Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.

Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.

Research Doctorate - Doctor of Philosophy (PhD) / A number of oncolytic picornaviruses are currently under evaluation as potential therapeutic agents for a range of human malignancies. In particular, a subset of naturally occurring human C-cluster enteroviruses; Coxsackievirus A13 (CVA13), Coxsackievirus A15 (CVA15), Coxsackievirus A18 (CVA18) and Coxsackievirus A21 (CVA21) and the human B-cluster enterovirus, Echovirus 1 (EV1), display promising pre-clinical oncolytic activity against a wide variety of neoplastic cells. CVA21 is currently under clinical evaluation for the control of melanoma, breast, prostate and head/neck cancer. The preferential targeting of cancer cells by this subset of viruses is based on extracellular capsid interactions with specific viral receptors (intercellular adhesion molecule-1 [ICAM-1], decay-accelerating factor [DAF] or integrin α2β1), on the surface of malignant cells. In the present study, the therapeutic potential of this subset of enteroviruses was evaluated as a novel treatment strategy for the control of human malignancies of the gastrointestinal system. In Chapter 3, the capacity of the aforementioned enteroviruses for oncolytic activity was assessed in a panel of in vitro human gastric cancer cell cultures. Flow cytometric analysis revealed low-to-medium levels of ICAM-1, in addition to abundant α2β1 and DAF expression on the surface of gastric cancer cell lines. Cell monolayer lytic infectivity assays demonstrated that, of the viruses under evaluation, EV1 displayed the most potent and widespread in vitro lytic activity against the gastric cancer cell lines. Monoclonal antibody blockade confirmed the specific integrin α2β1-mediated route of EV1 cell infection in the gastric cancer MKN-45 cell line. Subsequently, an in vivo dose ranging study assessing the efficacy of oncolytic EV1 was undertaken in an immune-compromised MKN-45-Luc mouse model of human gastric cancer peritoneal carcinomatosis (PC). In this model, an intra-peritoneal dose of as little as 1x103 TCID50 EV1 resulted in a significant reduction in peritoneal tumour burden. In Chapter 4, the oncolytic capacity of this enterovirus subset was further evaluated, as a potential therapeutic option for the control of colorectal cancer (CRC). Flow cytometric analysis of a panel of CRC cell lines demonstrated abundant levels of DAF and integrin α2β1, and low-to-moderate levels of ICAM-1 expression on the surface of CRC cells. Of the subset of viruses examined, a DAF-using variant of CVA21 (CVA21-DAFv) displayed the most potent and widespread oncolytic activity against in vitro CRC cell cultures. Consequently, the potential in vivo oncolytic capacity of CVA21-DAFv and the wild-type CVA21 was evaluated in three individual immune-compromised mouse sub-cutaneous xenograft models of human CRC. However, despite the immunohistochemical detection of ICAM-1/DAF on cells of the CRC xenografts, and the detection of infectious virus in the blood of treated tumour-bearing mice, a detectable reduction in tumour burden was not observed. On account of the varying degrees of oncolytic efficacy observed in colorectal and gastric cancers, global gene expression profiling was employed in Chapters 5 and 6, to further elucidate the molecular mechanisms of enterovirus-mediated tumour cell tropism and cell death. As the most extensively characterised virus in pre-clinical studies, and the only virus of this subset under current clinical evaluation, CVA21 was selected as the challenge virus for analysis of the transcriptional response to enterovirus infection. Malignant cells that displayed reproducible susceptibility to in vitro and in vivo lytic CVA21 challenge were necessary for extensive characterisation, therefore, melanoma SK-Mel-28 and breast cancer MDA-MB-231-Luc cell lines, rather than CRC cell lines, were utilised. In Chapter 5, the response of SK-Mel-28 and MDA-MB-231-Luc cell monolayers, and a supporting panel of malignant and normal cell lines, to in vitro CVA21 challenge was assessed. In Chapter 6, the transcriptional response of immune-compromised mouse SK-Mel-28 and MDA-MB-231-Luc xenograft cells to systemic CVA21 administration was characterised. The transcriptional response of cells propagated as in vitro monolayers differed markedly when compared to that of in vivo xenografts generated from the same cell lines. In Chapter 5, a delayed rate of CVA21 replication and cell lysis was observed in normal cell cultures, as compared to malignant cell lines. Gene expression profiling suggested that the normal human lung fibroblast cell line, MRC-5, mounted an interferon (IFN)-mediated innate immune response against CVA21 challenge, a phenomenon not observed following challenge of the malignant cell line panel. Such findings suggest a potential role for the functional status of the IFN-mediated innate immune system in the tumour cell tropism of oncolytic CVA21. Somewhat surprisingly, in Chapter 6, an IFN-mediated transcriptional response was observed in the SK-Mel-28/MDA-MB-231-Luc xenograft cells, potentially attributed to the ‘priming’ effects of in vivo endogenous murine IFN activity. Furthermore, in Chapters 5 and 6, the potential contributions of transcriptionally regulated genes, in respect to their biological roles in cell cycle regulation, apoptosis, oxidative stress, stimulation of anti-tumoural immunity, and inhibition of angiogenesis in CVA21-mediated oncolysis were considered. Moreover, in Chapter 6, a distinct genetic signature of infection was identified, comprising a total of 9 individual genes, significantly upregulated in response to infection in each xenograft model at 24 and 72 h following the systemic administration of CVA21. The identified genes involved in this core transcriptional response to infection may serve as effective molecular biomarkers for the evaluation of oncolytic CVA21 efficacy.

Research Doctorate - Doctor of Philosophy (PhD) / A number of oncolytic picornaviruses are currently under evaluation as potential therapeutic agents for a range of human malignancies. In particular, a subset of naturally occurring human C-cluster enteroviruses; Coxsackievirus A13 (CVA13), Coxsackievirus A15 (CVA15), Coxsackievirus A18 (CVA18) and Coxsackievirus A21 (CVA21) and the human B-cluster enterovirus, Echovirus 1 (EV1), display promising pre-clinical oncolytic activity against a wide variety of neoplastic cells. CVA21 is currently under clinical evaluation for the control of melanoma, breast, prostate and head/neck cancer. The preferential targeting of cancer cells by this subset of viruses is based on extracellular capsid interactions with specific viral receptors (intercellular adhesion molecule-1 [ICAM-1], decay-accelerating factor [DAF] or integrin α2β1), on the surface of malignant cells. In the present study, the therapeutic potential of this subset of enteroviruses was evaluated as a novel treatment strategy for the control of human malignancies of the gastrointestinal system. In Chapter 3, the capacity of the aforementioned enteroviruses for oncolytic activity was assessed in a panel of in vitro human gastric cancer cell cultures. Flow cytometric analysis revealed low-to-medium levels of ICAM-1, in addition to abundant α2β1 and DAF expression on the surface of gastric cancer cell lines. Cell monolayer lytic infectivity assays demonstrated that, of the viruses under evaluation, EV1 displayed the most potent and widespread in vitro lytic activity against the gastric cancer cell lines. Monoclonal antibody blockade confirmed the specific integrin α2β1-mediated route of EV1 cell infection in the gastric cancer MKN-45 cell line. Subsequently, an in vivo dose ranging study assessing the efficacy of oncolytic EV1 was undertaken in an immune-compromised MKN-45-Luc mouse model of human gastric cancer peritoneal carcinomatosis (PC). In this model, an intra-peritoneal dose of as little as 1x103 TCID50 EV1 resulted in a significant reduction in peritoneal tumour burden. In Chapter 4, the oncolytic capacity of this enterovirus subset was further evaluated, as a potential therapeutic option for the control of colorectal cancer (CRC). Flow cytometric analysis of a panel of CRC cell lines demonstrated abundant levels of DAF and integrin α2β1, and low-to-moderate levels of ICAM-1 expression on the surface of CRC cells. Of the subset of viruses examined, a DAF-using variant of CVA21 (CVA21-DAFv) displayed the most potent and widespread oncolytic activity against in vitro CRC cell cultures. Consequently, the potential in vivo oncolytic capacity of CVA21-DAFv and the wild-type CVA21 was evaluated in three individual immune-compromised mouse sub-cutaneous xenograft models of human CRC. However, despite the immunohistochemical detection of ICAM-1/DAF on cells of the CRC xenografts, and the detection of infectious virus in the blood of treated tumour-bearing mice, a detectable reduction in tumour burden was not observed. On account of the varying degrees of oncolytic efficacy observed in colorectal and gastric cancers, global gene expression profiling was employed in Chapters 5 and 6, to further elucidate the molecular mechanisms of enterovirus-mediated tumour cell tropism and cell death. As the most extensively characterised virus in pre-clinical studies, and the only virus of this subset under current clinical evaluation, CVA21 was selected as the challenge virus for analysis of the transcriptional response to enterovirus infection. Malignant cells that displayed reproducible susceptibility to in vitro and in vivo lytic CVA21 challenge were necessary for extensive characterisation, therefore, melanoma SK-Mel-28 and breast cancer MDA-MB-231-Luc cell lines, rather than CRC cell lines, were utilised. In Chapter 5, the response of SK-Mel-28 and MDA-MB-231-Luc cell monolayers, and a supporting panel of malignant and normal cell lines, to in vitro CVA21 challenge was assessed. In Chapter 6, the transcriptional response of immune-compromised mouse SK-Mel-28 and MDA-MB-231-Luc xenograft cells to systemic CVA21 administration was characterised. The transcriptional response of cells propagated as in vitro monolayers differed markedly when compared to that of in vivo xenografts generated from the same cell lines. In Chapter 5, a delayed rate of CVA21 replication and cell lysis was observed in normal cell cultures, as compared to malignant cell lines. Gene expression profiling suggested that the normal human lung fibroblast cell line, MRC-5, mounted an interferon (IFN)-mediated innate immune response against CVA21 challenge, a phenomenon not observed following challenge of the malignant cell line panel. Such findings suggest a potential role for the functional status of the IFN-mediated innate immune system in the tumour cell tropism of oncolytic CVA21. Somewhat surprisingly, in Chapter 6, an IFN-mediated transcriptional response was observed in the SK-Mel-28/MDA-MB-231-Luc xenograft cells, potentially attributed to the ‘priming’ effects of in vivo endogenous murine IFN activity. Furthermore, in Chapters 5 and 6, the potential contributions of transcriptionally regulated genes, in respect to their biological roles in cell cycle regulation, apoptosis, oxidative stress, stimulation of anti-tumoural immunity, and inhibition of angiogenesis in CVA21-mediated oncolysis were considered. Moreover, in Chapter 6, a distinct genetic signature of infection was identified, comprising a total of 9 individual genes, significantly upregulated in response to infection in each xenograft model at 24 and 72 h following the systemic administration of CVA21. The identified genes involved in this core transcriptional response to infection may serve as effective molecular biomarkers for the evaluation of oncolytic CVA21 efficacy.

abstract: Host organisms have evolved multiple mechanisms to defend against a viral infection and likewise viruses have evolved multiple methods to subvert the host's anti-viral immune response. Vaccinia virus (VACV) is known to contain numerous proteins involved in blocking the cellular anti-viral immune response. The VACV E3L protein is important for inhibiting the anti-viral immune response and deletions within this gene lead to a severe attenuation. In particular, VACV containing N-terminal truncations in E3L are attenuated in animal models and fail to replicate in murine JC cells. Monkeypox virus (MPXV) F3L protein is a homologue of the VACV E3L protein, however it is predicted to contain a 37 amino acid N-terminal truncation. Despite containing an N-terminal truncation in the E3L homologue, MPXV is able to inhibit the anti-viral immune response similar to wild-type VACV and able to replicate in JC cells. This suggests that MPXV has evolved another mechanism(s) to counteract host defenses and promote replication in JC cells. MPXV produces less dsRNA than VACV during the course of an infection, which may explain why MPXV posses a phenotype similar to VACV, despite containing a truncated E3L homologue. The development of oncolytic viruses as a therapy for cancer has gained interest in recent years. Oncolytic viruses selectively replicate in and destroy cancerous cells and leave normal cells unharmed. Many tumors possess dysregulated anti-viral signaling pathways, since these pathways can also regulate cell growth. Creating a mutation in the N-terminus of the VACV-E3L protein generates an oncolytic VACV that depends on dysregulated anti-viral signaling pathways for replication allowing for direct targeting of the cancerous cells. VACV-E3Ldel54N selectively replicates in numerous cancer cells lines and not in the normal cell lines. Additionally, VACV-E3Ldel54N is safe and effective in causing tumor regression in a xenograph mouse model. Lastly, VACV-E3Ldel54N was capable of spreading from the treated tumors to the untreated tumors in both a xenograph and syngeneic mouse model. These data suggest that VACV-E3Ldel54N could be an effective oncolytic virus for the treatment of cancer. / Dissertation/Thesis / Ph.D. Microbiology 2010

Despite improvements in chemotherapy and radical surgical debulking, peritoneal carcinomatosis (PC) remains among the most common causes of death for abdominal cancers. Immunotherapies have demonstrated efficacy in selected solid malignancies but their potential in PC is poorly explored. Here I report that intraperitoneal injection of an infected cell vaccine (ICV), consisting of autologous tumor cells infected ex-vivo with an oncolytic Maraba MG1 virus expressing interleukin-12 (IL-12), promotes the migration of activated natural killer (NK) cells to the peritoneal cavity in response to the secretion of interferon gamma-induced protein-10 (IP-10) from dendritic cells. This recruitment of cytotoxic, IFNγ-secreting NK cells is associated with a dramatic reduction in tumor burden and improved survival in a colon cancer model of PC. Even in mice with bulky PC (tumors >8 mm), a complete radiological response was demonstrated within 8-14 weeks, associated with 100% long-term survival. Importantly, these results were recapitulated in human lymphocytes exposed to human tumor cell lines infected with MG1-IL12. Finally, I demonstrate that MG1-IL12-ICV generates an effective CD4 and CD8 T cell response in mice following prophylactic immunization associated with the maturation of peritoneal dendritic cells and enrichment of tumor-specific peritoneal T cells. The research presented in this thesis suggests that an MG1-IL12-ICV is a promising therapy that could provide benefit to the thousands of patients diagnosed with PC each year.

Oncolytic Viruses (OVs) are often attenuated to increase their safety profile, however this can lead to reduced efficacy in heterogeneous malignancies and result in resistance to OV therapy. Our group utilizes small molecule enhancers of OV therapy termed viral sensitizers. These small molecules have been shown to enhance the replication and spread of oncolytic rhabdovirus VSVΔ51 in vitro and prolong survival in tumour-bearing mice. In this study, we evaluate the ef-fect of these viral sensitizers on the innate antiviral response in order to identify the mechanism of action responsible for their viral-sensitizing properties. Our previous data suggest that VSe1 and its structural analogues affect the type I IFN antiviral response and have the potential to af-fect cellular redox homeostasis. We hypothesized that VSe1 and its structural analogues potenti-ate VSV∆51 activity by inhibiting the type I IFN response via redox-mediated dysregulation. In this study, we demonstrate that the viral sensitizers inhibit the nuclear translocation and transcrip-tional activity of NFκB, which in turn dampens the expression of antiviral cytokines IFN-, TNFα and IL-6. We also provide evidence supporting the possibility that the NFκB inhibition may be a result of the formation of ROS intermediates by the viral sensitizers, which leads to re-duced nuclear translocation of NFκB subunits, thereby preventing NFκB-mediated cytokine production. Overall, this work contributes to the identification of the mechanism of action of our viral sensitizers and highlights the finding that oncolytic VSV infection can be enhanced through redox-mediated modulation of the innate antiviral response.

Oncolytic viruses (OV) are an emerging class of anticancer bio-therapeutics that induce antitumor immunity through selective replication in cancer cells. However, the efficacy of OVs as single agents remains limited. We postulate that resistance to oncolytic virotherapy results in part from the failure of tumor cells to be sufficiently infected. In this study, we provide evidence that in the context of sarcoma, a highly heterogeneous malignancy, the infection of tumors by different oncolytic viruses varies greatly. Similarly, for a given oncolytic virus, productive infection of tumors across patient samples varies by many orders of magnitude. To overcome this issue, we hypothesize that the infection of resistant tumors can be achieved through the use of selected small molecules. Here, we have identified two novel drug classes with the ability to improve the efficacy of OV therapy: fumaric and maleic acid esters (FMAEs) and vanadium compounds. FMAEs are enhancing infection of cancer cells by several oncolytic viruses in cancer cell lines and human tumor biopsies. The ability of FMAEs to enhance viral spread is due to their ability to inhibit type I IFN production and response, which is associated with their ability to block nuclear translocation of transcription factor NF-κB. Vanadium-based phosphatase inhibitors enhance OV infection of RNA viruses in vitro and ex vivo, in resistant cancer cell lines. Mechanistically, this involves subverting the antiviral type I IFN response towards a death-inducing and proinflammatory type II IFN response, leading to improved OV spread, increased bystander killing of cancer cells, and enhanced anti-tumor immune-stimulation. Both FMAEs and vanadium compounds improve therapeutic outcomes of OV treatment in syngeneic tumor models, leading to durable responses, even in models otherwise refractory to OV and drug alone. Overall, we showcased novel avenues for the development of improved immunotherapy strategies.

Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.

Glioblastoma is the most common primary brain tumour in adults. Despite the aggressive standard of care currently used, median patient survival following treatment is only 14 months. Innovative treatment options are needed for these patients. Recently, oncolytic viruses have emerged as promising immunotherapies for the treatment of solid tumours. Preliminary work in our lab has demonstrated that Farmington virus, a novel brain-safe oncolytic rhabdovirus, can be engineered to encode a tumour-associated antigen (TAA) to prime and boost antigen-specific adaptive immune responses. Since other rhabdoviruses share this boosting capacity, a heterologous rhabdovirus prime/boost regimen can be designed to combine two powerful oncolytics and a robust anti-TAA adaptive immune response. We evaluated Farmington’s ability to vaccinate against a self- glioblastoma antigen and two foreign glioblastoma-associated antigens. Farmington was able to vaccinate against the foreign antigens, leading to efficacy in prophylactic and therapeutic glioblastoma models. Additionally, treatment with heterologous rhabdoviruses demonstrated efficacy in an aggressive murine mammary carcinoma model. Herein, we demonstrate promising preliminary results for a novel glioblastoma therapeutic approach. Le glioblastome est la tumeur primaire la plus fréquente chez l’adulte. La survie moyenne des patients n’excède pas 14 mois malgré une prise en charge thérapeutique agressive. Par conséquent, la mise au point de traitements innovants et efficaces est une nécessité pour ces patients. Des avancées récentes ont mise en évidence l’intérêt des virus oncolytiques dans le traitement des tumeurs solides. Des travaux préliminaires réalisés au sein de notre laboratoire ont, en effet, démontré que le virus Farmington pouvait être modifié afin d’exprimer un antigène associé aux tumeurs (AAT), pour initier et potentialiser une réponse immunitaire adaptative spécifique. D’autres rhabdovirus possèdent des capacités de potentialisation immunitaire similaires et peuvent être utilisés en association avec le virus Farmington modifié pour amorcer et amplifier la réponse immunitaire oncolytique de l’hôte. Le but de ce projet était d’évaluer le potentiel du virus Farmington comme vaccin contre des antigènes tumoraux d’origine endogène ou exogène associés au glioblastome. Nos résultats ont montré que le virus Farmington a la capacité d’induire une réponse immunitaire prophylactique et thérapeutique contre les antigènes tumoraux exogènes dans des modèles de glioblastome. De plus, l’utilisation de rhabdovirus hétérologues s’est aussi révélée efficace pour le traitement de carcinome mammaire agressif chez la souris. Cette étude préliminaire apporte des résultats prometteurs pour le développement de nouvelles approches thérapeutiques efficaces dans le traitement du glioblastome.