Safety, Tumor Reduction, and Clinical Impact of Zika Virus Injection in Dogs with Advanced-Stage Brain Tumors

Abstract

Malignant brain tumors are among the most aggressive cancers with poor prognosis and no effective treatment. Recently, we reported the oncolytic potential of Zika virus infecting and destroying the human central nervous system (CNS) tumors in vitro and in immunodeficient mice model. However, translating this approach to humans requires pre-clinical trials in another immunocompetent animal model. Here, we analyzed the safety of Brazilian Zika virus (ZIKV^BR) intrathecal injections in three dogs bearing spontaneous CNS tumors aiming an anti-tumoral therapy. We further assessed some aspects of the innate immune and inflammatory response that triggers the anti-tumoral response observed during the ZIKV^BR administration in vivo and in vitro. For the first time, we showed that there were no negative clinical side effects following ZIKV^BR CNS injections in dogs, confirming the safety of the procedure. Furthermore, the intrathecal ZIKV^BR injections reduced tumor size in immunocompetent dogs bearing spontaneous intracranial tumors, improved their neurological clinical symptoms significantly, and extended their survival by inducing the destruction specifically of tumor cells, sparing normal neurons, and activating an immune response. These results open new perspectives for upcoming virotherapy using ZIKV to destroy and induce an anti-tumoral immune response in CNS tumors for which there are currently no effective treatments.

Keywords: CNS tumors; ZIKA virus; canine clinical trial; immune cytokine profile; virotherapy.

Graphical abstract

Figure 1

Safety of ZIKV^BR Intrathecal Injection in Dogs (A) Viral RNA copies of peripheral blood serum at 3, 6, and 9 days after ZIKV^BR intrathecal injection in Pitbull immunosuppressed dog. (B) Viral titer in testicle, kidney, prostate bladder, and CSF samples at euthanasia in Pitbull dog, 18 days after ZIKV^BR injection (∗∗∗∗p < 0.0001). (C and D) Representative images of frontal lobe (C) and medulla (D) CNS tissues IF immunolabeling for ZIKV^BR (red), and nuclei DAPI (blue). Scale bar, 20 μm. (E) Representative images of neurons from CNS tissues IF immunolabeling for ZIKV^BR (red), β3-tubulin cytoplasmatic (green) protein, and nuclei DAPI (blue). Yellow staining in neuron image (E) shows unspecific staining for lipofuscin accumulation. Scale bar, 20 μm. (F) H&E representative images of urologic tissues: prostate (Fʹ) and testicle (Fʹʹ). Scale bar, 100 μm. (G) Representative spleen tissue of H&E (Gʹ) and IHC immunolabeling for ZIKV^BR (brown) and hematoxylin (blue) (Gʹʹ). Scale bar, 100 μm. (H) Representative lymph node tissue of H&E (Hʹ) and IHC immunolabeling for ZIKV^BR (brown) and hematoxylin (blue) (Hʹʹ). Scale bar, 500 μm. The brown areas in H&E preparation of spleen (Gʹ) and lymph node tissue (Hʹ) suggests a hemosiderin accumulation. Canine ZIKV-IgG and ZIKV-IgM quantification by ELISA in peripheral blood serum (I) and CSF (J) dog samples. (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001, two-way ANOVA with Bonferroni multiple comparison test. Three technical replicas for each sample).

Figure 2

Tumor Remission after ZIKV^BR Treatment in Boxer Dog Bearing Oligodendroglioma (A) Representative transversal tumor MRI at days 0, 14, 21, 35, 60, 90, 120, and 150 after clinical ZIKV^BR oncolytic treatment protocol beginning. (B) Tumor volume growth kinetics quantified by MRI analysis. (C) Representative 3D MRI analyses of four different dimensions at day 0 and 60 after ZIKV^BR first injection. Scale bar, 1 cm. (D) Post-mortem macro images of CNS tumor. Scale bar, 1 cm. (E–M) Representative images of tumor (E and F), cerebellum (G), medulla (H), left testicle (I), and right testicle bearing leydigoma (J–M) tissues. IHC (E, I–K) staining for H&E and ZIKV^BR (brown, white arrowhead), and IF (F–H, L and M) Immunolabeling for ZIKV^BR (red, white arrowhead), β3-tubulin cytoplasmatic (green) protein and nuclei DAPI (blue). Scale bar, 100 μm (E, I, and K), 20 μm (F–H, L and M), and 200 μm (J).

Figure 3

Tumor Remission after ZIKV^BR Treatment in Dachshund Dog Bearing a Rare Intracranial Meningioma (A) Representative dorsal and transversal tumor MRI at days 0, 14, 21, 42, 52, and 78 after clinical ZIKV^BR oncolytic treatment protocol onset. (B) Tumor volume growth kinetics quantified by MRI analysis. (C) Representative 3D MRI analyses of four different dimensions at day 0 and 14 after ZIKV^BR first injection. Scale bar, 1 cm. (D) Post-mortem macro images of CNS tumor. Scale bar, 1 cm. (E–G) Representative images of tumor positive for ZIKV^BR (red), microglia marker (G) (green), and nuclei DAPI (blue). Scale bar, 10 μm (E) and 20 μm (F and G). (H) H&E representative image of the tumor highlighting the lymphocytes infiltration (white arrowhead) and necrosis area with calcium accumulation (black arrow). Scale bar, 100 μm.

Figure 4

Canine Cytokine Profile Generated by Brain Tumors in the Presence of ZIKV^BR in Both In Vivo and In Vitro Models (A–C) Cytokine profile in the serum and CSF of Pitbull (A), Boxer (B), and Dachshund (C) dogs at different time points after ZIKV^BR injection. (D) Schematic representation of canine monocyte and D-GBM non-contact co-culture assay. (E–L) Cytokines concentration in non-contact co-culture groups normalized by respective day mock condition. (M) Schematic representation of canine monocyte and D-GBM contact co-culture. (∗p < 0.05, t test in comparison with correspondent MOCK group. Two technical replicas for each two biologic replica). (N–P) Evaluation of monocytes activation with CD80 (N), CD83 (O), and CD64 (P) positive cell quantification by flow cytometer. Gating strategy for the evaluation of canine monocytes in the co-culture assay detailed in Figure S5. (Q) qRT-PCR analysis of Boxer and Dachshund tumor tissue for lymphocyte T cells (MHC, GRANZYME B, and PD1), tumor cell receptor PDL-1, inflammatory monocyte (CCR2), microglia (CX3CR1), macrophage M1 (CXCL10, CCR7, GATA3, STAT1, NOS2, and IDO1), macrophage M2 (ARG1 and SOC3), and NK cells (NKP30 and NK2D), normalized by endogenous expression (Beta-Actin).