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

Abstract

Background: Glioblastoma (GBM) is the most aggressive form of cancer of the central nervous system. Despite advances in immunotherapies and standard-of-care 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 immune-based therapies or established treatment modalities. Oncolytic viruses, by displaying conditionally enabled tumor cell-restricted 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 patient-derived 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 CT-2A GBM-bearing 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 M2-like macrophages and increasing the ratio of CD8+T cells to regulatory T cells. This reversion of immunosuppression linked to monocyte-derived 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.

Keywords: Central Nervous System Cancer; Immunotherapy; Intratumoral; Oncolytic virus; Tumor microenvironment - TME.

Figure 1. Infectivity and replication capacity of IBDV in murine GBM and patient-derived 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 post-infection 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). Two-way 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, non-significant; TCID50, tissue culture infectious dose 50.

Figure 2. Antiviral and pro-inflammatory gene expression triggered by IBDV infection. Gene expression analysis (RT-qPCR). Cancer cell monolayers (A–D), bone-marrow-derived macrophages and human microglia HCM3 (E–F) cells were infected with IBDV and NDV-LS at an MOI=3 for 16 hours. Expression levels for each individual gene were calculated as log10 of fold induction over mock-infected cells. Two-way 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, non-significant.

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. Two-way 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, non-significant; TMZ, temozolomide.

Figure 4. In vivo antitumor effect of IBDV. (A–B) Pathogenicity studies. (A) 6–8 weeks old C57BL/6 mice (N=3) were intravenously administered with either PBS or 10⁷ PFU of IBDV; H&E histochemistry of indicated organs processed 24 hours post systemic viral dissemination. (B) IBDV intracranial toxicity: body-weight progression of C57BL/6 mice (N=3) after exposure to a single intracranial injection of IBDV at the indicated dose. (C–E) Antitumor capacity of IBDV. (C) Schematic representation of the study. CT-2A tumor-bearing mice were intratumorally administered 10⁷ PFU of IBDV/NDV or PBS, once every other day, for a total of four doses. Tumor volume was monitored every 48 hours. Experimental end-point: 1,000 mm3 tumor volume. (D) Average of tumor volume per experimental group at the time of first death. Two-way ANOVA. *p<0.05, **p<0.01. (E) Individual tumor growth curves. (F) Survival analysis: log-rank (Mantel-Cox) test. ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; IBDV, infectious bursal disease virus; PBS, phosphate-buffered saline.

Figure 5. Immunostimulation of innate and adaptive immune response in TME on IBDV treatment in CT-2A murine GBM model. (A) Schematic representation of the study: CT-2A tumor-bearing mice were intratumorally treated every other day with a total of either one or three doses of 10⁷ PFU of IBDV. Mice were sacrificed 1 day after the first or third injection in which tumor masses (TME) were harvested and probed with primary-conjugated antibodies against main biomarkers of immune cells. (B) Analysis of main immune populations in TME: temporal distribution of immune cell populations within TME of IBDV/PBS-treated mice. (C–J) Analysis of myeloid and lymphoid immune cell populations in TME: changes in innate and adaptive immune response in IBDV-treated compared with PBS-treated mice were analyzed in both groups (day 1 and 5). Two-way ANOVA analysis: *p<0.05; ***p<0.001; ****p<0.0001. ANOVA, analysis of variance; dLN, draining lymph nodes; GBM, glioblastoma; IBDV, infectious bursal disease virus; PBS, phosphate-buffered saline; TAM, tumor-associated macrophage; TME, tumor microenvironment; Treg, regulatory T cell.

Figure 6. Immunophenotyping of tdLN in CT-2A murine GBM model. (A) Analysis of immune populations in tdLN: temporal distribution of immune cell populations within tdLN of IBDV/PBS-treated mice over the course of the treatment. (B–H) Analysis of myeloid and lymphoid immune cell populations in tdLN: changes in lymphoid and antigen-presenting cells were analyzed in IBDV-treated mice compared with PBS-treated mice in both groups (day 1 and 5). Two-way ANOVA analysis: *p<0.05; ***p<0.001; ****p<0.0001. ANOVA, analysis of variance; tdLN, tumor-lymph nodes; GBM, glioblastoma; IBDV, infectious bursal disease virus; PBS, phosphate-buffered saline; Treg, regulatory T cell.