Infusions of Epstein-Barr virus-specific cytotoxic T lymphocytes as post-remission therapy in high-risk post-transplant lymphoproliferative disorder patients: report of two cases.
Kim Nayoun,Sohn Hyun-Jung,Oh Joo Hyun,Jeon Young-Woo,Lee Hyun-Joo,Cho Hyun-Il,Chung Byung Ha,Yang Chul-Woo,Kim Tai-Gyu,Cho Seok-Goo
International journal of hematology
Conventional therapeutic approaches to post-transplant lymphoproliferative disorder (PTLD) occurring after solid-organ transplantation have shown only limited success in achieving durable response. Key factors driving the pathogenesis of PTLD include Epstein-Barr virus (EBV) reactivation and impaired immune surveillance due to prolonged immune suppression. Thus, EBV-specific cytotoxic T lymphocytes (EBV-CTLs) have emerged as an alternative therapeutic approach for the treatment of EBV-associated PTLD by enhancing EBV-specific immunity. We evaluated the safety and efficacy of EBV latent membrane proteins (LMP)-1- and 2-specific CTLs in two PTLD patients at high risk for relapse. Following diagnosis, patients were initially treated with a combination of chemotherapy and/or radiotherapy. Patients then received a total of eight doses of 2 × 10 EBV-CTLs/m. Following initial therapy, both patients achieved complete remission confirmed by FDG-PET/CT imaging. Post-remission therapy using adoptive transfer of EBV-CTLs was safe without immediate or late toxicities. Infusion of EBV-CTLs led to an overall reduction in plasma EBV levels in the peripheral blood, which was associated with long-term remission of both patients during a follow-up of more than 65 months. Further prospective studies with larger number of patients will be needed to confirm the role of EBV-CTLs as post-remission therapy in high-risk PTLD.
Intratumoral Cytotoxic T-Lymphocyte Density and PD-L1 Expression Are Prognostic Biomarkers for Patients with Colorectal Cancer.
Calik Ilknur,Calik Muhammet,Turken Gulistan,Ozercan Ibrahim Hanifi,Dagli Adile Ferda,Artas Gokhan,Sarikaya Burcu
Medicina (Kaunas, Lithuania)
Cytotoxic T-lymphocyte (CTL)-mediated inflammatory response to tumors plays a crucial role in preventing the progression of some cancers. Programmed cell death ligand 1 (PD-L1), a cell-surface glycoprotein, has been reported to repress T-cell-mediated immune responses against tumors. However, the clinical significance of PD-L1 in colorectal cancer (CRC) remains unclear. Our aim was to elucidate the prognostic significance of PD-L1 expression and CD8+ CTL density in CRC. : CD8 and PD-L1 immunostaining was conducted on 157 pathologic specimens from patients with CRC. The CD8+ CTL density and PD-L1 expression within the tumor microenvironment were assessed by immunohistochemistry. Tumor invasion (pT) was significantly correlated with intratumoral ( = 0.011) and peritumoral ( = 0.016) CD8+ CTLs density in the tumor microenvironment. In addition, there was a significant difference in the intensity of CD8+ CTLs between patients with and without distant metastases (intratumoral = 0.007; peritumoral = 0.037, T-test). Lymph node metastasis (pN) and TNM stage were significantly correlated with PD-L1 expression in CRC cells ( = 0.015, = 0.029, respectively). Multivariate analysis revealed a statistically significant relationship between the intratumoral CD8+ CTL density and disease-free survival (DFS) (hazard ratio [HR] 2.06; 95% confidence interval [CI]: 1.01-4.23; = 0.043). The DFS was considerably shorter in patients with a high expression of PD-L1 in cancer cells than those with a low expression (univariate HR 2.55; 95% CI 1.50-4.34; = 0.001; multivariate HR 0.48; 95% CI 0.28-0.82; = 0.007). Conversely, patients with high PD-L1 expression in tumor-infiltrating lymphocytes had a longer DFS in both univariate analysis (HR 0.25; 95% CI: 0.14-0.44; < 0.001) and multivariate analysis (HR 3.42; 95% CI: 1.95-6.01; < 0.001). : The CD8+ CTL density and PD-L1 expression are prognostic biomarkers for the survival of patients with CRC.
Frequency of Epstein-Barr virus-specific cytotoxic T lymphocytes in the blood of Southern Chinese blood donors and nasopharyngeal carcinoma patients.
Whitney Bruce M,Chan Anthony T C,Rickinson Alan B,Lee Steven P,Lin C K,Johnson Philip J
Journal of medical virology
Undifferentiated nasopharyngeal carcinoma is very common among Southern Chinese. While most patients have the disease detected and treated early, those who are diagnosed with advanced stages face a poor prognosis. Nasopharyngeal carcinoma is associated with latent Epstein-Barr virus (EBV); it was suggested previously that a cytotoxic T-lymphocyte (CTL)-based therapy targeting EBV proteins may offer a possible new form of treatment for this disease. The most likely target of this treatment is latent membrane protein 2 (LMP2). To define further the preexisting level of anti-EBV immunity in Chinese subjects, the frequency of peripheral blood mononuclear cells (PBMCs) responding to peptide epitopes was determined using an ELISPOT assay in 50 healthy control blood donors and in 26 patients newly diagnosed with nasopharyngeal carcinoma. A total of 7 LMP2, 2 LMP1, 1 EBNA3A, and 1 EBNA3B epitopes were used in a HLA-restricted manner. As reported previously for healthy virus carriers in western countries, it was found that in both groups the strongest responses were to epitopes in the EBNA proteins with weaker responses to the LMP epitopes. It was found that LMP2 epitopes were recognized in a greater percentage of both groups than previously reported, due most likely to the greater sensitivity of the ELISPOT method. However, patients with nasopharyngeal carcinoma demonstrated a weaker response than that displayed by healthy control subjects to several epitopes. The results demonstrate that LMP2 epitopes are recognized widely in an HLA-restricted manner in patients with nasopharyngeal carcinoma and that immunotherapy to boost preexisting immunity to these epitopes may offer a viable method to treat such patients or to protect against recurrence.
In vivo expansion of LMP 1- and 2-specific T-cells in a patient who received donor-derived EBV-specific T-cells after allogeneic stem cell transplantation.
Bollard Catherine M,Gottschalk Stephen,Huls M Helen,Molldrem Jeffrey,Przepiorka Donna,Rooney Cliona M,Heslop Helen E
Leukemia & lymphoma
Immunotherapy approaches with antigen-specific cytotoxic T lymphocytes (CTLs) have provided safe and effective prophylaxis and treatment of Epstein-Barr virus (EBV)-associated lymphomas arising after bone marrow transplantation. EBV is also associated with other malignancies including approximately 40% of cases of Hodgkin's disease, making this tumor another potential target for EBV-targeted immunotherapy. This study describes a patient with multiple relapsed EBV positive Hodgkin's Disease who received both autologous and allogeneic EBV CTL lines. After multiple chemotherapeutic and radiotherapy regimens including two autologous stem cell transplants, he received two doses of gene-marked autologous EBV-specific CTL which resulted in disease stabilization for 6 months. The gene-marked EBV-CTL persisted for 12 months in the peripheral blood after which he proceeded to unrelated donor stem cell transplant followed by immunotherapy with donor-derived EBV-specific CTL. Despite low levels of donor chimerism, the patient remains in complete remission 5 years post-allogeneic SCT. Comparison of the autologous and the donor-derived CTL lines showed that the donor line had specificity for two tumor-associated EBV antigens, latent membrane protein (LMP)1 and 2 compared to the autologous line, which only had specificity for LMP2 epitopes. Following infusion of the donor-derived CTL, functional analyses showed that T-cells reactive with both LMP1 and LMP2 epitopes expanded in the peripheral blood, suggesting that strategies to increase their frequency may result in a broader cytotoxic response against EBV+ Hodgkin tumors.
Adoptive T-cell transfer and chemotherapy in the first-line treatment of metastatic and/or locally recurrent nasopharyngeal carcinoma.
Chia Whay-Kuang,Teo Marissa,Wang Who-Whong,Lee Bernett,Ang Soo-Fan,Tai Wai-Meng,Chee Chit-Lai,Ng Joanna,Kan Rebecca,Lim Wan-Teck,Tan Sze-Huey,Ong Whee-Sze,Cheung Yin-Bun,Tan Eng-Huat,Connolly John E,Gottschalk Stephen,Toh Han-Chong
Molecular therapy : the journal of the American Society of Gene Therapy
The outcomes for patients with metastatic or locally recurrent Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) remain poor. Adoptive immunotherapy with EBV-specific cytotoxic T lymphocytes (EBV-CTLs) has proven clinical efficacy, but it has never been evaluated in the first-line treatment setting in combination with chemotherapy. To evaluate the safety and efficacy of a chemotherapy in combination with adoptive EBV-CTL transfer, we conducted a phase 2 clinical trial consisting of four cycles of gemcitabine and carboplatin (GC) followed by up to six doses of EBV-CTL. Thirty-eight patients were enrolled, and 35 received GC and EBV-CTL. GC-CTL therapy resulted in a response rate of 71.4% with 3 complete responses and 22 partial responses. With a median follow up of 29.9 months, the 2-year and 3-year overall survival (OS) rate was 62.9 and 37.1%, respectively. Five patients did not require further chemotherapy for more than 34 months since initiation of CTL. Infusion of CTL products containing T cells specific for LMP2 positively correlated with OS (hazard ratio: 0.35; 95% confidence interval: 0.14-0.84; P = 0.014). Our study achieved one of the best survival outcomes in patients with advanced NPC, setting the stage for a future randomized study of chemotherapy with and without EBV-CTL.
Quantitative study of cytotoxic T-lymphocyte immunotherapy for nasopharyngeal carcinoma.
Shengjun Wang,Yunbo Guo,Liyan Song,Jinming Li,Qinkai Deng
Theoretical biology & medical modelling
BACKGROUND:In clinical practice, the common strategy for immunotherapy of nasopharyngeal carcinoma (NPC) is to infuse cytotoxic T-lymphocyte (CTL) lines several times by intravenous injection, but it is difficult by laboratory research to investigate the relationship between treatment time-point, the amount of CTL added and the therapeutic effect. The objective of this study is to establish a mathematical model to study the therapeutic effect of different treatment time-points and amounts of CTL, and to predict the change in therapeutic effect when the percentage of EBV LMP2-specific CTL is increased from 10% to 20%. RESULTS:The concentration of epidermal growth factor receptor (EGFR) in the tumor cell cytomembranes increases after CTL is added. Concurrently, there is a marked downward trend of the phosphorylated transforming growth factor-α (TGFα)-EGFR complex in the tumor cell cytomembranes, which indicates restriction of tumor growth after CTL immunotherapy. The relationships among the time of addition of CTL, the amount of CTL added, different CTL specificities for LMP2 and the increment rate k of the total number of tumor cells were evaluated. CONCLUSIONS:The simulation results quantify the relationships among treatment time-points, amount of CTL added, and the corresponding therapeutic effect of immunotherapy for NPC.
In vitro anti-tumor immune response induced by dendritic cells transfected with EBV-LMP2 recombinant adenovirus.
Pan Ying,Zhang Jinkun,Zhou Ling,Zuo Jianmin,Zeng Yi
Biochemical and biophysical research communications
Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is a high-incidence tumor in southern China. Latent membrane proteins 2 (LMP2) is a subdominant antigen of EBV. The present study was to develop a dendritic cells (DCs)-based cancer vaccine (rAd-LMP2-DC) and to study its biological characteristics and its immune functions. Our results showed that LMP2 gene transfer did not alter the typical morphology of mature DC, and the representative phenotypes of mature DC (CD80, CD83, and CD86) were highly expressed in rAd-LMP2-DCs. The expression of LMP2 in rAd-LPM2-DCs was about 84.54%, which suggested efficient gene transfer. Transfected DCs markedly increased antigen-specific T-cell proliferation. The specific cytotoxicity against NPC cell was significantly higher than that in controls (p < 0.05), and enhanced with increased stimulations by transfected DCs. In addition, phenotypic analysis demonstrated that the LMP2-specific CTLs consisted of both CD4(+) and CD8(+) T cells. These results showed that development of DC-based vaccine by transfection with malignancy-associated virus antigens could elicit potent CTL response and provide a potential strategy of immunotherapy for EBV-associated NPC.
The generation and characterization of LMP2-specific CTLs for use as adoptive transfer from patients with relapsed EBV-positive Hodgkin disease.
Bollard Catherine M,Straathof Karin C M,Huls M Helen,Leen Alan,Lacuesta Kristine,Davis Alan,Gottschalk Stephen,Brenner Malcolm K,Heslop Helen E,Rooney Cliona M
Journal of immunotherapy (Hagerstown, Md. : 1997)
Cellular adoptive immunotherapy for virus-associated malignant disease is an attractive strategy, since viral antigens provide targets for specific T lymphocytes. In Epstein-Barr virus (EBV)-positive Hodgkin disease (HD), a limited number of EBV-encoded antigens such as the latent membrane antigens (LMP) 1 and 2 are expressed on the malignant Reed-Sternberg cells. The authors aimed to generate cytotoxic T lymphocytes (CTLs) from patients with relapsed HD by specifically targeting LMP2A. Patients with relapsed HD have highly immunosuppressive tumors and have been heavily pretreated with cytotoxic agents. As a result, monocytes and lymphocytes are numerically reduced and functionally impaired. Approaches using dendritic cells (DCs) as the sole antigen-presenting cell to expand LMP2-specific CTL lines in vitro have proved impractical. The authors now show how small amounts of patient peripheral blood can be used to produce DCs expressing LMP2 after Ad5F35 transduction, and how an initial reactivation of LMP2-specific CTLs can be followed by stimulation with lymphoblastoid cell lines overexpressing LMP2 from the same vector. Large numbers of LMP2-specific cytotoxic lymphocytes are produced that contain both CD4+ and CD8+ T cells (favoring long-term persistence in vivo) and recognize multiple LMP2 epitopes (minimizing the risk of tumor antigen loss variants). This approach is being used in a current clinical trial.
Specific cellular immune responses in mice immunized with DNA, adeno-associated virus and adenoviral vaccines of Epstein-Barr virus-LMP2 alone or in combination.
Wang Zhan,Yang Songmei,Zhou Ling,Du Haijun,Mo Wuning,Zeng Yi
Science China. Life sciences
Cellular immune responses, particularly those associated with CD3(+)CD8(+) cytotoxic T lymphocytes (CTL), are critical factors in controlling viral infection. Nasopharyngeal carcinoma (NPC) is closely associated with persistent Epstein-Barr virus (EBV) infection. NPC vaccine studies have focused on enhancing specific antiviral CTL responses. In this study, three vaccines capable of expressing the EBV-latent membrane protein 2 (LMP2) (a DNA vector, an adeno-associated virus (AAV) vector, and a replication-defective adenovirus serotype 5 (Ad5) vector) were respectively used to immunize female Balb/c mice (4-6 weeks old) at weeks 0, 2 and 4, either alone or in combination. Our results suggest that combined immunization with DNA, AAV, and adenovirus vector vaccines induced specific cellular immunity more effectively than any of these vectors alone or a combination of two of the three, constituting a sound vaccine strategy for the prevention and treatment of NPC.
Analysis of Epstein-Barr viral DNA load, EBV-LMP2 specific cytotoxic T-lymphocytes and levels of CD4+CD25+ T cells in patients with nasopharyngeal carcinomas positive for IgA antibody to EBV viral capsid antigen.
Mo Wu-ning,Tang An-zhou,Zhou Ling,Huang Guang-wu,Wang Zhan,Zeng Yi
Chinese medical journal
BACKGROUND:Epstein-Barr virus (EBV) is a herpesvirus commonly associated with several malignant diseases including nasopharyngeal carcinoma (NPC), which is a common cancer in Southeastern Asia. Previous studies showed that plasma levels of EBV-DNA might be a sensitive and reliable biomarker for the diagnosis, staging and evaluating of therapy for NPC. There are a few analyses of the levels of EBV-latent membrane protein 2 (LMP2)-specific cytotoxic T-lymphocytes (CTLs) in patients with NPC. This study was conducted to investigate the levels of EBV-LMP2-specific CTLs, EBV-DNA load and the level of CD4(+)CD25(+) T cells in such patients. METHODS:From February 2006 to April 2006, 62 patients with NPC, 40 healthy virus carriers positive for EBV viral capsid antigen (EBV-IgA-VCA) and 40 controls were enrolled in the study. We used a highly sensitive ELISPOT assay, real-time polymerase chain reaction (PCR) and flow cytometry to measure the EBV-LMP2-specific CTL response, the EBV DNA load and the level of CD4(+)CD25(+) T cells, respectively. RESULTS:The EBV-LMP2-specific CTL responses of the samples from the control, healthy virus carriers and patients with NPC were significantly different from the LMP2 epitopes, with the control and healthy virus carrier samples displaying a stronger response in three cases. There were significant differences in EBV DNA load in serum between NPC and the healthy groups; patients with NPC at stages III or IV had significantly higher viral loads compared with those at stages I or II. A significantly higher percentage of CD4(+)CD25(+) T lymphocytes were detected in the patients, compared with healthy virus carriers and healthy controls. Moreover, patients with advanced stages of NPC (III and IV) had significantly higher percentages than the patients with early stages (I and II). CONCLUSIONS:Patients with NPC are frequently unable to establish or maintain sufficient immunosurveillance to control proliferating B cells harboring EBV and to destroy the tumor cells that express immunodominant LMP2 proteins. Controlling the activity of CD4(+)CD25(+) T cells and elevating CD8(+) cells specific for LMP2 epitopes could be an effective immunotherapy for patients with NPC.
Early T Cell Recognition of B Cells following Epstein-Barr Virus Infection: Identifying Potential Targets for Prophylactic Vaccination.
Brooks Jill M,Long Heather M,Tierney Rose J,Shannon-Lowe Claire,Leese Alison M,Fitzpatrick Martin,Taylor Graham S,Rickinson Alan B
Epstein-Barr virus, a B-lymphotropic herpesvirus, is the cause of infectious mononucleosis, has strong aetiologic links with several malignancies and has been implicated in certain autoimmune diseases. Efforts to develop a prophylactic vaccine to prevent or reduce EBV-associated disease have, to date, focused on the induction of neutralising antibody responses. However, such vaccines might be further improved by inducing T cell responses capable of recognising and killing recently-infected B cells. In that context, EBNA2, EBNA-LP and BHRF1 are the first viral antigens expressed during the initial stage of B cell growth transformation, yet have been poorly characterised as CD8+ T cell targets. Here we describe CD8+ T cell responses against each of these three "first wave" proteins, identifying target epitopes and HLA restricting alleles. While EBNA-LP and BHRF1 each contained one strong CD8 epitope, epitopes within EBNA2 induced immunodominant responses through several less common HLA class I alleles (e.g. B*3801 and B*5501), as well as subdominant responses through common class I alleles (e.g. B7 and C*0304). Importantly, such EBNA2-specific CD8+ T cells recognised B cells within the first day post-infection, prior to CD8+ T cells against well-characterised latent target antigens such as EBNA3B or LMP2, and effectively inhibited outgrowth of EBV-transformed B cell lines. We infer that "first wave" antigens of the growth-transforming infection, especially EBNA2, constitute potential CD8+ T cell immunogens for inclusion in prophylactic EBV vaccine design.
EBV Infection and Glucose Metabolism in Nasopharyngeal Carcinoma.
Zhang Jun,Jia Lin,Tsang Chi Man,Tsao Sai Wah
Advances in experimental medicine and biology
To establish persistent infection in cells, viruses evolve strategies to alter host cellular pathways to regulate cell proliferation and energy metabolism which support viral infection. Epstein-Barr virus (EBV) undergoes both lytic and latent infection to achieve persistent and lifelong infection in human. EBV readily infects human B cells, driving their transformation to proliferative lymphoblastoid cell lines (LCL), and eventually establishes lifelong latent infection in memory B cells. In contrary, EBV undergoes lytic replication upon infection into normal epithelial cells which is essential for the replication of EBV genome and production of infectious viral particles for transmission through saliva. EBV shuttles between B cells and epithelial cells to complete its infection cycle. EBV infection is closely associated with nasopharyngeal carcinoma (NPC) and is present in practically 100% of undifferentiated NPC. In contrast to undergo lytic infection of normal pharyngeal epithelium, EBV establishes latent infection in NPC. The switch from lytic infection to latent infection may represent an early and essential step in the development of NPC. Recent studies in both B cells and NPC cells latently infected with EBV reveal alterations in cell metabolism to support persistent and latent EBV infection. Events underlying the switching of lytic to latent EBV infection in NPC cells are largely undefined. Molecular events and alterations of cell metabolism are likely to play crucial roles in switching EBV infection from lytic to latent in NPC cells. Latent EBV infection and expression of viral genes, including LMP1, LMP2, and possibly EBV-encoded micro RNAs, may play essential roles in alterations of cell metabolism to support NPC pathogenesis. Alteration of energy metabolism is an essential hallmark of cancer. The role of altered energy metabolism in host cells in modulating latent and lytic EBV infection in NPC cells is unclear. In this review, we will discuss the impact of genetic alterations in NPC to module cellular metabolism and its influence on latent infection and lytic reactivation of EBV infection in NPC cells. In particular, the role of EBV-encoded genes in driving glucose metabolism and their contribution to NPC pathogenesis will be discussed. This new perspective on the interplay between EBV infection and altered host metabolic pathways in NPC pathogenesis may offer novel and effective therapeutic strategies in the treatment of NPC and other EBV-associated malignancies.
Epstein-Barr Virus-Associated Malignancies: Roles of Viral Oncoproteins in Carcinogenesis.
El-Sharkawy Ahmed,Al Zaidan Lobna,Malki Ahmed
Frontiers in oncology
The Epstein-Barr virus (EBV) is the first herpesvirus identified to be associated with human cancers known to infect the majority of the world population. EBV-associated malignancies are associated with a latent form of infection, and several of the EBV-encoded latent proteins are known to mediate cellular transformation. These include six nuclear antigens and three latent membrane proteins (LMPs). In lymphoid and epithelial tumors, viral latent gene expressions have distinct pattern. In both primary and metastatic tumors, the constant expression of latent membrane protein 2A (LMP2A) at the RNA level suggests that this protein is the key player in the EBV-associated tumorigenesis. While LMP2A contributing to the malignant transformation possibly by cooperating with the aberrant host genome. This can be done in part by dysregulating signaling pathways at multiple points, notably in the cell cycle and apoptotic pathways. Recent studies also have confirmed that LMP1 and LMP2 contribute to carcinoma progression and that this may reflect the combined effects of these proteins on activation of multiple signaling pathways. This review article aims to investigate the aforementioned EBV-encoded proteins that reveal established roles in tumor formation, with a greater emphasis on the oncogenic LMPs (LMP1 and LMP2A) and their roles in dysregulating signaling pathways. It also aims to provide a quick look on the six members of the EBV nuclear antigens and their roles in dysregulating apoptosis.
Phase I trial of adoptively transferred tumor-infiltrating lymphocyte immunotherapy following concurrent chemoradiotherapy in patients with locoregionally advanced nasopharyngeal carcinoma.
Li Jiang,Chen Qiu-Yan,He Jia,Li Ze-Lei,Tang Xiao-Feng,Chen Shi-Ping,Xie Chuan-Miao,Li Yong-Qiang,Huang Li-Xi,Ye Shu-Bio,Ke Miao-La,Tang Lin-Quan,Liu Huai,Zhang Lu,Guo Shan-Shan,Xia Jian-Chuan,Zhang Xiao-Shi,Zheng Li-Min,Guo Xiang,Qian Chao-Nan,Mai Hai-Qiang,Zeng Yi-Xin
Adoptive cell therapy (ACT) for cancers using autologous tumor-infiltrating lymphocytes (TILs) can induce immune responses and antitumor activity in metastatic melanoma patients. Here, we aimed to assess the safety and antitumor activity of ACT using expanded TILs following concurrent chemoradiotherapy (CCRT) in patients with locoregionally advanced nasopharyngeal carcinoma (NPC). Twenty-three newly diagnosed, locoregionally advanced NPC patients were enrolled, of whom 20 received a single-dose of TIL infusion following CCRT. All treated patients were assessed for toxicity, survival and clinical and immunologic responses. Correlations between immunological responses and treatment effectiveness were further studied. Only mild adverse events (AEs), including Grade 3 neutropenia (1/23, 5%) consistent with immune-related causes, were observed. Nineteen of 20 patients exhibited an objective antitumor response, and 18 patients displayed disease-free survival longer than 12 mo after ACT. A measurable plasma Epstein-Barr virus (EBV) load was detected in 14 patients at diagnosis, but a measurable EBV load was not found in patients after one week of ACT, and the plasma EBV load remained undetectable in 17 patients at 6 mo after ACT. Expansion and persistence of T cells specific for EBV antigens in peripheral blood following TIL therapy were observed in 13 patients. The apparent positive correlation between tumor regression and the expansion of T cells specific for EBV was further investigated in four patients. This study shows that NPC patients can tolerate ACT with TILs following CCRT and that this treatment results in sustained antitumor activity and anti-EBV immune responses. A larger phase II trial is in progress.
Epstein-Barr Virus (EBV)-derived BARF1 encodes CD4- and CD8-restricted epitopes as targets for T-cell immunotherapy.
Kalra Mamta,Gerdemann Ulrike,Luu Jessica D,Ngo Minthran C,Leen Ann M,Louis Chrystal U,Rooney Cliona M,Gottschalk Stephen
BACKGROUND AIMS:EBV type II latency tumors, such as Hodgkin lymphoma (HL), Non-Hodgkin lymphoma (NHL) and nasopharyngeal carcinoma, express a limited array of EBV antigens including Epstein-Barr nuclear antigen (EBNA)1, latent membrane protein (LMP)1, LMP2, and BamH1-A right frame 1 (BARF1). Adoptive immunotherapy for these malignancies have focused on EBNA1, LMP1 and LMP2 because little is known about the cellular immune response to BARF1. METHODS:To investigate whether BARF1 is a potential T-cell immunotherapy target, we determined the frequency of BARF1-specific T-cell responses in the peripheral blood of EBV-seropositive healthy donor and patients with EBV-positive malignancies, mapped epitopes and evaluated the effector function of ex vivo-generated BARF1-specific T-cell lines. RESULTS:BARF1-specific T cells were present in the peripheral blood of 12/16 (75%) EBV-positive healthy donors and 13/20 (65%) patients with EBV-positive malignancies. Ex vivo expanded BARF1-specific T-cell lines contained CD4- and CD8-positive T-cell subpopulations, and we identified 23 BARF1 peptides, which encoded major histocompatibility complex class I- and/or II-restricted epitopes. Epitope mapping identified one human leukocyte antigen (HLA)-A*02-restricted epitope that was recognized by 50% of HLA-A*02, EBV-seropositive donors and one HLA-B*15(62)-restricted epitope. Exvivo expanded BARF1-specific T cells recognized and killed autologous, EBV-transformed lymphoblastoid cell lines and partially HLA-matched EBV-positive lymphoma cell lines. DISCUSSION:BARF1 should be considered as an immunotherapy target for EBV type II (and III) latency. Targeting BARF1, in addition to EBNA1, LMP1 and LMP2, has the potential to improve the efficacy of current T-cell immunotherapy approaches for these malignancies.
A recombinant modified vaccinia ankara vaccine encoding Epstein-Barr Virus (EBV) target antigens: a phase I trial in UK patients with EBV-positive cancer.
Taylor Graham S,Jia Hui,Harrington Kevin,Lee Lip Wai,Turner James,Ladell Kristin,Price David A,Tanday Manjit,Matthews Jen,Roberts Claudia,Edwards Ceri,McGuigan Lesley,Hartley Andrew,Wilson Steve,Hui Edwin P,Chan Anthony T C,Rickinson Alan B,Steven Neil M
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:Epstein-Barr virus (EBV) is associated with several cancers in which the tumor cells express EBV antigens EBNA1 and LMP2. A therapeutic vaccine comprising a recombinant vaccinia virus, MVA-EL, was designed to boost immunity to these tumor antigens. A phase I trial was conducted to demonstrate the safety and immunogenicity of MVA-EL across a range of doses. EXPERIMENTAL DESIGN:Sixteen patients in the United Kingdom (UK) with EBV-positive nasopharyngeal carcinoma (NPC) received three intradermal vaccinations of MVA-EL at 3-weekly intervals at dose levels between 5 × 10(7) and 5 × 10(8) plaque-forming units (pfu). Blood samples were taken at screening, after each vaccine cycle, and during the post-vaccination period. T-cell responses were measured using IFNγ ELISpot assays with overlapping EBNA1/LMP2 peptide mixes or HLA-matched epitope peptides. Polychromatic flow cytometry was used to characterize functionally responsive T-cell populations. RESULTS:Vaccination was generally well tolerated. Immunity increased after vaccination to at least one antigen in 8 of 14 patients (7/14, EBNA1; 6/14, LMP2), including recognition of epitopes that vary between EBV strains associated with different ethnic groups. Immunophenotypic analysis revealed that vaccination induced differentiation and functional diversification of responsive T-cell populations specific for EBNA1 and LMP2 within the CD4 and CD8 compartments, respectively. CONCLUSIONS:MVA-EL is safe and immunogenic across diverse ethnicities and thus suitable for use in trials against different EBV-positive cancers globally as well as in South-East Asia where NPC is most common. The highest dose (5 × 10(8) pfu) is recommended for investigation in current phase IB and II trials.
EBV-Directed T Cell Therapeutics for EBV-Associated Lymphomas.
McLaughlin Lauren P,Gottschalk Stephen,Rooney Cliona M,Bollard Catherine M
Methods in molecular biology (Clifton, N.J.)
Epstein Barr virus (EBV) is a human gamma herpes virus that establishes latency in B cells after primary infection. EBV generally only causes a mild, self-limiting viral illness but is also associated with several malignancies including posttransplantation lymphoproliferative disorder in the immunosuppressed host as well as Hodgkin and non-Hodgkin lymphoma in the immune competent host. The expression of EBV antigens by lymphoma has important applications as targets for adoptive T cell therapy. However, as many lymphomas only express subdominant EBV antigens that are less immunogenic, novel strategies are needed to manufacture EBV-specific T cell products specific for Latent Membrane Protein 1 (LMP1) and LMP2, which are expressed in lymphomas with type II and III latency. While several techniques for manufacturing EBV-CTLs are described in the literature, this chapter focuses on one method for generating Good Manufacturing Practice (GMP)-compliant EBV-specific T cell products that are enriched with LMP1 and LMP2.
Sustained complete responses in patients with lymphoma receiving autologous cytotoxic T lymphocytes targeting Epstein-Barr virus latent membrane proteins.
Bollard Catherine M,Gottschalk Stephen,Torrano Vicky,Diouf Oumar,Ku Stephanie,Hazrat Yasmin,Carrum George,Ramos Carlos,Fayad Luis,Shpall Elizabeth J,Pro Barbara,Liu Hao,Wu Meng-Fen,Lee Daniel,Sheehan Andrea M,Zu Youli,Gee Adrian P,Brenner Malcolm K,Heslop Helen E,Rooney Cliona M
Journal of clinical oncology : official journal of the American Society of Clinical Oncology
PURPOSE:Tumor cells from approximately 40% of patients with Hodgkin or non-Hodgkin lymphoma express the type II latency Epstein-Barr virus (EBV) antigens latent membrane protein 1 (LMP1) and LMP2, which represent attractive targets for immunotherapy. Because T cells specific for these antigens are present with low frequency and may be rendered anergic by the tumors that express them, we expanded LMP-cytotoxic T lymphocytes (CTLs) from patients with lymphoma using autologous dendritic cells and EBV-transformed B-lymphoblastoid cell lines transduced with an adenoviral vector expressing either LMP2 alone (n = 17) or both LMP2 and ΔLMP1 (n = 33). PATIENTS AND METHODS:These genetically modified antigen-presenting cells expanded CTLs that were enriched for specificity against type II latency LMP antigens. When infused into 50 patients with EBV-associated lymphoma, the expanded CTLs did not produce infusional toxicities. RESULTS:Twenty-eight of 29 high-risk or multiple-relapse patients receiving LMP-CTLs as adjuvant therapy remained in remission at a median of 3.1 years after CTL infusion. None subsequently died as a result of lymphoma, but nine succumbed to complications associated with extensive prior chemoradiotherapy, including myocardial infarction and secondary malignancies. Of 21 patients with relapsed or resistant disease at the time of CTL infusion, 13 had clinical responses, including 11 complete responses. T cells specific for LMP as well as nonviral tumor-associated antigens (epitope spreading) could be detected in the peripheral blood within 2 months after CTL infusion, but this evidence for epitope spreading was seen only in patients achieving clinical responses. CONCLUSION:Autologous T cells directed to the LMP2 or LMP1 and LMP2 antigens can induce durable complete responses without significant toxicity. Their earlier use in the disease course may reduce delayed treatment-related mortality.
Human Leukocyte Antigen (HLA) A*1101-Restricted Epstein-Barr Virus-Specific T-cell Receptor Gene Transfer to Target Nasopharyngeal Carcinoma.
Zheng Yong,Parsonage Greg,Zhuang Xiaodong,Machado Lee R,James Christine H,Salman Asmaa,Searle Peter F,Hui Edwin P,Chan Anthony T C,Lee Steven P
Cancer immunology research
Infusing virus-specific T cells is effective treatment for rare Epstein-Barr virus (EBV)-associated posttransplant lymphomas, and more limited success has been reported using this approach to treat a far more common EBV-associated malignancy, nasopharyngeal carcinoma (NPC). However, current approaches using EBV-transformed lymphoblastoid cell lines to reactivate EBV-specific T cells for infusion take 2 to 3 months of in vitro culture and favor outgrowth of T cells targeting viral antigens expressed within EBV(+) lymphomas, but not in NPC. Here, we explore T-cell receptor (TCR) gene transfer to rapidly and reliably generate T cells specific for the NPC-associated viral protein LMP2. We cloned a human leukocyte antigen (HLA) A*1101-restricted TCR, which would be widely applicable because 40% of NPC patients carry this HLA allele. Studying both the wild-type and modified forms, we have optimized expression of the TCR and demonstrated high-avidity antigen-specific function (proliferation, cytotoxicity, and cytokine release) in both CD8(+) and CD4(+) T cells. The engineered T cells also inhibited LMP2(+) epithelial tumor growth in a mouse model. Furthermore, transduced T cells from patients with advanced NPC lysed LMP2-expressing NPC cell lines. Using this approach, within a few days large numbers of high-avidity LMP2-specific T cells can be generated reliably to treat NPC, thus providing an ideal clinical setting to test TCR gene transfer without the risk of autoimmunity through targeting self-antigens.
Novel Immunotherapy Options for Extranodal NK/T-Cell Lymphoma.
Hu Boyu,Oki Yasuhiro
Frontiers in oncology
Extranodal NK/T-cell lymphoma (ENKTCL) is a highly aggressive mature NK/T-cell neoplasm marked by NK-cell phenotypic expression of CD3ε and CD56. While the disease is reported worldwide, there is a significant geographic variation with its highest incidence in East Asian countries possibly related to the frequent early childhood exposure of Epstein-Barr virus (EBV) and specific ethnic-genetical background, which contributes to the tumorigenesis. Historically, anthracycline-based chemotherapy such as CHOP (cyclophosphamide, adriamycin, vincristine, and prednisone) was used, but resulted in poor outcomes. This is due in part to intrinsic ENKTCL resistance to anthracycline caused by high expression levels of P-glycoprotein. The recent application of combined modality therapy with concurrent or sequential radiation therapy for early stage disease, along with non-anthracycline-based chemotherapy regimens consisting of drugs independent of P-glycoprotein have significantly improved clinical outcomes. Particularly, this neoplasm shows high sensitivity to l-asparaginase as NK-cells lack asparagine synthase activity. Even still, outcomes of patients with advanced stage disease or those with relapsed/recurrent disease are dismal with overall survival of generally a few months. Thus, novel therapies are needed for this population. Clinical activity of targeted antibodies along with antibody-drug conjugates, such as daratumumab (naked anti-CD38 antibody) and brentuximab vedotin (anti-CD30 antibody conjugated with auristatin E), have been reported. Further promising data have been shown with checkpoint inhibitors as high levels of programmed death-ligand 1 expression are observed in ENKTCL due to EBV-driven overexpression of the latent membrane proteins [latent membrane protein 1 (LMP1) and LMP2] with activation of the NF-κB/MAPK pathways. Initial case series with programmed death 1 inhibitors showed an overall response rate of 100% in seven relapsed patients including five with a complete response (CR). Furthermore, cellular immunotherapy with engineered cytotoxic T lymphocytes targeted against LMP1 and LMP2 have shown encouraging results with durable CRs as either maintenance therapy after initial induction chemotherapy or in the relapsed/refractory setting. In this paper, we review this exciting field of novel immunotherapy options against ENKTCL that hopefully will change the treatment paradigm in this deadly disease.
Novel ELISA for serodiagnosis of nasopharyngeal carcinoma based on a B cell epitope of Epstein-Barr virus latent membrane protein 2.
Cai Yiqi,Song Yiling,Cen Danwei,Zhang Chanqiong,Mao Shanshan,Ye Xiaoxian,Xiong Yirong,Jiang Pengfei,Chen Jun,Xue Xiangyang,Zhang Lifang,Zhu Guanbao
Epstein-Barr virus (EBV) is widespread and is associated with nasopharyngeal carcinoma (NPC). Serological detection of EBV is commonly used for screening, diagnosis and epidemiological surveys of NPC. In the present study, a novel B cell multi-epitope peptide fusion protein (EBV-LMP2-3B), which is composed of three B cell linear epitopes (RIEDPPFNSLL, TLNLT and KSLSSTEFIPN) of EBV latent membrane protein 2 (LMP2), was expressed in a prokaryotic expression system and purified using Ni-nitrilotriacetate-Sepharose. The immunogenicity and binding specificity of EBV-LMP2-3B were evaluated on the basis of antibody responses in immunized BALB/c mice, western blotting and indirect immunofluorescence assay. Evaluation of EBV-LMP2-3B as a serological diagnostic reagent was performed using an indirect ELISA in 198 patients with NPC and 102 healthy adults. These results revealed that EBV-LMP2-3B was able to eliminate the high-titer serum antibody response in BALB/c mice. Western blot analysis and indirect immunofluorescence assay confirmed that the mouse immune sera recognized the native LMP2. Compared with healthy adults, patients with NPC demonstrated significantly greater reactivity to EBV-LMP2-3B (P<0.05). Furthermore, it was possible to effectively detect specific IgG in sera from patients with NPC, with a sensitivity of 91.91% and specificity of 93.14%, representing an improvement over the traditional viral capsid antigen-IgA-based detection method with 59.59% sensitivity and 75.49% specificity. In conclusion, the EBV-LMP2-3B protein may be used as a serological diagnostic reagent to screen for and diagnose NPC.
The Safety and Immunological Effects of rAd5-EBV-LMP2 Vaccine in Nasopharyngeal Carcinoma Patients: A Phase I Clinical Trial and Two-Year Follow-Up.
Si Yongfeng,Deng Zhuoxia,Lan Guiping,Du Haijun,Wang Yongli,Si Jinyuan,Wei Jiazhang,Weng Jingjin,Qin Yangda,Huang Bo,Yang Yong,Qin Ying
Chemical & pharmaceutical bulletin
Epstein-Barr virus (EBV)-encoded latent membrane protein 2 (LMP2) promotes nasopharyngeal carcinoma (NPC) progression. Previously, we reported that the dendritic cells (DCs) transfected with EBV-LMP2 recombinant serotype 5 adenoviruses (rAd5) induced anti-tumor effect by eliciting cytotoxic T lymphocytes (CTLs)-mediated immune response in vitro and the adenoviral vaccine of EBV-LMP2 (rAd5-EBV-LMP2) stimulated antigen-specific cellular immunity in mice. However, the safety and immunological effect of rAd5-EBV-LMP2 vaccine in human still remained unknown. Here we conducted a single-center, non-randomized, open-label, single-arm phase I clinical trial to clarify this unsolved issue. A total of 24 patients with regional advanced NPC were sequentially enrolled into three dose level groups (2×10(9), 2×10(10), 2×10(11) vp). The rAd5-EBV-LMP2 vaccines were intramuscularly injected for four times within 28 d (D0, D7, D14, D28). Blood samples were harvested immediately before every vaccination, one week and one month after the last vaccination (D0, D7, D14, D28, D35, D58). All the vaccine inoculation-related toxicities presented as grade I/II adverse events. The most frequent systemic adverse reactions were fatigue (33.0%, 8/24), myalgia (29.2%, 7/24) and cough (29.2%, 7/24), while the most common regional adverse reaction was tenderness in the inoculation site (54.2%, 13/24). In addition, proportion of CD(3+)CD(4+) cells in peripheral blood was significantly increased in the high dose group (2×10(11) vp). The rAd5-EBV-LMP2 vaccine was generally well-tolerated and the high dose (2×10(11) vp) is recommended to be adopted in phase II studies. The long-term outcome of rAd5-EBV-LMP2 vaccine inoculation is required to be determined in following placebo-controlled trials.
In vitro evaluation of the therapeutic effectiveness of EBV-LMP2 recombinant adenovirus vaccine in nasopharyngeal carcinoma.
Ge Yuyang,Zhou Zhixiang,Wang Xiaoli,Zhou Yubai,Liu Wei,Teng Zhiping,Zeng Yi
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Immunotherapeutic strategies based on Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2) antigen-specific cytotoxic T lymphocytes (CTLs) have been proven to boost LMP2-specific CTL responses in patients with nasopharyngeal carcinoma (NPC). Such strategies can produce clinical benefits in some patients with NPC. Currently, the major challenge limiting the use of immunotherapy for NPC is its low clinical response rate. The efficacy of immunotherapy based on EBV-LMP2 specific CTLs depends mainly on their cytotoxic activity, but no studies have been conducted to elucidate this activity. In this study, laser confocal scanning microscopy (LCSM) and real-time cell analysis (RTCA) were used to evaluate the killing function and its underlying mechanism of LMP2-specific CTLs. LCSM showed that LMP2-specific CTLs recognize and kill target cells expressing viral escape protein LMP2, and that the killing rate is related to the number of CTLs adhering to the target cells. LMP2-specific CTL-mediated cytotoxicity is rate limited by the time required for effective contact and recognition between CTLs and target cells. RTCA showed that the protective effect of LMP2-specific CTLs required an appropriate effector-to-target ratio, and that LMP2-specific CTLs could not eradicate residual target cells at a low effector-to-target ratio. Moreover, our results revealed that LMP2-specific CTL responses involve two independent but complementary mechanisms: the perforin/granzyme and Fas/FasL pathways. Therefore, we have elucidated, for the first time, the selective cytotoxicity and mechanism by which LMP2-specific CTLs induced by the rAd-LMP2 vaccine kill target cells and have explored the killing mode and several key parameters of killing mediated by LMP2-specific CTLs. Our study will contribute to the knowledge of vaccines targeting EBV-LMP2 and to the improvement of immunotherapeutic strategies.
CD40L-adjuvanted DNA vaccine carrying EBV-LMP2 antigen enhances anti-tumor effect in NPC transplantation tumor animal.
Lei Lei,Li Jianhui,Liu Meiqing,Hu Xiaoming,Zhou Ya,Yang Shiming
Central-European journal of immunology
CD40L, a costimulatory molecule for dendritic cells (DCs) and B cells, can serve as an adjuvant for enhancing the specific immune response induced by DNA vaccine carrying tumor-associated antigens. In this study, we investigated the potential of CD40L as an adjuvant to enhance the anti-tumor effect mediated by a DNA vaccine based on the Epstein-Barr virus-latent membrane protein 2 (EBV-LMP2) antigen. The plasmids capable of expressing the fusion protein EBV-LMP2-CD40L were constructed. Expression vector pVAX1 and plasmid expressing the individual antigen EBV-LMP2 were used as control groups. These plasmids were used to immunize female BALB/c mice (4-6 weeks old) at days 0, 7 and 14. The results suggest that immunization with DNA vaccines carrying fusion gene EBV-LMP2-CD40L can induce specific immunity more effectively than the plasmid expression individual antigen EBV-LMP2. In order to evaluate the anti-tumor effect of this DNA vaccine, we constructed a tumor bearing mouse model. After immunization, the tumor bearing mouse model, DNA vaccination with EBV-LMP2-CD40L plasmid significantly inhibited tumor growth in the tumor bearing mouse model and enhanced the tumor inhibition rate. This study demonstrated that encoding the EBV-LMP2 tumor antigen within an EBV-LMP2-CD40L DNA vaccine generates an effective antitumor response against EBV tumor, which may be a promising method to improve the antitumor immunity of DNA vaccine.
Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines.
Sun Liying,Hao Yanzhe,Wang Zhan,Zeng Yi
Epstein-Barr virus (EBV) is related to a variety of malignant tumors, and its encoded protein, latent membrane protein 2 (LMP2), is an effective target antigen that is widely used to construct vector vaccines. However, the model cells carrying LMP2 have still not been established to assess the oncolytic effect of LMP2-related vaccines at present. In this study, TC-1-GLUC-LMP2 tumor cells were constructed as target cells to evaluate the anti-tumor effects of LMP2-assosiated vaccines. The results showed that both and () genes could be detected by polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) in TC-1-GLUC-LMP2 cells. Western blot results showed that the LMP2 and proteins were stably expressed in tumor cells for at least 30 generations. We mixed 5 × 10⁴ LMP2-specific mouse splenic lymphocytes with 5 × 10³ TC-1-GLUC-LMP2 target cells and found that the target cells were killed as the specific killing effect was obviously enhanced by the increased quantities of LMP2-peptide stimulated spleens. Furthermore, the tumor cells could not be observed in the mice inoculated TC-1-GLUC-LMP2 cells after being immunized with vaccine-LMP2, while the vaccine-NULL immunized mice showed that tumor volume gradually grew with increased inoculation time. These results indicated that the TC-1-GLUC-LMP2 cells stably expressing and produced tumors in mice, and that the LMP2-specific cytotoxic T lymphocyte (CTL) effectively killed the cells in vitro and in vivo, suggesting that TC-1-GLUC-LMP2 cells can be used as model cells to assess the immune and antitumor effects of LMP2-related vaccines.
Latent Membrane Protein 2 (LMP2).
Cen Osman,Longnecker Richard
Current topics in microbiology and immunology
LMP2A is an EBV-encoded protein with three domains: (a) an N-terminal cytoplasmic domain, which has PY motifs that bind to WW domain-containing E3 ubiquitin ligases and an ITAM that binds to SH2 domain-containing proteins, (b) a transmembrane domain with 12 transmembrane segments that localizes LMP2A in cellular membranes, and (c) a 27-amino acid C-terminal domain which mediates homodimerization and heterodimerization of LMP2 protein isoforms. The most prominent two isoforms of the protein are LMP2A and LMP2B. The LMP2B isoform lacks the 19-amino acid N-terminal domain found in LMP2A, which modulates cellular signaling resulting in a baseline activation of B cells and degradation of cellular kinases leading to the downregulation of normal B cell signaling pathways. These two seemingly contradictory processes allow EBV to establish and maintain latency. LMP2 is expressed in many EBV-associated malignancies. While its antigenic properties may be useful in developing LMP2-specific immunity, the LMP2A N-terminal motifs also provide a basis to target LMP2A-modulated cellular kinases for the development of treatment strategies.
Potential role of LMP2 as tumor-suppressor defines new targets for uterine leiomyosarcoma therapy.
Hayashi Takuma,Horiuchi Akiko,Sano Kenji,Hiraoka Nobuyoshi,Kasai Mari,Ichimura Tomoyuki,Sudo Tamotsu,Tagawa Yoh-Ichi,Nishimura Ryuichiro,Ishiko Osamu,Kanai Yae,Yaegashi Nobuo,Aburatani Hiroyuki,Shiozawa Tanri,Konishi Ikuo
Although the majority of smooth muscle neoplasms found in the uterus are benign, uterine leiomyosarcoma (LMS) is extremely malignant, with high rates of recurrence and metastasis. We earlier reported that mice with a homozygous deficiency for LMP2, an interferon (IFN)-γ-inducible factor, spontaneously develop uterine LMS. The IFN-γ pathway is important for control of tumor growth and invasion and has been implicated in several cancers. In this study, experiments with human and mouse uterine tissues revealed a defective LMP2 expression in human uterine LMS that was traced to the IFN-γ pathway and the specific effect of JAK-1 somatic mutations on the LMP2 transcriptional activation. Furthermore, analysis of a human uterine LMS cell line clarified the biological significance of LMP2 in malignant myometrium transformation and cell cycle, thus implicating LMP2 as an anti-tumorigenic candidate. This role of LMP2 as a tumor suppressor may lead to new therapeutic targets in human uterine LMS.
EBV load in whole blood correlates with LMP2 gene expression after pediatric heart transplantation or allogeneic hematopoietic stem cell transplantation.
Ruf Stephanie,Behnke-Hall Kachina,Gruhn Bernd,Reiter Alfred,Wagner Hans J
BACKGROUND:Epstein-Barr virus (EBV) is associated with posttransplant lymphoproliferative disease (PTLD), and EBV load measurement is an important tool to monitor transplant patients. Although EBV DNA quantification has high sensitivity to identify patients at risk for PTLD, it lacks specificity. We examined whether EBV gene expression in peripheral B cells can increase specificity or correlates with EBV load. METHODS:Altogether, 220 blood samples were collected from pediatric patients after heart transplantation (HTx, n=57), renal transplantation (n=1), or hematopoietic stem cell transplantation (n=21). In each blood sample, EBV load was quantified in whole blood, plasma, and B cells using qPCR. Additionally EBV gene expression (EBNA2, LMP1, LMP2, and BZLF1) in B cells was analyzed using relative quantitative RT-qPCR. RESULTS:Positive expression of at least one gene was detected in 112 (51%) of 220 samples. Patients with PTLD or chronic high viral loads after solid organ transplantation exhibited no homogeneous EBV gene expression pattern. Expression of LMP2, LMP1, or EBNA2 was only observed when EBV load exceeded 1000 copies/mL. A high correlation between the level of LMP2 expression and EBV load in B cells or whole blood was observed (ρ=0.72 or ρ=0.6, HTx population). CONCLUSION:The analysis of EBV gene expression in peripheral B cells does not provide additional information about patients' risk of developing PTLD. As EBV load in whole blood correlates well with LMP2 gene expression in EBV-infected B cells, EBV DNA quantification in whole blood alone seems to be a sufficient tool to monitor these patients.
Chimerically fused antigen rich of overlapped epitopes from latent membrane protein 2 (LMP2) of Epstein-Barr virus as a potential vaccine and diagnostic agent.
Lin Xiaoyun,Chen Shao,Xue Xiangyang,Lu Lijun,Zhu Shanli,Li Wenshu,Chen Xiangmin,Zhong Xiaozhi,Jiang Pengfei,Sename Torsoo Sophia,Zheng Yi,Zhang Lifang
Cellular & molecular immunology
Epstein-Barr virus (EBV) is prevalent throughout the world and is associated with several malignant diseases in humans. Latent membrane protein 2 (LMP2) of EBV plays a crucial role in the pathogenesis of EBV-associated tumors; therefore, LMP2 has been considered to be a potential immunodiagnostic and immunotherapeutic target. A multi-epitope-based antigen is a promising option for therapeutic vaccines and diagnoses of such malignancies. In this study, we systematically screened cytotoxic T lymphocyte (CTL), helper T cell (Th) and B-cell epitopes within EBV-LMP2 using bioinformatics. Based on the screen, two peptides rich in overlapping epitopes of both T cells and B cells were selected to construct a plasmid containing the sequence for a chimeric multi-epitope protein referred to as EBV-LMP2m, which is composed of LMP2aa195∼232 and LMP2aa419∼436. The EBV-LMP2m protein was expressed in E. coli BL21 (DE3) after prokaryotic codon optimization. Inoculation of the purified chimeric antigen in BALB/c mice induced not only high levels of specific IgG in the serum and secretory IgA in the vaginal mucus but also a specific CTL response. By using purified EBV-LMP2m as an antigen, the presence of specific IgG in the serum specimens of 202 nasopharyngeal carcinoma (NPC) patients was effectively detected with 52.84% sensitivity and 95.40% specificity, which represents an improvement over the traditional detection method based on VCA-IgA (60.53% sensitivity and 76.86% specificity). The above results indicate that EBV-LMP2m may be used not only as a potential target antigen for EBV-associated tumors but also a diagnostic agent for NPC patients.
Association between LMP2/LMP7 genetic variability and the metastasis risk of ovarian cancer in Chinese women in Beijing.
Song Li,Ma Nina,Han Lei,Yan Han,Yan Bo,Yuan Zhenyan,Cao Bangwei
LMP2/LMP7 gene (LMP, low molecular mass protein) perform a critical role in the foreign antigen processing via the major histocompatibility complex-I (MHC-I) complex CD8(+) cytotoxic T lymphocytes (CTL) pathway. This study was designed to investigate whether the sequence variants in LMP2/LMP7 gene would increase the risk of ovarian cancer in the Chinese population. Total of 235 patients with ovarian cancer and 338 normal controls were recruited. Two polymorphisms of LMP2-60 (Arg→His) and LMP7-145 (Gln→Lys) were identified by PCR-RFLP (RFLP, restriction fragment length polymorphism) method. The association of LMP2/LMP7 gene variations with ovarian cancer was assessed by logistic regression analysis. The results revealed that LMP7-145 Gln/Lys and Lys/Lys alleles were associated with the risk of ovarian cancer (P=0.002, OR=2.47; P<0.001, OR=3.23). Meanwhile, the relationship between the LMP7-145 polymorphism and the lymph node metastasis and tumor distant metastasis were also found. No statistical correlation between any of the LMP2-60 polymorphic genotypes and the ovarian cancer clinicopathological characteristics were observed (P>0.05). These results suggested that LMP7 genetic variant could increase the susceptibility to ovarian cancer development; especially increase the risk of lymph node and tumor distant metastasis.