RESUMO
OBJECTIVES: Pelareorep (reolysin), a Dearing strain of reovirus serotype 3, has demonstrated oncolytic activity as single agent and synergy with chemotherapy. We evaluated pelareorep, combined with standard second-line chemotherapy in patients with non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: This randomized phase II trial enrolled patients with advanced or metastatic NSCLC after first line chemotherapy. After a safety run-in, patients were randomized 1:1 to chemotherapy (pemetrexed [500â¯mg/m2, non-squamous], or docetaxel [75â¯mg/m2], day 1 every 21â¯days]) +/- pelareorep (4.5â¯×â¯1010 TCID50, days 1-3 every 21â¯days), stratified by EGFR mutation status. The primary outcome was progression free survival (PFS) of patients randomized to chemotherapyâ¯+â¯pelareorep vs. chemotherapy alone. Secondary outcomes included overall survival, objective response rate and exploratory translational analyses. RESULTS: Between October 2012 and August 2015, 166 patients were enrolled (14 to the safety run in). Pelareorep did not improve the PFS vs. single agent chemotherapy (median PFS 3.0 months, 95% confidence interval [CI] 2.6-4.1) vs. 2.8 months (95% CI 2.5-4.0), hazard ratio (HR) 0.90 (95% CI 0.65-1.25), Pâ¯=â¯0.53). Neither KRAS or EGFR mutation was associated with improved PFS, but STK11 mutations did appear to have an association with improved PFS (HR 0.29 [0.12-0.67); as did PIK3CA mutation (HR 0.45 [0.22-0.93]). The combination was tolerable, although associated with increased rates of neutropenic fever. CONCLUSION: The addition of pelareorep to second-line chemotherapy did not improve the PFS of patients with NSCLC. The three-day pelareorep schedule was tolerable. Further research is needed to evaluate the potential benefit in molecular subtypes of NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/terapia , Orthoreovirus Mamífero 3/imunologia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos , Infecções por Reoviridae/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Canadá , Terapia Combinada , Docetaxel/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pemetrexede/uso terapêutico , Recidiva , Terapia de Salvação , Adulto JovemRESUMO
Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease.IMPORTANCE Acute viral infections are thought to be cleared by the host with few lasting consequences. However, there may be much broader and long-lasting effects of viruses on immune homeostasis. Infection with reovirus, a common, nonpathogenic virus, triggers inflammation against innocuous food antigens, implicating this virus in the development of celiac disease, an autoimmune intestinal disorder triggered by exposure to dietary gluten. Using two reovirus strains that differ in the capacity to abrogate oral tolerance, we found that strain-specific differences in the capacity to replicate in the intestine inversely correlate with the capacity to induce apoptotic death of intestinal epithelial cells, providing a host-mediated process to restrict intestinal infection. This work contributes new knowledge about virus-host interactions in the intestine and establishes a foundation for future studies to define mechanisms by which viruses break oral tolerance in celiac disease.
Assuntos
Apoptose/imunologia , Células Epiteliais/imunologia , Mucosa Intestinal/imunologia , Orthoreovirus Mamífero 3/imunologia , Orthoreovirus de Mamíferos/imunologia , Infecções por Reoviridae/imunologia , Animais , Antígenos Virais/imunologia , Linhagem Celular , Cricetinae , Células Epiteliais/patologia , Células Epiteliais/virologia , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Camundongos , Infecções por Reoviridae/patologiaRESUMO
Epithelial cells are a major port of entry for many viruses, but the molecular networks which protect barrier surfaces against viral infections are incompletely understood. Viral infections induce simultaneous production of type I (IFN-α/ß) and type III (IFN-λ) interferons. All nucleated cells are believed to respond to IFN-α/ß, whereas IFN-λ responses are largely confined to epithelial cells. We observed that intestinal epithelial cells, unlike hematopoietic cells of this organ, express only very low levels of functional IFN-α/ß receptors. Accordingly, after oral infection of IFN-α/ß receptor-deficient mice, human reovirus type 3 specifically infected cells in the lamina propria but, strikingly, did not productively replicate in gut epithelial cells. By contrast, reovirus replicated almost exclusively in gut epithelial cells of IFN-λ receptor-deficient mice, suggesting that the gut mucosa is equipped with a compartmentalized IFN system in which epithelial cells mainly respond to IFN-λ that they produce after viral infection, whereas other cells of the gut mostly rely on IFN-α/ß for antiviral defense. In suckling mice with IFN-λ receptor deficiency, reovirus replicated in the gut epithelium and additionally infected epithelial cells lining the bile ducts, indicating that infants may use IFN-λ for the control of virus infections in various epithelia-rich tissues. Thus, IFN-λ should be regarded as an autonomous virus defense system of the gut mucosa and other epithelial barriers that may have evolved to avoid unnecessarily frequent triggering of the IFN-α/ß system which would induce exacerbated inflammation.
Assuntos
Células Epiteliais/imunologia , Mucosa Intestinal/imunologia , Leucócitos/imunologia , Infecções por Reoviridae/imunologia , Animais , Separação Celular , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Interferon-alfa/imunologia , Interferon beta/imunologia , Interferon gama/imunologia , Orthoreovirus Mamífero 3/imunologia , Camundongos , Camundongos Knockout , Reação em Cadeia da PolimeraseRESUMO
Tumor-associated immunosuppression aids cancer cells to escape immune-mediated attack and subsequent elimination. Recently, however, many oncolytic viruses, including reovirus, have been reported to overturn such immunosuppression and promote the development of a clinically desired antitumor immunity, which is known to promote favorable patient outcomes. Contrary to this existing paradigm, in this article we demonstrate that reovirus augments tumor-associated immunosuppression immediately following its therapeutic administration. Our data show that reovirus induces preferential differentiation of highly suppressive CD11b(+), Gr-1(+), Ly6C(high) myeloid cells from bone marrow hematopoietic progenitor cells. Furthermore, reovirus administration in tumor-bearing hosts drives time-dependent recruitment of CD11b(+), Gr-1(+), Ly6C(high) myeloid cells in the tumor milieu, which is further supported by virus-induced increased expression of numerous immune factors involved in myeloid-derived suppressor cell survival and trafficking. Most importantly, CD11b(+), Gr-1(+), Ly6C(high) myeloid cells specifically potentiate the suppression of T cell proliferation and are associated with the absence of IFN-γ response in the tumor microenvironment early during oncotherapy. Considering that the qualitative traits of a specific antitumor immunity are largely dictated by the immunological events that precede its development, our findings are of critical importance and must be considered while devising complementary interventions aimed at promoting the optimum efficacy of oncolytic virus-based anticancer immunotherapies.
Assuntos
Vetores Genéticos , Imunomodulação , Células Mieloides/imunologia , Células Mieloides/metabolismo , Neoplasias/imunologia , Vírus Oncolíticos , Fenótipo , Animais , Antígenos Ly/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Antígeno CD11b/metabolismo , Diferenciação Celular , Quimiotaxia/imunologia , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Humanos , Orthoreovirus Mamífero 3/genética , Orthoreovirus Mamífero 3/imunologia , Camundongos , Células Mieloides/citologia , Neoplasias/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/imunologia , Receptores de Quimiocinas/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Microambiente Tumoral/imunologiaRESUMO
The naturally occurring oncolytic virus (OV), reovirus, replicates in cancer cells causing direct cytotoxicity, and can activate innate and adaptive immune responses to facilitate tumour clearance. Reovirus is safe, well tolerated and currently in clinical testing for the treatment of multiple myeloma, in combination with dexamethasone/carfilzomib. Activation of natural killer (NK) cells has been observed after systemic delivery of reovirus to cancer patients; however, the ability of OV to potentiate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) is unexplored. This study elucidates the potential of oncolytic reovirus for the treatment of chronic lymphocytic leukaemia (CLL), both as a direct cytotoxic agent and as an immunomodulator. We demonstrate that reovirus: (i) is directly cytotoxic against CLL, which requires replication-competent virus; (ii) phenotypically and functionally activates patient NK cells via a monocyte-derived interferon-α (IFNα)-dependent mechanism; and (iii) enhances ADCC-mediated killing of CLL in combination with anti-CD20 antibodies. Our data provide strong preclinical evidence to support the use of reovirus in combination with anti-CD20 immunotherapy for the treatment of CLL.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Orthoreovirus Mamífero 3/imunologia , Vírus Oncolíticos/imunologia , Rituximab/imunologia , Rituximab/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/imunologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Efeito Citopatogênico Viral , Feminino , Humanos , Imunidade Inata , Fatores Imunológicos/imunologia , Fatores Imunológicos/uso terapêutico , Imunofenotipagem , Imunoterapia , Células Matadoras Naturais/imunologia , Leucemia Linfocítica Crônica de Células B/diagnóstico , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Replicação ViralRESUMO
Optimum clinical protocols require systemic delivery of oncolytic viruses in the presence of an intact immune system. We show that preconditioning with immune modulators, or loading virus onto carrier cells ex vivo, enhances virus-mediated antitumor activity. Our early trials of systemic reovirus delivery showed that after infusion reovirus could be recovered from blood cells--but not from plasma--suggesting that rapid association with blood cells may protect virus from neutralizing antibody. We therefore postulated that stimulation of potential carrier cells directly in vivo before intravenous viral delivery would enhance delivery of cell-associated virus to tumor. We show that mobilization of the CD11b(+) cell compartment by granulocyte macrophage-colony stimulating factor immediately before intravenous reovirus, eliminated detectable tumor in mice with small B16 melanomas, and achieved highly significant therapy in mice bearing well-established tumors. Unexpectedly, cytokine conditioning therapy was most effective in the presence of preexisting neutralizing antibody. Consistent with this, reovirus bound by neutralizing antibody effectively accessed monocytes/macrophages and was handed off to tumor cells. Thus, preconditioning with cytokine stimulated recipient cells in vivo for enhanced viral delivery to tumors. Moreover, preexisting neutralizing antibody to an oncolytic virus may, therefore, even be exploited for systemic delivery to tumors in the clinic.
Assuntos
Citocinas/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Vírus Oncolíticos/genética , Transdução Genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais , Antígeno CD11b/metabolismo , Citocinas/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Imunidade/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Orthoreovirus Mamífero 3/genética , Orthoreovirus Mamífero 3/imunologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/mortalidade , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Terapia Viral Oncolítica , Vírus Oncolíticos/imunologia , Receptores Fc/genética , Receptores Fc/metabolismo , Carga TumoralRESUMO
Flaviviruses, particularly Japanese encephalitis virus (JEV) and West Nile virus (WNV), are important causes of virus-induced central nervous system (CNS) disease in humans. We used microarray analysis to identify cellular genes that are differentially regulated following infection of the brain with JEV (P3) or WNV (New York 99). Gene expression data for these flaviviruses were compared to those obtained following infection of the brain with reovirus (type 3 Dearing), an unrelated neurotropic virus. We found that a large number of genes were up-regulated by all three viruses (using the criteria of a change of >2-fold and a P value of <0.001), including genes associated with interferon signaling, the immune system, inflammation, and cell death/survival signaling. In addition, genes associated with glutamate signaling were down-regulated in infections with all three viruses (criteria, a >2-fold change and a P value of <0.001). These genes may serve as broad-spectrum therapeutic targets for virus-induced CNS disease. A distinct set of genes were up-regulated following flavivirus infection but not following infection with reovirus. These genes were associated with tRNA charging and may serve as therapeutic targets for flavivirus-induced CNS disease. IMPORTANCE Viral infections of the central nervous system (CNS) are an important cause of morbidity and mortality. Treatment options for virus-induced CNS disease are limited, and for many clinically important neurotropic viruses, no specific therapy of proven benefit is currently available. We performed microarray analysis to identify genes that are differentially regulated in the brain following virus infection in order to identify pathways that might provide novel therapeutic targets for virus-induced CNS disease. Although several studies have described gene expression changes following virus infection of the brain, this report is the first to directly compare large-scale gene expression data from different viruses. We identified genes that are differentially regulated in infection of the brain with viruses from different families and those which appear to be specific to flavivirus infections.
Assuntos
Encéfalo/imunologia , Encéfalo/virologia , Vírus da Encefalite Japonesa (Subgrupo)/fisiologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Vírus do Nilo Ocidental/fisiologia , Aminoacil-tRNA Sintetases/biossíntese , Animais , Apoptose , Vírus da Encefalite Japonesa (Subgrupo)/imunologia , Ácido Glutâmico , Interferons/biossíntese , Orthoreovirus Mamífero 3/imunologia , Orthoreovirus Mamífero 3/fisiologia , Camundongos , Análise em Microsséries , Receptores de Interleucina-17/biossíntese , Transdução de Sinais , Regulação para Cima , Vírus do Nilo Ocidental/imunologiaRESUMO
Reovirus infection is a well-characterized experimental system for the study of viral pathogenesis and antiviral immunity within the central nervous system (CNS). We have previously shown that c-Jun N-terminal kinase (JNK) and the Fas death receptor each play a role in neuronal apoptosis occurring in reovirus-infected brains. Death-associated protein 6 (Daxx) is a cellular protein that mechanistically links Fas signaling to JNK signaling in several models of apoptosis. In the present study, we demonstrate that Daxx is upregulated in reovirus-infected brain tissue through a type I interferon-mediated mechanism. Daxx upregulation is limited to brain regions that undergo reovirus-induced apoptosis and occurs in the cytoplasm and nucleus of neurons. Cytoplasmic Daxx is present in Fas-expressing cells during reovirus encephalitis, suggesting a role for Daxx in Fas-mediated apoptosis following reovirus infection. Further, in vitro expression of a dominant negative form of Daxx (DN-Daxx), which binds to Fas but which does not transmit downstream signaling, inhibits apoptosis of reovirus-infected cells. In contrast, in vitro depletion of Daxx results in increased expression of caspase 3 and apoptosis, suggesting that Daxx plays an antiapoptotic role in the nucleus. Overall, these data imply a regulatory role for Daxx in reovirus-induced apoptosis, depending on its location in the nucleus or cytoplasm.
Assuntos
Apoptose , Proteínas de Transporte/biossíntese , Citoplasma/virologia , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Orthoreovirus Mamífero 3/patogenicidade , Neurônios/virologia , Proteínas Nucleares/biossíntese , Animais , Proteínas Correpressoras , Orthoreovirus Mamífero 3/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares , Regulação para CimaRESUMO
Human milk contains many bioactive components, including secretory IgA, oligosaccharides, and milk-associated proteins. We assessed the antiviral effects of several components of milk against mammalian reoviruses. We found that glucocerebroside (GCB) inhibited the infectivity of reovirus strain type 1 Lang (T1L), whereas gangliosides GD3 and GM3 and 3'-sialyllactose (3SL) inhibited the infectivity of reovirus strain type 3 Dearing (T3D). Agglutination of erythrocytes mediated by T1L and T3D was inhibited by GD3, GM3, and bovine lactoferrin. Additionally, α-sialic acid, 3SL, 6'-sialyllactose, sialic acid, human lactoferrin, osteopontin, and α-lactalbumin inhibited hemagglutination mediated by T3D. Using single-gene reassortant viruses, we found that serotype-specific differences segregate with the gene encoding the viral attachment protein. Furthermore, GD3, GM3, and 3SL inhibit T3D infectivity by blocking binding to host cells, whereas GCB inhibits T1L infectivity post-attachment. These results enhance an understanding of reovirus cell attachment and define a mechanism for the antimicrobial activity of human milk.
Assuntos
Proteínas do Capsídeo/imunologia , Orthoreovirus Mamífero 3/imunologia , Orthoreovirus Mamífero 3/patogenicidade , Leite Humano/imunologia , Orthoreovirus de Mamíferos/imunologia , Orthoreovirus de Mamíferos/patogenicidade , Polissacarídeos/imunologia , Animais , Proteínas do Capsídeo/genética , Bovinos , Feminino , Gangliosídeo G(M3)/imunologia , Gangliosídeos/imunologia , Genes Virais , Células HeLa , Testes de Inibição da Hemaglutinação , Interações Hospedeiro-Patógeno/imunologia , Humanos , Células L , Orthoreovirus Mamífero 3/classificação , Orthoreovirus Mamífero 3/genética , Camundongos , Leite Humano/virologia , Oligossacarídeos/imunologia , Orthoreovirus de Mamíferos/classificação , Orthoreovirus de Mamíferos/genética , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/prevenção & controle , Infecções por Reoviridae/virologia , Sorotipagem , Especificidade da Espécie , Ligação ViralRESUMO
Reovirus infection of the murine spinal cord (SC) was used as a model system to investigate innate immune responses during viral myelitis, including the activation of glia (microglia and astrocytes) and interferon (IFN) signaling and increased expression of inflammatory mediators. Reovirus myelitis was associated with the pronounced activation of SC glia, as evidenced by characteristic changes in cellular morphology and increased expression of astrocyte and microglia-specific proteins. Expression of inflammatory mediators known to be released by activated glia, including interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNF-α), chemokine (C-C motif) ligand 5 (CCL 5), chemokine (C-X-C motif) ligand 10 (CXCL10), and gamma interferon (IFN-γ), was also significantly upregulated in the SC of reovirus-infected animals compared to mock-infected controls. Reovirus infection of the mouse SC was also associated with increased expression of genes involved in IFN signaling, including IFN-stimulated genes (ISG). Further, reovirus infection of mice deficient in the expression of the IFN-α/ß receptor (IFNAR(-/-)) resulted in accelerated mortality, demonstrating that IFN signaling is protective during reovirus myelitis. Experiments performed in ex vivo SC slice cultures (SCSC) confirmed that resident SC cells contribute to the production of at least some of these inflammatory mediators and ISG during reovirus infection. Microglia, but not astrocytes, were still activated, and glia-associated inflammatory mediators were still produced in reovirus-infected INFAR(-/-) mice, demonstrating that IFN signaling is not absolutely required for these neuroinflammatory responses. Our results suggest that activated glia and inflammatory mediators contribute to a local microenvironment that is deleterious to neuronal survival.
Assuntos
Imunidade Inata , Orthoreovirus Mamífero 3/imunologia , Mielite/imunologia , Infecções por Reoviridae/imunologia , Transdução de Sinais/imunologia , Medula Espinal/imunologia , Animais , Citocinas , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Inflamação/virologia , Mediadores da Inflamação/imunologia , Camundongos , Camundongos Knockout , Mielite/genética , Mielite/patologia , Mielite/virologia , Neuroglia/imunologia , Neuroglia/patologia , Neuroglia/virologia , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologia , Infecções por Reoviridae/genética , Infecções por Reoviridae/patologia , Transdução de Sinais/genética , Medula Espinal/patologia , Medula Espinal/virologiaRESUMO
The trafficking of effector cells to sites of infection is crucial for antiviral responses. However, the mechanisms of recruitment of the interferon-γ-producing and cytotoxic CD56(+) T cells are poorly understood. Human mast cells are sentinel cells found in the skin and airway and produce selected proinflammatory mediators in response to multiple pathogen-associated signals. The role of human mast cell-derived chemokines in T-cell recruitment to virus infection was examined. Supernatants from primary human cord blood-derived mast cells (CBMCs) infected with mammalian reovirus were examined for chemokine production and utilized in chemotaxis assays. Virus-infected CBMCs produced several chemokines, including CCL3, CCL4, and CCL5. Supernatants from reovirus-infected CBMCs selectively induced the chemotaxis of CD8(+) T cells (10±1%) and CD3(+)CD56(+) T cells (19±5%). CD56(+) T-cell migration was inhibited by pertussis toxin (65±9%) and met-RANTES (56±7%), a CCR1/CCR5 antagonist. CD56(+) T cells expressed CCR5, but little CCR1. The depletion of CCL3, CCL4, and CCL5 from reovirus-infected CBMC supernatants significantly (41±10%) inhibited CD56(+) T-cell chemotaxis. This study demonstrates a novel role for mast cells and CCR5 in CD56(+) T-cell trafficking and suggests that human mast cells enhance immunity to viruses through the selective recruitment of cytotoxic effector cells to virus infection sites. These findings could be exploited to enhance local T-cell responses in chronic viral infection and malignancies at mast cell-rich sites.
Assuntos
Antígeno CD56/imunologia , Orthoreovirus Mamífero 3/imunologia , Mastócitos/imunologia , Linfócitos T/imunologia , Antígeno CD56/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Células Cultivadas , Quimiocina CCL3/imunologia , Quimiocina CCL3/metabolismo , Quimiocina CCL3/farmacologia , Quimiocina CCL4/imunologia , Quimiocina CCL4/metabolismo , Quimiocina CCL4/farmacologia , Quimiocina CCL5/imunologia , Quimiocina CCL5/metabolismo , Quimiocina CCL5/farmacologia , Quimiocinas/imunologia , Quimiocinas/metabolismo , Quimiocinas/farmacologia , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Meios de Cultivo Condicionados/farmacologia , Relação Dose-Resposta a Droga , Sangue Fetal/citologia , Citometria de Fluxo , Imunofluorescência , Interações Hospedeiro-Patógeno/imunologia , Humanos , Ligantes , Orthoreovirus Mamífero 3/fisiologia , Mastócitos/metabolismo , Mastócitos/virologia , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Linfócitos T/metabolismoRESUMO
An oncolytic virus is considered a targeted cancer therapy due to its ability to specifically target, replicate in and lyse cancer cells while leaving normal cells unharmed. Over the last few years several tumor selective oncolytic viruses have been developed. These include certain DNA viruses such as adenovirus that have been genetically manipulated to target specific cancer cells by exerting restrictions on the virus at the level of cell entry, viral gene transcription and viral protein translation. There are a variety of RNA viruses being studied as possible cancer therapies including reovirus. Reovirus is intrinsically oncolytic without the need for any genetic manipulation. The inherent oncolytic properties of this virus are derived from the fact that it specifically targets cells with an activated Ras pathway found in many cancer cells. REOLYSIN® is a proprietary formulation of human reovirus type 3 Dearing developed by Oncolytics Biotech Inc. and is the only therapeutic reovirus in clinical development. This review provides an overview of the development of REOLYSIN as a potential cancer therapeutic and the growing role of in situ based molecular pathology methods in providing clinical proof of concept and in guiding clinical development.
Assuntos
Imuno-Histoquímica , Orthoreovirus Mamífero 3/genética , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Replicação Viral , Animais , Anticorpos Antivirais/análise , Ensaios Clínicos como Assunto , Humanos , Masculino , Orthoreovirus Mamífero 3/imunologia , Camundongos , Neoplasias/virologia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/terapia , Neoplasias da Próstata/virologiaRESUMO
Soluble gp130 (sgp130) has been shown to suppress the inflammatory response of autoimmune pathologies; however, its effects on virus infection are not known. Here, we report that intraperitoneal treatment of mice with sgp130-Fc fusion protein at the time of oral reovirus serotype 3 infection resulted in altered morphopathological changes that were evident by less shortening of intestinal villi length and crypt depth after infection. That the effect mediated by sgp130 treatment was due to an increase in intestinal crypt cell proliferation was demonstrated by an increase in the number of crypt mitotic figures. This was further confirmed by increased immunoreactivity to the Cdc47 proliferation-associated antigen in crypts of sgp130-treated virus-infected mice compared to infected non-treated mice. These findings suggest that sgp130 may have a beneficial effect during intestinal virus infection by disrupting interleukin-6 trans-signalling, thereby reducing the local inflammatory response.
Assuntos
Receptor gp130 de Citocina/metabolismo , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Infecções por Reoviridae/patologia , Animais , Receptor gp130 de Citocina/imunologia , Feminino , Hiperplasia , Inflamação/imunologia , Inflamação/virologia , Mucosa Intestinal/metabolismo , Orthoreovirus Mamífero 3/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologiaRESUMO
PURPOSE: Reovirus is a naturally occurring oncolytic virus in clinical trials. Although tumor infection by reovirus can generate adaptive antitumor immunity, its therapeutic importance versus direct viral oncolysis is undefined. This study addresses the requirement for viral oncolysis and replication, and the relative importance of antitumor immunity and direct oncolysis in therapy. EXPERIMENTAL DESIGN: Nonantigen specific T cells loaded with reovirus were delivered i.v. to C57BL/6 and severe combined immunodeficient mice bearing lymph node and splenic metastases from the murine melanoma, B16ova, with assessment of viral replication, metastatic clearance by tumor colony outgrowth, and immune priming. Human cytotoxic lymphocyte priming assays were done with dendritic cells loaded with Mel888 cells before the addition of reovirus. RESULTS: B16ova was resistant to direct oncolysis in vitro, and failed to support reovirus replication in vitro or in vivo. Nevertheless, reovirus purged lymph node and splenic metastases in C57BL/6 mice and generated antitumor immunity. In contrast, reovirus failed to reduce tumor burden in severe combined immunodeficient mice bearing either B16ova or reovirus-sensitive B16tk metastases. In the human system, reovirus acted solely as an adjuvant when added to dendritic cells already loaded with Mel888, supporting priming of specific antitumor cytotoxic lymphocyte, in the absence of significant direct tumor oncolysis; UV-treated nonreplicating reovirus was similarly immunogenic. CONCLUSION: The immune response is critical in mediating the efficacy of reovirus, and does not depend upon direct viral oncolysis or replication. The findings are of direct relevance to fulfilling the potential of this novel anticancer agent.
Assuntos
Citotoxicidade Imunológica/fisiologia , Orthoreovirus Mamífero 3/fisiologia , Melanoma Experimental/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/fisiologia , Replicação Viral/fisiologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Imunoterapia Adotiva/métodos , Orthoreovirus Mamífero 3/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/virologia , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Vírus Oncolíticos/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/transplante , Linfócitos T Citotóxicos/virologia , Resultado do Tratamento , Replicação Viral/imunologiaRESUMO
PURPOSE: Early clinical trials are under way exploring the direct oncolytic potential of reovirus. This study addresses whether tumor infection by reovirus is also able to generate bystander, adaptive antitumor immunity. EXPERIMENTAL DESIGN: Reovirus was delivered intravenously to C57BL/6 mice bearing lymph node metastases from the murine melanoma, B16-tk, with assessment of nodal metastatic clearance, priming of antitumor immunity against the tumor-associated antigen tyrosinase-related protein-2, and cytokine responses. In an in vitro human system, the effect of reovirus infection on the ability of Mel888 melanoma cells to activate and load dendritic cells for cytotoxic lymphocyte (CTL) priming was investigated. RESULTS: In the murine model, a single intravenous dose of reovirus reduced metastatic lymph node burden and induced antitumor immunity (splenocyte response to tyrosinase-related protein-2 and interleukin-12 production in disaggregated lymph nodes). In vitro human assays revealed that uninfected Mel888 cells failed to induce dendritic cell maturation or support priming of an anti-Mel888 CTL response. In contrast, reovirus-infected Mel888 cells (reo-Mel) matured dendritic cells in a reovirus dose-dependent manner. When cultured with autologous peripheral blood lymphocytes, dendritic cells loaded with reo-Mel induced lymphocyte expansion, IFN-gamma production, specific anti-Mel888 cell cytotoxicity, and cross-primed CD8+ T cells specific against the human tumor-associated antigen MART-1. CONCLUSION: Reovirus infection of tumor cells reduces metastatic disease burden and primes antitumor immunity. Future clinical trials should be designed to explore both direct cytotoxic and immunotherapeutic effects of reovirus.
Assuntos
Antineoplásicos/uso terapêutico , Citotoxicidade Imunológica/imunologia , Neoplasias Experimentais/terapia , Terapia Viral Oncolítica/métodos , Infecções por Reoviridae/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Interferon gama/biossíntese , Oxirredutases Intramoleculares/imunologia , Metástase Linfática/patologia , Ativação Linfocitária/imunologia , Antígeno MART-1 , Orthoreovirus Mamífero 3/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/virologia , Reação em Cadeia da Polimerase , Linfócitos T Citotóxicos/imunologiaRESUMO
PURPOSE: To determine the safety and feasibility of daily i.v. administration of wild-type oncolytic reovirus (type 3 Dearing) to patients with advanced cancer, assess viral excretion kinetics and antiviral immune responses, identify tumor localization and replication, and describe antitumor activity. EXPERIMENTAL DESIGN: Patients received escalating doses of reovirus up to 3 x 10(10) TCID(50) for 5 consecutive days every 4 weeks. Viral excretion was assessed by reverse transcription-PCR and antibody response by cytotoxicity neutralization assay. Pretreatment and post-treatment tumor biopsies were obtained to measure viral uptake and replication. RESULTS: Thirty-three patients received 76 courses of reovirus from 1 x 10(8) for 1 day up to 3 x 10(10) TCID(50) for 5 days, repeated every four weeks. Dose-limiting toxicity was not seen. Common grade 1 to 2 toxicities included fever, fatigue, and headache, which were dose and cycle independent. Viral excretion at day 15 was not detected by reverse transcription-PCR at 25 cycles and only in 5 patients at 35 cycles. Neutralizing antibodies were detected in all patients and peaked at 4 weeks. Viral localization and replication in tumor biopsies were confirmed in 3 patients. Antitumor activity was seen by radiologic and tumor marker (carcinoembryonic antigen, CA19.9, and prostate-specific antigen) evaluation. CONCLUSIONS: Oncolytic reovirus can be safely and repeatedly administered by i.v. injection at doses up to 3 x 10(10) TCID(50) for 5 days every 4 weeks without evidence of severe toxicities. Productive reoviral infection of metastatic tumor deposits was confirmed. Reovirus is a safe agent that warrants further evaluation in phase II studies.
Assuntos
Orthoreovirus Mamífero 3 , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/análise , Estudos de Viabilidade , Feminino , Humanos , Injeções Intravenosas , Masculino , Orthoreovirus Mamífero 3/imunologia , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/imunologia , Terapia Viral Oncolítica/efeitos adversosRESUMO
Oncolytic viruses can exert their antitumor activity via direct oncolysis or activation of antitumor immunity. Although reovirus is currently under clinical investigation for the treatment of localized or disseminated cancer, any potential immune contribution to its efficacy has not been addressed. This is the first study to investigate the ability of reovirus to activate human dendritic cells (DC), key regulators of both innate and adaptive immune responses. Reovirus induced DC maturation and stimulated the production of the proinflammatory cytokines IFN-alpha, TNF-alpha, IL-12p70, and IL-6. Activation of DC by reovirus was not dependent on viral replication, while cytokine production (but not phenotypic maturation) was inhibited by blockade of PKR and NF-kappaB signaling. Upon coculture with autologous NK cells, reovirus-activated DC up-regulated IFN-gamma production and increased NK cytolytic activity. Moreover, short-term coculture of reovirus-activated DC with autologous T cells also enhanced T cell cytokine secretion (IL-2 and IFN-gamma) and induced non-Ag restricted tumor cell killing. These data demonstrate for the first time that reovirus directly activates human DC and that reovirus-activated DC stimulate innate killing by not only NK cells, but also T cells, suggesting a novel potential role for T cells in oncolytic virus-induced local tumor cell death. Hence reovirus recognition by DC may trigger innate effector mechanisms to complement the virus's direct cytotoxicity, potentially enhancing the efficacy of reovirus as a therapeutic agent.
Assuntos
Células Dendríticas/imunologia , Imunidade Inata , Células Matadoras Naturais/imunologia , Orthoreovirus Mamífero 3/imunologia , Neoplasias/imunologia , Linfócitos T/imunologia , Morte Celular/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Citocinas/imunologia , Células Dendríticas/virologia , Humanos , Imunidade Celular , NF-kappa B/imunologia , Transdução de Sinais/imunologia , Fatores de Tempo , Replicação Viral/imunologiaRESUMO
There is an emerging realization from animal models that the immune response may have both detrimental and beneficial therapeutic effects during cancer virotherapy. However, there is a dearth of clinical data on the immune response to viral agents in patients. During a recently completed phase I trial of intravenous reovirus type 3 Dearing (RT3D), heavily pretreated patients with advanced cancers received RT3D at doses escalating from 1 x 10(8) tissue culture infectious dose-50 (TCID(50)) on day 1 to 3 x 10(10) TCID(50) on 5 consecutive days of a 4 weekly cycle. A detailed analysis of the immune effects was conducted by collecting serial clinical samples for analysis of neutralizing anti-reoviral antibodies (NARA), peripheral blood mononuclear cells (PBMC) and cytokines. Significant increases in NARA were seen with peak endpoint titres >1/10 000 in all but one patient. The median fold increase was 250, with a range of 9-6437. PBMC subset analysis showed marked heterogeneity. At baseline, CD3+CD4+ T cells were reduced in most patients, but after RT3D therapy their numbers increased in 47.6% of patients. In contrast, most patients had high baseline CD3+CD8+ T-cell levels, with 33% showing incremental increases after therapy. In some patients, there was increased cytotoxic T-cell activation post-therapy, as shown by increased CD8+perforin/granzyme+ T-cell numbers. Most patients had high numbers of circulating CD3-CD56+ NK cells before therapy and in 28.6% this increased with treatment. Regulatory (CD3+CD4+CD25+) T cells were largely unaffected by the therapy. Combined Th1 and Th2 cytokine expression increased in 38% of patients. These data confirm that even heavily pretreated patients are capable of mounting dynamic immune responses during treatment with RT3D, although these responses are not clearly related to the administered virus dose. These data will provide the basis for future studies aiming to modulate the immune response during virotherapy.
Assuntos
Terapia Genética/métodos , Orthoreovirus Mamífero 3/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Citocinas/sangue , Feminino , Humanos , Imunidade Inata , Injeções Intravenosas , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/imunologia , Resultado do TratamentoRESUMO
Mammalian Orthoreoviruses are important models for studies of viral pathogenesis. In the rat lung, Reovirus strain type 3 Dearing (T3D) induces substantially more inflammation than does strain type 1 Lang (T1L). To better understand mechanisms underlying differences in the host inflammatory response elicited by T1L and T3D, we characterized cytokine expression patterns induced by those strains after infection of THP-1 monocyte cells. THP-1 cells were adsorbed with either viable or ultraviolet- inactivated T1L and T3D and assayed for mRNA and protein production of growth-regulated oncogene-alpha (GRO-alpha), interleukin-8 (IL-8), or tumor necrosis factor-alpha (TNF-alpha). T3D stimulated mRNA and protein production of all three cytokines, whereas T1L stimulated mRNA and protein production of IL-8 and TNF-alpha but not GRO-alpha. In each case, T3D induced greater cytokine mRNA and protein expression than did T1L. Nonviable virus did not stimulate detectable cytokine secretion, suggesting a requirement for viral RNA synthesis in cytokine induction by THP-1 cells. A greater percentage of THP-1 cells was infected with T1L than T3D as assessed by infectious center assay, and T1L achieved higher yields of infectious progeny than did T3D in infected THP-1 cells as determined by plaque assay. These strain-dependent differences in cytokine responses and corresponding replication patterns in monocyte cells parallel findings made in studies of rat models of pneumonia and provide clues about how Reovirus interfaces with the host innate immune response to produce pulmonary disease.
Assuntos
Citocinas/metabolismo , Inflamação/virologia , Orthoreovirus Mamífero 3/imunologia , Monócitos/virologia , Orthoreovirus de Mamíferos/imunologia , Linhagem Celular , Citocinas/genética , Humanos , Inflamação/imunologia , Orthoreovirus Mamífero 3/fisiologia , Orthoreovirus de Mamíferos/fisiologia , Especificidade da Espécie , Replicação ViralRESUMO
Respiratory enteric orphan virus (reovirus) has been used to study many aspects of the biology and genetics of viruses, viral infection, pathogenesis, and the immune response to virus infection. This report describes the functional activity of virus labeled with Alexa Fluor 488, a stable fluorescent dye. Matrix assisted laser desorption-time of flight analysis indicated that Alexa Fluor 488 labeled the outer capsid proteins of reovirus. Labeled virus bound to murine L929 fibroblasts as determined by flow cytometry and fluorescence microscopy, and the specificity of binding were demonstrated by competitive inhibition with non-labeled virus. Labeled reovirus induced apoptosis and cytopathic effect in infected L929 cells. Mice infected with labeled virus mounted robust serum antibody and CD8(+) T-cell responses, indicating that labeled virus retained immunogenicity in vivo. These results indicate that Alexa Fluor 488-labeled virus provides a powerful new tool to analyze reovirus infection in vitro and in vivo.