Silicification of organisms in silica-depositing environments can impact both their ecology and their presence in the fossil record. Although microbes have been silicified under laboratory and environmental conditions, viruses had not been, prior to this work. Bacteriophage T4 was successfully silicified under laboratory conditions that closely simulated those found in silica-depositing hot springs. Virus morphology was maintained during the short period of silicification (48 hours), and a clear elemental signature of silicon and phosphorus was detected by energy-dispersive X-ray spectrophotometry (EDX). However, the EDX signature of silicified virus was not sufficiently distinct from that of cell membrane or phosphate minerals for that technique to be used to discover viral remains in hot spring mineral deposits. Having shown that bacteriophage T4 can be silicified, it was then determined that the impact of silica exposure on infectivity varied widely between different viruses. The effect on infectivity did not appear to be related to virus size or morphology. In addition, the impact on infectivity was at least partially reversible, indicating that it was caused, at least in part, by occluding infection-related structures on the virus, rather than destruction or denaturation of the virus. Those viruses which showed a decline in infectivity with silica exposure also showed increased resistance to desiccation after being exposed to silica, which has implication for long-range virus dispersal. The desiccation resistance was proportional to the degree that silicification reduced infectivity in that virus. Desiccation resistance also declined with prolonged exposure to drying, suggesting that the mechanism was due to the silica coating helping to retain water rather than replacing the hydrogen bonding of water. Virus dispersal is critical for both the spread of disease and the diverse roles that viruses play in Earth ecology. However, the mechanisms of host-independent virus dispersal are poorly understood and hotly debated. These experiments showed that, under mild conditions, diverse viruses can be coated in silica and that silica coating provides some, if not most, viruses with remarkable desiccation tolerance. Virus silicification thus provides a potential mechanism for global dispersal of viruses that could not otherwise tolerate the desiccation of wind-borne transportation.

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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.

Conditional lethal mutants of poliovirus type 1 (Mahoney) were generated by treatment with the mutagen hydroxylamine. Temperature-sensitive mutants were selected by the replica plating technique at temperatures of 33°C (permissive) and 39°C (restrictive). New mutants were generated to achieve a larger population of mutants and also to generate additional RNA- mutants in this population. These mutants were characterized by two criteria: RNA synthesis and thermal stability. RNA synthesis is measured by the accumulation of labeled uridine incorporation into trichloroacetic acid (TCA) insoluble material. The thermal stability is determined by the difference in plaque forming units before and after treatment of the virion at 45°C. Complementation co-infections (5 MOI for each virus stock) were analyzed for the presence of the 150S virion particle of poliovirus after sedimentation through a linear sucrose gradient. Complementation is observed between RNA(+) mutants v.s. RNA(-) mutants, and between two RNA(-) mutants, but not between two RNA(+) mutants. Although reciprocal complementation has not been documented in this study some speculation on complementation is presented in this thesis.

published_or_final_version / Pathology / Master / Master of Philosophy

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