Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

In the last decade cancer immunotherapy has taken huge strides forward from bench to bedside and being approved as drugs. Cancer immunotherapy harnesses the power of patient’s own immune system to fight cancer. Approaches are diverse and include antibodies, therapeutic vaccines, adoptively transferred T-cells, immune checkpoint inhibitors, oncolytic viruses and immune cell activators such as toll-like receptor (TLR) agonists. Excellent clinical responses have been observed for certain cancers with checkpoint antibodies and chimeric antigen receptor (CAR)-engineered T-cells. It is however becoming evident that strategies need to be combined for broader effective treatment responses because cancers evolve to escape immune recognition. A conditionally replication-competent oncolytic adenovirus (Ad5PTDf35-[Δ24]) was engineered to secrete Helicobacter pylori Neutrophil Activating Protein (HP-NAP, a TLR-2 agonist) to combine viral oncolysis and immune stimulation. Treatment with Ad5PTDf35-[Δ24-sNAP] improved survival of mice bearing human neuroendocrine tumors (BON). Expression of HP-NAP in the tumor microenvironment promoted neutrophil infiltration, proinflammatory cytokine secretion and increased necrosis. We further studied the ability of HP-NAP to activate dendritic cells (DCs) a key player in priming T-cell responses. HP-NAP phenotypically matured and activated DCs to secrete the T-helper type-1 (Th-1) polarizing cytokine IL-12. HP-NAP-matured DCs were functional; able to migrate to draining lymph nodes and prime antigen-specific T-cell proliferation. CAR T-cells were engineered to secrete HP-NAP upon T-cell activation. Secreted HP-NAP was able to mature DCs, leading to a reciprocal effect on the CAR T-cells with improved cytotoxicity in vitro. Semliki Forest virus (SFV), an oncolytic virus with natural neuro-tropism was tagged with central nervous system (CNS)-specific microRNA target sequences for miR124, miR125 and miR134 to selectively attenuate virus replication in healthy CNS cells. Systemic infection of mice with the SFV4miRT did not cause encephalitis, while it retained its ability to replicate in tumor cells and cure a big proportion of mice bearing syngeneic neuroblastoma and gliomas. Therapeutic efficacy of SFV4miRT inversely correlated with type-I antiviral interferon response (IFN-β) mounted by tumor cells. In summary, combining immunotherapeutic strategies with HP-NAP is a promising approach to combat cancers and SFV4miRT is an excellent candidate for treatment of neuroblastomas and gliomas.

This thesis describes the design of a virotherapy strategy capable of destroying both breast cancer vasculature and tumour cells, using an oncolytic adenovirus expressing angiogenesis-regulating proteins. Five oncolytic adenoviruses were compared to identify the best virotherapy agent for breast cancer, including measurement of cytotoxicity in vitro, and replication, intra-tumoural spread and anticancer efficacy in vivo. The viruses tested were Ad-dl922-947 (targets G1-S checkpoint defects); Ad-Onyx-015 and Ad-Onyx-017 (target p53/mRNA nuclear export defects); Ad-vKH1 (targets Wnt pathway defects) and AdEHE2F (targets estrogen receptor/G1-S checkpoint/hypoxia signalling defects). AdEHE2F demonstrated optimal oncolytic activity and selectivity against breast cancer, accordingly this virus was engineered to express potent regulatory angiogenic proteins, namely soluble Flt1 and soluble Delta like-4 (Dll4). sFlt1 is the soluble extra-cellular domain of VEGFR1 and binds to and sequesters VEGF-A, thereby preventing VEGFR2 stimulation which is crucial to trigger angiogenesis. sDll4 is the soluble extracellular domain of Dll4 and has been previously shown to block Dll4/Notch signalling. Dll4/Notch signalling increases a chaotic and non-functional angiogenesis which ultimately delays tumour growth. Importantly, VEGF and Dll4 are the only angiogenesis genes reported to be haploinsufficient in vascular development and both have been shown to have a good anti-tumour effect. sFlt1 and sDll4 genes were substituted for the viral genes E3 6.7K/gp19K of AdEHE2F, thereby using endogenous adenoviral machinery to drive production. The activities of AdEHE2F viruses expressing either sFlt1 or sDll4 were compared in vitro and in vivo. sFlt1 (expressed from AdEHE2F) inhibited endothelial cell proliferation and sprouting whereas sDll4 increased proliferation and branching in vitro. In vivo AdEHE2F expressing sFlt1 or sDll4 both showed superior anticancer activity compared to parental AdEHE2F, indicating at least additive efficacy between virotherapy and regulatory angiogenic approaches.

Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

abstract: Combination therapy has shown to improve success for cancer treatment. Oncolytic virotherapy is cancer treatment that uses engineered viruses to specifically infect and kill cancer cells, without harming healthy cells. Immunotherapy boosts the body's natural defenses towards cancer. The combination of oncolytic virotherapy and immunotherapy is explored through deterministic systems of nonlinear differential equations, constructed to match experimental data for murine melanoma. Mathematical analysis was done in order to gain insight on the relationship between cancer, viruses and immune response. One extension of the model focuses on clinical needs, with the underlying goal to seek optimal treatment regimens; for both frequency and dose quantity. The models in this work were first used to estimate parameters from preclinical experimental data, to identify biologically realistic parameter values. Insight gained from the mathematical analysis in the first model, allowed for numerical analysis to explore optimal treatment regimens of combination oncolytic virotherapy and dendritic vaccinations. Permutations accounting for treatment scheduled were done to find regimens that reduce tumor size. Observations from the produced data lead to in silico exploration of immune-viral interactions. Results suggest under optimal settings, combination treatment works better than monotherapy of either type. The most optimal result suggests treatment over a longer period of time, with fractioned doses, while reducing the total dendritic vaccination quantity, and maintaining the maximum virotherapy used in the experimental work. / Dissertation/Thesis / Doctoral Dissertation Applied Mathematics for the Life and Social Sciences 2016

The use of oncolytic viruses (OVs) to selectively destroy cancer cells is poised to make a major impact in the clinic and potentially revolutionize cancer therapy. Pre-clinical and clinical studies have shown that OV therapy is safe, well-tolerated and effective in a broad range of cancers. Still, resistance due to tumour heterogeneity highlights areas for improvement in OV based therapeutics. Combining OVs and small molecules is a promising strategy to selectively enhance OV-mediated anti-tumour effects. To this end, we have previously identified the synthetic compound Viral Sensitizer 1 (VSe1) that enhances the spread of oncolytic vesicular stomatitis virus (VSVΔ51) in resistant cancer cell lines up to 1000-fold, resulting in synergistic cell killing and improved efficacy in vitro and in vivo. The electrophilic nature of VSe1 prompted us to investigate the scaffold to identify active analogs with more favourable physiochemical properties and explore structure-activity relationships (SAR). In vitro assays and a rational approach in the design of VSe1 analogs allowed us to identify functional groups that can be modified without hampering activity. Lead compounds created in this study based on a pyrrole scaffold increase OV growth up to 2000-fold in vitro and demonstrate remarkable selectivity for cancer cells over normal tissue ex vivo and in vivo. Compared to the parental VSe1, these small molecules also possess enhanced stability with reduced electrophilicity and are well-tolerated in animals, leading to reduced tumour burden and prolonged survival in vivo when used in combination with VSVΔ51. It was known from previous studies that VSe1 suppresses the type I interferon response generated by cancer cells to defend against viral infection. In this study, further investigation revealed that VSe1 and its analogs inhibit the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), resulting in dampened transcriptional expression and secretion of IFN-β and interferon stimulated genes, thereby increasing viral replication and spread. While these findings further elucidated the effect these compounds have on the innate antiviral response, the molecular mechanisms leading to NFκB inhibition remained unclear. We used the newly generated VSe1 analogs to perform ligand-based affinity capture studies leading to the identification of glutathione-s-transferases as interacting proteins, catalytically inhibited by VSe1 and to a lesser extent by its pyrrole analogs. Further inquiry revealed that VSe1 and its analogs cause an imbalance in cellular glutathione homeostasis and increase oxidative stress, which is associated with inhibition of the nuclear translocation of NFκB. However, further studies are required to assess whether these phenomena are directly or indirectly linked. Overall, this study highlights a novel approach to improving OV therapy by using a previously uncharacterized class of compounds that ultimately alter the innate cellular antiviral response through inhibition of NFκB.

Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

Oncolytic viruses (OVs) are novel biological agents that selectively infect and kill malignant cells. OVs can also generate anti-cancer immunity. Our lab exploited this phenomenon and developed an in vitro vaccine with infected leukemia cells with oncolytic virus vaccine – and named immunotherapy by leukemia-oncolytic virus (iLOV) – that provided in vivo protection in a murine model for acute lymphoblastic leukemia. This work further characterizes iLOV biology and the interaction of its immune responses. An in vitro immune response assay was optimized to detect and quantify the in vivo anti-leukemia immunity generated by iLOV. Anti-viral immunity is an obstacle for OV therapy. Although iLOV created anti-viral antibodies towards itself, these neutralizing antibodies did not hinder the vaccine’s ability to initiate complement or dendritic cell activation. We envision personalized versions of iLOV for leukemia patients in remission to prevent the possibility of relapse. This work highlights new advantages for infected cell vaccines and supports the progress of iLOV toward clinical testing.

Oncolytic viruses are novel candidates for cancer therapy and their efficacy relies on their capacity to overcome the host’s anti-viral barriers. In mammalian cells, the anti-viral response involves a protein-signaling cascade known as the interferon pathway, which alerts the immune system and limits the propagation of infection. Given that most cancer cells have defects in this pathway, they are susceptible to viral infection and responsive to oncolytic virotherapy. For reasons that remain unknown, many cancers are still refractory to oncolytic viruses, which suggests the existence of additional antiviral mechanisms. In this study, we investigate the potential involvement of an alternative antiviral pathway in cancer cells. Given that insects and plants rely on the RNA silencing pathway for their anti-viral protection, we investigated the presence of a similar mechanism in cancer cells. We found viral genome-derived small RNAs in various cancer cell lines upon infection, which is indicative of an RNA-mediated antiviral response. Also, various viruses encode suppressors of the RNA interference pathway. To determine if an oncolytic virus could benefit from such a factor, we engineered an oncolytic virus variant to encode the Nodamura virus B2 protein, a known inhibitor of RNA silencing-mediated immune responses. Using this virus, we observed enhanced cytotoxicity in 33 out of the 38 human cancer cell lines tested. Furthermore, our results show inhibition of viral genome cleavage and altered microRNA processing by our B2-expressing oncolytic virus. Taken together, our data suggests the blockade of RNA silencing antiviral pathways and/or antiviral microRNA processing improves the efficacy of our B2-encoding virus in a cell-line specific manner. Overall, our results establish the improved potential of our novel virus therapy and demonstrate for the first time the involvement of RNA pathways in the antiviral defense of cancer cells.

Oncolytic viruses (OVs) are emerging as a potent therapeutic platform for the treatment of malignant disease. The tumor cells inability to induce antiviral defences in response to a small cytokine known as interferon (IFN) is a common defect exploited by OVs. Heterogeneity in IFN signalling across tumors is therefore a pillar element of resistance to these therapies. I have generated a mathematical model and simulation platform to study the impact of IFN on OV dynamics in normal and cancerous tissues. In the first part of my thesis, I used this model to identify novel OV engineering strategies which could be implemented to overcome IFN based resistance in tumor tissues. From these simulations, it appears that a positive feedback loop, established by virus-mediated expression of an interferon-binding decoy receptor, could increase tumor cytotoxicity without compromising normal cells. The predictions set forth by this model have been validated both qualitatively and quantitatively in in-vitro and in-vivo models using two independent OV strains. This model has subsequently been used to investigate OV attenuation mechanisms, the impact of tumor cell heterogeneity, as well as drug-OV interactions. Following these results, it became apparent that selectivity should equally be observed when overwhelming the cell with a non replicating virus. While normal tissues will clear this pseudo-infection rapidly, owing to their high baseline in antiviral products at the onset of infection, tumor cells with defective anti-viral pathways should not have readily available biomachinery required to degrade this pro-apoptotic signal. Recapitulated by the mathematical model, non-replicating virus-derived particles generated by means of UV irradiation selectively kill tumor cells in cultured cell lines and patient samples, leading to long term cures in murine models. Taken together, this thesis uses a novel mathematical model and simulation platform to understand, design & improve oncolytic virus-based therapeutics.