Virus-induced Lymphoma Research Articles

Electrical storm due to Epstein-Barr virus-induced lymphoma of a transplanted heart: a case report

BackgroundCancers, and specifically lymphomas, are one of the main causes of morbidity and mortality after heart transplantation. Sixteen percent of heart transplant recipients develop cancer within 5 years and lymphomas represent 10% of these patients.Case summaryWe report the case of an Epstein–Barr Virus–induced primary cardiac lymphoma on the graft. The patient initially present an electrical storm quickly controlled under medical treatment. The multimodal exploration led us to the diagnosis of lymphoma. The lymphoma has spread quickly in spite of the numerous treatments we have tried and has resulted in arrhythmia complications.DiscussionThis case report highlights the challenging management of heart transplant lymphoma and its treatment. Primary cardiac lymphoma on the graft is highly rare with poor prognosis and arrhythmia complications.

Identification and Validation of Ikaros (IKZF1) as a Cancer Driver Gene for Marek's Disease Virus-Induced Lymphomas.

Marek’s disease virus (MDV) is the causative agent for Marek’s disease (MD), which is characterized by T-cell lymphomas in chickens. While the viral Meq oncogene is necessary for transformation, it is insufficient, as not every bird infected with virulent MDV goes on to develop a gross tumor. Thus, we postulated that the chicken genome contains cancer driver genes; i.e., ones with somatic mutations that promote tumors, as is the case for most human cancers. To test this hypothesis, MD tumors and matching control tissues were sequenced. Using a custom bioinformatics pipeline, 9 of the 22 tumors analyzed contained one or more somatic mutation in Ikaros (IKFZ1), a transcription factor that acts as the master regulator of lymphocyte development. The mutations found were in key Zn-finger DNA-binding domains that also commonly occur in human cancers such as B-cell acute lymphoblastic leukemia (B-ALL). To validate that IKFZ1 was a cancer driver gene, recombinant MDVs that expressed either wild-type or a mutated Ikaros allele were used to infect chickens. As predicted, birds infected with MDV expressing the mutant Ikaros allele had high tumor incidences (~90%), while there were only a few minute tumors (~12%) produced in birds infected with the virus expressing wild-type Ikaros. Thus, in addition to Meq, key somatic mutations in Ikaros or other potential cancer driver genes in the chicken genome are necessary for MDV to induce lymphomas.

Association of Bovine Leukemia Virus-Induced Lymphoma with BoLA-DRB3 Polymorphisms at DNA, Amino Acid, and Binding Pocket Property Levels

Bovine leukemia virus (BLV) causes enzootic bovine leucosis, a malignant B-cell lymphoma in cattle. The DNA sequence polymorphisms of bovine leukocyte antigen (BoLA)-DRB3 have exhibited a correlation with BLV-induced lymphoma in Holstein cows. However, the association may vary between different cattle breeds. Furthermore, little is known about the relationship between BLV-induced lymphoma and DRB3 at the amino acid and structural diversity levels. Here, we comprehensively analyzed the correlation between BLV-induced lymphoma and DRB3 at DNA, amino acid, and binding pocket property levels, using 106 BLV-infected asymptomatic and 227 BLV-induced lymphoma Japanese black cattle samples. DRB3*011:01 was identified as a resistance allele, whereas DRB3*005:02 and DRB3*016:01 were susceptibility alleles. Amino acid association studies showed that positions 9, 11, 13, 26, 30, 47, 57, 70, 71, 74, 78, and 86 were associated with lymphoma susceptibility. Structure and electrostatic charge modeling further indicated that binding pocket 9 of resistance DRB3 was positively charged. In contrast, alleles susceptible to lymphoma were neutrally charged. Altogether, this is the first association study of BoLA-DRB3 polymorphisms with BLV-induced lymphoma in Japanese black cattle. In addition, our results further contribute to understanding the mechanisms regarding how BoLA-DRB3 polymorphisms mediate susceptibility to BLV-induced lymphoma.

Lipid-Orchestrated Acceleration of Epstein-Barr Virus-Induced B-Cell Lymphoma Via the Secreted Phospholipase A <sub>2</sub>-Mediated Modification of Exosomes

Exosomes act as intercellular communicators by transferring protein and microRNA cargoes, yet the role of exosome lipids remains unclear. Here, we show that the pro-tumorigenic action of lymphoma-derived exosomes is augmented via secreted phospholipase A2(sPLA2)-driven lipid metabolism. Hydrolysis of exosomal phospholipids by group X sPLA2, which was induced in macrophages of Epstein-Barr virus (EBV) lymphoma, increased the production of fatty acids, lysophospholipids and their metabolites. sPLA2-treated exosomes were smaller, self-aggregated, showed better uptake, and increased cytokine expression and lipid mediator signaling in tumor associated macrophages. Pharmacological inhibition of endogenous sPLA2 suppressed lymphoma growth in EBV-infected humanized mice; treatment with sPLA2-modified exosomes reversed this phenotype. Further, sPLA2 expression in human large B-cell lymphomas inversely correlated with patient survival. Overall, the sPLA2-mediated exosome modification promotes tumor development.

Copy number variations in BOLA-DQA2, BOLA-DQB, and BOLA-DQA5 show the genomic architecture and haplotype frequency of major histocompatibility complex class II genes in Holstein cows.

Bovine major histocompatibility complex (MHC) class II region contains many genes. The bovine leukocyte antigen (BoLA)-DRB3 was reportedly associated with susceptibility of various phenotypes of infections including bovine leukemia virus-induced lymphoma. However, the association of the remaining genes with various phenotypes has not been clarified due to the complicated genomic structure of the MHC class II region. Thus, in this study, we elucidated the MHC class II genomic structure, including the novel alleles and copy number variations (CNVs). We determined the copy numbers of BOLA-DQA2 (DQA2), BOLA-DQB (DQB2), BOLA-DQA5 (DQA5), BLA-DQB (DQB1), LOC100848815 (DQA1), and BOLA-DRB3 (DRB3) in 127 unrelated Holstein cows by TaqMan copy number assay. The genomes were sequenced using target next-generation sequencing (NGS) based on multiplex polymerase chain reaction. Combining the results of the copy numbers and alleles, we identified the BoLA alleles directly without haplotype estimation. Pairwise linkage disequilibrium (LD) analysis between alleles and genes were performed. The CNVs of DQA2, DQB2, and DQA5 in Holstein cows were detected. The frequency of the whole gene deletion in DQA2, DQB2, and DQA5 was 35.4%, 93.7%, and 93.7%, respectively. After target NGS, we identified 37 alleles in the six genes. Fifteen novel alleles (40.5%) were not registered in the IPD-MHC Database. LD analysis showed strong LD among the DQB2*deletion, DQA5*deletion, and DRB3*27:03 alleles. Our findings will provide important insights into the identification of the BoLA genes associated with various infection-related phenotypes.

Lymphotropic Viruses EBV, KSHV and HTLV in Latin America: Epidemiology and Associated Malignancies. A Literature-Based Study by the RIAL-CYTED

The Epstein–Barr virus (EBV), Kaposi sarcoma herpesvirus (KSHV) and human T-lymphotropic virus (HTLV-1) are lymphomagenic viruses with region-specific induced morbidity. The RIAL-CYTED aims to increase the knowledge of lymphoma in Latin America (LA), and, as such, we systematically analyzed the literature to better understand our risk for virus-induced lymphoma. We observed that high endemicity regions for certain lymphomas, e.g., Mexico and Peru, have a high incidence of EBV-positive lymphomas of T/NK cell origin. Peru also carries the highest frequency of EBV-positive classical Hodgkin lymphoma (HL) and EBV-positive diffuse large B cell lymphoma, not otherwise specified (NOS), than any other LA country. Adult T cell lymphoma is endemic to the North of Brazil and Chile. While only few cases of KSHV-positive lymphomas were found, in spite of the close correlation of Kaposi sarcoma and the prevalence of pathogenic types of KSHV. Both EBV-associated HL and Burkitt lymphoma mainly affect young children, unlike in developed countries, in which adolescents and young adults are the most affected, correlating with an early EBV seroconversion for LA population despite of lack of infectious mononucleosis symptoms. High endemicity of KSHV and HTLV infection was observed among Amerindian populations, with differences between Amazonian and Andean populations.

BoLA-DRB3 Polymorphism is Associated with Differential Susceptibility to Bovine Leukemia Virus-Induced Lymphoma and Proviral Load.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis. However, less than 5% of BLV-infected cattle will develop lymphoma, suggesting that, in addition to viral infection, host genetic polymorphisms might play a role in disease susceptibility. Bovine leukocyte antigen (BoLA)-DRB3 is a highly polymorphic gene associated with BLV proviral load (PVL) susceptibility. Due to the fact that PVL is positively associated with disease progression, it is believed that controlling PVL can prevent lymphoma development. Thus, many studies have focused on the relationship between PVL and BoLA-DRB3. Despite this, there is little information regarding the relationship between lymphoma and BoLA-DRB3. Furthermore, whether or not PVL-associated BoLA-DRB3 is linked to lymphoma-associated BoLA-DRB3 has not been clarified. Here, we investigated whether or not lymphoma-associated BoLA-DRB3 is correlated with PVL-associated BoLA-DRB3. We demonstrate that two BoLA-DRB3 alleles were specifically associated with lymphoma resistance (*010:01 and *011:01), but no lymphoma-specific susceptibility alleles were found; furthermore, two other alleles, *002:01 and *012:01, were associated with PVL resistance and susceptibility, respectively. In contrast, lymphoma and PVL shared two resistance-associated (DRB3*014:01:01 and *009:02) BoLA-DRB3 alleles. Interestingly, we found that PVL associated alleles, but not lymphoma associated alleles, are related with the anti-BLV gp51 antibody production level in cows. Overall, our study is the first to demonstrate that the BoLA-DRB3 polymorphism confers differential susceptibility to BLV-induced lymphoma and PVL.

Marek's Disease Virus-Encoded MicroRNA 155 Ortholog Critical for the Induction of Lymphomas Is Not Essential for the Proliferation of Transformed Cell Lines.

MicroRNAs (miRNAs) are small noncoding RNAs with profound regulatory roles in many areas of biology, including cancer. MicroRNA 155 (miR-155), one of the extensively studied multifunctional miRNAs, is important in several human malignancies such as diffuse large B cell lymphoma and chronic lymphocytic leukemia. Moreover, miR-155 orthologs KSHV-miR-K12-11 and MDV-miR-M4, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and Marek's disease virus (MDV), respectively, are also involved in oncogenesis. In MDV-induced T-cell lymphomas and in lymphoblastoid cell lines derived from them, MDV-miR-M4 is highly expressed. Using excellent disease models of infection in natural avian hosts, we showed previously that MDV-miR-M4 is critical for the induction of T-cell lymphomas as mutant viruses with precise deletions were significantly compromised in their oncogenicity. However, those studies did not elucidate whether continued expression of MDV-miR-M4 is essential for maintaining the transformed phenotype of tumor cells. Here using an in situ CRISPR/Cas9 editing approach, we deleted MDV-miR-M4 from the MDV-induced lymphoma-derived lymphoblastoid cell line MDCC-HP8. Precise deletion of MDV-miR-M4 was confirmed by PCR, sequencing, quantitative reverse transcription-PCR (qRT-PCR), and functional analysis. Continued proliferation of the MDV-miR-M4-deleted cell lines demonstrated that MDV-miR-M4 expression is not essential for maintaining the transformed phenotype, despite its initial critical role in the induction of lymphomas. Ability to examine the direct role of oncogenic miRNAs in situ in tumor cell lines is valuable in delineating distinct determinants and pathways associated with the induction or maintenance of transformation in cancer cells and will also contribute significantly to gaining further insights into the biology of oncogenic herpesviruses.IMPORTANCE Marek's disease virus (MDV) is an alphaherpesvirus associated with Marek's disease (MD), a highly contagious neoplastic disease of chickens. MD serves as an excellent model for studying virus-induced T-cell lymphomas in the natural chicken hosts. Among the limited set of genes associated with MD oncogenicity, MDV-miR-M4, a highly expressed viral ortholog of the oncogenic miR-155, has received extensive attention due to its direct role in the induction of lymphomas. Using a targeted CRISPR-Cas9-based gene editing approach in MDV-transformed lymphoblastoid cell lines, we show that MDV-miR-M4, despite its critical role in the induction of tumors, is not essential for maintaining the transformed phenotype and continuous proliferation. As far as we know, this was the first study in which precise editing of an oncogenic miRNA was carried out in situ in MD lymphoma-derived cell lines to demonstrate that it is not essential in maintaining the transformed phenotype.

A 1-Year Prospective French Nationwide Study of Emergency Hospital Admissions in Children and Adults with Primary Immunodeficiency.

Patients with primary immunodeficiency (PID) are at risk of serious complications. However, data on the incidence and causes of emergency hospital admissions are scarce. The primary objective of the present study was to describe emergency hospital admissions among patients with PID, with a view to identifying "at-risk" patient profiles. We performed a prospective observational 12-month multicenter study in France via the CEREDIH network of regional PID reference centers from November 2010 to October 2011. All patients with PIDs requiring emergency hospital admission were included. A total of 200 admissions concerned 137 patients (73 adults and 64 children, 53% of whom had antibody deficiencies). Thirty admissions were reported for 16 hematopoietic stem cell transplantation recipients. When considering the 170 admissions of non-transplant patients, 149 (85%) were related to acute infections (respiratory tract infections and gastrointestinal tract infections in 72 (36%) and 34 (17%) of cases, respectively). Seventy-seven percent of the admissions occurred during winter or spring (December to May). The in-hospital mortality rate was 8.8% (12 patients); death was related to a severe infection in 11 cases (8%) and Epstein-Barr virus-induced lymphoma in 1 case. Patients with a central venous catheter (n = 19, 13.9%) were significantly more hospitalized for an infection (94.7%) than for a non-infectious reason (5.3%) (p = 0.04). Our data showed that the annual incidence of emergency hospital admission among patients with PID is 3.4%. The leading cause of emergency hospital admission was an acute infection, and having a central venous catheter was associated with a significantly greater risk of admission for an infectious episode.

Marek's Disease Virus Disables the ATR-Chk1 Pathway by Activating STAT3.

Oncogenic virus replication often leads to genomic instability, causing DNA damage and inducing the DNA damage response (DDR) pathway. The DDR pathway is a cellular pathway that senses DNA damage and regulates the cell cycle to maintain genomic stability. Therefore, the DDR pathway is critical for the viral lifecycle and tumorigenesis. Marek's disease virus (MDV), an alphaherpesvirus that causes lymphoma in chickens, has been shown to induce DNA damage in infected cells. However, the interaction between MDV and the host DDR is unclear. In this study, we observed that MDV infection causes DNA strand breakage in chicken fibroblast (CEF) cells along with an increase in the DNA damage markers p53 and p21. Interestingly, we showed that phosphorylation of STAT3 was increased during MDV infection, concomitantly with a decrease of Chk1 phosphorylation. In addition, we found that MDV infection was enhanced by VE-821, an ATR-specific inhibitor, but attenuated by hydroxyurea, an ATR activator. Moreover, inhibition of STAT3 phosphorylation by Stattic eliminates the ability of MDV to inhibit Chk1 phosphorylation. Finally, we showed that MDV replication was decreased by Stattic treatment. Taken together, these results suggest that MDV disables the ATR-Chk1 pathway through STAT3 activation to benefit its replication.IMPORTANCE MDV is used as a biomedical model to study virus-induced lymphoma due to the similar genomic structures and physiological characteristics of MDV and human herpesviruses. Upon infection, MDV induces DNA damage, which may activate the DDR pathway. The DDR pathway has a dual impact on viruses because it manipulates repair and recombination factors to facilitate viral replication and also initiates antiviral action by regulating other signaling pathways. Many DNA viruses evolve to manipulate the DDR pathway to promote virus replication. In this study, we identified a mechanism used by MDV to inhibit ATR-Chk1 pathways. ATR is a cellular kinase that responds to broken single-stranded DNA, which has been less studied in MDV infection. Our results suggest that MDV infection activates STAT3 to disable the ATR-Chk1 pathway, which is conducive to viral replication. This finding provides new insight into the role of STAT3 in interrupting the ATR-Chk1 pathway during MDV replication.

Multifunctional miR-155 Pathway in Avian Oncogenic Virus-Induced Neoplastic Diseases.

MicroRNAs (miRNAs) are small noncoding RNAs that fine-tune the responses of the cell by modulating the cell transcriptome and gene expression. MicroRNA 155 (miR-155) is a conserved multifunctional miRNA involved in multiple roles including the modulation of the immune responses. When deregulated, miR-155 can also contribute to cancer as has been demonstrated in several human malignancies such as diffuse large B cell lymphoma, chronic lymphocytic leukemia, as well as in Epstein–Barr virus (EBV)-induced B cell transformation. Avian oncogenic viruses such as Marek’s disease virus (MDV), avian leukosis virus (ALV), and reticuloendotheliosis virus (REV) that account for more than 90% of cancers in avian species, also make use of the miR-155 pathway during oncogenesis. While oncogenic retroviruses, such as ALV, activate miR-155 by insertional activation, acutely transforming retroviruses use transduced oncogenes such as v-rel to upregulate miR-155 expression. MDV on the other hand, encodes a functional miR-155 ortholog mdv1-miR-M4, similar to the miR-155 ortholog kshv-miR-K11 present in Kaposi’s sarcoma-associated herpesvirus (KSHV). We have shown that mdv1-miR-M4 is critical for the induction of MDV-induced lymphomas further demonstrating the oncogenic potential of miR-155 pathway in cancers irrespective of the diverse etiology. In this review, we discuss on our current understanding of miR-155 function in virus-induced lymphomas focusing primarily on avian oncogenic viruses.

B-lymphocyte stimulator: can we consider it a marker for severity of hepatitis C virus-induced B-cell non-Hodgkin lymphoma?

ObjectiveB-lymphocyte stimulator (BLyS) is a member of tumor necrosis factor family. BLyS is essential for the survival of normal and malignant B lymphocyte. Hepatitis C virus (HCV) infection likely represents the early event leading to BLyS upregulation. The aim of this study was to determine the relation between serum BLyS levels and the severity of HCV-related B-cell non-Hodgkin lymphoma (B-NHL).Patients and methodsSeventy-eight B-NHL patients both HCV-positive and HCV-negative and 20 patients with HCV infection without lymphoproliferative disorders, in addition to 20 age-matched and sex-matched controls, were included in the study. PCR for HCV, evaluation of levels of soluble BLyS protein in blood, bone marrow aspirate and biopsy with flow cytometry, and lymph node biopsy with immunophenotyping for CD5, CD23, CD10, CD20, and cyclinD1 were performed.ResultsThe serum BLyS levels were significantly higher in B-NHL patients and HCV patients without lymphoproliferative disorders compared with the control group. The serum BLyS levels were statistically significantly higher in aggressive lymphoma patients with HCV-positive infection compared with aggressive lymphoma patients with HCV-negative infection, but there was no statistically significant difference between BLyS levels in indolent B-NHL patients with or without HCV infections. Moreover, there was no statistically significant difference between BLyS levels in aggressive and indolent lymphoma patients with HCV-positive infection.ConclusionBLyS levels are increased in HCV-induced B-NHL but it cannot be considered as a marker for severity of the disease (indolent or aggressive). More studies are needed.

Prophylactic and therapeutic adenoviral vector-based multivirus-specific T-cell immunotherapy for transplant patients

Viral infections including cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus are a common and predictable problem in transplant recipients. While cellular immune therapies have been successfully used to tackle infectious complications in transplant recipients, manufacturing immunotherapies to address the multitude of possible pathogens can be technically challenging and labor-intensive. Here we describe a novel adenoviral antigen presentation platform (Ad-MvP) as a tool for rapid generation of multivirus-specific T-cells in a single step. Ad-MvP encodes 32 CD8+ T-cell epitopes from cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus as a contiguous polyepitope. We demonstrate that Ad-MvP vector can be successfully used for rapid in vitro expansion of multivirus-specific T-cells from transplant recipients and in vivo priming of antiviral T-cell immunity. Most importantly, using an in vivo murine model of Epstein-Barr virus-induced lymphoma, we also show that adoptive immunotherapy with Ad-MvP expanded autologous and allogeneic multivirus-specific T-cells is highly effective in controlling Epstein-Barr virus tumor outgrowth and improving overall survival. We propose that Ad-MvP has wide ranging therapeutic applications in greatly facilitating in vivo priming of antiviral T-cells, the generation of third-party T-cell banks as “off-the-shelf” therapeutics as well as autologous T-cell therapies for transplant patients.

Common Viral Integration Sites Identified in Avian Leukosis Virus-Induced B-Cell Lymphomas.

ABSTRACTAvian leukosis virus (ALV) induces B-cell lymphoma and other neoplasms in chickens by integrating within or near cancer genes and perturbing their expression. Four genes—MYC, MYB, Mir-155, and TERT—have previously been identified as common integration sites in these virus-induced lymphomas and are thought to play a causal role in tumorigenesis. In this study, we employ high-throughput sequencing to identify additional genes driving tumorigenesis in ALV-induced B-cell lymphomas. In addition to the four genes implicated previously, we identify other genes as common integration sites, including TNFRSF1A, MEF2C, CTDSPL, TAB2, RUNX1, MLL5, CXorf57, and BACH2. We also analyze the genome-wide ALV integration landscape in vivo and find increased frequency of ALV integration near transcriptional start sites and within transcripts. Previous work has shown ALV prefers a weak consensus sequence for integration in cultured human cells. We confirm this consensus sequence for ALV integration in vivo in the chicken genome.

A Systematic Analysis of miRNA Transcriptome in Marek’s Disease Virus-Induced Lymphoma Reveals Novel and Differentially Expressed miRNAs

Marek’s disease is a lymphoproliferative neoplastic disease of the chicken, which poses a serious threat to poultry health. Marek’s disease virus (MDV)-induced T-cell lymphoma is also an excellent biomedical model for neoplasia research. Recently, miRNAs have been demonstrated to play crucial roles in mediating neoplastic transformation. To investigate host miRNA expression profiles in the tumor transformation phase of MDV infection, we performed deep sequencing in two MDV-infected samples (tumorous spleen and MD lymphoma from liver), and two non-infected controls (non-infected spleen and lymphocytes). In total, 187 and 16 known miRNAs were identified in chicken and MDV, respectively, and 17 novel chicken miRNAs were further confirmed by qPCR. We identified 28 down-regulated miRNAs and 11 up-regulated miRNAs in MDV-infected samples by bioinformatic analysis. Of nine further tested by qPCR, seven were verified. The gga-miR-181a, gga-miR-26a, gga-miR-221, gga-miR-222, gga-miR-199*, and gga-miR-140* were down-regulated, and gga-miR-146c was up-regulated in MDV-infected tumorous spleens and MD lymphomas. In addition, 189 putative target genes for seven differentially expressed miRNAs were predicted. The luciferase reporter gene assay showed interactions of gga-miR-181a with MYBL1, gga-miR-181a with IGF2BP3, and gga-miR-26a with EIF3A. Differential expression of miRNAs and the predicted targets strongly suggest that they contribute to MDV-induced lymphomagenesis.

Kaposi's sarcoma herpesvirus lytic replication compromises apoptotic response to p53 reactivation in virus-induced lymphomas

Primary effusion lymphomas (PELs) are aggressive Kaposi's sarcoma herpesvirus (KSHV)-induced malignancies with median survival time <6 months post-diagnosis. Mutations in the TP53 gene seldom occur in PELs, suggesting that genetic alterations in the TP53 are not selected during PEL progression. We have reported that p53 reactivation by an inhibitor of the p53-MDM2 interaction, Nutlin-3, induces selective and massive apoptosis in PEL cells leading to efficient anti-tumor activity in a subcutaneous xenograft model for PEL. Here, we show compelling anti-tumor activity of Nutlin-3 in the majority of intraperitoneal PEL xenografts in vivo. Interestingly, our results demonstrate that spontaneous induction of viral lytic replication in tumors could drastically attenuate the p53-dependent apoptotic response to Nutlin-3. Moreover, viral reactivation compromised p53-dependent apoptosis in PEL cells treated with genotoxic anti-cancer agents doxorubicin and etoposide. We have recently demonstrated that the Ser/Thr kinases Pim 1 and 3 are required to trigger induction of the lytic replication cascade of KSHV. We have now assessed the ability of a novel Pim kinase inhibitor to restore the Nutlin-3-induced cytotoxicity in lytic PEL cells. PEL cells induced to lytic replication by phorbol esters showed 50% inhibition of active viral replication following treatment with the Pim kinase inhibitor. Importantly, co-treatment of these cells with the kinase inhibitor and Nutlin-3 resulted in a robust restoration of the Nutlin-3-induced cell death. These results highlight the potential impact of activation of viral lytic replication on disease progression and response to treatment in KSHV-induced lymphomas.

Moving Successful Virus-specific T-cell Therapy for Hematopoietic Stem Cell Recipients to Late Phase Clinical Trials

There are now multiple effective strategies for the activation and expansion, or direct selection of virus-specific T cells that may be able to eliminate a range of virus infections in the immunocompromised host. In this mini-review, we explain how the stage is set for their rigorous evaluation in large-scale clinical trials. Virus infections are the cause of 30% or more of transplant-related deaths in recipients of T-cells depleted, allogeneic hematopoietic stem cell transplants (HSCT).1 Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenoviruses (AdV) are the most common culprits, but other common viruses like parainfluenza virus, respiratory syncytial virus, influenza viruses, polyomaviruses, and human herpesvirus 6 together contribute significant morbidity and mortality (Figure 1).2,3,4 Small molecule therapies are often ineffectual, always costly and frequently produce significant adverse effects. Figure 1 Relative frequency of viral infections after HSCT. Adv, adenovirus; BK, JC, KI, and WU (polyomaviruses); CMV, cytomegalovirus; H1N1, influenza strain Hemagglutinnin 1 Neuraminidase 1); HHV, human herpes virus; HSCT, hematopoietic stem cell transplant; ... Virus-specific T-cells derived from stem cell donors can prevent and treat post-transplant viral infections, in the recipients for whom they were intended, and also in partially human leukocyte antigen (HLA)-matched, third party recipients.5,6,7 The low toxicity and long-term protection provided by virus-specific T-cells compares favorably with the significant toxicities and short-term effects of most antivirals.5,6,8,9,10 Is it time, therefore, to begin the transfer of T-cell manufacturing from academic phase I/II clinical trials into hospital or industry-supported facilities so that virus-specific T-cells can be made available to all high-risk HSCT recipients? Several barriers prevent the broader use of virus-specific T-cell therapies after stem cell transplantation. While T-cell therapies for EBV, CMV, and AdV have clearly demonstrated their safety and efficacy both as prophylaxis and as therapy, for many other viruses, the antigens that induce protective T-cells have yet to be identified. Moreover, these infections may occur in <5% of patients making it difficult to perform the rigorous comparative effectiveness studies that will be required to show lower overall cost, fewer adverse effects, and equivalent or superior efficacy. Before any of these barriers can be breached effectively by the academic institutions who are the major developers of these T-cells therapeutics, we must select and optimize manufacturing strategies that are robust and scalable and have the lowest possible cost. Finally, the leap to late phase trials cannot be accomplished by academic institutions alone, but requires partnership with industry. This article will largely deal with selection and optimization of virus-specific T-cell manufacturing strategies.

Herpesvirus telomerase RNA (vTR) with a mutated template sequence abrogates herpesvirus-induced lymphomagenesis.

Telomerase reverse transcriptase (TERT) and telomerase RNA (TR) represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR) on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV) as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5) by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1) that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2) that this strategy could be used to generate novel vaccine candidates against virus-induced lymphoma.

Structural characteristics and antiviral activity of multiple peptides derived from MDV glycoproteins B and H

BackgroundMarek's disease virus (MDV), which is widely considered to be a natural model of virus-induced lymphoma, has the potential to cause tremendous losses in the poultry industry. To investigate the structural basis of MDV membrane fusion and to identify new viral targets for inhibition, we examined the domains of the MDV glycoproteins gH and gB.ResultsFour peptides derived from the MDV glycoprotein gH (gHH1, gHH2, gHH3, and gHH5) and one peptide derived from gB (gBH1) could efficiently inhibit plaque formation in primary chicken embryo fibroblast cells (CEFs) with 50% inhibitory concentrations (IC50) of below 12 μM. These peptides were also significantly able to reduce lesion formation on chorioallantoic membranes (CAMs) of infected chicken embryos at a concentration of 0.5 mM in 60 μl of solution. The HR2 peptide from Newcastle disease virus (NDVHR2) exerted effects on MDV specifically at the stage of virus entry (i.e., in a cell pre-treatment assay and an embryo co-treatment assay), suggesting cross-inhibitory effects of NDV HR2 on MDV infection. None of the peptides exhibited cytotoxic effects at the concentrations tested. Structural characteristics of the five peptides were examined further.ConclusionsThe five MDV-derived peptides demonstrated potent antiviral activity, not only in plaque formation assays in vitro, but also in lesion formation assays in vivo. The present study examining the antiviral activity of these MDV peptides, which are useful as small-molecule antiviral inhibitors, provides information about the MDV entry mechanism.

7,3′,4′-Trihydroxyisoflavone, a Metabolite of the Soy Isoflavone Daidzein, Suppresses Ultraviolet B-induced Skin Cancer by Targeting Cot and MKK4

Nonmelanoma skin cancer is one of the most frequently occurring cancers in the United States. Chronic exposure to UVB irradiation is a major cause of this cancer. Daidzein, along with genistein, is a major isoflavone found in soybeans; however, little is known about the chemopreventive effects of daidzein and its metabolites in UVB-induced skin cancer. Here, we found that 7,3',4'-trihydroxyisoflavone (THIF), a major metabolite of daidzein, effectively inhibits UVB-induced cyclooxygenase 2 (COX-2) expression through the inhibition of NF-κB transcription activity in mouse skin epidermal JB6 P+ cells. In contrast, daidzein had no effect on COX-2 expression levels. Data from Western blot and kinase assays showed that 7,3',4'-THIF inhibited Cot and MKK4 activity, thereby suppressing UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays indicated that 7,3',4'-THIF competed with ATP to inhibit Cot or MKK4 activity. Topical application of 7,3',4'-THIF clearly suppressed the incidence and multiplicity of UVB-induced tumors in hairless mouse skin. Hairless mouse skin results also showed that 7,3',4'-THIF inhibits Cot or MKK4 kinase activity directly, resulting in suppressed UVB-induced COX-2 expression. A docking study revealed that 7,3',4'-THIF, but not daidzein, easily docked to the ATP binding site of Cot and MKK4, which is located between the N- and C-lobes of the kinase domain. Collectively, these results provide insight into the biological actions of 7,3',4'-THIF, a potential skin cancer chemopreventive agent.