EBV-positive (EBV+) malignancies outside the transplant setting. This is because of, in part, the heterogeneous expression and poor immunogenicity of the viral antigens expressed, namely LMPs 1 and 2, EBNA1, and BARF1 (type-2 [T2] latency). However, EBV lytic cycle proteins are also expressed in certain EBV+ malignancies and, because several EBV lytic cycle proteins are abundantly expressed, have oncogenic activity, and likely contribute to malignancy, we sought and identified viral lytic-cycle transcripts in EBV+ Hodgkin lymphoma biopsies. This provided the rationale for broadening the target antigen–specific repertoire of EBV-specific T cells (EBVSTs) for therapy. We stimulated, with both lytic and latent cycle proteins, peripheral blood mononuclear cells from healthy donors and patients with EBV+ lymphoma to produce broad repertoire (BR) EBVSTs. Compared with T2 antigen-specific EBVSTs, BR-EBVSTs more rapidly cleared autologous EBV+ tumors in NSG mice and produced higher levels of proinflammatory cytokines that should reactivate the immunosuppressive tumor microenvironment leading to epitope spreading. Our results confirm that lytic cycle antigens are clinically relevant targets for EBV+ lymphoma and underpin the rationale for integrating BR-EBVSTs as a therapeutic approach for relapsed/refractory EBV+ lymphoma (ClinicalTrials.gov identifier: NCT01555892 and NCT04664179), as well as for other EBV-associated malignancies.

Proteome-wide analysis of CD8+ T cell responses to EBV lytic infection

Epstein-Barr virus (EBV) is one of 9 Human herpesviruses that can develop lifelong persistence. These viruses provide an antigenically complex challenge that induce strong CD8+T cell immunity during primary infection and continue to shape this immunity through recurrent lytic reactivation. Here is described the first lytic proteome-wide analysis of CD8+T cell responses to EBV and the first to compare primary vs memory CD8+T cell responses to any human herpesvirus. Primary CD8+T cells were mitogenically expanded directly from the blood of infectious mononucleosis (IM) patients. Comparatively, memory CD8+T cells required pre-enrichment using autologous dendritic cells loaded with a lytically-infected EBV cell lysate and FACS selection based upon the activation marker 4-1BB. Enriched cells were then expanded in vitro as for IM cells. Preparations from 7 IM patients and 7 healthy carriers were screened against each of the 70 EBV lytic cycle proteins in combination with the donors’ HLA-I alleles. Multiple reactivities were identified across the full lytic cycle with 146 responses identified amongst the 7 IM patients and 96 amongst the 7 healthy carriers. However the distribution of responses varied between the 2 cohorts with primary responses targeting IE and a small group of E proteins whereas memory responses targeted all phases but with some prominent responses against L proteins. This infers that responses in primary infection therefore appear to be shaped by presentation on the infected cell surface prior to the activity of viral evasins. However long-term carriage appears to re-shape the virus-specific response.

Proteome-wide analysis of CD8+ T cell responses to EBV reveals differences between primary and persistent infection.

Human herpesviruses are antigenically rich agents that induce strong CD8+T cell responses in primary infection yet persist for life, continually challenging T cell memory through recurrent lytic replication and potentially influencing the spectrum of antigen-specific responses. Here we describe the first lytic proteome-wide analysis of CD8+ T cell responses to a gamma1-herpesvirus, Epstein-Barr virus (EBV), and the first such proteome-wide analysis of primary versus memory CD8+ T cell responses to any human herpesvirus. Primary effector preparations were generated directly from activated CD8+ T cells in the blood of infectious mononucleosis (IM) patients by in vitro mitogenic expansion. For memory preparations, EBV-specific cells in the blood of long-term virus carriers were first re-stimulated in vitro by autologous dendritic cells loaded with a lysate of lytically-infected cells, then expanded as for IM cells. Preparations from 7 donors of each type were screened against each of 70 EBV lytic cycle proteins in combination with the donor’s individual HLA class I alleles. Multiple reactivities against immediate early (IE), early (E) and late (L) lytic cycle proteins, including many hitherto unrecognised targets, were detected in both contexts. Interestingly however, the two donor cohorts showed a different balance between IE, E and L reactivities. Primary responses targeted IE and a small group of E proteins preferentially, seemingly in line with their better presentation on the infected cell surface before later-expressed viral evasins take full hold. By contrast, target choice equilibrates in virus carriage with responses to key IE and E antigens still present but with responses to a select subset of L proteins now often prominent. We infer that, for EBV at least, long-term virus carriage with its low level virus replication and lytic antigen release is associated with a re-shaping of the virus-specific response.

Identification of Epstein-Barr Virus Replication Proteins in Burkitt's Lymphoma Cells.

The working model to describe the mechanisms used to replicate the cancer-associated virus Epstein-Barr virus (EBV) is partly derived from comparisons with other members of the Herpes virus family. Many genes within the EBV genome are homologous across the herpes virus family. Published transcriptome data for the EBV genome during its lytic replication cycle show extensive transcription, but the identification of the proteins is limited. We have taken a global proteomics approach to identify viral proteins that are expressed during the EBV lytic replication cycle. We combined an enrichment method to isolate cells undergoing EBV lytic replication with SILAC-labeling coupled to mass-spectrometry and identified viral and host proteins expressed during the EBV lytic replication cycle. Amongst the most frequently identified viral proteins are two components of the DNA replication machinery, the single strand DNA binding protein BALF2, DNA polymerase accessory protein BMRF1 and both subunits of the viral ribonucleoside-diphosphate reductase enzyme (BORF2 and BaRF1). An additional 42 EBV lytic cycle proteins were also detected. This provides proteomic identification for many EBV lytic replication cycle proteins and also identifies post-translational modifications.

Microtubule depolymerization activates the Epstein–Barr virus lytic cycle through protein kinase C pathways in nasopharyngeal carcinoma cells

Elevated levels of antibodies against Epstein-Barr virus (EBV) and the presence of viral DNA in plasma are reliable biomarkers for the diagnosis of nasopharyngeal carcinoma (NPC) in high-prevalence areas, such as South-East Asia. The presence of these viral markers in the circulation suggests that a minimal level of virus reactivation may have occurred in an infected individual, although the underlying mechanism of reactivation remains to be elucidated. Here, we showed that treatment with nocodazole, which provokes the depolymerization of microtubules, induces the expression of two EBV lytic cycle proteins, Zta and EA-D, in EBV-positive NPC cells. This effect was independent of mitotic arrest, as viral reactivation was not abolished in cells synchronized at interphase. Notably, the induction of Zta by nocodazole was mediated by transcriptional upregulation via protein kinase C (PKC). Pre-treatment with inhibitors for PKC or its downstream signalling partners p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) abolished the nocodazole-mediated induction of Zta and EA-D. Interestingly, the effect of nocodazole, as well as colchicine and vinblastine, on lytic gene expression occurred only in NPC epithelial cells but not in cells derived from lymphocytes. These results establish a novel role of microtubule integrity in controlling the EBV life cycle through PKC and its downstream pathways, which represents a tissue-specific mechanism for controlling the life-cycle switch of EBV.

Control of Epstein-Barr virus infection in vitro by T helper cells specific for virion glycoproteins

Epstein-Barr virus (EBV) establishes lifelong persistent infections in humans by latently infecting B cells, with occasional cycles of reactivation, virus production, and reinfection. Protective immunity against EBV is mediated by T cells, but the role of EBV-specific T helper (Th) cells is still poorly defined. Here, we study the Th response to the EBV lytic cycle proteins BLLF1 (gp350/220), BALF4 (gp110), and BZLF1 and show that glycoprotein-specific Th cells recognize EBV-positive cells directly; surprisingly, a much higher percentage of target cells than those expressing lytic cycle proteins were recognized. Antigen is efficiently transferred to bystander B cells by receptor-mediated uptake of released virions, resulting in recognition of target cells incubated with <1 virion/cell. T cell recognition does not require productive infection and occurs early after virus entry before latency is established. Glycoprotein-specific Th cells are cytolytic and inhibit proliferation of lymphoblastoid cell lines (LCL) and the outgrowth of LCL after infection of primary B cells with EBV. These results establish a novel role for glycoprotein-specific Th cells in the control of EBV infection and identify virion proteins as important immune targets. These findings have implications for the treatment of diseases associated with EBV and potentially other coated viruses infecting MHC class II–positive cells.

Mosquito Salivary Gland Extracts Induce EBV-Infected NK Cell Oncogenesis Via CD4+ T Cells in Patients with Hypersensitivity to Mosquito Bites

Severe hypersensitivity to mosquito bites (HMB) is characterized by intense local skin reactions and systemic symptoms such as high fever, lymphadenopathy, and hepatosplenomegaly. Patients with HMB often have natural killer (NK) cell lymphocytosis associated with Epstein-Barr virus (EBV) infection. Here we investigated whether mosquito bites have any influence on the oncogenesis of EBV-infected NK cells. We examined six HMB patients with EBV-infected NK cell lymphocytosis. We first demonstrated that CD4+ T cells, but not NK cells, proliferated well in response to mosquito salivary gland extracts (SGE), especially to SGE of Aedes albopictus. When NK cells were cocultured with autologous CD4+ T cells stimulated by mosquito SGE, the expression of viral oncogene latent membrane protein 1 (LMP1) was remarkably enhanced. Next, we stimulated mononuclear cells of the patients with mosquito SGE, and NK cell counts were monitored for 28 d. The counts changed little from initial levels in the culture with mosquito SGE, whereas they decreased steadily in the culture without the extracts. Furthermore, we detected LMP1 mRNA in the skin lesion induced by mosquito SGE. These results suggest that mosquito bites can induce expression of the viral oncogene LMP1 in NK cells via mosquito antigen-specific CD4+ T cells, which is involved in the oncogenesis of NK cells in vivo.

Specificity of T cells in synovial fluid: high frequencies of CD8(+) T cells that are specific for certain viral epitopes.

CD8+ T cells dominate the lymphocyte population insynovial fluid in chronic inflammatory arthritis. It is known that theseCD8+ T cells are often clonally or oligoclonally expanded, but theirspecificity and their relevance to the pathogenesis of joint disease hasremained unclear. We found that as many as 15.5% of synovial CD8+ Tcells may be specific for a single epitope from an Epstein-Barr virus lyticcycle protein. The virus-specific T cells within the joint showed increasedexpression of markers of activation and differentiation compared with those inthe periphery, and retained their functional capacity to secreteproinflammatory cytokines on stimulation. These activated, virus-specificCD8+ T cells could therefore interact with synoviocytes, either bycell-cell contact or by a cytokine network, and play a 'bystander'role in the maintenance of inflammation in patients with arthritis.