abstract: This work advances structural and biophysical studies of three proteins important in disease. First protein of interest is the Francisella tularensis outer membrane protein A (FopA), which is a virulence determinant of tularemia. This work describes recombinant expression in Escherichia coli and successful purification of membrane translocated FopA. The purified protein was dimeric as shown by native polyacrylamide gel electrophoresis and small angle X-ray scattering (SAXS) analysis, with an abundance of β-strands based on circular dichroism spectroscopy. SAXS data supports the presence of a pore. Furthermore, protein crystals of membrane translocated FopA were obtained with preliminary X-ray diffraction data. The identified crystallization condition provides the means towards FopA structure determination; a valuable tool for structure-based design of anti-tularemia therapeutics. Next, the nonstructural protein μNS of avian reoviruses was investigated using in vivo crystallization and serial femtosecond X-ray crystallography. Avian reoviruses infect poultry flocks causing significant economic losses. μNS is crucial in viral factory formation facilitating viral replication within host cells. Thus, structure-based targeting of μNS has the potential to disrupt intracellular viral propagation. Towards this goal, crystals of EGFP-tagged μNS (EGFP-μNS (448-605)) were produced in insect cells. The crystals diffracted to 4.5 Å at X-ray free electron lasers using viscous jets as crystal delivery methods and initial electron density maps were obtained. The resolution reported here is the highest described to date for μNS, which lays the foundation towards its structure determination. Finally, structural, and functional studies of human Threonine aspartase 1 (Taspase1) were performed. Taspase1 is overexpressed in many liquid and solid malignancies. In the present study, using strategic circular permutations and X-ray crystallography, structure of catalytically active Taspase1 was resolved. The structure reveals the conformation of a 50 residues long fragment preceding the active side residue (Thr234), which has not been structurally characterized previously. This fragment adopted a straight helical conformation in contrast to previous predictions. Functional studies revealed that the long helix is essential for proteolytic activity in addition to the active site nucleophilic residue (Thr234) mediated proteolysis. Together, these findings enable a new approach for designing anti-cancer drugs by targeting the long helical fragment. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

Purpose: The objective of this thesis is to evaluate clinical studies that have used oncolytic viruses as treatment and to compare their treatment-outcomes on patients with malignant glioma. Method: This thesis is a systematic literature review where PubMed has been used as the database for data collection. Two searches were done using the search phrases oncolytic virus AND Glioma and oncolytic virus AND brain tumor. Several of the articles showed up multiple times in different searches. After having applied the inclusion criteria, ten of the seventeen articles were removed. Remaining were seven articles used for the thesis. Results: The study conducted by Forsythe et al., using reovirus showed the median overall survival (OS) to be 21 weeks and the median time to progression (TTP) was 4.3 weeks. The study conducted by Kicielinski et al., using REOLYSIN showed the median OS to be 140 days. Median TTP was 61 days. The study conducted by Geletneky et al., 2017 was the first dose-escalating clinical trial for the use of H-1 parvovirus. The median TTP was 111 days and the median OS was 464 days. The study conducted by Lang et al., DNX-2401 was used and in group A the median OS time was 9.5 months. In group B the median OS in the group was 13 months. In another example of an oncolytic adenovirus is ONYX-015, the median TTP after treatment for all patients was 46 days. The median OS for patients diagnosed with glioblastoma multiforme was 4.9 months and for patients with anaplastic astrocytoma and anaplastic oligodendroglioma was 11.3 months across. In a study conducted by Freeman et al. using newcastle disease virus, the OS ranged from 3-66 weeks from the start of treatment and TTP ranged from 2-53 weeks. The study conducted by Markert et al., the median OS from treatment with G207 was 7.5 months. The median TTP was around 2.5 months. Conclusion: Oncolytic viruses are promising agents for treatment against malignant gliomas. No definite outcomes of the treatment could be concluded, however, the median survival was extended in certain cases. The patients tolerated the oncolytic viruses well with no adverse effects correlated with the treatments. There are currently more virus vectors being tested as new developments are needed in this field.

In the past several years, cancer treatments using FDA approved immune checkpoint inhibitors have become more popular than common chemotherapeutic agents. However, the costs, risks, and benefits associated with these treatments are still being assessed. Currently, new therapies are being tested that utilize oncolytic viruses to treat solid tumor and metastatic cancers. Reovirus is a non-enveloped virus with a double capsid structure and a genome consisting of 10 segments of double-stranded RNA encoding eleven proteins, which has been shown to have effective oncolytic activity. There are various different strains of reoviruses that can produce cytopathic effects in mammalian host cells. Moreover, several studies have shown that reovirus can be administered in multiple ways and that administration may depend on the cancer type. This investigation examined the type 3 dearing strain of reovirus and whether different types of tumors would benefit from having a specific administration appropriate to their type such as intratumoral injections for solid tumors and intravenous administration for blood/metastatic cancers. Various clinical trials were assessed in which reovirus was administered intratumorally, intravenously at a maximum dose of 3x1010 TCID50, and in combinations with other cancer therapeutics. Reovirus was shown to be safe and well-tolerated across a variety of administrations and cancer morphologies. Moreover, along with its cytopathic effects, reovirus was shown to have potent immune system stimulating effects. Overall, intratumoral administration was preferred effective for solid tumor cancers while intravenous administration was preferred for blood and metastatic cancers.

No description available.

Respiratory virus infections are associated with asthma pathogenesis and exacerbations in children and adults. Unfortunately, it remains largely unknown whether innate and adaptive T cell anti-viral immunity differs in allergic disease versus health. Here, we established a short-term primary cell culture system using human peripheral blood mononuclear cells (PBMC) optimized for measuring immune responses to reovirus, respiratory syncytial virus (RSV) and metapneumovirus (MPV) based on virus-specific cytokine and chemokine production. The prevalence and intensity of innate and adaptive responses in children and adult populations was addressed. Using this in vitro model of human anti-viral immunity, we tested our global hypothesis that asthmatics mount anti-viral cytokine responses to respiratory viruses that differ from those of healthy individuals. MPV and RSV, although both ubiquitous and leading to very high levels of infection, seroconversion and clinically similar presentation in the population, evoke distinct innate and adaptive T cell-dependent cytokine responses. Reovirus induced exceptionally strong IFN recall responses concomitant with intense IL-10 production, which were independent of viral replication in PBMC. Surprisingly, despite Type 1 cytokine production dominated adaptive immune responses in both asthmatic and non-asthmatic individuals, asthmatics exhibited significantly stronger pro-inflammatory IFNγ and IL-10 production towards virus stimulation than non-asthmatic children and adults. Moreover, children with current AHR, regardless of asthmatic status, exhibit a greater frequency and intensity of IFNγ responses towards pneumoviruses than do non-AHR counterparts. Conversely, expression of chemokine CCL5 was substantially weaker in asthmatics, and was further decreased in children with AHR and familial history of asthma. This pattern of enhanced pro-inflammatory and deficient anti-viral CCL5 responses towards pneumoviruses in children with markers of symptomatic asthma or AHR may underlie the enhanced sensitivity of these children to experience breathing difficulties following infection with respiratory viruses. Furthermore, we have clearly demonstrated a gene by environment interaction, whereby ETS exposure in children with familial asthma results in suppressed anti-viral IFNγ and IL-10 production. Therefore, we have attempted to determine whether genetic variation affects the intermediate phenotype of anti-viral immunity, in the population and dependent on clinical status. In summary, we have demonstrated that asthma, childhood exposures and genetics shape anti-viral cytokine responses in human. These findings have a substantial impact for physicians deciding the contextually appropriate treatment for asthma symptoms in their patients and could have implications for experimentation relating to mechanisms of disease, clinical practice and development of appropriate therapeutics. / October 2007

Viruses are obligate parasites that use the host cellular machinery to produce progeny virions. The host responds to this invading pathogen by induction of the immune system; however, the virus employs a variety of strategies to overcome these attacks. The complexity of the virus-host interaction is of great interest to researchers with aims to both characterize the relationship and target steps of the viral life cycle to hinder infection. Many targeted tactics employ single protein analysis; however, approaches that examine the whole set of virus/host interactions are available. Transcriptional alterations within host cells have been determined for many virus- host interactions by micro-array techniques; however little is known about the effects on cellular proteins. This study uses a quantitative mass spectrometric-based method, SILAC, to study differences in a host cell's proteome with infection by a virus. Mammalian reoviruses and herpes simplex viruses are prototypical viruses commonly studied to determine virus life cycle and interactions with hosts. Using three strains of reoviruses and one HSV1 strain, cells were infected to identify differentially regulated proteins at different times. Thousands of proteins were identified for each virus type, some up or down regulated after infection. Biological functions and network analyses were performed using online networking tools. These pathway analyses indicated numerous processes including cell death and inflammatory response are affected by T1L reovirus infection. Comparing reovirus strains revealed a greater overall proteomic change in host function when infected with the more pathogenic T3DC strain. For the HSV infection, host proteins altered during the different immediate early, earlyand late phases of infection helped characterize the host-virus interaction parallel to the virus life cycle. Overall, my study has characterized proteomic changes in different virus infection systems, identifying numerous novel cellular functional pathways and specific proteins altered during virus infections, specifically the secretogranin II protein that had opposite types of regulation in reoviruses and HSV and was examined for its effects on virus replication. Further studies on the novel proteomic characteristics may provide greater understanding to the complex virus-host interactome, leading to possible antiviral targets. / October 2015

Réovirus, connu sous le nom REOLYSIN®, est présentement à l'étude à titre d'agent oncolytique. Or, la spécificité du virus pour les cellules cancéreuses pourrait être optimisée par une modification au niveau de la protéine d'attachement σ1. La présente étude vise à démontrer qu'une telle amélioration est possible par l'utilisation de la méthode nouvellement décrite de génétique inverse. Par cette technique, il est possible d'ajouter un polypeptide d'une longueur de quarante acides aminés à l'extrémité C-terminale de σ1. Il est aussi possible d'engendrer des virus mutés en leur site d'activité mucinolytique. Les virus nouvellement créés démontrent une efficacité de réplication diminuée, mais demeurent infectieux. Contrairement aux méthodes traditionnellement utilisées avec réovirus, la méthode de génétique inverse permet de conserver les mutations engendrées, par substitution ou addition, au cours des cycles de réplication. Une telle étude démontre qu'il serait possible de modifier le tropisme de réovirus. / Reovirus, also known under the tradename REOLYSIN®, is currently under clinical trial as an oncolytic agent. The specificity of the virus for transformed (cancerous) cells could be improved by modifications of the σ1 attachment protein. This study presents two ways to achieve this improvement by using a newly developed method of reverse genetics for double-stranded RNA viruses. As a proof of concept, we engineered the addition of an exogenous polypeptide of forty amino-acids to the C-terminal end of σ1. Subsequently, we created viruses with compromised mucinolytic activities by introducing amino-acids substitutions in the appropriate genetic sites. A decreased replicative efficacy was observed in all experimentally generated viruses, despite maintaining their infectious capacity. Reverse genetics allows the generation of viruses that retain their mutations throughout successive replicative cycles, which was impossible with traditionnal techniques. This project demonstrates the feasibility of tropism modification by using the reverse genetics method.

Respiratory virus infections are associated with asthma pathogenesis and exacerbations in children and adults. Unfortunately, it remains largely unknown whether innate and adaptive T cell anti-viral immunity differs in allergic disease versus health. Here, we established a short-term primary cell culture system using human peripheral blood mononuclear cells (PBMC) optimized for measuring immune responses to reovirus, respiratory syncytial virus (RSV) and metapneumovirus (MPV) based on virus-specific cytokine and chemokine production. The prevalence and intensity of innate and adaptive responses in children and adult populations was addressed. Using this in vitro model of human anti-viral immunity, we tested our global hypothesis that asthmatics mount anti-viral cytokine responses to respiratory viruses that differ from those of healthy individuals. MPV and RSV, although both ubiquitous and leading to very high levels of infection, seroconversion and clinically similar presentation in the population, evoke distinct innate and adaptive T cell-dependent cytokine responses. Reovirus induced exceptionally strong IFN recall responses concomitant with intense IL-10 production, which were independent of viral replication in PBMC. Surprisingly, despite Type 1 cytokine production dominated adaptive immune responses in both asthmatic and non-asthmatic individuals, asthmatics exhibited significantly stronger pro-inflammatory IFNγ and IL-10 production towards virus stimulation than non-asthmatic children and adults. Moreover, children with current AHR, regardless of asthmatic status, exhibit a greater frequency and intensity of IFNγ responses towards pneumoviruses than do non-AHR counterparts. Conversely, expression of chemokine CCL5 was substantially weaker in asthmatics, and was further decreased in children with AHR and familial history of asthma. This pattern of enhanced pro-inflammatory and deficient anti-viral CCL5 responses towards pneumoviruses in children with markers of symptomatic asthma or AHR may underlie the enhanced sensitivity of these children to experience breathing difficulties following infection with respiratory viruses. Furthermore, we have clearly demonstrated a gene by environment interaction, whereby ETS exposure in children with familial asthma results in suppressed anti-viral IFNγ and IL-10 production. Therefore, we have attempted to determine whether genetic variation affects the intermediate phenotype of anti-viral immunity, in the population and dependent on clinical status. In summary, we have demonstrated that asthma, childhood exposures and genetics shape anti-viral cytokine responses in human. These findings have a substantial impact for physicians deciding the contextually appropriate treatment for asthma symptoms in their patients and could have implications for experimentation relating to mechanisms of disease, clinical practice and development of appropriate therapeutics.

Reovirus is internalized after interaction of the outer proteins μ1, σ1 and σ3 with the host cell. Proteolysis of σ3 and cleavage of μ1 (into δ and φ) eventually leads to the formation of a more infectious subviral particle named “ISVP”. The infectious entry of viruses, but not of ISVPs, can be blocked using various entry inhibitors and therefore, suggests that there is a threshold of σ3 digestion required to allow particle to bypass entry blockers. By combining protease and detergent to the digestion of virions, data from this work showed distinct particles generated along the transition pathway. In addition, studies involving flow cytometry and specific antibodies (anti-μ1) showed that between virus and ISVP there is a gradual yet heterogeneous particle proteolysis that is directly related to the virus infectivity. The findings and approaches taken for this thesis work can possibly be extended for studying other non-enveloped viruses. Moreover, it may help to shed some light on the development of safe and effective oncolytic agents.

Respiratory virus infections are associated with asthma pathogenesis and exacerbations in children and adults. Unfortunately, it remains largely unknown whether innate and adaptive T cell anti-viral immunity differs in allergic disease versus health. Here, we established a short-term primary cell culture system using human peripheral blood mononuclear cells (PBMC) optimized for measuring immune responses to reovirus, respiratory syncytial virus (RSV) and metapneumovirus (MPV) based on virus-specific cytokine and chemokine production. The prevalence and intensity of innate and adaptive responses in children and adult populations was addressed. Using this in vitro model of human anti-viral immunity, we tested our global hypothesis that asthmatics mount anti-viral cytokine responses to respiratory viruses that differ from those of healthy individuals. MPV and RSV, although both ubiquitous and leading to very high levels of infection, seroconversion and clinically similar presentation in the population, evoke distinct innate and adaptive T cell-dependent cytokine responses. Reovirus induced exceptionally strong IFN recall responses concomitant with intense IL-10 production, which were independent of viral replication in PBMC. Surprisingly, despite Type 1 cytokine production dominated adaptive immune responses in both asthmatic and non-asthmatic individuals, asthmatics exhibited significantly stronger pro-inflammatory IFNγ and IL-10 production towards virus stimulation than non-asthmatic children and adults. Moreover, children with current AHR, regardless of asthmatic status, exhibit a greater frequency and intensity of IFNγ responses towards pneumoviruses than do non-AHR counterparts. Conversely, expression of chemokine CCL5 was substantially weaker in asthmatics, and was further decreased in children with AHR and familial history of asthma. This pattern of enhanced pro-inflammatory and deficient anti-viral CCL5 responses towards pneumoviruses in children with markers of symptomatic asthma or AHR may underlie the enhanced sensitivity of these children to experience breathing difficulties following infection with respiratory viruses. Furthermore, we have clearly demonstrated a gene by environment interaction, whereby ETS exposure in children with familial asthma results in suppressed anti-viral IFNγ and IL-10 production. Therefore, we have attempted to determine whether genetic variation affects the intermediate phenotype of anti-viral immunity, in the population and dependent on clinical status. In summary, we have demonstrated that asthma, childhood exposures and genetics shape anti-viral cytokine responses in human. These findings have a substantial impact for physicians deciding the contextually appropriate treatment for asthma symptoms in their patients and could have implications for experimentation relating to mechanisms of disease, clinical practice and development of appropriate therapeutics.