Survival for patients with advanced cancer has remained dismal, and there is a need for new treatments. In this context viral immune therapy is a promising novel strategy. Intravenous delivery confers advantages as it enables simultaneous treatment of primary tumour and any metastatic deposits but host defences limit Vaccinia virus's (VV) ability to infect tumour after systemic administration. Although Vaccinia virus can potentially be delivered systemically as it can evade both complement and neutralising antibodies, our investigations have revealed that VV cannot effectively infect tumour cells in immunocompetent mice after systemic delivery. Strikingly, we observed that if macrophages were depleted in the mice using clodronate liposomes, VV infection of tumours was dramatically enhanced. However, clodronate liposomes non-selectively deplete macrophages and potentially diminish any beneficial macrocytic activity in the tumour microenvironment unrelated to viral clearance. Consequently, a more clinically appropriate agent is needed. Macrophages recognise and ingest pathogenic microorganisms through phagocytosis, a process for which several lines of evidence have highlighted an important role for phosphatidylinositol 3-kinases. Accordingly, in these investigations I have evaluated the effect of selective PI3K inhibitors on macrophage phagocytosis in vitro and demonstrated that IC87114 (a PI3 kinase delta inhibitor) is effective at reducing uptake of VV by macrophages, confirming this finding in transgenic macrophages with a mutant of the PI3 kinase delta isoform knocked in. Subsequently, it was confirmed that IC87114 affects attachment of the virus to macrophages but plays no role in internalisation of the virus. In cancer cells cultured in isolation, the inhibitor has no direct cytotoxic effect and when combined with VV, in the same in vitro system, there is no change in the amount of cell death compared to VV alone treated controls. Biodistribution studies have established that IC87114 combined with VV results in statistically significantly higher levels of virus detected in tumours compared to the groups treated with VV alone, with similarly limited off-target effects. Finally, three different efficacy studies have demonstrated statistically significantly superior tumour responses in the VV+IC87114 group. In conclusion, PI3k delta blockade is an effective strategy for enhancing systemic delivery of VV in a preclinical model and could be a useful adjuvant in VV clinical trials.

Similar to cancer cells, HIV-infected cells differ from HIV-uninfected cells in that they have altered interferon signaling pathways, the apparent reason for the selectivity of certain oncolytic viruses (OVs). Therefore, it was hypothesized that use of an OV, such as recombinant Maraba virus (MG1), may be a potential approach to eliminate latently-infected cells constituting the HIV reservoir while sparing HIV-uninfected cells. This was studied in U1, ACH-2, OM-10 and J1.1 cells and their respective HIV-uninfected parent cell lines in addition to CD4+CD25-HLADR- cells from HIV-infected individuals on effective antiretroviral therapy. Although MG1 infected and killed latently HIV-infected U1 cells to a greater degree than the HIV-uninfected parent U937 cells, this was not observed in the other HIV-infected cell lines and their respective parent cell lines. Furthermore, results from primary cells suggest that MG1 alone does not appear to eliminate cells which comprise the major HIV reservoir. Challenges of studying the HIV reservoir and priorities for future studies examining the use of OVs as a potential strategy to eliminate the HIV reservoir are discussed.

Similar to cancer cells, HIV-infected cells differ from HIV-uninfected cells in that they have altered interferon signaling pathways, the apparent reason for the selectivity of certain oncolytic viruses (OVs). Therefore, it was hypothesized that use of an OV, such as recombinant Maraba virus (MG1), may be a potential approach to eliminate latently-infected cells constituting the HIV reservoir while sparing HIV-uninfected cells. This was studied in U1, ACH-2, OM-10 and J1.1 cells and their respective HIV-uninfected parent cell lines in addition to CD4+CD25-HLADR- cells from HIV-infected individuals on effective antiretroviral therapy. Although MG1 infected and killed latently HIV-infected U1 cells to a greater degree than the HIV-uninfected parent U937 cells, this was not observed in the other HIV-infected cell lines and their respective parent cell lines. Furthermore, results from primary cells suggest that MG1 alone does not appear to eliminate cells which comprise the major HIV reservoir. Challenges of studying the HIV reservoir and priorities for future studies examining the use of OVs as a potential strategy to eliminate the HIV reservoir are discussed.

Current treatment methods for Acute Leukemia (AL) only provide temporary therapeutic efficacy as most patients will experience relapse within 2 years following first remission. Our lab has determined that vaccination with autologous cells infected with oncolytic virus MG1 can provide durable cures in a pre-clinical mouse model of AL. However, the mechanism(s) by which the infected cell vaccine (ICV) stimulates T cell dependent anti-tumour immunity and provides protection against tumour growth is unknown. This thesis was aimed to determine 1) what antigen presenting cell populations are activated post ICV immunization and 2) what T cell subsets are important in developing anti-tumour immunity during ICV immunization. My thesis has demonstrated that ICV immunization is more effective at inducing in vivo dendritic cell activation compared to irradiated L1210 cells alone and this activation may be a reason as to why we see improved anti-tumour efficacy in our ICV model. In addition, we have determined that CD4 T cells play an essential anti-leukemic role during ICV immunization and that neutralizing antibody production is a CD4 T cell dependent mechanism. Our data also demonstrates that both CD4 and CD8 T cell populations from ICV immunized mice provide a leukemia-specific anti-tumour immune response. Taken together, this data suggests that CD4 T cells may be acting as helper T cells to aid in the robust activation of leukemia-specific anti-tumour CD8 T cells. Our pre-clinical data characterizing the immune response has improved our understanding of the mechanism(s) which contribute to the efficacy of the ICV and will help provide a rationale framework with which to begin translating this treatment to clinical trials.

Similar to cancer cells, HIV-infected cells differ from HIV-uninfected cells in that they have altered interferon signaling pathways, the apparent reason for the selectivity of certain oncolytic viruses (OVs). Therefore, it was hypothesized that use of an OV, such as recombinant Maraba virus (MG1), may be a potential approach to eliminate latently-infected cells constituting the HIV reservoir while sparing HIV-uninfected cells. This was studied in U1, ACH-2, OM-10 and J1.1 cells and their respective HIV-uninfected parent cell lines in addition to CD4+CD25-HLADR- cells from HIV-infected individuals on effective antiretroviral therapy. Although MG1 infected and killed latently HIV-infected U1 cells to a greater degree than the HIV-uninfected parent U937 cells, this was not observed in the other HIV-infected cell lines and their respective parent cell lines. Furthermore, results from primary cells suggest that MG1 alone does not appear to eliminate cells which comprise the major HIV reservoir. Challenges of studying the HIV reservoir and priorities for future studies examining the use of OVs as a potential strategy to eliminate the HIV reservoir are discussed.

Oncolytic viruses (OVs) have been engineered or selected for cancer cell-specific infection; however, we have found that following intravenous administration of vesicular stomatitis virus (VSV), tumour cell killing rapidly extends far beyond the initial sites of infection. This Bystander Effect is due to the virus’ ability to specifically target tumour vasculature through tumour-specific infection of tumour endothelium and the induction of an inflammatory response resulting in tumour-restricted coagulation, acute vascular disruption, apoptosis and necrosis of the tumour core. VSV-infected tumours, reconstructed in three-dimensions from serial histological sections, revealed that the majority of the tumour mass lacks significant blood flow in contrast to uninfected tumours, which exhibit relatively uniform perfusion. VSV infection rapidly induced intravascular coagulation within 6 hours of intravenous administration. The induction of coagulation was dependent on neutrophils and could be prevented with inhibitors of the coagulation pathway. Normal vasculature was not infected by VSV and no increase in coagulation was observed. Vascular collapse was also observed with the oncolytic poxvirus, JX-594, in patients and preclinical models. Biopsies from patients enrolled in a dose escalation trial for JX-594 were immunoreactive for vaccinia antigens and transgene products in high dose cohorts. Tumour-associated vessels from patients treated with JX-594 were infected with JX-594 and expressed virally encoded transgenes. A decrease in blood flow was also observed 5 days post infection. Several viruses, VSV, JX-594, vvDD, Maraba, and Sindbis, were able to rapidly induce widespread bystander cell death in a subset of mouse models. Tumours responded to OV therapy in three ways, and the type of response was determined by two factors - susceptibility to infection and the heterogeneity of the tumour microenvironment. Heterogeneity correlated with E-cadherin expression. Among tumours that supported viral replication, cancers with low E-cadherin expression were susceptible to vascular collapse. E-cadherin positive tumours were susceptible to infection and direct cell killing but resistant to vascular disruption or bystander cell death. If poorly-differentiated tumours were resistant to infection, no acute cell killing was observed. These histological subtypes provide a potential framework for the rational selection of patients, the integration of combination therapies and the creation of designer viruses to improve the success of OV therapy.

Oncolytic viruses (OVs) are a class of cancer therapy that is currently undergoing clinical trials on its way to full regulatory approval. At present, the downstream processing of OVs relies on a combination of chromatography and membrane-based processes to remove process-related (e.g. host-cell proteins and nucleic acids) and product-related impurities (e.g. aggregated virus particles). This thesis explores various methods that can potentially be used to address the challenges associated with downstream processing during the production of OVs. To this end, the Rhabdoviral vector, which is currently undergoing clinical trials (phase I/II) for use in treating advanced or metastatic solid tumors, was selected as a promising oncolytic virus. One potential improvement in the downstream process that was investigated was the use of monolithic column chromatography for Rhabdovirus purification. Two monolithic anion-exchange columns (2 and 6 µm pore size) and one hydrophobic interaction column (6 µm pore size) were used to examine how column pore size affects virus recovery and contaminant removal. This investigation ultimately inspired the development of a purification process based on monolithic hydrophobic interaction column chromatography. Furthermore, this work is also the first to investigate how additives, namely glycerol, impact the hydrophobic interaction chromatography of virus particles. The developed process could be readily implemented for the scaled-up purification of the Rhabdoviral vector. Another challenge associated with the downstream processing of OVs is membrane fouling, which is characterized by a dramatic rise in transmembrane pressure (TMP) and low virus recovery. Indeed, membrane fouling poses a significant challenge, as some recent studies have reported that it can result in viral vector titer losses of over 80%. One critical use of membranes in downstream processing is for the sterile filtration of OVs, which is a required final step that is conducted right before vialing and involves passing the virus particles through a validated sterile filter. One of the main objectives of this thesis was to develop a fundamental understanding of the sterile filtration process and to optimize it in order to achieve higher throughput and lower losses, which are both essential to the large-scale production of OVs. To this end, a dead-end sterile filtration setup was designed, and various commercially available filters were evaluated to examine how membrane morphology affects fouling and product recovery. The results of these tests showed that double-layered composite filters enabled higher virus recovery and filtration capacity compared to single-layered sterile filters. Another cause of membrane fouling is the aggregation of virus particles, which is mediated by various interactions in the solution. To study this, the above-described setup was re-designed to create an effective procedure that utilizes minimal volumes of virus solution, while also enabling the rapid assessment of microscale filtration performance and a comprehensive understanding of virus-virus and virus-membrane interactions. This setup was used to study how different additives, including various proteins (bovine serum albumin and α-lactalbumin) and polymers (polyethylene glycol and polyvinylpyrrolidone), affect the microfiltration of the Rhabdoviral vector and, consequently, the TMP profile. Furthermore, the correlation between the membrane fouling rate (via TMP profiles) and virus recovery was also investigated. This investigation revealed that proteins significantly increase virus transmission and that polymers are incapable of mimicking the effects of the proteins. To explain this phenomenon, a theory based on the biophysical structure of proteins, mainly heterogenicity in charge distribution, was proposed. Moreover, membrane surface modification tests were conducted using bovine serum albumin, with the results indicating that this approach has considerable potential for enhancing virus transmission. Due to the similarities between the test setup and actual downstream processing unit operations, the results from this part of the thesis could be easily and accurately applied to process optimization. / Thesis / Candidate in Philosophy / There is considerable interest in the development of oncolytic viruses for cancer immunotherapy. Indeed, at the time of this thesis’ writing, a Canadian team of researchers is conducting the world’s first clinical trial using a combination of two viruses to kill cancer cells and stimulate an immune response. The process of manufacturing oncolytic viruses is generally divided into two major steps: upstream processing and downstream processing. While upstream processing focuses on virus propagation, downstream processing aims at removing process-related and product-related impurities. However, research into downstream process design and optimization has largely been neglected in favour of a focus on upstream processing, aimed at increasing bioreactor yields and achieving high viral titers. Consequently, downstream processing has become the main bottleneck in virus manufacturing processes, accounting for as much as 70% production costs. This thesis aims to identify and develop a fundamental understanding of the main challenges associated with the downstream processing of oncolytic viruses and to investigate methods for addressing them. Specifically, the present work focuses on the purification and final sterile filtration steps in the manufacturing of oncolytic Rhabdoviral vectors.

Oncolytic viruses can be genetically modified to limit their replication in normal cells rendering them a cancer specific treatment. In addition, they can induce a 'danger signal' in the form of pathogen- and damage-associated molecular patterns leading to anti-tumour immunity. Furthermore, they can be armed with various immunomodulatory molecules to further enhance anti-tumour immunity. In this project I aim to exploit these qualities to develop a translatable cancer vaccine. Virus-infected cancer cells were injected subcutaneously in a prime/boost regimen. Dying cancer cells will release the required danger signal leading to dendritic cell activation and cross-presentation of tumour associated antigens to T cells to elicit an anti-tumour immune response. Our results in the murine pancreatic cancer model showed that vaccination with virusinfected DT6606 cells induced tumour specific immunity capable of protecting vaccinated animals against re-challenge with tumour cells. The highest level of interferon gamma production, a surrogate marker of anti-tumour immunity, was achieved when animals were primed with adenovirus-infected cells. There was no significant difference between various boost groups. To enhance the safety of the proposed protocol a secondary treatment was introduced to arrest the proliferation of tumour cells prior to injection. Our results confirmed that secondary treatment with mitomycin does not affect the induction of tumour specific immunity and it does not affect the release of pathogen-associated molecular patterns in the form of viral proteins and DNA. To test our vaccination regimen in head and neck squamous cell carcinoma (HNSCC) we develop a clinically relevant mouse model using SCC7, B4B8 and LY2 cells to replicate various clinical scenarios including locally advancing disease and post excision locoregional recurrence. Vaccinating mice with HNSCC cells pre-infected with our recently developed tumour-targeted triple-deleted adenovirus (AdTD) resulted in a cell-specific antitumour immune response. In addition, it resulted in an increase in effector memory T-cells of both CD4+ and CD8+ phenotypes. Efficacy studies showed our vaccination can significantly slow down the growth rate of tumours in locally advancing disease. This led to increase survival of the vaccinated mice although it did not reach statistical significance. To further enhance the efficacy of our vaccination regimen, we aimed to increase T cell trafficking to the tumour site. CCL25 is a gut homing chemokine. Priming T cells in the presence of CCL25 will lead to upregulation of the surface expression of α4β7 integrin. The latter is a ligand of MAdCAM-1, a cell adhesion molecule highly expressed in the gut and pancreatic tumours. The α4β7/MAdCAM-1 interaction results in preferential homing of activated T cells to these organs. We hypothesised that vaccinating mice with pancreatic tumour cells pre-infected with a CCL25-armed adenovirus will lead to increased T cell trafficking to pancreatic tumours leading to enhanced efficacy. Although we achieved encouraging results in our pilot experiment, we did not detect any significant increase in α4β7 expression once we added a secondary treatment to the vaccination protocol. Similarly, efficacy experiments in the pancreatic cancer transgenic KPC mice did not show any difference in survival between AdTD-CCL25 and the control virus although both groups showed a trend towards increased survival compared to naïve mice. In conclusion, Virus-infected cancer cell vaccine is a potentially promising immunotherapeutic strategy that can be combined with traditional cancer therapies to increase survival of HNSCC and pancreatic cancer patients.

HIV infection represents a major health and socioeconomic challenge worldwide. Despite significant advances in therapy, a cure for HIV continues to be elusive. The design of novel curative strategies will require targeting and elimination of cells that constitute the latent HIV-1 reservoir. However, such an approach is impeded by the inability to distinguish latently HIV-infected cells from uninfected cells. The type-I interferon (IFN-I) response is an integral antiviral defense mechanism, but is impaired at multiple levels during productive HIV infection. Interestingly, similar global impairments in IFN-I signaling have been observed in various human cancers. This led to the development of IFN-sensitive oncolytic viruses, including the recombinant Vesicular Stomatitis Virus (VSV 51) and Maraba virus (MG1), as virotherapy designed to treat various cancers. Based on this, it was hypothesized that IFN-I signaling is impaired in latently HIV-infected cells (as observed in productively infected cells) and that VSV 51 and MG1 may be able to exploit such intracellular defects to target and eliminate latently HIV-infected cells, while sparing healthy cells. First, using cell line models of HIV-1 latency, intracellular defects in IFN-I responses, including impaired IFN / production and expression of IFNAR1, MHC-I, ISG15, and PKR, were demonstrated to represent an important feature of latently HIV-infected cells. Consistent with this, the latently HIV-infected cell lines were observed to have a greater sensitivity to VSV 51 and MG1 infection, and MG1-mediated killing, than the HIV-uninfected parental cells. Next, the ability of oncolytic viruses to kill latently HIV-infected human primary cells was demonstrated using an in vitro resting CD4+ T cell model of latency. Interestingly, while both VSV 51 and MG1 infection resulted in a significant reduction in inducible p24 expression, a dose-dependent decrease in integrated HIV-1 DNA was only observed following MG1 infection. In keeping with this, MG1 infection of memory CD4+ T cells from HIV-1 infected individuals on HAART also resulted in a significant decrease in inducible HIV-1 gag RNA expression. By targeting an intracellular pathway that is impaired in latently HIV-infected cells, the findings presented in this dissertation highlight a novel, proof-of-concept approach to eliminate the latent HIV-1 reservoir. Given that VSV 51 and MG1 are currently being studied in cancer clinical trials, there is significant potential to translate this work to in vivo studies.

Oncolytic viruses promise to significantly improve current cancer treatments through their tumour-selective replication and multimodal attack against cancer cells. However, one of the biggest setbacks for oncolytic virus therapies is the intravenous delivery of the virus, as it can be cleared by neutralizing antibodies (nAbs) from the bloodstream before it reaches the tumour cells. In our group, we have succeeded in developing aptamers to vesicular stomatitis virus (VSV), as well as to rabbit anti-VSV polyclonal neutralizing antibodies (nAbs). We tested these aptamers’ biological activity with a cell-based plaque forming assay and found that the aptamers prevented in vitro neutralization of VSV by nAbs and increased the virus infection rate of transformed cells up to 77%. In line with this approach, we enhanced the delivery of oncolytic viruses by selecting aptamers to the CT26 colon carcinoma cell line. The binding of aptamer pools has been tested on flow cytometry and the best pools were subjected to high throughput sequencing. Selected aptamers were linked to anti-VSV aptamers and applied for target delivery of the virus to cancer cells. Development of this aptamer-based technology aims to improve viral anti-cancer therapies, with a potential to be applied as treatment for patients affected with cancer. Finally, in collaboration with a group from Erlangen University, we performed an aptamer selection using capillary electrophoresis and cell-SELEX. The target, the extracellular domain of human CD83, is a maturation marker for dendritic cells and is involved in the regulation of the immune system. Selected aptamer sequences bound selectively to mature dendritic cells, in comparison to immature dendritic cells, and thus hold promise to be applied for further studies leading to a better understanding of CD83’s mechanism of action.