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Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, persistently infects over 90% of the human adult population and is associated with several human cancers. To establish life-long infection, EBV tampers with the induction of type I interferon (IFN I)-dependent antiviral immunity in the host. How various EBV genes help orchestrate this crucial strategy is incompletely defined. Here, we reveal a mechanism by which the EBV nuclear antigen 3A (EBNA3A) may inhibit IFNß induction. Using proximity biotinylation we identify the histone acetyltransferase P300, a member of the IFNß transcriptional complex, as a binding partner of EBNA3A. We further show that EBNA3A also interacts with the activated IFN-inducing transcription factor IRF3 that collaborates with P300 in the nucleus. Both events are mediated by the N-terminal domain of EBNA3A. We propose that EBNA3A limits binding of IRF3 to the IFNß promoter, thereby hampering downstream IFN I signaling. Collectively, our findings suggest a new mechanism of immune evasion by EBV, affected by its latency gene EBNA3A.
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BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a tumour entity with unmet medical need. To assess the therapeutic potential of oncolytic virotherapy (OVT) against PDAC, different oncolytic viruses (OVs) are currently investigated in clinical trials. However, systematic comparisons of these different OVs in terms of efficacy against PDAC and biomarkers predicting therapeutic response are lacking. METHODS: We screened fourteen patient-derived PDAC cultures which reflect the intra- and intertumoural heterogeneity of PDAC for their sensitivity to five clinically relevant OVs, namely serotype 5 adenovirus Ad5-hTERT, herpes virus T-VEC, measles vaccine strain MV-NIS, reovirus jin-3, and protoparvovirus H-1PV. Live cell analysis, quantification of viral genome/gene expression, cell viability as well as cytotoxicity assays and titration of viral progeny were conducted. Transcriptome profiling was employed to identify potential predictive biomarkers for response to OV treatment. FINDINGS: Patient-derived PDAC cultures showed individual response patterns to OV treatment. Twelve of fourteen cultures were responsive to at least one OV, with no single OV proving superior or inferior across all cultures. Known host factors for distinct viruses were retrieved as potential biomarkers. Compared to the classical molecular subtype, the quasi-mesenchymal or basal-like subtype of PDAC was found to be more sensitive to H-1PV, jin-3, and T-VEC. Generally, expression of viral entry receptors did not correlate with sensitivity to OV treatment, with one exception: Expression of Galectin-1 (LGALS1), a factor involved in H-1PV entry, positively correlated with H-1PV induced cell killing. Rather, cellular pathways controlling immunological, metabolic and proliferative signaling appeared to determine outcome. For instance, high baseline expression of interferon-stimulated genes (ISGs) correlated with relative resistance to oncolytic measles virus, whereas low cyclic GMP-AMP synthase (cGAS) expression was associated with exceptional response. Combination treatment of MV-NIS with a cGAS inhibitor improved tumour cell killing in several PDAC cultures and cells overexpressing cGAS were found to be less sensitive to MV oncolysis. INTERPRETATION: Considering the heterogeneity of PDAC and the complexity of biological therapies such as OVs, no single biomarker can explain the spectrum of response patterns. For selection of a particular OV, PDAC molecular subtype, ISG expression as well as activation of distinct signaling and metabolic pathways should be considered. Combination therapies can overcome resistance in specific constellations. Overall, oncolytic virotherapy is a viable treatment option for PDAC, which warrants further development. This study highlights the need for personalised treatment in OVT. By providing all primary data, this study provides a rich source and guidance for ongoing developments. FUNDING: German National Science Foundation (Deutsche Forschungsgemeinschaft, DFG), German Cancer Aid (Deutsche Krebshilfe), German National Academic Scholarship Foundation (Studienstiftung des deutschen Volkes), Survival with Pancreatic Cancer Foundation.
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Biomarcadores Tumorais , Terapia Viral Oncolítica , Vírus Oncolíticos , Neoplasias Pancreáticas , Humanos , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/metabolismo , Perfilação da Expressão Gênica , Linhagem Celular Tumoral , Sobrevivência Celular , Células Tumorais CultivadasRESUMO
Despite decades of research, the prognosis of high-grade pediatric brain tumors (PBTs) remains dismal; however, recent cases of favorable clinical responses were documented in clinical trials using oncolytic viruses (OVs). In the current study, we employed four different species of OVs: adenovirus Delta24-RGD, herpes simplex virus rQNestin34.5v1, reovirus R124, and the non-virulent Newcastle disease virus rNDV-F0-GFP against three entities of PBTs (high-grade gliomas, atypical teratoid/rhabdoid tumors, and ependymomas) to determine their in vitro efficacy. These four OVs were screened on 14 patient-derived PBT cell cultures and the degree of oncolysis was assessed using an ATP-based assay. Subsequently, the observed viral efficacies were correlated to whole transcriptome data and Gene Ontology analysis was performed. Although no significant tumor type-specific OV efficacy was observed, the analysis revealed the intrinsic biological processes that associated with OV efficacy. The predictive power of the identified expression profiles was further validated in vitro by screening additional PBTs. In summary, our results demonstrate OV susceptibility of multiple patient-derived PBT entities and the ability to predict in vitro responses to OVs using unique expression profiles. Such profiles may hold promise for future OV preselection with effective oncolytic potency in a specific tumor, therewith potentially improving OV responses.
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Reovirus type 3 Dearing (Reo), manufactured for clinical application as pelareorep, is an attractive anticancer agent under evaluation in multiple phase 2 clinical trials for the treatment of solid tumors. It elicits its anticancer efficacy by inducing both oncolysis and intratumoral T-cell influx. Because most people have been preexposed to Reo, neutralizing antibodies (NAb) are prevalent in patients with cancer and might present a barrier to effective Reo therapy. Here, we tested serum of patients with cancer and healthy controls (n = 100) and confirmed that Reo NAbs are present in >80% of individuals. To investigate the effect of NAbs on both the oncolytic and the immunostimulatory efficacy of Reo, we established an experimental mouse model with Reo preexposure. The presence of preexposure-induced NAbs reduced Reo tumor infection and prevented Reo-mediated control of tumor growth after intratumoral Reo administration. In B cell-deficient mice, the lack of NAbs provided enhanced tumor growth control after Reo monotherapy, indicating that NAbs limit the oncolytic capacity of Reo. In immunocompetent mice, intratumoral T-cell influx was not affected by the presence of preexposure-induced NAbs and consequently, combinatorial immunotherapy strategies comprising Reo and T-cell engagers or checkpoint inhibitors remained effective in these settings, also after a clinically applied regimen of multiple intravenous pelareorep administrations. Altogether, our data indicate that NAbs hamper the oncolytic efficacy of Reo, but not its immunotherapeutic capacity. Given the high prevalence of seropositivity for Reo in patients with cancer, our data strongly advocate for the application of Reo as part of T cell-based immunotherapeutic strategies.
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Neoplasias , Terapia Viral Oncolítica , Vírus Oncolíticos , Reoviridae , Humanos , Animais , Camundongos , Anticorpos Neutralizantes , Anticorpos Antivirais , Neoplasias/terapia , Neoplasias/etiologia , Linfócitos T , ImunoterapiaRESUMO
Antibody-mediated delivery of immunogenic epitopes to redirect virus-specific CD8+ T-cells towards cancer cells is an emerging and promising new therapeutic strategy. These so-called antibody-epitope conjugates (AECs) rely on the proteolytic release of the epitopes close to the tumor surface for presentation by HLA class I molecules to eventually redirect and activate virus-specific CD8+ T-cells towards tumor cells. We fused the immunogenic EBV-BRLF1 epitope preceded by a protease cleavage site to the C-terminus of the heavy and/or light chains of cetuximab and trastuzumab. We evaluated these AECs and found that, even though all AECs were able to redirect the EBV-specific T-cells, AECs with an epitope fused to the C-terminus of the heavy chain resulted in higher levels of T-cell activation compared to AECs with the same epitope fused to the light chain of an antibody. We observed that all AECs were depending on the presence of the antibody target, that the level of T-cell activation correlated with expression levels of the antibody target, and that our AECs could efficiently deliver the BRLF1 epitope to cancer cell lines from different origins (breast, ovarian, lung, and cervical cancer and a multiple myeloma). Moreover, in vivo, the AECs efficiently reduced tumor burden and increased the overall survival, which was prolonged even further in combination with immune checkpoint blockade. We demonstrate the potential of these genetically fused AECs to redirect the potent EBV-specific T-cells towards cancer in vitro and in vivo.
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Imunoconjugados , Neoplasias , Humanos , Linfócitos T CD8-Positivos , Epitopos , Herpesvirus Humano 4/genética , Neoplasias/terapia , Epitopos de Linfócito TRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy which shows unparalleled therapeutic resistance due to its genetic and cellular heterogeneity, dense stromal tissue, and immune-suppressive tumour microenvironment. Oncolytic virotherapy has emerged as a new treatment modality which uses tumour-specific viruses to eliminate cancerous cells. Non-human primate adenoviruses of the human adenovirus B (HAdV-B) species have demonstrated considerable lytic potential in human cancer cells as well as limited preexisting neutralizing immunity in humans. Previously, we have generated a new oncolytic derivative of the gorilla-derived HAdV-B AdV-lumc007 named 'GoraVir'. Here, we show that GoraVir displays oncolytic efficacy in pancreatic cancer cells and pancreatic-cancer-associated fibroblasts. Moreover, it retains its lytic potential in monoculture and co-culture spheroids. In addition, we established the ubiquitously expressed complement receptor CD46 as the main entry receptor for GoraVir. Finally, a single intratumoural dose of GoraVir was shown to delay tumour growth in a BxPC-3 xenograft model at 10 days post-treatment. Collectively, these data demonstrate that the new gorilla-derived oncolytic adenovirus is a potent oncolytic vector candidate that targets both pancreatic cancer cells and tumour-adjacent stroma.
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Therapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects of these antibody conjugates require optimization to increase their efficacy. Here we evaluated different strategies to conjugate an EBV epitope (YVL/A2) preceded by a protease cleavage site to the antibodies cetuximab and trastuzumab. Three approaches were taken: chemical conjugation (i.e. a thiol-maleimide reaction) to reduced cysteine side chains, heavy chain C-terminal enzymatic conjugation using sortase A, and genetic fusions, to the heavy chain (HC) C-terminus. All three conjugates were capable of T-cell activation and target-cell killing via proteolytic release of the EBV epitope and expression of the antibody target was a requirement for T-cell activation. Moreover, AECs generated with a second immunogenic epitope derived from CMV (NLV/A2) were able to deliver and redirect CMV specific T-cells, in which the amino sequence of the attached peptide appeared to influence the efficiency of epitope delivery. Therefore, screening of multiple protease cleavage sites and epitopes attached to the antibody is necessary. Taken together, our data demonstrated that multiple AECs could sensitize cancer cells to virus-specific T cells.
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Infecções por Citomegalovirus , Imunoconjugados , Neoplasias , Humanos , Epitopos , Peptídeos , Anticorpos , Peptídeo Hidrolases , Neoplasias/terapiaRESUMO
The absence of T cells in the tumor microenvironment of solid tumors is a major barrier to cancer immunotherapy efficacy. Oncolytic viruses, including reovirus type 3 Dearing (Reo), can recruit CD8+ T cells to the tumor and thereby enhance the efficacy of immunotherapeutic strategies that depend on high T-cell density, such as CD3-bispecific antibody (bsAb) therapy. TGF-ß signaling might represent another barrier to effective Reo&CD3-bsAb therapy due to its immunoinhibitory characteristics. Here, we investigated the effect of TGF-ß blockade on the antitumor efficacy of Reo&CD3-bsAb therapy in the preclinical pancreatic KPC3 and colon MC38 tumor models, where TGF-ß signaling is active. TGF-ß blockade impaired tumor growth in both KPC3 and MC38 tumors. Furthermore, TGF-ß blockade did not affect reovirus replication in both models and significantly enhanced the Reo-induced T-cell influx in MC38 colon tumors. Reo administration decreased TGF-ß signaling in MC38 tumors but instead increased TGF-ß activity in KPC3 tumors, resulting in the accumulation of α-smooth muscle actin (αSMA+) fibroblasts. In KPC3 tumors, TGF-ß blockade antagonized the antitumor effect of Reo&CD3-bsAb therapy, even though T-cell influx and activity were not impaired. Moreover, genetic loss of TGF-ß signaling in CD8+ T cells had no effect on therapeutic responses. In contrast, TGF-ß blockade significantly improved therapeutic efficacy of Reo&CD3-bsAb in mice bearing MC38 colon tumors, resulting in a 100% complete response. Further understanding of the factors that determine this intertumor dichotomy is required before TGF-ß inhibition can be exploited as part of viroimmunotherapeutic combination strategies to improve their clinical benefit. Significance: Blockade of the pleiotropic molecule TGF-ß can both improve and impair the efficacy of viro-immunotherapy, depending on the tumor model. While TGF-ß blockade antagonized Reo&CD3-bsAb combination therapy in the KPC3 model for pancreatic cancer, it resulted in 100% complete responses in the MC38 colon model. Understanding factors underlying this contrast is required to guide therapeutic application.
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Neoplasias do Colo , Neoplasias Pancreáticas , Camundongos , Animais , Linfócitos T CD8-Positivos , Fator de Crescimento Transformador beta , Neoplasias Pancreáticas/tratamento farmacológico , Imunoterapia , Microambiente TumoralRESUMO
Reoviruses are used as oncolytic viruses to destroy tumor cells. The concomitant induction of anti-tumor immune responses enhances the efficacy of therapy in tumors with low amounts of immune infiltrates before treatment. The reoviruses should provoke immunogenic cell death (ICD) to stimulate a tumor cell-directed immune response. Necroptosis is considered a major form of ICD, and involves receptor-interacting protein kinase 1 (RIPK1), RIPK3 and phosphorylation of mixed-lineage kinase domain-like protein (MLKL). This leads to cell membrane disintegration and the release of damage-associated molecular patterns that can activate immune responses. Reovirus Type 3 Dearing (T3D) can induce necroptosis in mouse L929 fibroblast cells and mouse embryonic fibroblasts. Most human tumor cell lines have a defect in RIPK3 expression and consequently fail to induce necroptosis as measured by MLKL phosphorylation. We used the human colorectal adenocarcinoma HT29 cell line as a model to study necroptosis in human cells since this cell line has frequently been described in necroptosis-related studies. To stimulate MLKL phosphorylation and induce necroptosis, HT29 cells were treated with a cocktail consisting of TNFα, the SMAC mimetic BV6, and the caspase inhibitor Z-VAD-FMK. While this treatment induced necroptosis, three different reovirus T3D variants, i.e., the plasmid-based reverse genetics generated virus (T3DK), the wild-type reovirus T3D isolate R124, and the junction adhesion molecule-A-independent reovirus mutant (jin-1) failed to induce necroptosis in HT29 cells. In contrast, these viruses induced MLKL phosphorylation in murine L929 cells, albeit with varying efficiencies. Our study shows that while reoviruses efficiently induce necroptosis in L929 cells, this is not a common phenotype in human cell lines. This study emphasizes the difficulties of translating the results of ICD studies from murine cells to human cells.
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Orthoreovirus Mamífero 3 , Humanos , Animais , Camundongos , Orthoreovirus Mamífero 3/metabolismo , Necroptose/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Fibroblastos/metabolismo , Linhagem Celular Tumoral , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Apoptose/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy which shows unparalleled therapeutic resistance. Oncolytic viruses have emerged as a new treatment approach and convey their antitumor activity through lysis of cancer cells. The therapeutic efficacy of oncolytic viruses is largely dependent on the induction of immunogenic cell death (ICD) and the subsequent antitumor immune responses. However, the concurrent generation of antiviral immune responses may also limit the a virus' therapeutic window. GoraVir is a new oncolytic adenovirus derived from the Human Adenovirus B (HAdV-B) isolate AdV-lumc007 which was isolated from a gorilla and has demonstrated excellent lytic activity in both in vitro and in vivo models of PDAC. In this study, we characterized the immunostimulatory profile of cancer cell death induced by GoraVir and the concerted cellular antiviral responses in three conventional pancreatic cancer cell lines. While GoraVir was shown to induce late apoptotic/necrotic cell death at earlier time points post infection than the human adenovirus type 5 (HAdV-C5), similar levels of ICD markers were expressed. Moreover, GoraVir was shown to induce ICD not dependent on STING expression and regardless of subsequent antiviral responses. Together, these data demonstrate that GoraVir is an excellent candidate for use in oncolytic virotherapy.
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Adenovírus Humanos , Carcinoma Ductal Pancreático , Vírus Oncolíticos , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/terapia , Morte Celular , Adenoviridae/genética , Carcinoma Ductal Pancreático/terapia , Antivirais , Neoplasias PancreáticasRESUMO
The adenoviruses (AdVs) isolated from humans are taxonomically grouped in seven different species in the Mastadenovirus genus (HAdV-A through G). AdVs isolated from apes are often included in one of the human AdV species. Here we describe the sequence analyses of ten new AdVs that are related to the HAdV-C species and that were isolated from healthy western lowland gorillas, bonobos, chimpanzees, and orangutans kept in Dutch zoos. We analyzed these viruses and compared their genome sequences to those of human- and ape-derived AdV sequences in the NCBI GenBank database. Our data demonstrated that the ape-derived viruses clustering to HAdV-C are markedly distinct from the human HAdV-C species in the size and nucleotide composition (%GC) of their genome, differ in the amino-acid sequence of AdV proteins, and have longer RGD-loops in their penton-base proteins. The viruses form three well-separated clades (the human, the gorilla, and the combined group of the bonobo and chimpanzee viruses), and we propose that these should each be given species-level ranks. The Ad-lumc005 AdV isolated from orangutans was found to be very similar to the gorilla AdVs, and bootstrap inference provided evidence of recombination between the orangutan AdV and the gorilla AdVs. This suggests that this virus may not be a genuine orangutan AdV but may have been transferred from a gorilla to an orangutan host.
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Adenovírus Humanos , Hominidae , Mastadenovirus , Adenoviridae/genética , Adenovírus Humanos/genética , Animais , Gorilla gorilla , Hominidae/genética , Humanos , Pan troglodytes , Filogenia , PongoRESUMO
A recent Nature Medicine article reported a phase II single-arm trial assessing the efficacy of a triple-mutated, third-generation oncolytic herpes simplex virus type 1 in patients with recurrent or residual glioblastoma. We discuss the results and highlight the potential of locally administered virus-based therapies to fight these lethal tumors.
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Glioblastoma , Herpesvirus Humano 1 , Terapia Viral Oncolítica , Vírus Oncolíticos , Glioblastoma/terapia , Herpesvirus Humano 1/genética , Humanos , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Padrão de CuidadoRESUMO
BACKGROUND: Many solid tumors do not respond to immunotherapy due to their immunologically cold tumor microenvironment (TME). We and others found that oncolytic viruses (OVs), including reovirus type 3 Dearing, can enhance the efficacy of immunotherapy by recruiting CD8+ T cells to the TME. A significant part of the incoming CD8+ T cells is directed toward reovirus itself, which may be detrimental to the efficacy of OVs. However, here we aim to exploit these incoming virus-specific T cells as anticancer effector cells. METHODS: We performed an in-depth characterization of the reovirus-induced T-cell response in immune-competent mice bearing pancreatic KPC3 tumors. The immunodominant CD8+ T-cell epitope of reovirus was identified using epitope prediction algorithms and peptide arrays, and the quantity and quality of reovirus-specific T cells after reovirus administration were assessed using high-dimensional flow cytometry. A synthetic long peptide (SLP)-based vaccination strategy was designed to enhance the intratumoral frequency of reovirus-specific CD8+ T cells. RESULTS: Reovirus administration did not induce tumor-specific T cells but rather induced high frequencies of reovirus-specific CD8+ T cells directed to the immunodominant epitope. Priming of reovirus-specific T cells required a low-frequent population of cross-presenting dendritic cells which was absent in Batf3-/- mice. While intratumoral and intravenous reovirus administration induced equal systemic frequencies of reovirus-specific T cells, reovirus-specific T cells were highly enriched in the TME exclusively after intratumoral administration. Here, they displayed characteristics of potent effector cells with high expression of KLRG1, suggesting they may be responsive against local reovirus-infected cells. To exploit these reovirus-specific T cells as anticancer effector cells, we designed an SLP-based vaccination strategy to induce a strong T-cell response before virotherapy. These high frequencies of circulating reovirus-specific T cells were reactivated on intratumoral reovirus administration and significantly delayed tumor growth. CONCLUSIONS: These findings provide proof of concept that OV-specific T cells, despite not being tumor-specific, can be exploited as potent effector cells for anticancer treatment when primed before virotherapy. This is an attractive strategy for low-immunogenic tumors lacking tumor-specific T cells.
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Neoplasias , Terapia Viral Oncolítica , Vírus Oncolíticos , Animais , Linfócitos T CD8-Positivos , Imunoterapia , Camundongos , Terapia Viral Oncolítica/métodos , Microambiente TumoralRESUMO
Gastrointestinal (GI) cancers are characterized by extensive tumor stroma that both promotes tumor progression and acts as a physical barrier for adjacent tumor cells, limiting the effect of current treatment modalities. Oncolytic virotherapy is currently investigated in clinical trials as a novel therapeutic agent for different malignancies of the GI tract, but it is largely unknown whether these viruses can also target the tumor stroma. Here, we investigated the tropism of two commonly studied OVs, adenovirus and reovirus, towards primary GI fibroblasts from human oesophageal, gastric, duodenal and pancreatic carcinomas (N = 36). GI fibroblasts were susceptible to type 3 Dearing (T3D) strain R124 and bioselected mutant reovirus (jin-3) infection but not oncolytic adenovirus (Ad5-Δ24). Efficient infection and apoptosis of human and mouse GI cancer-derived fibroblasts by these reoviruses was partially dependent on the expression of the reovirus entry receptor, Junctional Adhesion Molecule-A (JAM-A). Moreover, human GI cancer organoid-fibroblast co-cultures showed higher overall infectivity when containing JAM-A expressing fibroblasts as compared to JAM-A negative fibroblasts, indicating a potential role of JAM-A expressing fibroblasts for viral dissemination. We further show that JAM-A is not only necessary for efficient reovirus infection of fibroblasts but also partially mediates reovirus-induced apoptosis, dependent on signaling through the C-terminal PDZ-domain of JAM-A. Altogether, our data show the presence of JAM-A expressing fibroblasts in both human and murine GI cancers that are amenable to infection and induction of apoptosis by reovirus, extending the potential anti-cancer actions of reovirus with stromal targeting.
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Fibroblastos Associados a Câncer , Molécula A de Adesão Juncional , Neoplasias , Terapia Viral Oncolítica , Reoviridae , Humanos , Camundongos , Animais , Reoviridae/genética , Trato GastrointestinalRESUMO
The use of human adenoviruses (hAds) as oncolytic agents has demonstrated considerable potential. However, their efficacy in clinical studies is generally moderate and often varies between patients. This may, in part, be attributable to variable pre-existing neutralizing immunity in patients, which can impact the antitumor efficacy and lead to response heterogeneity. Our aim was to isolate new Ads for the development of oncolytic vectors with low prevalence of neutralizing immunity in the human population. To this end, we isolated a collection of new nonhuman primate (nhp) Ads from stool samples of four great ape species held captive. We elected 12 isolates comprising the broadest genetic variability for further characterization. For three new nhpAds, all classified as the human adenovirus B (HAdV-B) species, no neutralizing activity could be detected when exposed to a preparation of immunoglobulins isolated from a pool of >1,000 donors as a surrogate of population immunity. In addition, the nhpAds of the HAdV-B species showed enhanced oncolytic potency compared to nhpAds of the HAdV-C species as well as to human adenovirus type 5 (HAdV-C5) in vitro when tested in a panel of 29 human cancer cell lines. Next-generation sequencing of the viral genomes revealed higher sequence similarity between hAds and nhpAds of HAdV-B compared to HAdV-C, which might underlie the differences in oncolytic ability. As a proof-of-concept, the Rb-binding domain of the E1A protein of the gorilla-derived HAdV-B nhpAd-lumc007 was deleted, thereby creating a new oncolytic derivative, which demonstrated increased oncolytic potential compared to HAdV-C5. Collectively, our data demonstrate that nhpAds of the HAdV-B species can serve as an alternative for the development of potent oncolytic Ad vectors with limited pre-existing neutralizing immunity in humans.
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Adenovírus Humanos , Neoplasias , Adenoviridae/genética , Adenovírus Humanos/genética , Animais , Genoma Viral , Humanos , Neoplasias/genética , Neoplasias/terapia , Primatas/genéticaRESUMO
Treatment of castration-resistant prostate cancer remains a challenging clinical problem. Despite the promising effects of immunotherapy in other solid cancers, prostate cancer has remained largely unresponsive. Oncolytic viruses represent a promising therapeutic avenue, as oncolytic virus treatment combines tumour cell lysis with activation of the immune system and mounting of effective anti-tumour responses. Mammalian Orthoreoviruses are non-pathogenic human viruses with a preference of lytic replication in human tumour cells. In this study, we evaluated the oncolytic efficacy of the bioselected oncolytic reovirus mutant jin-3 in multiple human prostate cancer models. The jin-3 reovirus displayed efficient infection, replication, and anti-cancer responses in 2D and 3D prostate cancer models, as well as in ex vivo cultured human tumour slices. In addition, the jin-3 reovirus markedly reduced the viability and growth of human cancer cell lines and patient-derived xenografts. The infection induced the expression of mediators of immunogenic cell death, interferon-stimulated genes, and inflammatory cytokines. Taken together, our data demonstrate that the reovirus mutant jin-3 displays tumour tropism, and induces potent oncolytic and immunomodulatory responses in human prostate cancer models. Therefore, jin-3 reovirus represents an attractive candidate for further development as oncolytic agent for treatment of patients with aggressive localised or advanced prostate cancer.
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Orthoreovirus Mamífero 3 , Terapia Viral Oncolítica , Vírus Oncolíticos , Neoplasias da Próstata , Reoviridae , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Mamíferos , Vírus Oncolíticos/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , Reoviridae/genéticaRESUMO
Type 1 diabetes (T1D) is an autoimmune disease characterized by autoreactive T cell-mediated destruction of the insulin-producing pancreatic ß-cells. Increasing evidence suggest that the ß-cells themselves contribute to their own destruction by generating neoantigens through the production of aberrant or modified proteins that escape central tolerance. We recently demonstrated that ribosomal infidelity amplified by stress could lead to the generation of neoantigens in human ß-cells, emphasizing the participation of nonconventional translation events in autoimmunity, as occurring in cancer or virus-infected tissues. Using a transcriptome-wide profiling approach to map translation initiation start sites in human ß-cells under standard and inflammatory conditions, we identify a completely new set of polypeptides derived from noncanonical start sites and translation initiation within long noncoding RNA. Our data underline the extreme diversity of the ß-cell translatome and may reveal new functional biomarkers for ß-cell distress, disease prediction and progression, and therapeutic intervention in T1D.
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Inflamação , Células Secretoras de Insulina/metabolismo , Biossíntese de Proteínas/genética , RNA Longo não Codificante/genética , Autoimunidade/genética , Biomarcadores/análise , Biomarcadores/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Perfilação da Expressão Gênica/métodos , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Células Secretoras de Insulina/patologia , Pancreatite/genética , Pancreatite/metabolismo , Pancreatite/patologia , Iniciação Traducional da Cadeia Peptídica/genética , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismo , Análise de Sequência de RNA/métodos , TranscriptomaRESUMO
Oncolytic viruses are promising agents for cancer therapy because they selectively infect and kill tumor cells, and because they trigger immune responses that can boost anticancer immunity. Key to the latter process is the production of type I interferons (IFN-Is) that can turn noninflamed "cold" tumors into "hot" ones. Besides this desired anticancer effect, IFN-Is are antiviral and successful oncolytic virotherapy thus relies on tightly controlled IFN-I levels. This requires a profound understanding of when and how tumor cells induce IFN-I in response to specific viruses. In this study, we uncovered two key factors that augment IFN-I production in transformed human myeloid cells infected with a tumor-selective reovirus. Viral replication and IFN-α/ß receptor (IFNAR) signaling progressively reinforced the levels of IFN-I expressed by infected cells. Mechanistically, both augmented the activation of interferon regulatory factor 3, a key transcription factor for IFNß expression. Our findings imply that reovirus-permissive tumor cells themselves are a major source of IFN-I expression. As tumors can perturb the IFNAR pathway for their own survival, reovirus-exposed IFNAR-unresponsive tumors may need additional therapeutic intervention to promote the secretion of sufficient IFN-I into the tumor microenvironment. Our increased understanding of the parameters that affect reovirus-induced IFN-I levels could aid in the design of tailored virus-based cancer therapies.
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Interferon Tipo I , Humanos , Interferon Tipo I/genética , Interferon-alfa/genética , Interferon beta/genética , Transdução de Sinais , Replicação ViralRESUMO
Lentiviral vectors have become popular tools for stable genetic modification of mammalian cells. In some applications of lentiviral vector-transduced cells, infectious-lentiviral particles should be absent. Quantification of the free-vector particles that remain from the inoculum can be difficult. Therefore a formula was established that yields an estimation of the 'Reduction Ratio.' This ratio represents the loss of titer based on a number of vector-inactivating effects. In this study, we evaluated several parameters and assumptions that were used in the current formula. We generated new data on the stability and trypsin sensitivity of lentiviral vectors pseudotyped with eight heterologous envelope proteins and the loss of vectors by washing or passaging the cell cultures. Our data demonstrate that the loss of virus titer under the influence of trypsin as well as the half-life of the particles in tissue culture medium is dependent on the vector's envelope protein. While VSV-G-envelope-pseudotyped particles were unsensitive to trypsin, the titer of vectors pseudotyped with other envelope proteins decreased 2-110-fold. The half-life in culture medium ranged from 8 to 40 h for the different envelope-pseudotyped vectors, with 35 h for VSV-G-envelope-pseudotyped vector particles. Additionally, we found that removal of the culture medium from Ø35 mm to Ø10 cm dishes reduces the amount of vector particles in the culture by 50-fold and 20-fold, respectively. Together these data can be used to more precisely estimate the maximum number of free lentiviral vector particles in cell cultures.
Assuntos
Vetores Genéticos , Lentivirus , Animais , Vetores Genéticos/genética , Lentivirus/genética , Transdução Genética , Proteínas do Envelope Viral/genéticaRESUMO
BACKGROUND: T-cell-engaging CD3-bispecific antibodies (CD3-bsAbs) are promising modalities for cancer immunotherapy. Although this therapy has reached clinical practice for hematological malignancies, the absence of sufficient infiltrating T cells is a major barrier for efficacy in solid tumors. In this study, we exploited oncolytic reovirus as a strategy to enhance the efficacy of CD3-bsAbs in immune-silent solid tumors. METHODS: The mutant p53 and K-ras induced murine pancreatic cancer model KPC3 resembles human pancreatic ductal adenocarcinomas with a desmoplastic tumor microenvironment, low T-cell density and resistance to immunotherapy. Immune-competent KPC3 tumor-bearing mice were intratumorally injected with reovirus type 3 Dearing strain and the reovirus-induced changes in the tumor microenvironment and spleen were analyzed over time by NanoString analysis, quantitative RT-PCR and multicolor flow cytometry. The efficacy of reovirus in combination with systemically injected CD3-bsAbs was evaluated in immune-competent mice with established KPC3 or B16.F10 tumors, and in the close-to-patient human epidermal growth factor receptor 2 (HER2)+ breast cancer model BT474 engrafted in immunocompromised mice with human T cells as effector cells. RESULTS: Replication-competent reovirus induced an early interferon signature, followed by a strong influx of natural killer cells and CD8+ T cells, at the cost of FoxP3+ Tregs. Viral replication declined after 7 days and was associated with a systemic activation of lymphocytes and the emergence of intratumoral reovirus-specific CD8+ T cells. Although tumor-infiltrating T cells were mostly reovirus-specific and not tumor-specific, they served as non-exhausted effector cells for the subsequently systemically administered CD3-bsAbs. Combination treatment of reovirus and CD3-bsAbs led to the regression of large, established KPC3, B16.F10 and BT474 tumors. Reovirus as a preconditioning regimen performed significantly better than simultaneous or early administration of CD3-bsAbs. This combination treatment induced regressions of distant lesions that were not injected with reovirus, and systemic administration of both reovirus and CD3-bsAbs also led to tumor control. This suggests that this therapy might also be effective for metastatic disease. CONCLUSIONS: Oncolytic reovirus administration represents an effective strategy to induce a local interferon response and strong T-cell influx, thereby sensitizing the tumor microenvironment for subsequent CD3-bsAb therapy. This combination therapy warrants further investigation in patients with non-inflamed solid tumors.