BY RABIES VIRUS INFECTION

[0001] CROSS REFERENCE TO PRIORITY APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No.

62/379,681 filed August 25, 2016, the disclosure contents of which are hereby incorporated by reference in their entirety.

[0003] GOVERNMENT SUPPORT

[0004] This invention was made with government support under AI093369 awarded by the

National Institutes of Health. The government has certain rights in the invention.

[0005] FIELD OF INVENTION

[0006] This invention is generally related to therapeutic modulation of the tumor-promoting immune type 2 microenvironment by induction of a type 1 response to an attenuated rabies virus, vaccines that promote such modulation, and methods of enacting such modulation to treat malignant glioma.

[0007] BACKGROUND OF THE INVENTION

[0008] Standard of care treatments for malignant glioma offer poor prognosis contributing to an interest in immunotherapeutic strategies. While certain early phase trials of various cell based vaccines and checkpoint inhibitors have shown some promise, most have failed to improve long term survival of patients with highly malignant glioblastoma multiforme (GBM), and none have been approved as standard treatment (1). These studies have reaffirmed that the immunomodulatory nature of the glioma tumor microenvironment (TME) is a key hurdle that must be overcome for successful immunotherapy.

[0009] Infiltrating tumor associated macrophages (TAM) are undoubtedly a major contributor to the immunomodulatory nature of the malignant glioma TME (2). These TAM closely resemble M2 macrophages in phenotype, factor expression and function, and are likely to arise from monocytes polarized in the periphery in response to M-CSF and other factors prior to their infiltration into tumor tissue (3-5). TAM elaborate numerous products that can contribute to tumor promotion including growth factors, angiogenic factors, such as VEGF, as well as anti -inflammatory cytokines including IL-10 and TGF-β (6-8). In addition, TAM express elevated levels of PD-L1 and decreased levels of costimulatory markers and MHC class II, which together result in inhibited antitumor T cell function (9, 10). [00010] Evidence that glioma malignancy is driven by the infiltration and intratumoral activity of cells either resembling M2 monocytes or with similar functional properties comes from studies in animal models and glioma patients. In the murine orthotopic GL261 glioma model, increasing the ratio of CDl lb+ spleen cells implanted with tumor cells accelerates tumor growth (11). In humans, astrocytoma malignancy is closely associated with the levels of M2 TAM; high levels of intratumoral and systemic M2 cells correlate with poor prognosis and resistance to therapy (12-15), and inhibition of M2 polarization inhibits glioma progression (16, 17).

[00011] Although the CNS is considered somewhat immunologically privileged, immunity can be readily generated to brain tumor antigens in animal models. Mice immunized in the flank with GL261 tumor cells are protected against a subsequent intracranial (i.e.) challenge though GL261-cell specific IgGl isotype antibodies generated in response to the immunization, revealing a type 2 immune bias (18). Similarly, sera from GBM patients obtained prior to the onset of therapy generally contain tumor-reactive IgG2/IgG4 antibodies suggesting that there has been type 2 /Th2 immune recognition of tumor antigens (19,20). This is consistent with the polarization of monocytes to M2 and tumor progression rather than therapeutic anti-tumor immunity which is considered to require a type 1/Thl response. Type 1 immunity is associated with the activation of Thl CD4+ and CD8+ T cells, the production of cytokines such as IFN-γ, TNFa, and IL-12, enhanced Ml- polarization of monocytes/macrophages, and a reduction in Treg activity, which in the case of tumor antigens can result in cell infiltration into tumor tissues and tumor cell destruction through a variety of mechanisms (21-25). The use of a dendritic cell (DC)-based vaccine to provoke Thl anti-tumor immunity in glioma patients has shown therapeutic promise (26). In a mouse model of glioma, promoting a Thl anti -tumor response via the adenovirus mediated intratumoral expression of IL-12 has been shown to enhance T cell infiltration and tumor cytotoxicity, survival, and long lasting protection (27). Therapies that shift both the myeloid and T cell response, toward Ml and Thl, respectively, can further enhance these anti-tumor effects (17). However, such therapies are dependent upon delivering the immune effectors into tumor tissues which may be problematic particularly at early, treatable stages in glioma formation where the blood brain barrier (BBB) may still be relatively intact.

[00012] SUMMARY OF THE INVENTION

[00013] The inventions and embodiments described herein are directed towards methods and therapeutics that alter the brain tumor microenvironment from a state that supports tumor growth to one that is tumor destructive. For example, a first embodiment is directed towards a method to alter the brain tumor microenvironment from a state that supports tumor growth to a state that is tumor destructive by inducing innate type 1 immune mechanisms generally throughout central nervous system tissues that result in the acquisition of tumor destructive properties by astrocytic tumor cells and tumor associated macrophages. This is associated with phenotypic and functional transformation of the tumor cells including the expression of inducible nitric oxide and the production of cytotoxic free radicals and a change in the phenotypic and functional characteristics of tumor associated macrophages from tumor-promoting anti-inflammatory M2 to inflammatory, tumor-destructive Ml macrophages. As an example this state change can be accomplished by the administration of a live-attenuated rabies virus vaccine strain to healthy CNS tissues and is independent of the infection of tumor or tumor-associated cells.

[00014] A further method is directed towards inducing an overproduction of nitric oxide (NO) through a type 1 immune response comprising administering to a patient having a cancerous cell in the brain an effective amount of a RABV virus. Preferably the virus is administered intranasally or intratumorally.

[00015] A further embodiment is directed towards generating a localized increase in nitric oxide (NO) production and generating an increase in toxic radicals, as compared to a control; wherein said localized increase in NO and increase in toxic radicals is generated by administering a live-attenuated rabies virus vaccine to CNS tissues.

[00016] A method to treat malignant human astrocytomas by the infection of tissues surrounding the tissues with apathogenic, attenuated rabies virus followed by inactivation of the virus by the application of virus neutralizing antibodies.

[00017] A further embodiment is directed towards a method to convert the tumor microenvironment in malignant astrocytomas from a growth-promoting to tumor destructive state by immunological intervention, comprising administering to a patient an effective amount of an attenuated rabies virus sufficient to generate an immune response to convert the tumor microenvironment. Preferably, the converted tumor microenvironment contains at least a two-fold increase in CD4+ cells/mm ² as compared to control, or at least a two-fold increase in IFN-γ mRNA as compared to control, or at least a two- fold change in the concentration of IFN-β mRNA, as compared to control, or at least a two-fold increase in the expression of iNOS mRNA as compared to control, or at least a two-fold change in the concentration of TNF mRNA, as compared to control, or at least a two-fold reduction in the concentration of TGF-P2 mRNA, as compared to control, or combinations thereof. [00018] A further embodiment is directed towards a method to treat malignant human astrocytomas in a patient by the infection of tissues surrounding the astrocytoma with apathogenic, attenuated rabies virus; comprising administering to said patient an effective amount of said attenuated rabies virus intranasally or intratumorally into the astrocytoma.

[00019] In preferred embodiments, the method to treat malignant human astrocytomas results in wherein said infection of said tissues surrounding astrocytoma increases the amount of at least one of CD4+, IFN-γ, IFN-β, TNFa or iNOS expression in said tissues.

[00020] A further embodiment is directed towards a method of converting astrocytoma cells in a human patient from a transformed state to a cytotoxic anti-tumor state comprising administering an effective amount of an attenuated rabies virus to said patient. In preferred embodiments, the transformed state contains at least a two-fold increase in CD4+ cells/mm ² as compared to control, or at least a two-fold increase in IFN-γ mRNA as compared to control, or at least a two- fold change in the concentration of IFN-β mRNA, as compared to control, or at least a two-fold increase in the expression of iNOS mRNA as compared to control, or at least a two-fold change in the concentration of TNFa mRNA, as compared to control, or at least a two-fold reduction in the concentration of TGF-P2 mRNA, as compared to control, or combinations thereof.

[00021] A further embodiment is directed towards a method of converting tumor infiltrating, tumor-promoting M2 macrophages to a tumor destructive Ml state; comprising administering to a patient an effective amount of an attenuated rabies virus.

[00022] In preferred embodiments, the tumor destructive Ml state is identified by at least a two-fold increase in CD4+ cells/mm ² as compared to control, or at least a two-fold increase in IFN- γ mRNA as compared to control, or at least a two- fold change in the concentration of IFN-β mRNA, as compared to control, or at least a two-fold increase in the expression of iNOS mRNA as compared to control, or at least a two-fold change in the concentration of TNFa mRNA, as compared to control, or at least a two-fold reduction in the concentration of TGF-P2 mRNA, as compared to control, or combinations thereof.

[00023] BRIEF DESCRIPTION OF THE FIGURES

[00024] FIGS. 1 A and B depict that RABV infection prolongs survival of tumor-bearing mice via a Thl-dependent mechanism. Mice were stereotactically injected i.e. with 10 ⁵ GL261 cells and then given i.n. PBS (dashed line) or 10 ⁵ focus forming units (FFU) SPBN-GAS (solid line) on day 8 and euthanized when moribund. (A) C57BL/6 mouse survival presented over time, (n=10) per group. Data is representative of two experiments. Statistically significant differences between tumor bearing mice and infected tumor bearing mice measured by Mantel-Cox test and is denoted as: **p< 0.01. (B) Type 1 immunodeficient Tbet-/- mouse survival data are presented as percent survival over time. Tumor alone (n=8), tumor + RABV (n=9), difference not significant.

[00025] FIGS. 2 A-E depict that GL261 tumors become necrotic early after RABV infection.

H&E staining was performed on C57BL/6 (A) and Tbet-/- (B) mouse brains at 12 d.p.t. for each cohort of infection controls, tumor alone, and tumor + RABV infection. Scale bar represents ΙΟΟμπι. (C) Percent necrosis was calculated in ImageJ as total necrotic area/ tumor area (pixels) from 2-6 sections/ brain, (n=2) for each condition in C56BL/6 mice and Tbet-/- mice at 12 and 22 d.p.t. Mann- Whitney test: *p< 05, **p< 01, ***p< 001 compared between infected and uninfected mice and WT and Tbet-/- mice. Mann-Whitney test comparing 22 d.p.t. time point to 12 d.p.t. time points: #p< 05, ##p< 01, ###p<.001. (D) Ki67 (white) staining of C57BL/6 and (E) Tbet-/- mouse brain tumors at 12 d.p.t. showing less proliferation in the tumors from treated fully immunocompetent C57BL/6 mice Images representative of 6 sections per mouse and 2 mice per condition. White scale bars represent 50μπι.

[00026] FIGS. 3 A-C depict that RABV does not infect tumor or tumor-bearing cortex despite route of infection. (A) Pattern of RABV spread assessed by immunofluorescence staining in infected tumor bearing mice 15 d.p.t. Labeling of NeuN (medium grey), RABV N protein (white), and DAPI (dark grey). Images representative of 2 mice/condition and 6 sections/ mouse. Dashed white line and "T" indicates tumor border, scale bar represents 50μπι. (B) Mice with and without tumor were infected i.n. 8 d.p.t. and CNS tissue was collectedl2 and 22 d.p.t. for virus qPCR detection, (n=5). (C) Tumor bearing C57BL/6 mice were infected 12 d.p.t. i.n with 105 FFU RABV (n=4), or i.e. in LCX (n=5) or RCX (n=5) with 104 FFU RABV and CNS tissue was collected 22 d.p.t. Viral mRNA expressed as mean ± SEM copies mRNA /1000 L13 in the LCX, RCX, or tumor. Statistical significance determined by Wilcoxon matched-pairs signed rank test where *p< 05, ***p< 001. Statistical difference between matching section in respective treatment groups measured by Mann- Whitney test and indicated by: #p< 05.

[00027] FIGS. 4 A-F depict that Enhanced CD4+ T cell accumulation occurs in glioma tissue of RABV infected mice. Immunofluorescence staining performed 12 d.p.t in WT (A) and Tbet-/- (B) mice in infected controls and tumor bearing mice with and without infection. Immunolabeling of CD4+ cells (white) and DAPI (dark grey) at lOx where scale bar represents ΙΟΟμπι. (C) Quantification of CD4+ cells was performed with ImageJ with data presented as total CD4+ cells /mm2 tumor area. Immunofluorescence images converted to grey scale and quantification data are representative of 2 mice/ condition and 6 sections/ mouse. (D) qPCR analysis of tumor tissue from C57BL/6 and Tbet-/- tumor-bearing mice with and without infection performed 12 d.p.t and expressed as CD4 mRNA copies/ 100 LI 3. n=5 for each condition in C57BL/6 and Tbet-/- mice, differences NS. (E) Immunofluorescence staining of Ki67 (medium grey), CD4 (white) and DAPI (dark grey) staining of C57BL/6 and (F) Tbet-/- mouse brain tumors at 12 d.p.t. Fluorescent Images converted to grey scale are representative of 6 sections per mouse and 2 mice per condition. Images captured at 40x, white scale bars represent 50μιη.

[00028] FIGS. 5 A-E depict that RABV infection of GL261 -bearing mice does not enhance tumor-specific antibody production. (A) qPCR analysis of CNS tissue was performed 12 d.p.t. in C57BL/6 and Tbet ^_/" mice, data expressed as mean ± SEM copies κ-L chain/ 100000 LI 3. Statistical differences between cohorts were not detected. (B) GL261 cell-reactive IgGl and (C) IgG2A antibody measured via cell-based ELISA and presented as absorbance mean ± SEM in naive mice, at infection time point, and 22 d.p.t. No statistically significant differences observed. (D) RABV- specific IgGl and (E) IgG2A antibody isotyping performed via ELISA. Statistically significant differences between naive and infected cohorts at 22 d.p.t. determined by Mann-Whitney t test with *p< 05, ***p< 01. n= 5 for mock tumor groups, n=10 for infected and uninfected tumor-bearing cohorts. Background absorbance indicated by dashed line.

[00029] FIGS. 6 A-H depict that the TME of C57BL/6 RABV- infected mice exhibit an early proinflammatory shift. (A) IFNy mRNA expression in C57BL/6 and Tbet ^_/" mice at 12 d.p.t. presented as mean ± SEM copies mRNA/100 L13. (B) IFN-β expression fold change shown as mean ± SEM normalized to L13. (C) CXCLIO mRNA expression as mean ± SEM copies/ 100 L13. (D) TNFa expression mean ± SEM mRNA copies/ 100000 LI 3. (E) TGF-P2 expression in tumors of infected and uninfected C57BL6 and Tbet ^_/" mice 12 d.p.t., represented as fold change. (F) iNOS mRNA expression presented as mean ± SEM copies mRNA/ 100000 L13. Statistically significant difference measured with qPCR data were determined with Mann-Whitney test and indicated by: *p< 05, **p< 01, ****p<.0001. (G) Fluorescent staining in tumors of C57BL/6 and (H) Tbet ^_/" mice 12 d.p.t. with and without RABV infection for the vascular endothelial cell marker CD31 (white), iNOS (medium grey) and DAPI (dark grey).

[00030] FIGS. 7 A and B show that infection of tumor bearing mice causes a change in the pattern of cytokines and chemokines produced by tumor and tumor associated cells. (A). Heat map representation of cytokine levels from ex vivo supernatant produced by cells from tumor tissues excised 12 d.p.t. from C57BL/6 mice with and without RABV infection, as measured by Luminex. Significantly different expression of individual cytokines determined by permutation test. Statistically significant differences indicated by *p< 05, **p< 01, ****p< 0001. (B) PCA projection of Luminex data clustered by sample groups for tumor supernatant of infected (light grey) and uninfected (dark grey) tumor bearing mice. N= 5 mice per condition, replicates averaged for heat map expression and biological replicates reported in PCA analyses.

[00031] FIGS. 8 A-D depict that RAB V infection alters TAM polarization in GL261 tumors.

(A) Immunofluorescent staining converted to greyscale at 22 d.p.t. for the M2 marker CD206 (white), CD1 lb (medium grey) and DAPI (dark grey) in tumors of infected and uninfected C57BL/6 and (B) Tbet-/- mice. (C) Tumors stained for the Ml marker CD1 lc (white), F4/80 (medium grey) and DAPI (dark grey) in WT and (D) Tbet-/- infected and uninfected mice at 22 d.p.t. Images representative of 6 sections per mouse and 2 mice per condition. Scale bar indicates 50μπι.

[00032] FIGS. 9 A-D depict that GL261 cells produce antiviral factors that inhibit their infection. (A) Cytokines present in GL261 or NA cell-conditioned media after 6-48 hour cultures plotted in pg/ml. (B) Infection of NA cells measured in FFU. NA or GL261 24 or 72 hour cell- conditioned media applied to NA cells inhibit ability of virus to infect and spread in NA cells. (C) SPBN-GAS titration in NA and GL261 cells 48 hours post-infection, stained for RABV N protein (light grey), counterstained with Evans Blue (dark grey), 1 : 10 dilution shown for NA and GL261 cells at lOx magnification. (D) Percent of virally-infected NA cells and GL261 cells after virus titration performed at 48hr. TNTC represents "too numerous to count".

[00033] FIGS. 10 A-C depict expression of mRNAs encoding the adhesion molecule ICAM-

1 and the cytotoxic T cell marker CD8 are elevated in tumor tissues by attenuated rabies virus infection but the tumor infiltrating effectors do not have increased cytolytic activity as reflected by unchanged granzyme b mRNA levels. qPCR analysis of CNS tissue was performed 12d.p.t. in C57BL/6 and Tbef ^/_ mice and expressed as mean +/- SEM copies (A) ICAM-1, (B) CD8, (C) granzyme b (GZMb) mRNA copies/ 100000 L13. Statistical differences between cortical and tumor tissues of different treatment cohorts measured by Mann Whitney test and represented by: p< 05.

[00034] FIG. 11 depicts GL261 cells upregulate inflammatory iNOS expression in response to treatment with the type 1 cytokine IFNy in vitro. Immunofluorescent staining for iNOS (light grey) in GL261 cells treated with 0, 10, 100, or 1000 Units of recombinant mouse IFNy for 24hr. Images presented at 40x where the white bars represent 50 μπι. Data representative of two independent experiments.

[00035] FIGS. 12 A-F depict the expression of macrophage polarization marker mRNAs in GL261 tumors is altered by the response to RABV infection. qRT-PCR analysis of tumor tissue was performed 22 days post tumor implantation (14 days p.i.) in C57BL/6 mice and data is expressed as the mean relative expression +/- SEM of (A) CD206, (C) Retnla/FIZZl, (D) Argl, Yml, and (F) CD38. (C) CD163 data presented as mean +/- SEM mRNA copies/ 1000 L13. N = 10 mice/group. Statistical differences were assessed by the Mann-Whitney test and are indicated by: *p< 05, **p< 01, and ****p<.0001.

[00036] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[00037] Immunotherapeutic strategies for malignant glioma have to overcome the immunomodulatory activities of M2 monocytes that appear in the circulation and as tumor associated macrophages (TAM). M2 cell products contribute to the growth-promoting attributes of the tumor microenvironment (TME) and bias immunity towards type 2, away from the type 1 mechanisms with anti-tumor properties. Type 2 immunity is driven by T-helper 2 cells (CD4- positive, using the GATA-3 transcription factor) producing factors such as IL-4, IL-5, IL-10, IL-13 and is primarily associated with repair mechanisms, antibody production and monocyte/macrophage polarization to anti-inflammatory, M2. Type 1 immunity is driven by T-helper 1 cells (CD4- positive, using the T-bet transcription factor) producing factors such as IFN-γ, IL-2, T Fa, with IFN-γ being considered as the prototypical factor, and is associated with anti-viral and anti-tumor cytotoxic immune mechanisms through the activation, differentiation, and/or expansion of CD8- positive cytotoxic T cells, Natural Killer Cells, and monocyte/macrophage polarization to inflammatory, iNOS -expressing Ml . With typical tumor applications, a type 2 response is generated which sends M2 macrophages and other cells to "repair" or aid the cancerous tissues, as if an injury had occurred. Generating a type 1 response, instead of a type 2 response, will instead generate factors that will drive cell destruction and aid in tumor necrosis.

[00038] For example, in a situation where a cancerous mass is found in the brain, excision of some or all of the cells is indicated, yet, many times all of the tumor mass cannot be removed. Direct inoculation of the tumor with RABV, as described herein, would enable a type 1 response, not in the tumor cells themselves, but in the cells surrounding the tumor, and advantageously create a TME that destroys cells, including the tumor cells.

[00039] To drive type 1 immunity in CNS tissues we infected GL261 tumor-bearing mice with attenuated rabies virus (RABV). These neurotropic viruses spread to CNS tissues trans- axonally where they induce a strong type 1 immune response that involves Thl, CD8 and B cell entry across the blood brain barrier and virus clearance in the absence of overt sequelae. Intranasal infection with attenuated RABV prolonged the survival of mice bearing established GL261 brain tumors. Despite the failure of virus spread to the tumor, infection resulted in significantly enhanced tumor necrosis, extensive CD4 T cell accumulation and high levels of the proinflammatory factors IFNy, T Fa, and iNOS in the TME merely 4 days after infection, before significant virus spread or the appearance of RABV-specific immune mechanisms in CNS tissues. While the majority of the infiltrating CD4 cells appeared functionally inactive, the proinflammatory changes in the TME later resulted in the loss of accumulating M2 and increased Ml TAM. By contrast, mice deficient in the Thl transcription factor Tbet did not gain any survival advantage from RABV infection, exhibiting only limited tumor necrosis and no change in TME cytokines or TAM phenotype, highlighting the importance of type 1 mechanisms in this process and the likelihood that a minor, but important subset of the infiltrating CD4 T cells were functionally active Thl cells. As is known to those of skill in the art, the Tbet negative mice are deficient in the cytolytic pathway, and thus the underlying immune system does not generate a full response. Accordingly, the RABV infection works with the immune system to generate the type 1 immune response.

[00040] The difficulty in delivering type 1 immune effectors into CNS tissues can be overcome by the use of virus infection. In addition to the IL-12 adenoviral construct described above, the utility for glioma therapy of several oncolytic viruses that typically induce a type 1 immune responses has been assessed. However the focus of these studies has been on the capacity of these viruses to infect tumor cells and their associated oncolytic properties, and the effects of the associated type 1 immune mechanisms have not been thoroughly examined (28, 29).

[00041] To determine if type 1 immune mechanisms induced by virus infection may impact glioma growth in the absence of direct tumor cell infection and lysis, we have used the attenuated, neurotropic rabies virus (RABV) SPBN-GAS. This RABV strain contains multiple attenuating mutations in its glycoprotein that prohibit its reversion to pathogenicity, ensuring its safety even in immunocompromised animals (30, 31). Such viruses spread to the brain via axons, bypassing the BBB, then replicate in neurons and astrocytes causing minimal cell death (32). Unlike pathogenic strains, attenuated RABV trigger changes in the neurovasculature that allow immune effector entry into CNS tissues and do not cause the pathological inflammation associated with most virus infections of CNS tissue (33). In normal mice this results in the production of high levels of type 1 cytokines and virus neutralizing antibody in the CNS tissues and, ultimately, clearance of the virus without histological or clinical evidence of pathology. Type 1 immune mechanisms are central to virus clearance from the CNS as mice lacking Tbet, the transcription factor associated with the development of Thl cells, have a severe deficit in this process despite developing a strong RABV- specific Th2 response (31, 34-36). To determine if the induction of a type 1 response in CNS tissues would alter glioma growth, we infected congenic C57BL/6 and Tbet-/- mice bearing intracranial GL261 tumors with SPBN-GAS, monitoring the immune responses to tumor and viral antigens, cytokine production in the TME, and tumor growth.

[00042] Infection with attenuated RABV extends the survival of normal but not Thl deficient mice bearing i.e. GL261 tumors.

[00043] To determine if attenuated RABV infection, known to generate a strong type 1 immune response in CNS tissue, would prolong survival of mice with glioma, mice were implanted in the cerebral cortex with 105 GL261 cells, a dose that results in close to 100% tumor growth, and then intranasally (i.n.) infected with 105 f.f.u. of SPBN-GAS eight days later. At this time the tumor cells are growing exponentially and there is no intervention that can cause survival of the animals due to the extent of the tumor relative to the size of the mouse brain. Nevertheless, tumor-bearing C57BL/6 mice lived significantly longer when infected with RABV (Fig. 1A). This improved longevity evidently required the Thl arm of the immune response, as there was no benefit from infection in glioma-bearing Tbet-/- mice (Fig. IB). Neither wild type (WT) nor Tbet-/- mice exhibited clinical symptoms of RABV infection, and all mice had substantial tumor burdens at sacrifice.

[00044] RABV infection causes the early onset of tumor necrosis.

[00045] To gain insight into why RABV infection prolongs survival of C57BL/6 but not Tbet-

/- mice implanted with GL261 cells, we assessed brain and tumor tissues by histopathology at 12 and 22 days after tumor cell implantation. Differences in tumor tissues between the groups of mice were evident by gross examination with only tumors from infected WT mice exhibiting extensive hemorrhage (not shown). Consistent with their increased survival, tumor tissues from C57BL/6 mice that received SPBN-GAS were considerably more necrotic by 4 days after infection (12 days after GL261 cell implantation) than those from uninfected C57BL/6 or infected and uninfected Tbet-/- mice (Fig. 2A). Despite increasing necrotic areas in tumors from uninfected wild-type mice due to the extensive size of the tumors, infection continued to be associated with greater tumor necrosis in these animals over the next 10 days (Fig. 2C). A slight elevation in tumor necrosis was detected in Tbet-/- mice as a consequence of infection at the early time point but no difference between infected and uninfected Tbet-/- mice was detected later (Fig. 2B- C). Notably, tumor necrosis remained significantly less in these animals than in C57BL/6 mice at 22 days after tumor implantation regardless of treatment. In support of the concept the that tumors in RABV-infected C57BL/6 mice are less viable, staining for Ki67, a marker expressed by proliferating cells, revealed less tumor cell proliferation in these animals by comparison with uninfected counterparts and the Tbet-/- cohort (Fig. 2D-E).

[00046] RABV does not spread to tumor-bearing cortex or tumor, likely due to secretion of antiviral factors by the GL261 cells.

[00047] Tumor necrosis is evident in infected C57BL/6 mice as little as 4 days post-infection

(12 days post-tumor cell implantation) which is considerably before SPBN-GAS given i.n. spreads through the cortex tissues of normal mice (31) suggesting the possibility that the presence of the glioma enhances virus spread. However, the pattern of staining for virus that emerged in tumor- bearing mice indicates otherwise. When virus becomes detectable in brain tissues by immunofluorescent staining for nucleoprotein at 7 days post-infection it is observed predominantly in the left cortex (LCX) as opposed to the right tumor-bearing hemisphere (RCX) or the tumor itself (Fig. 3A). Analysis of tissues for RABV nucleoprotein mRNA confirms this observation. RABV- infected mice that had undergone implantation surgery but received an i.e. injection of PBS without cells had equal levels of viral mRNA in the LCX and RCX at all time points. When necrosis is observed in tumor tissues from mice infected 4 days previously, the level of viral mRNA in the CNS tissue is very low and undetectable in the tumors of either C57BL/6 or Tbet-/- mice. By 14 days post-infection, when virus is replicating at higher levels in the CNS, viral message was higher in the LCX compared to RCX, and the levels in tumor tissues are significantly lower than in the right cortex, and in turn, significantly lower than those measured in the left cortex (Fig. 3B). These results suggested that the tumor may in fact be inhibiting virus spread to the area. To address this possibility, tumor-bearing and control mice were infected with 104 f. f. u. SPBN-GAS in either the left or right cortex, 12 days after tumor implantation in the right cortex (Fig. 3D). Infection in either cortex resulted in a similar trend to i.n. infection where virus preferably replicated in the non-tumor-bearing LCX and only minimally in tumor tissues.

[00048] To test the hypothesis derived from the in vivo studies that products of GL261 cells may inhibit RABV replication and spread, we collected GL261 cell supernatants from cultures at various time points, measured secreted factors using a Luminex assay, and added the conditioned media to infected mouse neuroblastoma (NA) cells. Supernatants from NA cells, typically used to grow RABV, were used as controls. Compared to NA cells, GL261 cells secrete high levels of cytokines with anti-viral properties including RANTES, CXCL10, and Lif (Figure 9A). Moreover, GL261 -conditioned media was found to inhibit virus infection and spread in NA cells (Figure 9B). Finally, we compared the infectivity of SPBN-GAS for NA and GL261 cells finding that the latter are highly resistant to infection with the virus as shown by staining (Figure 9C) and enumeration of stained, infected cells (Figure 9D).

[00049] RABV infection promotes CD4 but not cytolytic CD8 T cell infiltration into glioma tissues.

[00050] Attenuated RABV infection is known to promote the infiltration of immune cells specific for non-viral antigens into CNS tissues (34). Tumor tissues from infected C57BL/6 and, to a lesser extent, Tbet-/- mice show elevated levels of CD4+ T cell accumulation by immunofluorescence than non-infected controls at 4 days post-infection (Fig. 4A- C). CD4+ T cells were not seen in brain tissues from infected animals without tumors at this time point, and there were no CD4+ T cells present in the cortex surrounding the GL261 tumor in either infected WT or Tbet- /- mice. However, when tumor tissues were assessed for CD4 mRNA levels, no difference between infected and non-infected animals was seen (Fig. 4D). Consistent with this finding, Ki67 staining revealed that the majority of CD4+ cells in tumor tissue are not actively proliferating in either C57BL/6 or Tbet-/- tumor-bearing mice regardless of whether or not they had been given RABV (Fig. 4 E-F).

[00051] RABV infection results in the enhanced expression of ICAM on neurovascular endothelial cells which would be expected to support immune cell infiltration into CNS tissues (38). Infection increased ICAM mRNA expression in the tumors of C57BL/6 mice but not Tbet-/- mice (Figure 10). However, no increase in CD8 or GZMB expression in tumors of either C57BL/6 or Tbet-/- mice 4 days following RABV infection was detected (Figure 10). Similarly, expression of the NK cell marker NKp46 was not significantly altered in tumors of infected WT or Tbet-/- animals (data not shown). Comparison of sections of tumor tissue from the different groups of mice stained for NKp46 and CD8 also failed to detect any increase in the numbers of NK or CD8 T cells as a consequence of RABV infection (data not shown).

[00052] RABV infection of glioma- bearing mice has no impact on the tumor cell-specific humoral response.

[00053] GL261 -specific antibodies, which evidently contribute to immune protection against tumor growth in the mouse GL261 model (18), provide insight into the class and magnitude of the tumor-specific immune response. We therefore assessed GL261 and RABV- specific antibody production in the tumor-bearing mice following infection. Significantly higher levels of mRNA specific for the antibody κ-light chain were detected in tumor tissues from infected C57BL/6 mice by comparison with uninfected tumor-bearing animals 4 days after virus infection (12 days post tumor implantation) as well as both infected and uninfected tumor-bearing Tbet-/- mice (Fig. 5A). Levels of mRNA for the B cell marker CD 19 were also increased in the tumor tissues of only RAB V- infected C57BL/6 mice and staining of tumor sections revealed a slight increase in the numbers of CD 19+ cells of these animals (data not shown). However, the development and isotypes of serum GL261 -specific antibodies in GL261 tumor-bearing C57BL/6 mice, analyzed using a cell-based ELISA, were unaltered by infection with RABV, becoming significant 22 days after cell implantation and remaining predominantly IgGl (Fig. 5B and C). On the other hand, the presence of tumor significantly reduced the levels of IgG2A RABV-specific antibodies elicited by virus infection without altering the relatively low levels of virus-specific IgGl detectable 14 days after infection (Fig. 5D and E).

[00054] RABV infection of tumor-bearing mice results in a Thl -dependent proinflammatory shift in the tumor microenvironment.

[00055] The elevated numbers of infiltrating CD4+ T cells and increase in mRNA specific for antibody κ-light chain in tumor tissues suggests that there may be an impact on the immune status of the TME 4 days after RABV infection despite low levels of virus replication in CNS tissues at this time. To test this hypothesis, we first assessed tumor tissues from control and RABV-infected C57BL/6 and Tbet-/- mice for levels of immunologically-relevant mRNAs known to be induced in the CNS tissues of normal mice at the onset of rabies infection. At 12 days after tumor cell implantation we observed significantly higher levels of IFNy mRNA expression in the tumor tissues of C57BL/6 mice that received RABV 4 days previously but not in tumor tissues from uninfected mice, Tbet-/- mice (Fig. 6A), or in the left cortex or right cortical tissues surrounding the tumors (data not shown). IFN-β mRNA levels were increased in tumor tissues from the same cohort of animals (Fig. 6B) as were levels of mRNA encoding CXCL10 (Fig. 6C) and TNFa (Fig. 6D). Of note, the levels of these mRNA in the tumor tissues from infected C57BL/6 mice are higher than those seen in similarly infected mice without tumors at this early time-point in the infection (data not shown). In contrast, levels of mRNA specific for the immunomodulatory cytokine TGF-P2 in tumor tissues were lowered by infection (Fig. 6E).

[00056] Attenuated RABV infection is known to induce the expression of iNOS, an enzyme responsible for the production of nitric oxide and associated cytotoxic radicals, primarily by cells closely associated with the neurovascular unit as opposed to infiltrating M2 macrophages (33). iNOS mRNA levels appeared to be selectively elevated in the tumor tissues of RABV-infected C57BL/6 but not Tbet-/- mice (Fig. 6F). To provide further insight into the nature of the iNOS+ cells, sections from the cortex and tumor tissues of C57BL/6 and Tbet-/- mice, either uninfected or infected 4 days previously, were stained for iNOS and CD31, a marker that is normally found on endothelial cells but can be expressed by intratumoral macrophages (39). In the tumor bearing mice CD31+ cells can be seen throughout the sections from both mouse strains, but substantial iNOS staining is only seen in tissues from RABV-infected C57BL/6 mice, localizing with CD31+ vasculature in the cortex (not shown) and appearing in the cytoplasm and nucleus of CD31- cells in the tumor tissue (Fig. 6G-H). To determine whether or not macrophages are responsible for the iNOS expression in the tumors of RABV-infected C57BL/6 mice, tumor tissues from these animals were co-stained for iNOS and the macrophage markers F4/80 (Fig. 61) and CD1 lb (Fig. 6J). While there was evidence of macrophage infiltration into tumor tissues at this early time point, iNOS expression localized to a different cell type, which we speculate based on morphology and the absence of macrophage markers for (CD1 lb and F4/80) is primarily the GL261 tumor cell. To investigate the possibility that the production of IFNy in the TME of RABV-infected C57BL/6 mice may promote iNOS expression in GL261 cells, we incubated the cells with increasing concentrations of mouse recombinant IFNy in vitro and stained the cells for iNOS. In culture, untreated GL261 cells exhibited low but detectable nuclear and cytoplasmic iNOS staining that increased in intensity with the addition of increasing concentrations of IFNy (Figure 11) indicating the likelihood that one of the anti-tumor mechanisms triggered by the shift in the innate immune environment of the CNS by attenuated rabies virus infection is by the induction of the tumor cells to produce cytotoxic radicals.

[00057] To provide further insight into the changes in the tumor tissues caused by cell infiltration and any functional changes in the GL261 tumor resident cells, supernatants from overnight cultures of dispersed cells from tumor tissues, excised from both 4-day infected and uninfected C57BL/6 mice 12 days post GL261 cell implantation, were analyzed for mouse cytokine/chemokine production by Luminex. A heat map representation and PC A of the data reveal distinct differences in the factors secreted by cells from the tumor tissues of infected versus uninfected mice, with those from the former producing significantly higher levels of a number of primarily type 1 cytokines including G-CSF, IFN-γ, IL-6, IL-17, CXCL10, MIP-2 and TNFa (Fig. 7A-B). Statistically significant differences cytokine/chemokine levels between sera obtained at the same time from infected and uninfected tumor-bearing mice were not detected (data not shown).

[00058] RABV infection causes a shift in TAM from M2 to Ml . [00059] At 15 days post-GL261 cell implantation similar numbers of CD206+ macrophages had accumulated in the tumor tissues regardless of whether or not the mice had been infected (data not shown). However when sections of tumors obtained from similar mice at 22 days post- implantation were stained for the macrophage markers CDl lb and F4/80, the M2 marker CD206, and CDl lc, which tends to be expressed at low levels on M2 macrophages and high levels on Ml macrophages, different patterns emerged for infected versus uninfected mice. While the majority of the CDl lb+ cells apparent in the tumor tissues of uninfected C57BL/6 mice co-expressed CD206, the majority of the CDl lb+ cells in tumors from infected C57BL/6 mice did not (Fig. 8 A). CDl lb and CD206 were expressed by different cell populations in tumor tissues from Tbet-/- mice but these were unchanged by infection (Fig.8B). When F4/80 was used to identify macrophages and CDl lc used to examine the cell subsets, TAM in uninfected C57BL/6 mice were negative for CDl lc, but positive for CDl lc in tumor tissues from infected mice (Fig. 8C). In contrast, tumor tissues from uninfected Tbet-/- mice contained large numbers of F4/80+/CDl lc+ populations with infection resulting in lower numbers of these cells and cohorts of cells that were either F4/80+ with low levels of CDl lc or individually positive for each marker (Fig. 8D). Analysis of mRNA from tumor tissue at this late time point (22 days post- implantation) also showed significant reductions in the expression of genes associated with M2 cells including the phenotypic markers CD 163 and CD206. The expression of genes encoding the M2 products resistin like alpha (Retnla/FIZZl) and Argl, were also reduced although the reduction in Argl was not statistically significant. In contrast levels of expression of Yml which is also a product of M2 cells, were increased in tumors of infected mice. In addition to CDl lc, mRNA encoding CD38, a marker expressed by a variety of activated immune cells including Ml macrophages, was found at significantly higher levels in tumors of RABV- infected mice (Figure 12).

[00060] Discussion

[00061] Infection of normal mice with attenuated neurotropic RABV drives the production of proinflammatory type 1 cytokines, the expression of iNOS -dependent radicals, and the accumulation of CD4 Thl cells in brain tissue, which are initially detectable 6 to 8 days after intranasal instillation of the virus (34). On the other hand, malignant gliomas, including those resulting from GL261 cell implantation, are characterized by the induction of type 2 immunity and the recruitment of antiinflammatory M2 monocytes into the TME (3-5). In this study we show that infection of GL261 glioma-bearing C57BL/6 mice with attenuated rabies causes a profound change in the immune bias of the TME from type 2 to type 1 which is associated with extensive tumor necrosis and results in prolonged survival.

[00062] Because RABV does not spread to tumor tissues the TME alterations that occur as a consequence of RABV infection likely result from immunological signals originating in infected tissues distant to the tumor that trigger the production of proinflammatory cytokines in the tumor, ultimately leading to tumor cell death. We observe increased IFNy, TNFa and iNOS within the tumor tissue merely 4 days after RABV infection in C57BL/6 but not in Thl -deficient Tbet-/- mice. The outcome is consistent with prior reports that TNFa and IFNy provide a therapeutic benefit in glioma, their expression promoting upregulation of iNOS which activates cell death cascades through the activity of its product nitric oxide (40-41). Accordingly, tumor-bearing uninfected C57BL/6 and uninfected as well as infected Tbet-/- mice, where there is minimal expression of proinflammatory cytokines, have lower iNOS expression and show little tumor necrosis. Whereas GL261 cells are positive for GFAP only at the tumor margin (data not shown and previously observed (42)) the iNOS-positive cells were scattered throughout the tumor parenchyma in infected mice generating a Thl response. Astrocytes can be triggered to express iNOS by IFNy and TNFa and iNOS-positive astrocytes have been observed in inflammatory lesions in mice with experimental allergic encephalomyelitis (43). Moreover, as shown here GL261 can be induced to express iNOS in vitro by IFNy treatment in vitro. Our data therefore suggest that GL261 cells, transformed astrocytes, can also express iNOS in response to IFNy in vivo, and suggest that these are the predominant iNOS expressing cell in tumors undergoing necrosis as a consequence of the innate immune response to attenuated RABV.

[00063] Corresponding with the early onset of tumor necrosis 4 days after infection 12 days following GL261 cell implantation, increased CD4+ T cells are observed in the tumor tissues of mice capable of generating a type 1 antiviral response and likely contribute to the antitumor effect and altered TME. The presence of these CD4+ cells in tumor but not surrounding tissue, along with their appearance merely four days after infection, suggests that they are not RABV-specific. In non- tumor bearing mice intranasally infected with attenuated RABV this is at the point when virus first appears in CNS tissues and it takes several additional days for antigen-specific cells to appear in the CNS. Moreover, virus spread to not only tumor tissues but also the surrounding parenchyma is limited. Therefore we consider that the CD4+ T cells are entering tumor tissues nonspecifically as a consequence of the local production of proinflammatory cytokines such as TNFa and the effects of free radical activity in the tumor, processes that have been implicated in the activation of vasculature and effector entry into CNS tissue (34, 44, 45). While the early onset of the CD4 cell entry, proinflammatory factor upregulation, and necrosis occur concomitantly and are likely related, there is a disparity between the high numbers of CD4+ T cells observed in the tumors and relatively low levels of CD4 mRNA in in tumor tissues. A similar phenomenon has been observed in Tbet-/- mice clearing attenuated RABV where there is substantial recruitment of Th2 CD4 cells to the CNS but the cells express low levels of activation markers and CD4 mRNA unlike the Thl CD4 T cells that enter the CNS tissues of C57BL/6 in response to the virus (31). This phenomenon has not been reported for other neuroimmune processes which are generally associated with Thl or Thl 7 cell infiltration.

[00064] Consequently, we speculate that the current results may reflect a CD4 infiltrate that is predominantly Th2, as is the natural response to GL261 antigens (18), and a common mechanism where Th2 cells largely lose transcriptional activity in CNS tissues. While this concept is supported by the lack of Ki67 expression by the tumor infiltrating CD4 cells it remains to be validated in other models. While the activity of Thl CD4 cells that enter tumor tissues is expected to be inhibited by the TME (46, 47) we expect that the anti -inflammatory mechanisms responsible are counteracted by the response to attenuated RABV. The fact that the therapeutic effect of attenuated rabies virus infection is only seen in mice that can mediate a Thl response suggests that elements of the type 1 response are responsible, possibly including the activity of a limited subset of tumor antigen-specific Thl cells in tumor tissues underlying more extensive Th2 accumulation. Alternatively, NK or CD8 T cells in the tumors of infected C57BL/6 mice, while not increased in number, may have acquired enhanced activity.

[00065] A later effect of the high expression levels of type one factors in the TME of C57BL/6 mice infected with attenuated RABV, subsequent to the onset of necrosis, is a change in the TAM population. Approximately 10 days after the onset of tumor necrosis and the proinflammatory shift in the TME there is a profound reduction in expression by TAM of the M2 marker CD206+ and an increase in the expression of CDl lc which is generally considered an Ml marker (48, 49). Together with the data from our studies of macrophage subset gene expression in tumor tissues, this suggests that RABV infection induces a shift in TAM away from a more typical M2 subset towards a less differentiated or Ml phenotype. Although the latter bear the Ml phenotype marker CDl lc, we were unable to determine whether or not they express the functional Ml marker iNOS due to the extensive expression of this enzyme in the infected tissues. Tbet-/- mice, which have large populations of both CD206+ M2 and CDl lc+ Ml -like cells within their GL261 tumors, do not display any alterations in the polarization of these cells as a consequence of RABV infection reaffirming that this is a type 1 -dependent process. Polarization of macrophages away from the M2 phenotype can block glioma progression. Specifically, administration of a CSF-IR inhibitor resulted in the loss of M2 cells, and though this was associated with an increase in macrophages with phagocytic functions, Ml-related gene expression was not otherwise increased (16). Ml macrophages are known to produce certain of the type 1 factors that we observe in the infected animals, as well as iNOS which can directly contribute to tumor cell lysis through the production of nitric oxide and associated cytotoxic radicals (50). While iNOS is expressed in the tumor tissues 4 days after RABV infection of mice that had GL261 cells implanted 8 days previously, it does not appear to be expressed by the macrophages at this time. Consequently, TAMs are unlikely to be contributing to iNOS- dependent tumor necrosis at its onset.

[00066] TAM are a heterogeneous continuum of highly plastic macrophage subsets that carry out diverse functions and respond to changes within the tumor (5, 51). While we cannot rule out the possibility that M2 TAM selectively undergo cell death and are replaced by infiltrating Ml macrophages, our data is consistent with the concept that macrophage populations in the glioma TME can be re-educated by the local cytokine milieu, as described by other investigators (16, 52. 53). The precise mechanisms responsible for the TAM re-polarization observed here are unknown, yet are undoubtedly dependent on type 1 immune processes acting in the TME as TAM polarization occurs in the absence of any differences in serum cytokine levels between infected and uninfected tumor-bearing mice.

[00067] Interestingly, Thl- dependent TME modulation, iNOS expression, tumor necrosis,

TAM polarization, and the increased survival of RABV-infected tumor-bearing C57BL/6 mice occurs without direct infection of the tumor, a requirement for oncolytic virus therapy. RABV replicates predominantly in the non- tumor bearing hemisphere and is largely excluded from the tumor, presumably due to antiviral factors secreted by GL261 cells. Also distinct from other immunotherapeutic strategies, the Thl -dependant changes in the TME are not accompanied by an enhanced Thl - biased glioma-specific peripheral response as measured by serum antibody. Instead, antibody isotype analysis indicates a shift in the RABV-specific response towards type 2 in the presence of a glioma. This bias has little impact on the pathogenicity of the virus as it is safe and readily cleared in mice that lack a range of immune components, including Tbet (31).

[00068] The failure of RABV infection to promote systemic Thl anti-glioma humoral immunity may be due to the transient nature of the antiviral response. This may contribute to the fact that the mice eventually succumb to their tumor. However, we do not yet know whether tumor growth, which was severely curtailed, or other factors relevant to the anti -tumor immune response are responsible for the animals' demise. At death the extent of necrotic tissue in the brains of the animals was substantial. Conceivably, an adjustment in the timing of infection or concomitant vaccination with a type 1 -biased glioma vaccine may improve the long-term outcome.

[00069] In summary, infection of GL261 tumor- bearing mice with attenuated, neurotropic

RABV prolongs survival and increases tumor necrosis via a Thl -dependent proinflammatory shift in the tumor microenvironment. Striking antitumor effects are achieved despite the limited ability of RABV to infect GL261 tumor cells. The change in the TME associated with the onset of tumor necrosis is accompanied by the expression of ΠΤΝΓγ, T Fa, and iNOS and followed by the replacement of tumor-supportive M2 with potentially destructive Ml cells. Mice lacking the Tbet transcription factor responsible for generating type 1 immunity did not exhibit increased survival, tumor necrosis, a proinflammatory TME modulation, or a beneficial change in the TAM population, indicating a Thl dependent mechanism is responsible for these antitumor effects. Accordingly, such modifications are warranted for human applications, wherein infection with attenuated, neurotropic RABV leads to modulation of the TME has therapeutic implications for brain tumor immunotherapy.

[00070] RABV are widely tested in human populations, being a virus useful for delivering vector loads into patients. Thus, the vaccination of human subjects with attenuated RABV is likely safe, efficacious, and a possibility in the near future due to the growing understanding that current inactivated rabies vaccines induce a type 2 response that is unable to clear a wildtype rabies virus infection from CNS tissues. Thus current inactivated vaccines fail to prevent the deaths of infected individuals whose post-exposure prophylaxis is delayed. Work on the development of live- attenuated rabies virus vaccines for human use therefore continues and these are extensively used in animal vaccination without adverse consequences. Due to the failure of standard of care and many experimental approached to treat malignant astrocytomas, a number of oncolytic viral therapies are being developed, but there are safety concerns due to the generally tissue destructive attributes of these viruses. While descended from a highly lethal virus, attenuated rabies viruses are available with a wide variety of redundant attenuating characteristics including double attenuating mutations in the glycoprotein, the inclusion of multiple copies of attenuated glycoprotein genes, and the inclusion of genes that encode cytokines that promote early immune responses such as IFNy. These viruses are highly crippled in their capacity to spread yet induce strong, virus-clearing immunity. Moreover, virus-neutralizing antibodies, the key effector of rabies virus clearance are readily available as a therapeutic if it is necessary to limit the infection. In extensive use in a wide variety of animal species, properly attenuated rabies viruses have proven safe, even if the virus reaches CNS tissue. For example, viruses attenuated with the approaches used for the virus used in our studies are safe for use in 5 day old mice, which have severely developmentally compromised immune systems.

[00071] For patients with inoperable or recurrent high grade astrocytomas with poor prognosis the certainty of death may far outweigh the risk of any sequelae due to the infection. The preferred path forward for use in humans would be to instill a highly attenuated rabies virus into the vicinity of the tumor with the understanding that it will minimally replicate in non-tumor tissues surrounding the lesion and cause the requisite state change in the tumor microenvironment. A GAS- attenuated virus expressing the human IFNy gene may be most appropriate for these purposes. Four to 5 days after infection, instillation of virus neutralizing antibodies may be used to promote the clearance of the virus. Alternatively it may be possible to mimic the effects of the infection by instilling into the tumor a cocktail of the immune factors induced by infection with attenuated rabies virus with the consequence of changing the state of the transformed astrocytes and tumor associated macrophages. Our observations have revealed that 1) changes in the tumor microenvironment and corresponding tumor necrosis occur early after infection with attenuated RABV, specifically before virus is readily detectable in the CNS, and 2) these apparent antitumor effects are dependent on a pronounced type 1 immune response. Therefore by providing the tumor microenvironment by direct application with the factors selectively induced by attenuated RABV infection within the CNS should be able to induce similar robust antitumor effects in GBM patients in the absence of RABV infection.

[00072] Mice have been extensively used to develop rabies virus vaccines and therapeutic reagents. Unlike many other viruses that can infect human CNS tissues, rabies viruses cause essentially similar infections and immune responses in all mammals. Research indicates that infection with attenuated RABV would likely have similar effects in patients with GBM as observed in the mouse GL261 model. Having been used for decades, the GL261 model is among the most widely used to test proposed therapies for glioma including immunotherapies.

[00073] The GL261 model reflects the human disease with regard to the nature of the transformed cells, effects of gene mutations, systemic effects of disease, and features of the local tumor microenvironment including the infiltrative pattern, tumor stem cell populations, and the presence of similar immune cell populations. For example, data from previous studies has demonstrated that patients with GBM have high levels of circulating Th2 factors and tumor derived microvesicles that polarize normal monocytes towards an M2 phenotype, all of which have been found in mice inoculated with GL261 cells. Furthermore, both human GBM and mouse GL261 tumors themselves appear to be highly infiltrated by M2 cells which not only inhibit tumor elimination, but actively aid in tumor growth. The RABV infection model utilized in this application changes the response to brain tumor cells observed systemically as well as within the local tumor microenvironment which based on our knowledge of rabies immunity in humans is likely to be the case for subjects with high grade astrocytomas including GBM.

[00074] Preferable treatment methods and route of administration in human patients is accomplished via intranasal application or via direct application into a tumor or resection cavity. For example, the virus can be formulated in a vaccine or therapeutic into a nasal delivery vehicle as is known to those of ordinary skill in the art. Furthermore, viral applications, for example, in a direct inoculation are known to those of ordinary skill in the art. These applications allow for the most direct introduction of the viral loads to the TME. While delivery through the general circulatory system may be possible, it may require high loads of virus to enact the same response, due to the body killing off many of the viruses before they target the TME of interest. Accordingly, a standard injection into fatty or muscular tissue, or direct introduction intravenously may be used in some instances, but is not the preferred route of introduction.

[00075] A particular method and strategy includes administration of the RABV into the patient, generating the immune response and generating the transformation in the TME to enable the change to a toxic microenvironment that is destructive to tumor cells. Then providing a therapeutic that selectively attacks the RABV, resulting in a modified TME, for destruction of the tumor cells, but reducing the viral load in the patient.

[00076] In certain instances, it is important to determine efficacy of treatment. We can collect data from circulating cells or blood, as well as biopsy, to compare levels of certain biomarkers as described herein. For example, when comparing to a control, we mean that the control is either a healthy normal patient, having levels of the biomarker of interest, or the control can be the patient themselves, with the control being levels of the biomarker before the RABV introduction. Comparison to a control allows for quantification of the changes in the TME. Furthermore, calculation of the changes will evidence the change in TME or the need for further viral loads to generate the response necessary in the TME.

[00077] A further method of treatment of a brain cancer is administering to a patient in need thereof, an effective amount of a combination of therapeutic components comprising IFNy, T Fa, and iNOS, suitable for changing the state of the transformed astrocytes and tumor associated macrophages. Therefore by providing the tumor microenvironment by direct application with the factors selectively induced by attenuated RABV infection within the CNS can induce a robust antitumor effects in GBM patients in the absence of RABV infection.

[00078] Materials and Methods

[00079] Mice

[00080] Eight to ten week old male wild-type C57BL/6 mice and Tbet-/- mice on a C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME), or raised at Thomas Jefferson University from The Jackson Laboratory founder animals. All procedures were conducted in accordance with Public Health Service Policy on Humane Care and Use of Laboratory Animals under protocols approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University (Animal Welfare Assurance Number A3085-01).

[00081] GL261 maintenance and implantation

[00082] The GL261 cell line was acquired from the National Cancer Institute. Cells were grown in RPMI supplemented with 10% fetal bovine serum (AFBS, Corning Inc., Corning, NY), 4mM L-glutamine (Thermo Fisher, Waltham, MA), 50ug/ml gentamicin (Thermo Fisher, Waltham, MA), and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich, Saint-Louis, MO) at 37°C in 5% C02. Prior to implantation GL261 cells were harvested with 0.25% Trypsin (Corning Inc., Corning, NY), then washed and suspended in 4°C PBS. For i.e. implantations mice were anesthetized with isoflurane (Vedco, St. Joseph, MO) and 105 GL261 cells in 2μ1 PBS were stereotactically injected in the right cerebral cortex, 1mm anterior to the bregma and 1mm to the right of the midline at a depth of 3mm. Normal control and RABV-infected control mice received surgeries during which PBS alone was injected.

[00083] Quantitative real-time PCR

[00084] At 12 and 22 days after tumor implantation, CNS tissues from GL261 -implanted animals were dissected, homogenized in TriReagent (MRC, Cincinnati, OH) by passaging through a 20-gauge needle and total RNA was extracted using the RNeasy minikit (Qiagen, Valencia, CA) according to manufacturer's protocol. cDNA was synthesized using oligo dT primers, dNTP, and Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Madison, WI). Quantitative real-time PCR (qPCR) was performed using iQ Supermix or iQ SYBRR Green Supermix (Bio-Rad Laboratories, Hercules, CA), for specific mRNAs with a Bio-Rad iCycler (BioRad, Hercules, CA). The Primer/probe sets (IDT, Coralville, IA) used for LI 3, SPBN-GAS, CD4, CD8, κ-L chain, IFN- β and IFN-γ were described previously (31) with additional sets listed in Table 1. All probes are dual labeled 5'-6-FAM and 3'-BHQ-l . Sample mRNA copy numbers were normalized to the housekeeping gene LI 3.

[00085] Table I. Genes selected for qrtPCR

Genes:

CXCL10

GZMB ICAM-1

iNOS

TGF-p2

TNFa

Note: the forward primers, reverse primers, and probes are known in the art. For example, they are published by E.K. Bongiorno, et al.; J. Immunol, 1, May, 2017 (www.j immunol . org/ content/ early/2011104/29/kimmunol .1601444).

[00086] Viral infection and in vitro viral inhibition curves

[00087] SPBN-GAS, a variant of the SAD B19 virus with two mutations in the glycoprotein that attenuate the virus and prevent reversion to a pathogenic strain, was propagated in NA cells as described elsewhere (30). Mice were anesthetized with isoflurane and infected with 105 focus forming units (f.f.u.) intranasally (i.n.) or 104 f.f.u. in the left or right cortex. Virus titration in both NA and GL261 cells was performed in 96-well plates when cells were approximately 80% confluent. Virus was added to media in ten-fold dilutions. To assess viral spread inhibition, GL261 cells were grown in supplemented RPMI which was removed at various time points. Cellular debris was removed by centrifugation and conditioned media was applied in two-fold serial dilutions to NA cells that were then infected with virus at 104 f.f.u. /ml. Alternatively, conditioned media was applied directly to NA cells and virus was serially titrated. For both virus assays, plates were incubated for 48hr at 34°C, then fixed in 80% acetone at 4°C. FITC-conjugated anti-RABV RNP (Fujirebio Diagnostics, Malvern, PA) was applied with Evans Blue counterstain (J.T. Baker, Phillipsburg, NJ). Virus f.f.u. were counted using a fluorescent microscope.

[00088] ELISA and cell-based ELISA

[00089] RABV-specific antibody isotypes were assessed by ELISA. Serum samples were collected 0, 8 and 12 days after infection and incubated on UV-inactivated RABV ERA-coated plates. Antibody titers were determined using secondary antibodies specific for IgG as well as IgGl and IgG2a as described previously (31). A cell-based ELISA was used to detect the level and isotype of GL261 -specific antibodies as described previously (18). Antibody isotypes were detected with alkaline phosphatase-conjugated anti-mouse IgG (1 : 1000), IgGl, IgG2a or IgG2b (1 :2000) (MP Biomedicals, Santa Ana, CA).

[00090] Histochemistry and immunofluorescence staining

[00091] Whole brains were snap frozen in Tissue-Tek O.C.T. compound at 12 and 22 days after implantation (Sakura Finetex, Torrence, CA) fixed in 95% ethanol, rinsed in water, stained with Mayer's hematoxylin solution and Eosin Y solution (Sigma Aldrich, St. Louis, MO), dehydrated and mounted with Richard-Allen Scientific™ Mounting Medium (Therm oFisher, Waltham, MA). Immunofluorescent staining was performed on sections fixed in cold methanol for 10 min at -20°C, rinsed in PBS and incubated with primary antibodies, diluted in PBS containing 2% BSA, 5% goat serum, and 0.25% Triton X-100, overnight at 4°C. Antibodies for RABV nucleoprotein, NeuN, and CD4 have been described previously (31) and additional reagents are listed in Table 2. Slides were then incubated with fluorescence-conjugated secondary antibodies and mounted with Vectashield® Hard Set™ mounting medium (Vector Laboratories, Inc., Burlingame, CA) containing DAPI. Brightfield and fluorescent images were acquired with a Leica DM6000 microscope with the Leica Application Suite v4 program (Leica Microsystems, Switzerland). Image brightness and contrast were adjusted using Photoshop CS5 software.

[00092] Table II. Primary and secondary Abs for immunohistochemistry

Target Tag Clone Host Isotype Dilution Supplier and reference

Primary Abs

CD l ib PE Ml/70 Rat IgG2b 1/200 BD Biosciences #553311 CDl lc APC HL3 Hamster IgGl 1/200 BD Biosciences #550261

ER- AbD Serotec

CD31 MP12 Rat IgG2a 1/1000 #MCA2388GA

Alexa Fluor AbD Serotec

CD206 647 MR5D3 Rat IgG2a 1/100 #MCA2235A647 F4/80 C1-A3-1 Rat IgG2b 1/100 AbD Serotec #MCA497 iNOS N20 Rabbit IgG 1/100 Santa Cruz #sc-651 Ki67 APC 16A8 Rat IgG2a 1/100 Biolegend #652405 Secondary Anti- Alexa Fluor

rabbit 488 Goat 1/200 Life Technologies #A 11008

Alexa Fluor

Anti-rat 555 Goat 1/1000 Life Technologies #A21434

[00093] In vitro iNOS induction

[00094] GL261 cells were cultured in 4- well Nunc™ Lab-Tek™ chamber slides (Therm oFisher, Rochester, NY) until confluent and treated with 0, 10, 100, or 1000 Units of recombinant mouse interferon-gamma (BD Bioscience, san Jose, CA) for 24 hours. Supernatant was removed from chamber wells and cells were washed once with PBS before fixation with ice-cold methanol for 10 minutes and staining as described above.

[00095] Luminex

[00096] Serum and tumors were collected from mice 12 days after GL261 implantation.

Tumors were homogenized with a 20 gauge syringe and passed through a 70μπι strainer, then cultured overnight in GL261 media and culture conditions. The level of relevant factors in serum and tumor supernatant was measured using MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panels (Millipore, Darmstadt, Germany) according to manufacturer protocol. Duplicate samples were analyzed by a FlexMAP 3D (Luminex, Austin, TX).

[00097] Statistical Analysis

[00098] Experimental data were plotted and statistical analyses were performed using Prism 5.01 (Graph Pad, Inc., San Diego, CA). Survival curves were assessed for significance with the Mantel-Cox test. ELISA, qPCR, and in vitro experiments were analyzed using the Mann-Whitney test. Statistically significant differences between groups are generally denoted as follows: * (p<0.05), ** (p<0.01) and *** (p<0.001). Principle component analysis (PCA) and heat maps were generated to determine patterns in cytokine expression across samples in MeV (37). Luminex mean fluorescent intensity (MFI) values were median centered across each cytokine. Three component 3D PCA plots were calculated for tumor samples and sera samples separately. Statistically significant differences in individual cytokines were determined by Student's t test with p-values based on permutation (critical p value = .05).