RESUMEN
Epstein-Barr virus (EBV) infects more than 95% of adults worldwide and is closely associated with various malignancies. Considering the complex life cycle of EBV, developing vaccines targeting key entry glycoproteins to elicit robust and durable adaptive immune responses may provide better protection. EBV gHgL-, gB- and gp42-specific antibodies in healthy EBV carriers contributed to sera neutralizing abilities in vitro, indicating that they are potential antigen candidates. To enhance the immunogenicity of these antigens, we formulate three nanovaccines by co-delivering molecular adjuvants (CpG and MPLA) and antigens (gHgL, gB or gp42). These nanovaccines induce robust humoral and cellular responses through efficient activation of dendritic cells and germinal center response. Importantly, these nanovaccines generate high levels of neutralizing antibodies recognizing vulnerable sites of all three antigens. IgGs induced by a cocktail vaccine containing three nanovaccines confer superior protection from lethal EBV challenge in female humanized mice compared to IgG elicited by individual NP-gHgL, NP-gB and NP-gp42. Importantly, serum antibodies elicited by cocktail nanovaccine immunization confer durable protection against EBV-associated lymphoma. Overall, the cocktail nanovaccine shows robust immunogenicity and is a promising candidate for further clinical trials.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Infecciones por Virus de Epstein-Barr , Glicoproteínas , Nanovacunas , Animales , Femenino , Humanos , Ratones , Adyuvantes Inmunológicos/administración & dosificación , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/prevención & control , Infecciones por Virus de Epstein-Barr/virología , Glicoproteínas/inmunología , Glicoproteínas/administración & dosificación , Herpesvirus Humano 4/inmunología , Linfoma/inmunología , Linfoma/virología , Nanovacunas/inmunologíaRESUMEN
Treatment options for Epstein-Barr virus (EBV)-cancers are limited, underscoring the need for new therapeutic approaches. We have previously shown that EBV-transformed cells and cancers lack homologous recombination (HR) repair, a prominent error-free pathway that repairs double-stranded DNA breaks; instead, EBV-transformed cells demonstrate genome-wide scars of the error-prone microhomology-mediated end joining (MMEJ) repair pathway. This suggests that EBV-cancers are vulnerable to synthetic lethal therapeutic approaches that target MMEJ repair. Indeed, we have previously found that targeting PARP, an enzyme that contributes to MMEJ, results in the death of EBV-lymphoma cells. With the emergence of clinical resistance to PARP inhibitors and the recent discovery of inhibitors of Polymerase theta (POLθ), the polymerase essential for MMEJ, we investigated the role of POLθ in EBV-lymphoma cells. We report that EBV-transformed cell lines, EBV-lymphoma cell lines, and EBV-lymphomas in AIDS patients demonstrate greater abundance of POLθ, driven by the EBV protein EBNA1, compared to EBV-uninfected primary lymphocytes and EBV-negative lymphomas from AIDS patients (a group that also abundantly expresses POLθ). We also find POLθ enriched at cellular DNA replication forks and exposure to the POLθ inhibitor Novobiocin impedes replication fork progress, impairs MMEJ-mediated repair of DNA double-stranded breaks, and kills EBV-lymphoma cells. Notably, cell killing is not due to Novobiocin-induced activation of the lytic/replicative phase of EBV. These findings support a role for POLθ not just in DNA repair but also DNA replication and as a therapeutic target in EBV-lymphomas and potentially other EBV-cancers as EBNA1 is expressed in all EBV-cancers.IMPORTANCEEpstein-Barr virus (EBV) contributes to ~2% of the global cancer burden. With a recent estimate of >200,000 deaths a year, identifying molecular vulnerabilities will be key to the management of these frequently aggressive and treatment-resistant cancers. Building on our earlier work demonstrating reliance of EBV-cancers on microhomology-mediated end-joining repair, we now report that EBV lymphomas and transformed B cell lines abundantly express the MMEJ enzyme POLθ that likely protects cellular replication forks and repairs replication-related cellular DNA breaks. Importantly also, we show that a newly identified POLθ inhibitor kills EBV-cancer cells, revealing a novel strategy to block DNA replication and repair of these aggressive cancers.
Asunto(s)
ADN Polimerasa theta , ADN Polimerasa Dirigida por ADN , Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Humanos , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , Infecciones por Virus de Epstein-Barr/virología , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades , Linfoma/virología , Linfoma/tratamiento farmacológico , Linfoma/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Roturas del ADN de Doble Cadena , Mutaciones Letales Sintéticas , Replicación del ADN/efectos de los fármacosRESUMEN
After infection of B cells, Epstein-Barr virus (EBV) engages host pathways that mediate cell proliferation and transformation, contributing to the propensity of the virus to drive immune dysregulation and lymphomagenesis. We found that the EBV protein EBNA2 initiates nicotinamide adenine dinucleotide (NAD) de novo biosynthesis by driving expression of the metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in infected B cells. Virus-enforced NAD production sustained mitochondrial complex I activity, to match adenosine triphosphate (ATP) production with bioenergetic requirements of proliferation and transformation. In transplant patients, IDO1 expression in EBV-infected B cells, and a serum signature of increased IDO1 activity, preceded development of lymphoma. In humanized mice infected with EBV, IDO1 inhibition reduced both viremia and lymphomagenesis. Virus-orchestrated NAD biosynthesis is therefore a druggable metabolic vulnerability of EBV-driven B cell transformation, opening therapeutic possibilities for EBV-related diseases.
Asunto(s)
Adenosina Trifosfato , Linfocitos B , Transformación Celular Viral , Infecciones por Virus de Epstein-Barr , Antígenos Nucleares del Virus de Epstein-Barr , Herpesvirus Humano 4 , Indolamina-Pirrol 2,3,-Dioxigenasa , NAD , Animales , Humanos , Ratones , Adenosina Trifosfato/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proliferación Celular , Complejo I de Transporte de Electrón/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Linfoma/virología , NAD/metabolismo , Proteínas Virales , ViremiaRESUMEN
Epstein-Barr virus (EBV) is responsible for many B cell lymphoproliferative disorders (LPD) spanning subclinical infection to immunodeficiency-related neoplasms. EBV establishes a latent infection in the host B cell as defined histologically by the expression of EBV latent membrane proteins and nuclear antigens. Herein, we characterize the latency patterns of immunodeficiency-related neoplasms including post-transplant lymphoproliferative disorders (PTLD) and therapy-related LPD (formerly iatrogenic) with latent membrane protein-1 (LMP-1) and EBV nuclear antigen-2 (EBNA-2) immunohistochemistry. The latency pattern was correlated with immunodeficiency and dysregulation (IDD) status and time from transplant procedure. 38 cases of EBV+ PTLD in comparison to 27 cases of classic Hodgkin lymphoma (CHL) and diffuse large B cell lymphoma (DLBCL) arising in either the therapy-related immunodeficiency setting (n = 12) or without an identified immunodeficiency (n = 15) were evaluated for EBV-encoded small RNAs by in situ hybridization (EBER-ISH) and for LMP-1 and EBNA-2 by immunohistochemistry. A full spectrum of EBV latency patterns was observed across PTLD in contrast to CHL and DLBCL arising in the therapy-related immunodeficiency setting. Polymorphic-PTLD (12 of 16 cases, 75 %) and DLBCL-PTLD (9 of 11 cases, 82 %) showed the greatest proportion of cases with latency III pattern. Whereas, EBV+ CHL in an immunocompetent patient showed exclusively latency II pattern (13 of 13 cases, 100 %). The majority of EBV+ PTLD occurred by three years of transplant procedure date and were enriched for latency III pattern (21 of 22 cases, 95 %). Immunohistochemical identification of EBV latency by LMP-1 and EBNA-2 can help classify PTLD in comparison to other EBV+ B cell LPD and lymphomas arising in therapy-related immunodeficiency and non-immunodeficiency settings.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Antígenos Nucleares del Virus de Epstein-Barr , Herpesvirus Humano 4 , Enfermedad de Hodgkin , Linfoma de Células B Grandes Difuso , Trastornos Linfoproliferativos , Proteínas de la Matriz Viral , Proteínas Virales , Latencia del Virus , Humanos , Trastornos Linfoproliferativos/virología , Trastornos Linfoproliferativos/patología , Trastornos Linfoproliferativos/diagnóstico , Herpesvirus Humano 4/aislamiento & purificación , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/complicaciones , Masculino , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Femenino , Adulto , Persona de Mediana Edad , Proteínas de la Matriz Viral/metabolismo , Enfermedad de Hodgkin/virología , Enfermedad de Hodgkin/patología , Linfoma de Células B Grandes Difuso/virología , Linfoma de Células B Grandes Difuso/patología , Anciano , Adulto Joven , Adolescente , Inmunohistoquímica , Niño , Linfoma/virología , Linfoma/patología , Hibridación in SituRESUMEN
The pronounced growth in livestock populations since the 1950s has altered the epidemiological and evolutionary trajectory of their associated pathogens. For example, Marek's disease virus (MDV), which causes lymphoid tumors in chickens, has experienced a marked increase in virulence over the past century. Today, MDV infections kill >90% of unvaccinated birds, and controlling it costs more than US$1 billion annually. By sequencing MDV genomes derived from archeological chickens, we demonstrate that it has been circulating for at least 1000 years. We functionally tested the Meq oncogene, one of 49 viral genes positively selected in modern strains, demonstrating that ancient MDV was likely incapable of driving tumor formation. Our results demonstrate the power of ancient DNA approaches to trace the molecular basis of virulence in economically relevant pathogens.
Asunto(s)
Pollos , Herpesvirus Gallináceo 2 , Enfermedad de Marek , Animales , Pollos/virología , Herpesvirus Gallináceo 2/clasificación , Herpesvirus Gallináceo 2/genética , Herpesvirus Gallináceo 2/patogenicidad , Linfoma/virología , Enfermedad de Marek/historia , Enfermedad de Marek/virología , Virulencia/genética , FilogeniaRESUMEN
Circular RNAs (circRNAs) are a recently rediscovered class of functional noncoding RNAs that are involved in gene regulation and cancer development. Next-generation sequencing approaches identified circRNA fragments and sequences underlying circularization events in virus-induced cancers. In the present study, we performed viral circRNA expression analysis and full-length sequencing in infections with Marek's disease virus (MDV), which serves as a model for herpesvirus-induced tumorigenesis. We established inverse PCRs to identify and characterize circRNA expression from the repeat regions of the MDV genome during viral replication, latency, and reactivation. We identified a large variety of viral circRNAs through precise mapping of full-length circular transcripts and detected matching sequences with several viral genes. Hot spots of circRNA expression included the transcriptional unit of the major viral oncogene encoding the Meq protein and the latency-associated transcripts (LATs). Moreover, we performed genome-wide bioinformatic analyses to extract back-splice junctions from lymphoma-derived samples. Using this strategy, we found that circRNAs were abundantly expressed in vivo from the same key virulence genes. Strikingly, the observed back-splice junctions do not follow a unique canonical pattern, compatible with the U2-dependent splicing machinery. Numerous noncanonical junctions were observed in viral circRNA sequences characterized from in vitro and in vivo infections. Given the importance of the genes involved in the transcription of these circRNAs, our study contributes to our understanding and complexity of this deadly pathogen. IMPORTANCE Circular RNAs (circRNAs) were rediscovered in recent years both in physiological and pathological contexts, such as in cancer. Viral circRNAs are encoded by at least two human herpesviruses, the Epstein Barr virus and the Kaposi's Sarcoma-associated herpesvirus, both associated with the development of lymphoma. Marek's disease virus (MDV) is a well-established animal model to study virus-induced lymphoma but circRNA expression has not been reported for MDV yet. Our study provided the first evidence of viral circRNAs that were expressed at key steps of the MDV lifecycle using genome-wide analyses of circRNAs. These circRNAs were primarily found in transcriptional units that corresponded to the major MDV virulence factors. In addition, we established a bioinformatics pipeline that offers a new tool to identify circular RNAs in other herpesviruses. This study on the circRNAs provided important insights into major MDV virulence genes and herpesviruses-mediated gene dysregulation.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Herpesvirus Gallináceo 2 , Enfermedad de Marek , ARN Circular , Animales , Pollos , Estudio de Asociación del Genoma Completo , Herpesvirus Gallináceo 2/genética , Herpesvirus Gallináceo 2/patogenicidad , Linfoma/virología , Enfermedad de Marek/virología , Proteínas Oncogénicas Virales/genética , ARN Circular/genética , ARN no Traducido/genética , Virulencia/genéticaRESUMEN
Epstein-Barr virus (EBV) is reportedly the first identified human tumor virus, and is closely related to the occurrence and development of nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), and several lymphomas. PD-L1 expression is elevated in EBV-positive NPC and GC tissues; however, the specific mechanisms underlying the EBV-dependent promotion of PD-L1 expression to induce immune escape warrant clarification. EBV encodes 44 mature miRNAs. In this study, we find that EBV-miR-BART11 and EBV-miR-BART17-3p upregulate the expression of PD-L1 in EBV-associated NPC and GC. Furthermore, EBV-miR-BART11 targets FOXP1, EBV-miR-BART17-3p targets PBRM1, and FOXP1 and PBRM1 bind to the enhancer region of PD-L1 to inhibit its expression. Therefore, EBV-miR-BART11 and EBV-miR-BART17-3p inhibit FOXP1 and PBRM1, respectively, and enhance the transcription of PD-L1 (CD274, http://www.ncbi.nlm.nih.gov/gene/29126 ), resulting in the promotion of tumor immune escape, which provides insights into potential targets for EBV-related tumor immunotherapy.
Asunto(s)
Herpesvirus Humano 4/genética , MicroARNs/genética , Carcinoma Nasofaríngeo/inmunología , Neoplasias Nasofaríngeas/inmunología , Neoplasias Gástricas/inmunología , Escape del Tumor/inmunología , Antígeno B7-H1/metabolismo , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Herpesvirus Humano 4/inmunología , Humanos , Linfoma/inmunología , Linfoma/virología , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/virología , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Neoplasias Gástricas/virología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Escape del Tumor/genética , Microambiente Tumoral/inmunologíaRESUMEN
Marek's disease virus (MDV) is a member of the genus Mardivirus in the subfamily Alphaherpesvirinae. There are three different serotypes of MDV designated as MDV-1 (Gallid herpesvirus type 2), MDV-2 (Gallid herpesvirus type 3), and MDV-3 (Meleagrid herpesvirus 1, herpesvirus of turkeys, HVT). MDV-1 is the only serotype that induces Marek's disease (MD), a lymphoproliferative disorder resulting in aggressive T-cell lymphomas and paralytic symptoms. In the lymphomas and lymphoblastoid cell lines (LCL) derived from them, MDV establishes latent infection with limited viral gene expression. The latent viral genome in LCL can be activated by co-cultivation with chicken embryo fibroblast (CEF) monolayers. MSB-1, one of the first MDV-transformed LCL established from the splenic lymphoma, is distinct in harboring both the oncogenic MDV-1 and non-oncogenic MDV-2 viruses. Following the successful application of CRISPR/Cas9 editing approach for precise knockdown of the MDV-1 genes in LCL, we describe here the targeted deletion of MDV-2 glycoprotein B (gB) in MSB-1 cells. Due to the essential nature of gB for infectivity, the production of MDV-2 plaques on CEF was completely abolished in the MDV-2-gB-deleted MSB-1 cells. Our study has demonstrated that the CRISPR/Cas9 system can be used for targeted inactivation of the co-infecting MDV-2 without affecting the MDV-1 in the MSB-1 cell line. Successful inactivation of MDV-2 demonstrated here also points toward the possibility of using targeted gene editing as an antiviral strategy against pathogenic MDV-1 and other viruses infecting chickens. IMPORTANCE Marek's disease (MD) is a lymphoproliferative disease of chickens characterized by rapid-onset lymphomas in multiple organs and by infiltration into peripheral nerves, causing paralysis. Lymphoblastoid cell lines (LCL) derived from MD lymphomas have served as valuable resources to improve understanding of distinct aspects of virus-host interactions in transformed cells including transformation, latency, and reactivation. MDV-transformed LCL MSB-1, derived from spleen lymphoma induced by the BC-1 strain of MDV, has a unique feature of harboring an additional non-pathogenic MDV-2 strain HPRS-24. By targeted deletion of essential gene glycoprotein B from the MDV-2 genome within the MSB-1 cells, we demonstrated the total inhibition of MDV-2 virus replication on co-cultivated CEF, with no effect on MDV-1 replication. The identified viral genes critical for reactivation/inhibition of viruses will be useful as targets for development of de novo disease resistance in chickens to avian pathogens.
Asunto(s)
Herpesvirus Gallináceo 3 , Linfoma , Enfermedad de Marek , Proteínas del Envoltorio Viral , Animales , Sistemas CRISPR-Cas , Línea Celular , Embrión de Pollo , Pollos , Herpesvirus Gallináceo 3/genética , Linfoma/veterinaria , Linfoma/virología , Proteínas del Envoltorio Viral/genéticaRESUMEN
The open reading frame 50 (ORF50) protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is the master regulator essential for initiating the viral lytic cycle. Previously, we have demonstrated that the ORF50 protein can cooperate with Sp3 to synergistically activate a set of viral and cellular gene promoters through highly conserved ORF50-responsive elements that harbor a Sp3-binding motif. Herein, we show that Sp3 undergoes proteolytic cleavage during the viral lytic cycle, and the cleavage of Sp3 is dependent on caspase activation. Since similar cleavage patterns of Sp3 could be detected in both KSHV-positive and KSHV-negative lymphoma cells undergoing apoptosis, the proteolytic cleavage of Sp3 could be a common event during apoptosis. Mutational analysis identifies 12 caspase cleavage sites in Sp3, which are situated at the aspartate (D) positions D17, D19, D180, D273, D275, D293, D304 (or D307), D326, D344, D530, D543, and D565. Importantly, we noticed that three stable Sp3 C-terminal fragments generated through cleavage at D530, D543, or D565 encompass an intact DNA-binding domain. Like the full-length Sp3, the C-terminal fragments of Sp3 could still retain the ability to cooperate with ORF50 protein to activate specific viral and cellular gene promoters synergistically. Collectively, our findings suggest that despite the proteolytic cleavage of Sp3 under apoptotic conditions, the resultant Sp3 fragments may retain biological activities important for the viral lytic cycle or for cellular apoptosis. IMPORTANCE The ORF50 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is the key viral protein that controls the switch from latency to lytic reactivation. It is a potent transactivator that can activate target gene promoters via interacting with other cellular DNA-binding transcription factors, such as Sp3. In this report, we show that Sp3 is proteolytically cleaved during the viral lytic cycle, and up to 12 caspase cleavage sites are identified in Sp3. Despite the proteolytic cleavage of Sp3, several resulting C-terminal fragments that have intact zinc-finger DNA-binding domains still retain substantial influence in the synergy with ORF50 to activate specific gene promoters. Overall, our studies elucidate the caspase-mediated cleavage of Sp3 and uncover how ORF50 utilizes the cleavage fragments of Sp3 to transactivate specific viral and cellular gene promoters.
Asunto(s)
Caspasas/metabolismo , Infecciones por Herpesviridae/metabolismo , Herpesvirus Humano 8/fisiología , Factor de Transcripción Sp3/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Apoptosis , Caspasas/genética , Regulación Viral de la Expresión Génica , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/fisiopatología , Infecciones por Herpesviridae/virología , Herpesvirus Humano 8/genética , Interacciones Huésped-Patógeno , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Linfoma/genética , Linfoma/metabolismo , Linfoma/fisiopatología , Linfoma/virología , Alineación de Secuencia , Factor de Transcripción Sp3/química , Factor de Transcripción Sp3/genética , Transactivadores/genética , Transactivadores/metabolismo , Latencia del VirusRESUMEN
Few data exist on Epstein-Barr virus (EBV) prevalence across the full spectrum of lymphoma subtypes, particularly in sub-Saharan Africa. The objective of our study was to test the presence of EBV in a nationally representative sample of malignant lymphomas diagnosed in the Butaro Cancer Center of Excellence (BCCOE) in Rwanda. Of 102 Hodgkin (HL) and 378 non-Hodgkin lymphomas (NHL) diagnosed in BCCOE between 2012 and 2018, 52 HL and 207 NHL were successfully tested by EBV-encoding RNA in situ hybridization. EBV prevalence was 54% in HL, being detected in all classical HL subtypes: mixed-cellularity (n = 3/8), nodular-sclerosis (n = 7/17) and lymphocyte-rich (n = 2/3). EBV prevalence was 9% in NHL, being 10% among 158 B-cell NHL, 3% among 35 T-cell NHL and the single NK-cell NHL was EBV-positive. Among B-cell NHL, EBV was present in the majority of Burkitt (n = 8/13), and was also rarely detected in follicular (n = 1/4) and acute B-cell lymphoblastic (n = 1/45) lymphomas. Five of the 45 (11%) diffuse large B-cell lymphomas (DLBCLs) were EBV-positive, including three out of five plasmablastic lymphoma (PBL). Of 39 HL and 163 NHL of known human immunodeficiency virus (HIV) status, 2 (5%) and 14 (9%) were HIV-positive, respectively, of which only four were also EBV-positive (2 PBL, 2 HL). In summary, we report rare regional-level data on the association of EBV with classical HL, Burkitt and DLBCLs, and report sporadic detection in other subtypes possibly related to EBV. Such data inform the burden of disease caused by EBV and can help guide application of future advances in EBV-specific prevention and therapeutics.
Asunto(s)
Herpesvirus Humano 4/aislamiento & purificación , Linfoma/virología , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Linfoma/clasificación , Linfoma/etiología , Masculino , Persona de Mediana Edad , ARN Viral/análisis , Rwanda , Factores de Tiempo , Adulto JovenRESUMEN
Hepatitis B virus (HBV) reactivation is a common complication in chronic or resolved HBV infection patients undergoing immunosuppressive chemotherapy. Furthermore, few articles have been published regarding the risk of HBV reactivation in lymphoma patients receiving chimeric antigen receptor (CAR) T-cell therapy and anti-HBV prophylaxis. Few guidelines or clear optimal strategies are available for managing these patients. Here, we present two cases of patients who underwent CAR-T-cell cocktail therapy with anti-CD19 and anti-CD22 CAR (CAR19/22) T cell for lymphoma. Patients had previous history of HBV infection, and blood tests on initial admission indicated positive results for hepatitis B surface antigen (HBsAg), antibody to hepatitis B core antigen (anti-HBc), and antibody to hepatitis B e antigen (anti-HBe), while serum HBV DNA level was undetectable. Therefore, two patients received entecavir as antiviral prophylactic therapy during their entire treatment. They were diagnosed with HBV reactivation based on positive serum HBV DNA test results, 2 weeks after CAR-T-cell infusion. Liver function assay indicated elevated levels of alanine transaminase (ALT) and aspartate transaminase (AST), combined with increased levels of total bilirubin (TBIL) and direct bilirubin (DBIL). Subsequently, they received anti-HBV treatment with entecavir and tenofovir. As a result, their serum HBV DNA copies and AST/ALT levels returned to normal after 1 week. These cases show that there is a risk of HBV reactivation in lymphoma patients with CAR-T-cell therapy despite entecavir preventive therapy, and combination treatment of entecavir and tenofovir may be an effective treatment option for such patients with HBV reactivation.
Asunto(s)
Antivirales/uso terapéutico , Guanina/análogos & derivados , Hepatitis B/prevención & control , Inmunoterapia Adoptiva , Infección Latente/prevención & control , Linfoma/terapia , Femenino , Guanina/uso terapéutico , Virus de la Hepatitis B , Humanos , Infusiones Intravenosas , Linfoma/virología , Masculino , Persona de Mediana Edad , Receptores Quiméricos de Antígenos , Insuficiencia del TratamientoRESUMEN
Recently, two cases of complete remission of classical Hodgkin lymphoma (cHL) and follicular lymphoma (FL) after SARS-CoV-2 infection were reported. However, the precise molecular mechanism of this rare event is yet to be understood. Here, we hypothesize a potential anti-tumor immune response of SARS-CoV-2 and based on a computational approach show that: (i) SARS-CoV-2 Spike-RBD may bind to the extracellular domains of CD15, CD27, CD45, and CD152 receptors of cHL or FL and may directly inhibit cell proliferation. (ii) Alternately, upon internalization after binding to these CD molecules, the SARS-CoV-2 membrane (M) protein and ORF3a may bind to gamma-tubulin complex component 3 (GCP3) at its tubulin gamma-1 chain (TUBG1) binding site. (iii) The M protein may also interact with TUBG1, blocking its binding to GCP3. (iv) Both the M and ORF3a proteins may render the GCP2-GCP3 lateral binding where the M protein possibly interacts with GCP2 at its GCP3 binding site and the ORF3a protein to GCP3 at its GCP2 interacting residues. (v) Interactions of the M and ORF3a proteins with these gamma-tubulin ring complex components potentially block the initial process of microtubule nucleation, leading to cell-cycle arrest and apoptosis. (vi) The Spike-RBD may also interact with and block PD-1 signaling similar to pembrolizumab and nivolumab- like monoclonal antibodies and may induce B-cell apoptosis and remission. (vii) Finally, the TRADD interacting "PVQLSY" motif of Epstein-Barr virus LMP-1, that is responsible for NF-kB mediated oncogenesis, potentially interacts with SARS-CoV-2 Mpro, NSP7, NSP10, and spike (S) proteins, and may inhibit the LMP-1 mediated cell proliferation. Taken together, our results suggest a possible therapeutic potential of SARS-CoV-2 in lymphoproliferative disorders.
Asunto(s)
COVID-19/metabolismo , Linfoma/inmunología , SARS-CoV-2/inmunología , Anticuerpos Monoclonales/inmunología , Antineoplásicos/farmacología , Sitios de Unión , COVID-19/complicaciones , Glicoproteínas/metabolismo , Glicoproteínas/ultraestructura , Humanos , Inmunidad/inmunología , Linfoma/terapia , Linfoma/virología , Modelos Teóricos , Simulación del Acoplamiento Molecular , Unión Proteica , Dominios Proteicos , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Proteínas Viroporinas/metabolismo , Proteínas Viroporinas/ultraestructuraRESUMEN
Among the mechanisms leading to progression to Adult T-cell Leukaemia/Lymphoma in Human T-cell Leukaemia Virus type 1 (HTLV-1)-infected subjects, the contribution of stromal components remains poorly understood. To dissect the role of fibroblasts in HTLV-1-mediated lymphomagenesis, transcriptome studies, cytofluorimetric and qRT-PCR analyses of surface and intracellular markers linked to plasticity and stemness in coculture, and in vivo experiments were performed. A transcriptomic comparison between a more lymphomagenic (C91/III) and the parental (C91/PL) cell line evidenced hyperactivation of the PI3K/Akt pathway, confirmed by phospho-ELISA and 2-DE and WB analyses. C91/III cells also showed higher expression of mesenchymal and stemness genes. Short-term coculture with human foreskin fibroblasts (HFF) induced these features in C91/PL cells, and significantly increased not only the cancer stem cells (CSCs)-supporting CD10+GPR77+ HFF subpopulation, but also the percentage of ALDH1bright C91/PL cells. A non-cytotoxic acetylsalicylic acid treatment decreased HFF-induced ALDH1bright C91/PL cells, downregulated mesenchymal and stemness genes in cocultured cells, and delayed lymphoma growth in immunosuppressed mice, thus hindering the supportive activity of HFF on CSCs. These data suggest that crosstalk with HFF significantly intensifies the aggressiveness and plasticity of C91/PL cells, leading to the enrichment in lymphoma-initiating cells. Additional research is needed to better characterize these preliminary findings.
Asunto(s)
Fibroblastos/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Linfoma/genética , Células Madre Neoplásicas/metabolismo , Animales , Antiinflamatorios no Esteroideos/farmacología , Aspirina/farmacología , Línea Celular , Células Cultivadas , Técnicas de Cocultivo , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Virus Linfotrópico T Tipo 1 Humano/fisiología , Humanos , Células Jurkat , Linfoma/tratamiento farmacológico , Linfoma/virología , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/virología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The avian α-herpesvirus known as Marek's disease virus (MDV) linearly integrates its genomic DNA into host telomeres during infection. The resulting disease, Marek's disease (MD), is characterized by virally-induced lymphomas with high mortality. The temporal dynamics of MDV-positive (MDV+) transformed cells and expansion of MD lymphomas remain targets for further understanding. It also remains to be determined whether specific host chromosomal sites of MDV telomere integration confer an advantage to MDV-transformed cells during tumorigenesis. We applied MDV-specific fluorescence in situ hybridization (MDV FISH) to investigate virus-host cytogenomic interactions within and among a total of 37 gonad lymphomas and neoplastic splenic samples in birds infected with virulent MDV. We also determined single-cell, chromosome-specific MDV integration profiles within and among transformed tissue samples, including multiple samples from the same bird. Most mitotically-dividing cells within neoplastic samples had the cytogenomic phenotype of 'MDV telomere-integrated only', and tissue-specific, temporal changes in phenotype frequencies were detected. Transformed cell populations composing gonad lymphomas exhibited significantly lower diversity, in terms of heterogeneity of MDV integration profiles, at the latest stages of tumorigenesis (>50 days post-infection (dpi)). We further report high interindividual and lower intraindividual variation in MDV integration profiles of lymphoma cells. There was no evidence of integration hotspots into a specific host chromosome(s). Collectively, our data suggests that very few transformed MDV+ T cell populations present earlier in MDV-induced lymphomas (32-50 dpi), survive, and expand to become the dominant clonal population in more advanced MD lymphomas (51-62 dpi) and establish metastatic lymphomas.
Asunto(s)
Herpesvirus Gallináceo 2/genética , Linfoma/genética , Enfermedad de Marek/genética , Enfermedades de las Aves de Corral/genética , Animales , Carcinogénesis/genética , Pollos/genética , Pollos/virología , Herpesvirus Gallináceo 2/patogenicidad , Interacciones Huésped-Patógeno/genética , Hibridación Fluorescente in Situ , Linfoma/etiología , Linfoma/patología , Linfoma/virología , Enfermedad de Marek/complicaciones , Enfermedad de Marek/patología , Enfermedad de Marek/virología , Enfermedades de las Aves de Corral/virología , Neoplasias del Bazo/etiología , Neoplasias del Bazo/genética , Neoplasias del Bazo/patología , Linfocitos T/virología , Telómero/genética , Telómero/virología , Integración Viral/genéticaRESUMEN
Epstein-Barr virus (EBV) and cytomegalovirus (CMV) are viruses globally distributed that have been associated with the development and prognosis of many pathologies, including hematological diseases. This study aimed to characterize the epidemiological profile of EBV infection and the infection-correlated hepatic manifestations in patients with hematological diseases of the northern Brazilian state of Amazonas. A total of 228 patients were serologically tested for the presence of anti-EBV and anti-CMV IgG antibodies through an enzyme-linked immunosorbent assay. The coinfection with CMV, sociodemographic and laboratory records of all patients were also assessed. The overall prevalence observed among the study population for EBV infection and EBV/CMV coinfection was 85.09% (95% CI: 0.80-0.90) and 78.51% (95% CI: 0.73-0.84), respectively. The age group 31-40 years old were more susceptible to EBV/CMV coinfection (95% CI: 1.59-93.41, p = 0.011), while young people aged 1-10 years old were less affected for both EBV infection (CI 95%; 0.66-0.91, p = 0.001) and EBV/CMV coinfection (95% CI: 0.52-0.81, p < 0.0001). High serum levels of the liver biomarker ferritin were associated with EBV infection (95% CI: 1.03-1.54, p = 0.031) and EBV/CMV coinfection (95% CI: 1.02-1.70, p = 0.038). Our findings indicated that the elevated prevalence of EBV infection is not associated with the hematological diseases or transfusion rates, but with the socioeconomic status of the study population. Also, this study suggests that the EBV infection and its coinfection with CMV are related to the increase of serum ferritin levels.
Asunto(s)
Anemia/epidemiología , Anticuerpos Antivirales/sangre , Infecciones por Citomegalovirus/epidemiología , Infecciones por Virus de Epstein-Barr/epidemiología , Ferritinas/sangre , Leucemia/epidemiología , Linfoma/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anemia/inmunología , Anemia/patología , Anemia/virología , Biomarcadores/sangre , Transfusión Sanguínea/estadística & datos numéricos , Brasil/epidemiología , Niño , Preescolar , Coinfección , Citomegalovirus/crecimiento & desarrollo , Citomegalovirus/patogenicidad , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/patología , Infecciones por Citomegalovirus/virología , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/patología , Infecciones por Virus de Epstein-Barr/virología , Femenino , Herpesvirus Humano 4/crecimiento & desarrollo , Herpesvirus Humano 4/patogenicidad , Humanos , Inmunoglobulina G/sangre , Lactante , Recién Nacido , Leucemia/inmunología , Leucemia/patología , Leucemia/virología , Hígado/inmunología , Hígado/patología , Hígado/virología , Linfoma/inmunología , Linfoma/patología , Linfoma/virología , Masculino , Persona de Mediana Edad , Prevalencia , Clase SocialRESUMEN
Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with the development of hematopoietic cancers of B-lymphocyte origin, including AIDS-related non-Hodgkin lymphoma (AIDS-NHL). Primary infection of B-cells with EBV results in their polyclonal activation and immortalization. The transferrin receptor 1 (TfR1), also known as CD71, is important for iron uptake and regulation of cellular proliferation. TfR1 is highly expressed in proliferating cells, including activated lymphocytes and malignant cells. We developed a mouse/human chimeric antibody targeting TfR1 (ch128.1/IgG1) that has previously shown significant antitumor activity in immunosuppressed mouse models bearing human malignant B-cells, including multiple myeloma and AIDS-NHL cells. In this article, we examined the effect of targeting TfR1 to inhibit EBV-driven activation and growth of human B-cells in vivo using an immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ [NOD/SCID gamma (NSG)] mouse model. Mice were implanted with T-cell-depleted, human peripheral blood mononuclear cells (PBMCs), either without EBV (EBV-), or exposed to EBV in vitro (EBV+), intravenously via the tail vein. Mice implanted with EBV+ cells and treated with an IgG1 control antibody (400 µg/mouse) developed lymphoma-like growths of human B-cell origin that were EBV+, whereas mice implanted with EBV+ cells and treated with ch128.1/IgG1 (400 µg/mouse) showed increased survival and significantly reduced inflammation and B-cell activation. These results indicate that ch128.1/IgG1 is effective at preventing the growth of EBV+ human B-cell tumors in vivo, thus, indicating that there is significant potential for agents targeting TfR1 as therapeutic strategies to prevent the development of EBV-associated B-cell malignancies. SIGNIFICANCE: An anti-TfR1 antibody, ch128.1/IgG1, effectively inhibits the activation, growth, and immortalization of EBV+ human B-cells in vivo, as well as the development of these cells into lymphoma-like tumors in immunodeficient mice.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Linfocitos B/inmunología , Infecciones por Virus de Epstein-Barr/complicaciones , Inmunoglobulina G/inmunología , Linfoma/tratamiento farmacológico , Receptores de Transferrina/inmunología , Linfocitos T/inmunología , Animales , Apoptosis , Linfocitos B/metabolismo , Linfocitos B/patología , Proliferación Celular , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4 , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos , Linfoma/patología , Linfoma/virología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales CultivadasRESUMEN
Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that typically causes asymptomatic infection but can promote B lymphoid tumors in the immune suppressed. In vitro, EBV infection of primary B cells stimulates glycolysis during immortalization into lymphoblastoid cell lines (LCLs). Lactate export during glycolysis is crucial for continued proliferation of many cancer cells-part of a phenomenon known as the "Warburg effect"- and is mediated by monocarboxylate transporters (MCTs). However, the role of MCTs has yet to be studied in EBV-associated malignancies, which display Warburg-like metabolism in vitro. Here, we show that EBV infection of B lymphocytes directly promotes temporal induction of MCT1 and MCT4 through the viral proteins EBNA2 and LMP1, respectively. Functionally, MCT1 was required for early B cell proliferation, and MCT4 up-regulation promoted acquired resistance to MCT1 antagonism in LCLs. However, dual MCT1/4 inhibition led to LCL growth arrest and lactate buildup. Metabolic profiling in LCLs revealed significantly reduced oxygen consumption rates (OCRs) and NAD+/NADH ratios, contrary to previous observations of increased OCR and unaltered NAD+/NADH ratios in MCT1/4-inhibited cancer cells. Furthermore, U-13C6-glucose labeling of MCT1/4-inhibited LCLs revealed depleted glutathione pools that correlated with elevated reactive oxygen species. Finally, we found that dual MCT1/4 inhibition also sensitized LCLs to killing by the electron transport chain complex I inhibitors phenformin and metformin. These findings were extended to viral lymphomas associated with EBV and the related gammaherpesvirus KSHV, pointing at a therapeutic approach for targeting both viral lymphomas.
Asunto(s)
Linfoma/metabolismo , Linfoma/virología , Transportadores de Ácidos Monocarboxílicos/antagonistas & inhibidores , Linfocitos B/virología , Línea Celular Tumoral , Proliferación Celular , Infecciones por Virus de Epstein-Barr/virología , Glucosa/metabolismo , Glutatión/metabolismo , Herpesvirus Humano 4/fisiología , Herpesvirus Humano 8/fisiología , Humanos , Ácido Láctico/metabolismo , Linfoma/patología , Metformina/farmacología , NAD/metabolismo , Consumo de Oxígeno , Fenformina/farmacología , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia ArribaRESUMEN
Subpopulations of B-lymphocytes traffic to different sites and organs to provide diverse and tissue-specific functions. Here, we provide evidence that epigenetic differences confer a neuroinvasive phenotype. An EBV+ B cell lymphoma cell line (M14) with low frequency trafficking to the CNS was neuroadapted to generate a highly neuroinvasive B-cell population (MUN14). MUN14 B cells efficiently infiltrated the CNS within one week and produced neurological pathologies. We compared the gene expression profiles of viral and cellular genes using RNA-Seq and identified one viral (EBNA1) and several cellular gene candidates, including secreted phosphoprotein 1/osteopontin (SPP1/OPN), neuron navigator 3 (NAV3), CXCR4, and germinal center-associated signaling and motility protein (GCSAM) that were selectively upregulated in MUN14. ATAC-Seq and ChIP-qPCR revealed that these gene expression changes correlated with epigenetic changes at gene regulatory elements. The neuroinvasive phenotype could be attenuated with a neutralizing antibody to OPN, confirming the functional role of this protein in trafficking EBV+ B cells to the CNS. These studies indicate that B-cell trafficking to the CNS can be acquired by epigenetic adaptations and provide a new model to study B-cell neuroinvasion associated CNS lymphoma and autoimmune disease of the CNS, including multiple sclerosis (MS).
Asunto(s)
Linfocitos B/patología , Linfocitos B/virología , Neoplasias del Sistema Nervioso Central/virología , Epigénesis Genética , Infecciones por Virus de Epstein-Barr/patología , Animales , Linfocitos B/metabolismo , Transformación Celular Viral/fisiología , Neoplasias del Sistema Nervioso Central/metabolismo , Neoplasias del Sistema Nervioso Central/patología , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4 , Linfoma/metabolismo , Linfoma/patología , Linfoma/virología , Ratones , Osteopontina/metabolismoRESUMEN
Immune checkpoint inhibitor (ICI) immunotherapies have vastly improved therapeutic outcomes for patients with certain cancer types, but these responses only manifest in a small percentage of all cancer patients. The goal of the present study was to improve checkpoint therapy efficacy by utilizing an engineered vaccinia virus to improve the trafficking of lymphocytes to the tumor, given that such lymphocyte trafficking is positively correlated with patient checkpoint inhibitor response rates. We developed an oncolytic vaccinia virus (OVV) platform expressing manganese superoxide dismutase (MnSOD) for use as both a monotherapy and together with anti-PD-L1. Intratumoral OVV-MnSOD injection in immunocompetent mice resulted in inflammation within poorly immunogenic tumors, thereby facilitating marked tumor regression. OVV-MnSOD administration together with anti-PD-L1 further improved antitumor therapy outcomes in models in which these monotherapy approaches were ineffective. Overall, our results emphasize the value of further studying these therapeutic approaches in patients with minimally or non-inflammatory tumors.
Asunto(s)
Antígeno B7-H1/antagonistas & inhibidores , Inhibidores de Puntos de Control Inmunológico/farmacología , Linfocitos Infiltrantes de Tumor/virología , Linfoma/terapia , Viroterapia Oncolítica , Superóxido Dismutasa/metabolismo , Virus Vaccinia/enzimología , Animales , Antígeno B7-H1/inmunología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Linfocitos Infiltrantes de Tumor/enzimología , Linfocitos Infiltrantes de Tumor/inmunología , Linfoma/enzimología , Linfoma/inmunología , Linfoma/virología , Ratones Endogámicos C57BL , Superóxido Dismutasa/genética , Carga Tumoral , Microambiente Tumoral/inmunología , Virus Vaccinia/genética , Virus Vaccinia/patogenicidadRESUMEN
The risk of malignant lymphomas is markedly increased in HIV-infected persons even in the era of effective combination antiretroviral therapy. Major risk factors are the depth of CD4-cell nadir and HIV viremia. R-CHOP remains treatment of choice for diffuse large B-cell lymphoma. In HIV-Burkitt lymphoma CODOX-M/IVAC proved superior to EPOCH in a large retrospective study. Standard treatment for plasmablastic or primary effusion lymphoma has not yet been defined. Favourable results have been reported with a stage adapted treatment for HIV-Hodgkin lymphoma. Patients with relapsed/refractory HIV-lymphoma should undergo autologous stem cell transplantation if indicated. Successful anti-CD19 CAR T-cell therapy was also reported in HIV-infected patients with refractory B-cell lymphoma.