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Lyophilization maximises vaccines’ stability, shelf life; maintains chemical or biological functions; enables easier transportation and storage compared to a cold chain. Success of vesicular stomatitis virus (VSV) based vaccines has been highlighted by its increased adoption by pharmaceutical companies and most importantly, the recently approved vaccine for high consequence Ebola virus (EBOV). Lujo virus (LUJV) and Lassa virus (LASV), both arenaviruses, are still without licensed vaccines, however, researchers are exploring VSV-vectored vaccines. Many of these highly pathogenic viruses are endemic in tropical regions like Africa where slight breach in cold chain may affect stability of vaccines. It was established that slight changes in cold chain conditions do not affect efficacy of VSV-based vaccines, however, it is imperative to optimise thermostability of VSV-based vaccines using lyophilization. The primary hypothesis of this study is that lyophilized VSV-LASV and VSV-EBOV vaccines exposed to sub-optimal temperature will retain infectivity in cell culture and protect against LASV challenge in inbred Hartley guinea pigs. Different combinations of commercially available stabilisers were used to assess the thermostability of VSV-based vaccines at 4, 21 and 37 °C of storage temperatures. Lyophilized VSV-LASV and VSV-EBOV were recoverable in Vero cells, the recovery titer showed an inverse relationship with exposed temperature and duration of storage of 1, 7, 35 and 90 days. Furthermore, lyophilized VSV-LASV affords 100% protection and sterilising immunity against LASV infection in inbred Hartley guinea pigs.

Blastomycosis is a pulmonary disease caused by Blastomyces dermatitidis, a dimorphic fungus endemic to Manitoba and northwestern Ontario. Immunosuppression is a major risk factor affecting disease susceptibility, yet host immunity is not well understood. Genetic immunodeficiencies can also influence disease, with variants in IL6, GATA2, and VDBP shown to influence susceptibility. However, additional genetic factors in disease susceptibility and severity remain undetected. Our study seeks to establish what is known about susceptibility to blastomycosis and explore genetic risk factors in a case-control cohort. We conducted a scoping review to establish current knowledge on blastomycosis immunity and a literature review to identify candidate genes influencing susceptibility to fungal and mycobacterial infections. Exomes from 18 blastomycosis cases and 9 controls were sequenced, variants were identified, and filtered according to best practices. We performed candidate gene prioritization and variant aggregation to identify genetic associations and explored the full exome dataset. The scoping review included 58 articles on susceptibility to blastomycosis. TNF-a, GM-CSF, CD4+ deficiency, and the IL-12-IFN-y pathway had the most evidence as susceptibility factors. The literature review identified 86 candidate genes relevant to fungal and mycobacterial infections. One hundred and three genetic variants in 42 candidate genes were identified in the exome dataset. No variants associated with susceptibility were identified in a single-variant analysis although two non-synonymous variants in TYK2 were enriched among cases suggesting a possible role in susceptibility. Gene-based association analysis found TLR1 and GATA2 enriched in controls (p = 0.024 and 0.051, respectively) suggesting a possible protective effect, although GATA2 has previously been associated with blastomycosis susceptibility. Gene cluster analysis identified genetic variants in genes of chromatin remodeling

Prions are infectious proteins that cause rare but invariably fatal neurodegenerative diseases in humans and other mammals. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common form of human prion disease, with an annual prevalence of 1-2 cases per million individuals. Though extremely rare, sCJD is devastating. This disease is rapid, with most patients dying within 5 months from the onset of symptoms and no available treatments. sCJD is heterogeneous, with diverse clinical signs, brain lesion profiles, and disease durations. It is believed this variability is attributed to different prion strains, which are influenced by host genetics and the shape of the misfolded protein. Strain diversity is poorly understood and remains to be explored experimentally. For these reasons, we have developed methodologies to exploit strain-specific properties and applied them in a retrospective analysis of 31 sCJD cases in Canada. We characterized the biochemical properties of the pathogenic prion proteins from these cases using thermal denaturation assays, capillary-electrophoresis immunoassays, and protein seeding kinetic assays. Atypical cases were inoculated into a novel animal model to analyze strain-specific properties such as incubation periods, clinical signs, and brain lesions. Within our cohort of 31 sCJD patients, we identified two atypical sCJD cases and two cases of a rare strain of sCJD called variably-protease sensitive prionopathy. We propose the two atypical sCJD cases represent novel strains of sCJD in Canada. Additionally, we have developed a sCJD strain baseline through which atypical cases could readily be identified in the future. Understanding prion strains has implications for the detection of zoonotic transmission as well as for the development of therapeutics.

Prion diseases are rare neurodegenerative disorders that are deadly, incurable, and caused by misfolded prion proteins (PrPSc) in the brain leading to neuropathological changes and rapid cognitive decline. The paucity of current approaches to prion disease research has hampered progress and so here we address two technical challenges that have limited research tools. Firstly, human prion isolates are incompatible with most cellular prion infection models, limiting their clinical relevance. Secondly, genome-wide transcriptional studies of bulk brain tissue cannot resolve the contribution of different brain cell types to prion pathogenesis. This makes it difficult to understand the relationship between prion accumulation, pathogenesis, and neurotoxicity. In the first part of this thesis we set out to develop novel in vitro cellular models of human prion infection using two approaches: (a) the prion organotypic slice culture assay (POSCA) and (b) an immortalized human neural progenitor cell line (ReN). In these two experimental paradigms, prion replication was sensitively tracked over several weeks through measurements of amyloid seeding activity with real time quaking induced conversion (RT-QuIC). Deer mouse cerebellar slice cultures and PrPC-overexpressing ReN cultures were found to resist infection with human prion isolates. While we were unable to establish an in vitro model of human prion infection, our findings reflect the inherent difficulty of establishing clinically relevant cellular models. In the second part of this thesis, we compared prion-elicited transcriptional responses between mouse brain cell types to shed further insight into prion neuropathology. Using bulk RNA sequencing, gene expression was examined longitudinally in microdissected brain tissues from an in vivo mouse model of RML Scrapie infection. We then applied single-cell RNA sequencing to brain cells isolated at the clinical endpoint of RML Scrapie infection. A single-cell atlas of nearly 15

At the onset of the COVID-19 pandemic, researchers around the globe joined forces to study the evolutionary biology of SARS-CoV-2 and identify approaches to minimize its spread in the population. Whole genome sequencing (WGS) quickly became the gold standard for monitoring SARS-CoV-2 viral evolution and how it may impact disease severity, transmission, and vaccine efficacy. As a result, researchers demonstrated concerted efforts to develop, optimize, and validate methods for WGS of the novel virus. This research herein optimized wet-lab sequencing protocols developed by the international research group using reverse transcription PCR-tiling and nanopore sequencing technologies to sequence the whole genome of SARS-CoV-2. As a part of the Canadian COVID Genomics Network (CanCOGeN), the work presented in this thesis outlines materials, methods, and results that contributed to the optimization and validation of a Canadian-specific SARS-CoV-2 WGS approach. To achieve this goal, the investigation included identifying the most economical reverse transcriptase, DNA polymerase, PCR primer schemes, library preparation conditions, and comparison of Nanopore and Illumina sequencing platforms. The optimized WGS protocol was shared with the CanCOGeN partners across Canada to increase Canada’s SARS-CoV-2 sequencing capacity towards a sustainable national genomic surveillance program. Throughout this project, the WGS protocol was validated to ensure that emerging variants of concern were detectable. In summary, the current research contributed to operationalizing the first national genomic surveillance program for viral pathogens in Canada. The developed protocol remains in use across Canadian partners and contributes data to support evidence-based decisions for implementing public health measures. Thanks to our work through the CanCOGeN project and its network of expertise, Canada is well-positioned and prepared to perform genomic surveillance of emerging and re-emerging pathogens

Influenza A virus (IAV) alters the expression of many host cellular proteins in the infected cells; many of these proteins interact with fibronectin. Proteasome subunit alpha type-2 (PSMA2), Chloride intracellular channel protein 1(CLIC1) and Heat Shock Protein Family A (Hsp70) member 5 (HSPA5) are three fibronectin interacting proteins that were highly expressed in IAV-infected cells. PSMA2 is part of the 20S proteasome complex, involved in proteolytic modification and recycling of cellular proteins. CLIC1 is a chloride channel that regulates intracellular pH, cell volume and trans-epithelial ion transport. HSPA5 helps to translocate nascent proteins to endoplasmic reticulum and subsequent folding. In this study, I have investigated the importance of PSMA2, CLIC1 and HSPA5 in IAV replication cycle. The knockdown (KD) of PSMA2, CLIC1 and HSPA5 in A549 cells, caused a significant reduction in extracellular progeny IAV. Although CLIC1 KD did not affect viral protein translation, IAV-nucleoprotein (NP) was accumulated in HSPA5 KD and PSMA2 KD cells. Viral RNAs were significantly higher in CLIC1 KD cells after IAV infection, but transcription was unaffected in PSMA2 KD and HSPA5 KD cells. The results indicate that PSMA2, HSPA5 and CLIC1 are critical host factors for IAV and are possibly involve in the terminal stages of viral replication. Further proteomic analysis to understand the role of PSMA2 KD in IAV infection at the proteomic level revealed that NRF2-mediated oxidative stress response signaling, was inhibited by IAV infection but was significantly activated by PSMA2 KD. Reactive oxygen species (ROS) level was significantly inhibited in wild-type cells but significantly increased in PSMA2 KD cells after IAV infection. However, IAV infection also caused significantly higher nuclear translocation of NFR2, which was inhibited in the PSMA2 KD cells. Treatment with ROS scavenger was able to reduce the inhibitory impact of PSMA2 KD. This indicates that PSMA2 is required