Infectious bursal disease virus (IBDV) as a novel oncolytic virotherapy in glioblastoma

1 TurPlanellsV, etal. J Immunother Cancer 2025;13:e011741. doi:10.1136/jitc-2025-011741 Open access Infectious bursal disease virus (IBDV) as a novel oncolytic virotherapy in glioblastoma Vicent TurPlanells,1,2,3 Yonina Bykov,1 Gloria Dawodu,1 Noemi GarcíaRomero,4 Sara IzpuraLuis,2,3 Leticia PérezRodríguez,5 Sergio RiusRocabert,2,3 Irina PalacínAliana,3 Javier ArranzHerrero,2,3,6 Inmaculada MárquezLeiva,6 Alvaro MonagoSanchez,4 MariaLuisa del Rio,7 JoseIgnacio RodriguezBarbosa ,7 Jordi CanoOchando,5,6 Adolfo GarcíaSastre,1,8,9,10,11,12 Daniel LozanoOjalvo,13 Estanislao NistalVillan ,2,3 Angel AyusoSacido,4,14,15 Sara CuadradoCastano 1,10,12,16 To cite: TurPlanellsV, BykovY, DawoduG, etal. Infectious bursal disease virus (IBDV) as a novel oncolytic virotherapy in glioblastoma. Journal for ImmunoTherapy of Cancer 2025;13:e011741. doi:10.1136/ jitc-2025-011741 ►Additional supplemental material is published online only. To view, please visit the journal online (https:// doi. org/ 10. 1136/ jitc2025011741). Accepted 13 October 2025 For numbered affiliations see end of article. Correspondence to Dr Sara CuadradoCastano; sara. cuadrado@ mssm. edu Dr Estanislao NistalVillan; estanislao. nistalvillan@ ceu. es Dr Angel AyusoSacido; ayusosa@ vithas. es Original research © Author(s) (or their employer(s)) 2025. Reuse permitted under CC BYNC. No commercial reuse. See rights and permissions. Published by BMJ Group. ABSTRACT Background Glioblastoma (GBM) is the most aggressive form of cancer of the central nervous system. Despite advances in immunotherapies and standardofcare treatments for GBMs, clinical outcomes remain limited— owing to the immunosuppressive tumor microenvironment and the intrinsic resistance of GBM to conventional approaches. As a result, there is growing interest in rational combination strategies, particularly those pairing oncolytic viruses with immunebased therapies or established treatment modalities. Oncolytic viruses, by displaying conditionally enabled tumor cellrestricted replication, while stimulating antitumor immune responses and leaving healthy tissue unharmed, have the potential to reshape the therapeutic landscape in GBM and aid in achieving more durable benefits for patients. This study investigates the use of infectious bursal disease virus (IBDV) as a potential virotherapy for GBM. Methods and results In vitro, IBDV infects and replicates within murine GBM cells and patientderived GBM stem cells, inducing direct oncolysis and activating proinflammatory gene expression programs. IBDV also enhances the cytolytic activity of temozolomide (TMZ) in treated GBM cells, complementing TMZ chemotherapeutic activity. In vivo, treatment with IBDV in CT2A GBMbearing syngeneic mice significantly reduced tumor growth and improved survival compared with control mice. Intratumoral administration of IBDV induces a deep remodeling of the tumor immune microenvironment, reducing immunosuppressive M2like macrophages and increasing the ratio of CD8+T cells to regulatory T cells. This reversion of immunosuppression linked to monocytederived macrophages has been confirmed on experimental ex vivo infections of explants derived from human GBM donors. Conclusion These findings support further consideration of IBDV as a novel virotherapeutic agent for GBM. WHAT IS ALREADY KNOWN ON THIS TOPIC ⇒Glioblastoma (GBM) is the most aggressive primary brain tumor, with limited treatment options and a median survival of only 15 months despite standard care with surgery, radiotherapy, and temozolomide (TMZ). Resistance to TMZ and the immunosuppressive tumor microenvironment (TME) present significant challenges to current treatments. Oncolytic viruses (OVs) have shown promise as a therapeutic strategy by selectively infecting tumor cells and activating potent immune responses. WHAT THIS STUDY ADDS ⇒This study introduces infectious bursal disease virus (IBDV) as a novel oncolytic virotherapy for GBM. IBDV effectively infects and replicates in patientderived glioblastoma stem cells and murine GBM cells, inducing tumor cell death and immune activation. The virus synergizes with TMZ to enhance cytotoxic effects and remodels the TME by reducing immunosuppressive cells, such as M2like macrophages and regulatory T cells, while increasing cytotoxic CD8+T cells. These findings establish IBDV as a safe and potent virotherapy candidate. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY ⇒IBDV’s ability to complement TMZ, reduce immunosuppression, and activate antitumor immunity positions it as a promising addition to GBM treatment regimens. Its nonhuman origin and that avoids preexisting immunity issues seen with other OVs in the general population. This study lays the groundwork for further preclinical and clinical research on IBDV, potentially leading to innovative combination therapies that improve patient with GBM outcomes and inform policies on oncolytic virotherapy integration into standard cancer care. Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2025-011741 on 4 November 2025. Downloaded from https://jitc.bmj.com on 6 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 2TurPlanellsV, etal. J Immunother Cancer 2025;13:e011741. doi:10.1136/jitc-2025-011741 Open access INTRODUCTION Glioblastoma (GBM) is a diffuse astrocyticoligodendroglial tumor. It is the most aggressive malignant primary brain tumor, which accounts for 77%–81% of all primary malignant central nervous system (CNS) tumors.1 It is classified as a Grade 4 glioma in the latest version of the WHO (CNS5), based on molecular and histopathological features.2 GBM is characterized by cytologically malignant, highly mitotic, and necrosisprone neoplasms, typically associated with a high proliferation rate and rapid disease progression both before and after surgery, leading to a fatal outcome. Current treatment consists of a combination of surgery, radiotherapy, and chemotherapy with the DNA alkylating agent temozolomide (TMZ). However, despite advances in clinical diagnosis and the development of novel therapies, no effective treatment exists, and the overall survival remains at 15 months.3 Several factors contribute to the failure of current treatments, including TMZ resistance pathways,4 high invasive and infiltrative potential, significant intratumoral and intertumoral heterogeneity, limited access to chemotherapeutic drugs like TMZ to the CNS due to the presence of the bloodbrain barrier, low permeability of blood vessels within the tumor mass,5 a highly immunosuppressive tumor microenvironment (TME),6 and the presence of a tumor cell subpopulation called cancer stem cells (CSCs).7 The study of CSCs has gained relevance due to their stem celllike properties and their implications in tumor development and cancer prognosis. CSCs are responsible for GBM tumor dissemination through brain parenchyma,8 contributing to glioma genesis, as well as drug resistance, metastasis, and tumor recurrence.9 Additionally, various mutations have been identified in these tumors. Particularly interesting are the heterozygous or homozygous deletions at Ch9p21.10 These deletions involve the genetic loss of the cyclindependent kinase inhibitor 2A gene (CDKN2A).11 The type I interferon (IFN) gene cluster is in the same chromosomal region as CDKN2A, and codeletion of both, CDKN2A and the type I IFN cluster, is common.12 13 IFN activity functions as one of the primary immune defense mechanisms against viral infections. The absence of IFN type I cluster may render these tumors more permissive to viral replication and potentially to oncolytic therapies.14 Therefore, it is crucial to develop new therapeutic approaches that address both the limited access to the CNS and the genetic characteristics of the tumors. Oncolytic viruses (OVs) represent a novel class of multimechanistic therapeutic agents for cancer treatment. OVs display conditionally enabled, tumor cellrestricted replication while stimulating antitumor immune responses and leaving healthy tissue unharmed.15 The susceptibility of cancer cells to support viral replication depends on unique molecular features mainly related to dysfunction in antiviral signaling pathways, while OVs remain inactive or unable to replicate in normal, healthy cells—where the antiviral defense machinery remains intact.16 17 The death of tumor cells leads to the release of tumor antigens, cytokines and chemokines that stimulate the recruitment and activation of innate cells, leading to a deep remodeling of the immunosuppressive TME; importantly, the recruitment and activation of antigenpresenting cells (APCs) capable of presenting both tumor and viral epitopes is critical to achieve T cellspecific tumor elimination.18 Numerous preclinical and earlyphase clinical studies (phase I and II) have explored both DNA and RNA OVs.19 A recent example is the conditional approval of an attenuated type I herpes simplex virus (Delytact) in Japan to treat GBM.20 As of today, the major challenges facing OVs in GBM treatment are linked to the highly immunosuppressive microenvironment of GBM often limits viral infection, spread, and the induction of robust antitumor immune responses, as well as preexisting antiviral immunity that can lead to rapid immune clearance of the virus and reduce treatment efficacy.21 22 Infectious bursal disease virus (IBDV) is a bisegmented doublestranded RNA virus that belongs to the genus Avibirnavirus within the family Birnaviridae. IBDV primarily infects young domestic chickens, targeting immature IgMbearing B lymphocytes localized in the bursa of Fabricius through several proposed surface proteins, including surface immunoglobulin M, integrin α4β1, and heat shock protein 90,23 24 which facilitate viral attachment, internalization, and uncoating. IBDV can also infect macrophages and monocytes, although to a lesser extent.25 As a result, chickens develop immunosuppression, making them more susceptible to opportunistic and secondary infections that can exacerbate disease severity. To date, there have been no reports of IBDV causing infections in mammals, including humans.26 Previous studies have explored whether IBDV can infect and replicate in mammalian cell lines—under laboratory conditions—demonstrating that IBDV typically does not establish productive infection or cytopathic effect in mammalian cells, and many IBDV strains require adaptation through serial passages to replicate efficiently in mammalian cells.27–29 In the case of IBDV, this restriction is likely due to the incompatibility of viral entry mechanisms, the absence of the required host factors for replication, and strong mammalian antiviral defense. Yet, to date, only one report has shown IBDV ability to infect human cervix adenocarcinoma HeLa cells.30 In this work, we investigate the potential of IBDV as OV. As of today, several avian viruses are under investigation for cancer therapy, including Newcastle disease virus (APMV1, Avian avulavirus 1, aka Avian paramyxovirus 1),31 32 avian reoviruses,33 avian avulavirus (APMV4)34 and certain avian influenza strains.35 Research into avian viruses as cancer therapeutics is robust and expanding, as their inherent oncolytic features, combined with host restrictions and lack of preexisting immunity in humans, are major advantages to be used as therapeutics in human cancer. Furthermore, IBDV has also been investigated for its potential broadspectrum antiviral effects Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2025-011741 on 4 November 2025. Downloaded from https://jitc.bmj.com on 6 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 3 TurPlanellsV, etal. J Immunother Cancer 2025;13:e011741. doi:10.1136/jitc-2025-011741 Open access in nononcologic contexts, including the postinfection treatment of acute and chronic viral diseases, reinforcing its safety and therapeutic versatility beyond cancer.36 Here, we have studied responses elicited by IBDV in different models of GBM. In addition to inducing tumor cell oncolysis, IBDV stimulates antiviral and proinflammatory immune responses in different glioma cells. In vitro, the susceptibility of GBM cells to IBDV infection and killing is further boosted in combination with the chemotherapeutic TMZ—current standard of care in GBM management. In preclinical studies in mice, intratumoral administration of IBDV delays tumor growth, concomitant to a robust immune cell remodeling at the TME. In human explant studies, short exposure to IBDV demonstrates a reduction of the immunosuppressive nature of GBM TME. MATERIAL AND METHODS Cell lines and viruses Murine glioma cell lines CT2A (ATCC cat# SCC194) and GL261 (ATCC cat# ACC 802) were maintained in Dulbecco’s Modified Eagle Medium (DMEM)/F12 supplemented with 10% FBS (Fetal Bovine Serum), 1% antibiotics, and 2 mM of Lglutamine. Human patientderived primary GBM cells (GBM18 and GBM27) were obtained according to GarcíaRomero, GonzálezTejedo et al, and subcultured in supplemented DMEM/F12 (Gibco, 11039) containing nonessential amino acids (10 mM; Gibco, 11140), Hepes (1 M; Gibco, 15630), Dglucose (45%; Sigma, St. Louis, Missouri, USA; G8769), BSAF5 (7.5%; Gibco, 15260), sodium pyruvate (100 mM; Gibco, 11360), Lglutamine (200 mM; Gibco, 25030), antibioticantimycotic (100×; Gibco, 15240), N2 supplement (100×; Gibco, 17502), hydrocortisone (1 µg/µL; Sigma, H0135), triiodothyronine (100 µg/mL; Sigma, T5516), EGF (25 ng/µL; Sigma, E9644), bFGF (25 ng/µL; Sigma, F0291), and heparin (1 µg/µL; Sigma, H3393). QM7 cells (ATCC cat# CRL1962) were cultured in DMEM supplemented with 10% FBS. Murine BMDMs (Bone MarrowDerived Macrophages) were isolated from the femur, tibia, and fibula of bone marrow of 8weekold C57BL/6 mice. Bone marrow was liberated through the injection of phosphatebuffered saline (PBS) and clogs were disaggregated mechanically by a syringe. Afterwards, cells were centrifuged for 5 min at 1,500 rpm and the supernatant was discarded. Finally, cells were resuspended in RPMI (Roswell Park Memorial Institute medium) medium supplemented with GMCSF (GranulocyteMacrophage ColonyStimulating Factor) (Gibco, Waltham, Massachusetts, USA) (30 ng/mL) before plating. After 4 days, cell medium was replaced with a fresh stock for 3 more days to complete cell differentiation before viral infections. The human microglia HMC3 cell line (ATCC cat# CRL3304) was maintained in DMEM supplemented with 10% FBS. Cells were maintained at 37°C with 5% of CO2 and 90% humidity. IBDV, Soroa strain, and serotype I, being low pathogenic in birds, were provided by Professor José F Rodríguez (CNBCSIC, Spain); viral stocks were propagated in QM7 cells and clearedpurified following the protocol previously described.37 The recombinant Newcastle disease virus LaSota strain NDVLSL289A, was propagated in 9dayold chickenembryonated eggs and clearpurified by ultracentrifugation in 30% sucrose gradient. Fluorescence microscopy and image acquisition Cancer cells were infected at the indicated multiplicity of infection (MOI) for 24 hours. Cell fixation was performed using 2% paraformaldehyde in PBS for 10 min. Cell membrane permeabilization was carried out using 0.2% TritonPBS for 10 min and blocked in PBS 1% BSA (Bovine Serum Albumin) for 1 hour. Specific antibodies used for indirect immune detection are described in online supplemental table 1. Images were taken using an EVOS FL cell imaging system (Thermo Fisher Scientific). Virus replication kinetics and tittering assays Supernatants of IBDVinfected GBM cells at corresponding time points were tittered in QM7 cells plated the day before in 96well plates in DMEM supplemented medium. IBDV supernatants were diluted by 1/10 dilutions in DMEM supplemented medium and left on QM7 cells for 72 hours. Negative control wells had GBM supernatants without IBDV infection. The cytopathic effect was analyzed by optical microscopy 72 hours postinfection. Tissue culture infectious dose 50 (TCID50/mL) viral titers were calculated using the ReedMuench method.38 Viability assays An (3- (4,5dimethylthiazol2yl)- 5- (3carboxymethoxy phenyl)- 2- (4sulfophenyl)- 2Htetrazolium) MTS assay was used to assess cell viability. A total of 1×10⁴ cells per well were plated in a 96well plate containing complete medium and allowed to adhere for 24 hours. After attachment, cells were treated with TMZ or infected with IBDV or NDVLS viruses for the corresponding experimental conditions. PBS was used as a control, with five replicate wells per condition. After the designated treatment time, 20 µL of MTS solution (5 mg/mL) was added to each well. The plates were then incubated at 37°C, protected from light, for 1.5 hours. Following incubation, the solutions in the wells were mixed, and absorbance was measured at 450 nm (for tetrazolium salts) and 630 nm (for background) using a microplate reader (Varioskan Lux). Relative cell viability was calculated as the percentage of absorbance compared with the control group. Western blots Protein was extracted from the cultured cells with radioimmunoprecipitation lysis buffer on ice and centrifuged at 12,000×g for 15 min at 4°C to eliminate cell debris. The total protein concentration was determined using a Bicinchoninic Acid (BCA) protein assay kit, and whole lysates mixed with 5× sodium dodecyl sulfate (SDS) Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2025-011741 on 4 November 2025. Downloaded from https://jitc.bmj.com on 6 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 4TurPlanellsV, etal. J Immunother Cancer 2025;13:e011741. doi:10.1136/jitc-2025-011741 Open access loading buffer were denatured with a 5 min incubation at 95°C. Then, equal amounts of protein were separated by SDSpolyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Blocked membranes were then incubated with their corresponding antibodies described in online supplemental table 1. The horseradish peroxidase immune complexes were detected using an enhanced chemiluminescence kit (Alkali Scientific) in a ChemiDoc imaging system (BioRad). Transcription analysis by qRT-PCR Cells were mock treated or infected with the specified virus at an MOI of 3 Plack formation units (PFU)/cell in 500 µL of OptiMEMI. After allowing virus adsorption for 1 hour, the cells were incubated with an additional 1,500 µL of supplemented media. Total RNA was isolated using a Qiagen RNeasy Minikit (catalog no.74106, Qiagen) at the indicated time postinfection. Complementary DNA (cDNA) synthesis was performed using the Maxima First Strand cDNA Synthesis Kit for quantitative Realtime reverse transcriptionpolymerase chain reaction (RTPCR) (catalog no. K1671, Thermo Fisher Scientific). Mean nfold expression levels of cDNA from three individual biological samples were normalized to housekeeping gene levels and calibrated to mocktreated samples according to the 2−ΔΔCt method.39 Results are expressed as relative cycle threshold (Ct) values to the mocktreated group. Human and murine primer sequences are compiled in online supplemental table 2. Histological staining Spleen, bone marrow, lymph nodes, and liver were harvested in 9weekold C57BL/6 mice after 24 hours of one IBDV (107 PFU) tail vein injection for systemic administration (n=3). PBS was used for negative controls. Tissue fragments were fixed in a 4% buffered formalin solution, pH 7.2, for 24 hours. The buffer was changed to ethanol solution 70% and organs were embedded in paraffin. Paraffin blocks of each organ were cut in a microtome (3 µm slices) and mounted on slides prior to routine histological analysis (H&E). Histological slides were qualitatively analyzed under light microscopy. Systemic and intracranial virus delivery studies 9weekold C57BL/6 mice (Charles River Laboratories, Wilmington, Massachusetts, USA) were stereotactically injected with viral doses of 104, 105, and 106 PFU into the striatum of the right hemisphere (0 mm anterior and 2.5 mm lateral to the bregma; 3.5 mm intraparenchymal). PBS was used as a control vehicle. Mice weight was monitored weekly for 6 weeks. Procedures used on mice were approved by and performed according to the guidelines of the institutional animalcare committee of Universidad Francisco Vitoria under the accession PROEX 142.8/24. The protocol for biodistribution studies was approved by Universidad de Leon under the access OEBAULE001–2023. Tumor model Tumor progression and survival studies were performed following Institutional Animal Care and Use Committee (IACUC) guidelines and have been approved by the IACUC of Icahn School of Medicine at Mount Sinai (IACUC2014–0234). 6–8 weeks old of age female C57BL/6J mice used in our in vivo studies were purchased from Jackson Laboratory. 1.5×106 CT2A cells were intradermally engrafted on the right hind leg and tumors were allowed to grow to 50 mm3 before treatment initiation. Tumorbearing mice were treated by intratumoral injection of IBDV, NDV, or PBS with viral doses of 1×107 PFU/50 µL PBS. Intratumoral injections were administered every other day for a total of four treatment doses or as specified for downstream analysis or tissue samples. Tumor volume was monitored every 48 hours, and every 24 hours as tumor volumes approached the experimental endpoint of 1,000 mm3. Mice were humanely euthanized on the day when the volume exceeded the predefined endpoint or at any sign of distress, including tumor ulceration. Tumor measurement was determined using a digital caliper, and the total volume was calculated using the formula: tumor volume (V)=L2×W, where L (tumor length) is the larger diameter and W (tumor width) is the smallest diameter. Human GBM explants study Human GBMisocitrate dehydrogenase (IDH) wildtype tumor biopsy was obtained from the Biobank of Hospital Universitario Puerta de Hierro Majadahonda/Instituto de Investigación Sanitaria Puerta de HierroSegovia de Arana. Ethical approval for the use of these samples was granted by the institutional review board of HM Hospitals (CEIm No: 23.06.2206GHM). All patients provided written informed consent prior to enrollment, and all procedures were conducted in accordance with the principles of the Declaration of Helsinki. Fresh tumor samples were placed in icecold HibernateA medium supplemented with penicillinstreptomycin immediately after resection and processed within 2 hours of surgery. GBM tissue was cut into 20–30 mm³ fragments under a dissection microscope, washed three times with icecold PBS, and then placed in 24well plates containing specialized growth medium (DMEMF12, Neurobasal, nonessential amino acids, N2 supplement, B27 supplement, human insulin, penicillinstreptomycin, and 2mercaptoethanol). The tissue slices were maintained at 37°C in a humidified atmosphere with 5% CO₂. Explants were subjected to TMZ treatment (1 mM) and/or IBDV infection (10⁷ PFU) and cultured for 72 hours prior to downstream processing and flow cytometry analysis. Flow cytometry Murine tumors and tumordraining lymph nodes (tdLN) were excised from terminated animals at the indicated time points and tissues were processed for downstream analysis by flow cytometry. Briefly, samples were incubated in FCblocking antibody Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2025-011741 on 4 November 2025. Downloaded from https://jitc.bmj.com on 6 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 5 TurPlanellsV, etal. J Immunother Cancer 2025;13:e011741. doi:10.1136/jitc-2025-011741 Open access (BD #553141) diluted in FluorescenceActivated Cell Sorting FACS buffer (1% BSA, 0.5 mM EDTA in PBS) for 20 min on ice, then resuspended in Fixable Viability Dye eFluor 780 (Thermo Fisher #65086514; 1:3000) in PBS for 10 min at room temperature. Samples were incubated with corresponding extracellular antibodies prepared in FACS buffer for 30 min. Samples were then fixed according to manufacturer’s instructions using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher, Ref. 00552300). Intracellular staining was performed in permeabilization wash buffer (eBioscience, Ref. 008333−56/88882400). Antibodies used for intracellular and extracellular staining of isolated immune cells are itemized in (online supplemental table 1). Samples were run on an LSRFortessa X20 flow cytometer (BD); immunophenotyping analysis was performed using FlowJo V.10 software. Single-cell isolation from GBM explants for spectral flow cytometry analysis GBM tumor biopsy specimens were preserved in MACS Tissue Storage Solution (Miltenyi Biotec, Bergisch Gladbach, Germany), maintained at 48°C, and processed within 3 hours after collection. To obtain singlecell suspensions, samples were enzymatically digested in 10 mL of RPMI with Ca2+/ Mg2 +(Gibco) containing 2% FBS (ATCC, Manassas, Virginia, USA), 0.5 mg/mL DNase I (grade II, from bovine pancreas; Roche, Penzberg, Germany), and 0.5 mg/mL collagenase IV (Gibco) for 40 min at 37°C with continuous shaking of 150 rpm. Tissues were disrupted, passed through a 70 mm cell strainer, and washed two times with RPMI medium (Gibco). Isolated cells were centrifuged and directly used for phenotyping. Immune phenotyping of GBM biopsy specimens was performed by staining the cells for spectral flow cytometry analyses using the flow antibodies listed in online supplemental materials, and samples were acquired with a Cytek Aurora 5laser cytometer (Cytek Biosciences, Fremont, California, USA). Immunophenotyping analysis was performed using FlowJo V.10 software. Statistical analysis Data analysis was performed using GraphPad Prism V.10 package. Oneway analysis of variance (ANOVA) or twoway ANOVA was used to compare multiple groups with one or two independent variables, respectively. Results are expressed as mean value±SEM or ±SD as indicated. Comparisons of survival curves were performed using the logrank (MantelCox) test. Survival analysis was carried out using the KaplanMeier method. P values>0.05 were considered statistically nonsignificant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. RESULTS Patient-derived GSCs and murine GBM models support IBDV replication While the cell cycle and immune responses to IBDV have been vastly covered in chicken models, our current understanding of the capacity of IBDV to infect and drive antiviral responses in mammalian cancer cells is limited to a single study performed using HeLa cells.40 To assess the ability of IBDV to infect brain cancer cells, murine gliomas (CT2A, GL261) and patientderived glioblastoma stem cells (GSCs), monolayers were exposed to IBDV for 24 hours. Indirect immunodetection targeting the viral protein VP3 revealed that IBDV is capable of efficiently infecting both human and murine cancer cells (figure 1A). Furthermore, all tested cancer cell types supported productive viral replication, as demonstrated by the cumulative recovery of infectious viral particles over the course of 120 hours (figure 1B,C). Additionally, IBDV displayed differences in replication kinetics and viral titers in a cellspecific manner: in murine GBMs, earlier accumulation of VP3 protein in GL261 cells (online supplemental figure S1) correlated with an increase in viral titers over time when compared with CT2A cells (figure 1B). In the case of human GSCs (figure 1C), there was a strong correlation between the capability of the infected cells to induce typeI IFN responses during viral infection and limit IBDV production (GBM18), as compared with GBM 27, which lacked the type I IFN cluster14 and rendered a higher viral yield. These data present evidence of the distinct celldependent behavior of IBDV in different mammalian brain cancer cells. Additionally, the cytopathic effect of IBDV infection was evaluated in different GBM cells, including the GSCs GBM18 and GBM27. Microscopy analysis at 120 hours postinoculation revealed a direct correlation between higher MOIs and an increased cytopathic effect, noticed by the presence of cellular debris and small vesicles (figure 1D,E). Remarkably, IBDV effectively disrupted GBM27 sphere architecture at an MOI as low as 1. MTS assay was used to evaluate the impact of the infection on cell viability at 24, 48, and 72 hours postinfection (hpi) with cells now exposed to a fixed MOI of 3 (figure 1F–I). The kinetics of the cytopathic effect of IBDV were compared with those of the NDVLS as a reference.41 The results showed that IBDV significantly reduced the viability of human and murine GBM cells at 72 hpi compared with NDVLS in murine CT2A and GL261. In CT2A and GL261 cells, a rebound effect was observed, where the cell viability increased at this time point, allowing for resumed growth and counteracting the effects of NDVLS infection (figure 1F,G). In GBM 18 and GBM 27 cells, both viruses reach their peak cytotoxic effect at 72 hpi, with similar kinetics (figure 1H,I). In contrast, experimental infection of HFF1 human fibroblast cell line cells showed no detectable viral replication at 72 hpi, with titers remaining low (10¹–10² TCID₅₀/mL), as shown in online supplemental figure S2B. Viability assays conducted at 48 and 72 hpi—time points when Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2025-011741 on 4 November 2025. Downloaded from https://jitc.bmj.com on 6 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 6TurPlanellsV, etal. J Immunother Cancer 2025;13:e011741. doi:10.1136/jitc-2025-011741 Open access Figure 1 Infectivity and replication capacity of IBDV in murine GBM and patientderived GSC cells. (A) Indirect Immunofluorescence microscopy: immunodetection of IBDV’s VP3 protein on GBMs 24 hours post viral exposure (MOI=3). Images were taken at 40× magnification. (B, C) IBDV’s replication kinetics—growth curves: viral titers were calculated by TCID50/mL method; initial input=MOI 0.01 (n=3). (D, E) Cytopathic effect: brightfield microscopy of GBMs monolayers 120 hours postinfection with IBDV at specified MOI. 20× magnification. (F, G, H, I)Viability analysis: cell viability was assessed via standard MTS assay at indicated times post infection (24, 48, and 72 hours). Twoway ANOVA analysis: *p<0.05; ***p<0.001; ****p<0.0001. ANOVA, analysis of variance; GBM, glioblastoma; GSC, glioblastoma stem cell; IBDV, infectious bursal disease virus; MOI, multiplicity of infection; ns, nonsignificant; TCID50, tissue culture infectious dose 50. Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2025-011741 on 4 November 2025. Downloaded from https://jitc.bmj.com on 6 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 7 TurPlanellsV, etal. J Immunother Cancer 2025;13:e011741. doi:10.1136/jitc-2025-011741 Open access IBDV induces cytotoxic effects in GBM cells—showed no decrease in fibroblast viability (online supplemental figure S2C). These findings confirm IBDV’s capability to effectively infect, replicate and induce oncolysis in vitro across different brain cancer cells, including patientderived GSCs and murine GBM cell lines with minimal impact in survival on healthy cells. IBDV infection triggers type-I interferon and pro-inflammatory cytokine release in GBMs Viruses with therapeutic potential against diverse tumors have been demonstrated to stimulate antiviral responses in infected cells. This primarily involves the induction of typeI IFN and cytokine release and ultimately promotes the activation of cell death programs.42 43 To study the intrinsic immunological response of GBM cells to IBDV infection, murine and human GBMs, including the human GSCs GBM18 and 27, were infected with IBDV at an MOI=3 for 16 hours. Subsequently, we analyzed the expression of IFNs (IFNB1, IL28) and the IFNstimulated gene MX1 as well as proinflammatory (IL1B, IL6) by RTqPCR. The clinical candidate NDVLS virus was included as reference for comparative purposes. Both murine and human GBM infected cells trigger a moderate type I IFN response, characterized by the expression of IFNB1 and MX1 genes, together with induction of typeIII IFN (IL28B) cytokines and proinflammatory genes (figure 2A–D), similarly to the effect elicited by NDVLS infection. During natural infection, IBDV has been shown to exert modulatory effects on cells of the monocytemacrophage lineage (MoMacs), with IBDVinfected MoMacs releasing chemotactic, proinflammatory and immunoregulatory cytokines.44 This delays the resolution of the viral infection and associated pathology in its natural host. In GBM, tumorassociated macrophages (TAMs) and microglia are the most abundant immune cell populations in the TME and the major drivers of immunosuppression and progression of the disease.45 Due to their relevance in GBM biology, IBDVtriggered proinflammatory gene expression signatures in mammalian murine bonemarrowderived macrophages (M1Mo) and HCM3 human microglia myeloidderived cells were analyzed by RTqPCR 72 hours after exposure to the virus (figure 2E,F). IBDV stimulates MX1 gene expression in both cell types and, consistently with the responses observed in GBMs, to a lower extent than NDVLS, highlighting IBDV’s moderate inflammatory behavior. Gene expression analysis of infected HFF1 human fibroblast cell line revealed that it activates an antiviral response, with expression of IFN-β and Mx1 detected at 24 hpi (online supplemental figure S2D). These latest results further support the lack of antagonism of typeI IFN signaling of IBDV in healthy human cells. Overall, these findings indicate that IBDV infection induces a moderate immunostimulatory effect across different GBM cells, including patientderived and murine lines, as well as in mouse and human myeloid cells and that mammalian healthy cells can efficiently counteract the replication of IBDV through the activation of antiviral signaling pathways. Combination of IBDV and TMZ exhibits synergistic impact on GBMs survival TMZ is the standard firstline treatment for patients newly diagnosed with gliomas.46 However, approximately 50% of GBMs exhibit resistance to TMZ, which can be attributed to both intrinsic and acquired resistance mechanisms.4 TMZinduced cell death presents differences in the murine and human GBM models under the current study (figure 3A–C). Specifically, the human GBM27 cell line was the most refractory to TMZ treatment, while GBM18 exhibited a dosedependent response. In contrast, murine GBM cells (CT2A) showed the greatest reduction in cell viability at lower TMZ doses. Considering these phenotypes—the differential sensitivity to TMZ by GBMs, we next investigated whether TMZ treatment could alter the susceptibility of GBM cells to IBDV infection (figure 3D–F). At 0.5 mM, the viability of GBM cell lines and GSCs decreases to around 60% with TMZ administration alone, having a similar impact on cell viability as IBDV infection only. Interestingly, concomitant administration of TMZ and IBDV significantly reduced viability when compared with each treatment individually. Followup highest singleagent statistical analysis confirmed a synergistic effect of both treatments reducing GBM cell viability, with a remarkable impact on GBM27 cells. Data to assess the combinatory effect of both treatments is compiled in (figure 3G–I and online supplemental figure S3A). Furthermore, sustained exposure to TMZ and IBDV demonstrated a dual effect in GBMs: the reduction in cell viability exerted by the combination was accompanied by enhanced IBDV replication (online supplemental figure S3B and C), as indicated by a significant increase in viral release at the peak of replication (72 hpi). This boost in viral production was not a reflection of a diminished antiviral responses, as indicated by the levels of expression of IFN-β and MX1 genes (online supplemental figure S3D and E). These results highlight the complementary and synergistic interaction between TMZ and IBDV in GBM models that exhibit different sensitivity to the chemotherapeutic TMZ. The clear effects demonstrated in vitro merit further investigation to clarify its mechanisms and clinical implications. Intratumoral administration of IBDV results in control over tumor growth and extended survival in CT-2A tumor-bearing mice Our in vitro studies demonstrated that IBDV can induce both immunostimulatory and cytotoxic responses in various GBMs, exhibiting a profile similar to that of the clinical OV candidate NDVLS. To assess the feasibility of using IBDV as a therapeutic Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2025-011741 on 4 November 2025. Downloaded from https://jitc.bmj.com on 6 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 8TurPlanellsV, etal. J Immunother Cancer 2025;13:e011741. doi:10.1136/jitc-2025-011741 Open access for GBM, we first conducted safety studies in immunocompetent C57Bl/J6 mice through both systemic and intracranial administration of IBDV (figure 4A,B). Histological analysis of tissue samples, 24 hours after systemic inoculation of a high dose of IBDV (107 PFU), revealed no virusassociated pathology in any Figure 2 Antiviral and proinflammatory gene expression triggered by IBDV infection. Gene expression analysis (RTqPCR). Cancer cell monolayers (A–D), bonemarrowderived macrophages and human microglia HCM3 (E–F) cells were infected with IBDV and NDVLS at an MOI=3 for 16 hours. Expression levels for each individual gene were calculated as log10 of fold induction over mockinfected cells. Twoway ANOVA analysis: *p<0.05; ***p<0.001; ****p<0.0001. ANOVA, analysis of variance; IBDV, infectious bursal disease virus; IFN, interferon; IL, interleukin; MOI, multiplicity of infection; mRNA, messenger RNA; ns, nonsignificant. Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2025-011741 on 4 November 2025. Downloaded from https://jitc.bmj.com on 6 November 2025 by guest. Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies. 9 TurPlanellsV, etal. J Immunother Cancer 2025;13:e011741. doi:10.1136/jitc-2025-011741 Open access Figure 3 Combinatory effect of TMZ with IBDV. (A, B, C) TMZ cytotoxicity analysis: at 72 hours, cell viability was measured via standard MTS assay compared with a Mock control after treatment with different concentrations of TMZ (DMSO control concentrations correspond to the final concentrations of TMZ used in each condition). (D, E, F) Combination effect of TMZ and IBDV: GBM cell viability was measured after treatment with 0.5 mM of TMZ, IBDV infection (MOI 1), or both conditions together. (G, H, I) HSA statistical test: the HSA statistical analysis was performed based on the effect of drug combinations relative to the effect of each treatment alone to assess additive, synergistic or antagonistic effects. DMSO was used as the vehicle for TMZ, and final DMSO concentrations in each well correspond to those of the TMZ treatments (eg, 2 mM TMZ corresponds to 2% DMSO). Equivalent DMSO concentrations were included in vehicle controls. Twoway ANOVA analysis: *p<0.05; ***p<0.001; ****p<0.0001. ANOVA, analysis of variance; GBM, glioblastoma; HSA, highest single agent; IBDV, infectious bursal disease virus; MOI, multiplicity of infection; ns, nonsignificant; TMZ, temozolomide. Journal for ImmunoTherapy of Cancer: first published as 10.1136/jitc-2025-011741 on 4 November 2025. 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