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<p>Coronaviruses are the largest known RNA viruses and infect a wide range of hosts. Human coronaviruses traditionally have been known to be the cause of the common cold and have been vastly understudied due to low morbidity and mortality. The emergence of SARS-CoV and MERS-CoV has altered the landscape of coronavirus research and proven the deadly capabilities of human coronaviruses. With two recent zoonotic events, it is increasingly important to understand the molecular biology of human coronaviruses. The coronavirus nucleocapsid protein is an essential structural protein that complexes with the viral genome. Though nucleocapsid formation is the protein’s major role, it has also been found to have other functions and effects during infection. The following research aimed to examine how the human coronavirus nucleocapsid protein affects the innate immune response <em>in vitro</em>. Modulation of the type I interferon response by the nucleocapsid was first investigated and the nucleocapsids were shown to have the ability to block interferon signalling. Additionally, the nucleocapsid protein was found to cause a dysregulation of transcription factor NFKB1. We propose a novel mechanism of this NFKB1 negative regulation interference. Taken together, we have further characterized the significant role of the coronavirus nucleocapsid protein in innate immune evasion.</p> / Doctor of Philosophy (Medical Science)

Background: Life-prolonging tumour removal surgery is associated with increased metastasis and disease recurrence. Natural Killer (NK) cells are critical for the anti-tumour immune response. Postoperatively, NK cell cytotoxicity and interferon-gamma (IFNγ) production are profoundly suppressed and this dysfunction has been linked to increased metastases/poor patient outcomes. NK cell activity depends on the integration of signals through receptors and can be modulated by soluble factors, including transforming growth factor- beta (TGFβ). The postoperative period is characterized by the expansion of myeloid-derived suppressor cells (sxMDSCs), which inhibit NK cell effector functions. I hypothesize that impaired NK cell IFNγ production is due to altered signaling pathways caused by sxMDSC-derived TGFβ. Methods: Postoperative changes in NK cell receptor expression, receptor-dependent phosphorylation of downstream targets, and rIL-2/12-stimulated IFNγ production were assessed using newly developed whole blood assays utilizing peripheral blood samples from cancer surgery patients. Isolated healthy NK cells were incubated in the presence of healthy/baseline/postoperative day (POD) 1 plasma or isolated sxMDSCs and NK cell phenotype and function were assessed. NK cells were also cultured with plasma in the presence/absence of a TGFβ blocking monoclonal antibody (mAb) or a TGFβ RI small molecule inhibitor (smi). Single-cell RNA-sequencing was performed on six colorectal cancer surgery patients at baseline and on POD1. S6 phosphorylation was used as a proxy for mammalian target of rapamycin complex (mTORC) 1 activity to investigate the mechanism of TGFβ-mediated NK cell dysfunction. Results: Intracellular NK cell IFNγ, activating receptors CD132 (IL-2R), CD212 (IL-12R), NKG2D, and DNAM-1, and the phosphorylation of downstream targets STAT5, STAT4, p38 MAPK, and S6 were significantly reduced on POD1. TGFβ was increased in patient plasma on POD1. The dysfunctional phenotype could be phenocopied in healthy NK cells through the addition of rTGFβ1 or by incubation with POD1 plasma. This dysfunctional phenotype could be prevented with the addition of an anti-TGFβ mAb or a TGFβ RI smi in culture. RNA-sequencing revealed a reduction in transcripts associated with mTOR effector functions, suggesting an impairment in mTOR. S6 phosphorylation was maintained with the addition of TGFβ-specific therapies. The hyporesponsive NK cell phenotype was reproduced upon culture of healthy NK cells with sxMDSCs and sxMDSCs were shown to produce soluble TGFβ in culture. Conclusion: Surgically stressed NK cells display a dysfunctional phenotype, which could be prevented in vitro through the addition of TGFβ-specific blocking therapies. sxMDSCs produced TGFβ and co- incubation induced dysfunction in healthy NK cells. The recovery of impaired S6 phosphorylation with TGFβ-specific therapies suggests that TGFβ is inducing NK cell dysfunction via inhibition of mTORC1 activity. The perioperative period of immunosuppression presents a window of opportunity for novel therapeutics to prevent metastases and cancer recurrence among cancer surgery patients.

Evidence suggests that the neurotropic parasite Toxoplasma gondii may play a role in the development of cognitive impairments. My hypothesis was that congenital exposure to T. gondii would lead to detectable age and sex related differences in behavior and neurotransmitter levels in mice. The neurotransmitter dopamine and commonly used anti-schizophrenic agents were evaluated against T. gondii in human fibroblast cells. Dopamine caused a significant increase in tachyzoite numbers at 250 nM but not 100 nM and the drugs valproic acid, fluphenazine, thioridazine and trifluoperazine inhibited T. gondii development. The effects T. gondii infection had on behavior were examined using a congenital mouse model. Previous work demonstrated maternal immune stimulation (MIS) with interferon gamma (INF-g) resulted in decreased fetal mortality from congenital T. gondii infections; therefore I examined the effects of INF- g treatment of mothers to determine if protection from the behavioral effects of T. gondii occurred in their offspring. No differences in concentrations of neurotransmitters in the brains of congenitally infected mice were observed. I found that mice infected with T. gondii developed adult onset behavior impairments with decreased rate of learning, increased activity and decreased memory, indicating cognitive impairment for male mice and not female mice. My findings support the evidence T. gondii is a factor in the development of cognitive impairments. My results for T. gondii exposed male mice are consistent with the convention that males have more cognitive impairments in the prodromal stage of schizophrenia. MIS with IFN-g had a minimal effect on behavior post sexual maturity but had a greater effect on pre sexual maturity female mice which exhibited difficulties with spatial memory, coordination and the ability to process stimuli. The results indicate the behavior alterations from IFN- g are transient. When MIS is given prior to congenital infection with T. gondii, we detected no behavior deficits in any group of mice, including male mice post sexual maturity. Based on the results of my study, I must reject the hypothesis that neurotransmitter levels are influenced by congenital toxoplasmosis and accept the hypothesis that congenital T. gondii infection caused cognitive impairments in male mice post sexual maturity. / Ph. D.

No / 1 In this study we examined the signalling events that regulate lipopolysaccharide (LPS)-stimulated induction of interferon regulatory factor (IRF)-1 in human umbilical vein endothelial cells (HUVECS). 2 LPS stimulated a time- and concentration-dependent increase in IRF-1 protein expression, an effect that was mimicked by the cytokine, tumour necrosis factor (TNF)-¿. 3 LPS stimulated a rapid increase in nuclear factor kappa B (NFKB) DNA-binding activity. Preincubation with the NFKB pathway inhibitors, N-¿-tosyl-L-lysine chloromethyl ketone (TLCK) or pyrrolidine dithiocarbamate (PDTC), or infection with adenovirus encoding IKB¿, blocked both IRF-1 induction and NFKB DNA-binding activity. 4 LPS and TNF¿ also stimulated a rapid activation of gamma interferon activation site/gamma interferon activation factor (GAS/GAF) DNA-binding in HUVECs. Preincubation with the Janus kinase (JAK)-2 inhibitor, AG490 blocked LPS-stimulated IRF-I induction but did not affect GAS/ GAF DNA-binding. 5 Preincubation with TLCK, PDTC or infection with I¿Ba adenovirus abolished LPS-stimulated GAS/GAF DNA-binding. 6 Incubation of nuclear extracts with antibodies to RelA/p50 supershifted GAS/GAF DNA-binding demonstrating the involvement of NF¿B isoforms in the formation of the GAS/GAF complex. 7 These studies show that NF¿B plays an important role in the regulation of IRF-1 induction in HUVECs. This is in part due to the interaction of NF¿B isoforms with the GAS/GAF complex either directly or via an intermediate protein.

Cancer and hepatitis viruses are two of the leading causes of death worldwide. Recombinant Interferon alpha2b (IFNa2b) is used as an immunotherapeutic drug for cancers, hepatitis viruses and several other viral diseases. Interferons are produced in low quantity naturally and production cost for recombinant IFNa2b in E.coli is very high. Since prokaryotes cannot form disulfide bonds, additional techniques have to be employed to create a functional form of IFNa2b. The average cost per patient for treatment with recombinant IFNa2b is $26,000 per year. Around 800 million people in the world are infected with Hepatitis C virus and most of them cannot afford the treatment costs. Producing recombinant IFNa2b in tobacco chloroplasts will overcome these problems and make the drug affordable for many people. A recombinant IFNa2b chloroplast vector was introduced into the tobacco cultivars Petit Havana (model) and LAMD-609 (low nicotine hybrid plants) in the Daqjell lab by particle gun bombardment. In this research, second-generation transgenic plants with the IFNa2b gene are subjected to various experiments to study the levels of IFNa2b expression. The psbA regulatory sequences present in the chloroplast vector are known to enhance protein expression in the presence of light. To analyze this effect and to find optimal growth conditions for maximal IFN a2b production, continuous light studies were performed. These results can be vital for mass production of IFNa2b.

Les anticorps monoclonaux (AcM) ont été utilisés pour traiter des cancers dès le début des années 1980, en particulier lors du travail pionnier de l’équipe de Ronald Levy dans le traitement des lymphomes. Ces traitements ont pendant longtemps été considérés comme une sérothérapie passive à effet immédiat et à court terme. Cependant, au cours de ces dernières années, le concept d’un effet « vaccinal » des anticorps à usage thérapeutique en oncologie a peu à peu vu le jour du fait de réponses cliniques à long terme observées chez certains patients et de différentes études précliniques. En 2010, notre équipe a démontré que des souris immunocompétentes injectées avec les cellules tumorales EL4-huCD20 et traitées avec un AcM anti-huCD20 générait une réponse immunitaire anti-tumorale à long-terme par le biais de mécanismes dépendants de la région constante de l’anticorps et de lymphocytes T CD4+. Mon travail de thèse a donc porté sur l’analyse des mécanismes cellulaires et moléculaires par lesquels le traitement par un AcM anti-CD20 génère une immunité cellulaire adaptative anti-tumorale. J’ai ainsi pu montrer que le traitement des souris avec l’AcM anti-CD20 conduit à une expansion de lymphocytes Th1 producteurs d’IFN-γ, à l’apparition de lymphocytes T CD4+ effecteurs mémoires spécifiques des cellules tumorales CD20+, et au blocage de l’expansion de lymphocytes Tregs induite par les cellules tumorales. Le rôle central dans la protection anti-tumorale et la genèse d’une réponse adaptative anti-tumorale joué par l’axe IL-12/IFN-γ et leurs principales sources cellulaires, cellules dendritiques (DCs) et cellules NK, a été démontré par des expériences de neutralisation de ces cytokines, qui provoque une importante diminution du nombre de Th1 spléniques, de déplétion des cellules NK, ainsi que par des analyses phénotypiques qui ont permis d’identifier des DCs activées par le traitement - comme le montre l’expression accrue des molécules de classe II du CMH et de co-stimulation CD80 et CD86 - comme une importante source cellulaire de l’IL-12. Enfin, nous avons pu montrer qu’un variant de l’IL-2, liant préférentiellement le récepteur de l’IL-2By et faiblement le récepteur de l’IL-2aBy exprimé majoritairement par les Tregs, permettait l’obtention d’une protection anti-tumorale accrue d’animaux porteurs de tumeurs et traités par l’AcM anti-CD20. En conclusion, nous avons démontré qu’un contexte immunitaire pro-tumoral façonné par la présence d’une tumeur en développement peut être inversé par le traitement par un anticorps anti-tumoral, aboutissant à un contexte anti-tumoral. Qu’une telle réponse immunitaire adaptative cellulaire puisse être observée chez des patients atteints de lymphomes, traités par un anticorps anti-CD20, reste encore à être déterminé. / Monoclonal antibodies have been used to treat cancers since the early 1980s, in particular with the pioneer work of Ronald Levy for the treatment of lymphomas. Those treatments have been considered for a long time as a passive serotherapy with immediate and short term actions. Yet, recently, the idea of a vaccine effect of therapeutic antibodies in oncology have appeared, after preclinical studies and clinic observations suggesting a long term immune response in patients. In 2010, our team demonstrated that immunocompetent mice injected with EL4-huCD20 tumor cells and treated with anti-huCD20 monoclonal antibody generated a long term anti-tumor immune response linked with mechanisms dependent on constant part of antibodies and CD4+ T cells. My PhD work was based on the analysis of cellular and molecular mechanisms by which the treatment by an anti-CD20 mAb generates a cellar adaptive anti-tumor immunity. I could show that the treatment of mice with anti-CD20 antibody lead to the expansion of Th1 lymphocytes IFN-γ producers, to the apparition of effector memory CD4+ T cells specific for CD20 antigen, and to the blockade of the expansion of Treg cells induced by tumor cells. The key role of an adaptive anti-tumor immune response played by IL-12/IFN- γ and their main cellular sources, dendritic cells and NK cells, in the anti-tumor protection and genesis, has been demonstrated by experiments of cytokine neutralization, provoking an important decrease of splenic Th1 number, by NK depletion and by phenotypic analysis that allowed the identification of DCs activated by the treatment – as it is shown by the increased expression of MHC-II and CD80 and CD86 costimulation molecules, - as an important cellular source of IL-12. Finally, we could show that a variant of IL-2, binding preferentially IL-2By with a lower affinity for the IL-2aBy receptor mainly expressed by Tregs, could induce an increased anti-tumor protection of tumor-bearing animals treated with anti-CD20 mAb. In conclusion, we have demonstrated that a pro-tumor immune contexture affected by a growing tumor can be modified by an anti-tumor antibody leading to an anti-tumor contexture. That such cellular adaptive immune response could be observed in lymphoma patients treated with anti-CD20 still need to be determined.

The Interferon (IFN) family of cytokines are produced in direct response to virus infection and they constitute the first line of defence against virus infection by inducing hundreds of interferon stimulated genes (ISGs) which act in concert to establish the so-called “antiviral state”. Influenza A viruses and parainfluenza virus type 5 (PIV5) are both small negative strand RNA viruses that must circumvent their hosts’ interferon (IFN) response for replication. However, the ways in which these viruses interact with the IFN system are very different. Although PIV5 replication is initially severely impaired in cells in a pre-existing IFN-induced antiviral state, it manages to overcome the antiviral state by targeting an essential component of type I IFN signalling, STAT1, for degradation. Thus the cells cannot maintain the antiviral state indefinitely without continuous signalling. Consequently, the virus resumes its normal replication pattern after 24-48 hours post-infection. In clear contrast, influenza virus fails to establish its replication in the majority of infected cells (90-95%) with a pre-existing IFN-induced antiviral state, although a few cells are still able to produce viral antigens. To further investigate how influenza virus interacts with cells in a pre-existing IFN-induced antiviral state, I have used in situ hybridization to follow the fate of input and progeny genomes in cells that have, or have not, been treated with IFN prior to infection. Here I show for the first time that IFN pre-treatment blocks the nuclear import of influenza A virus genome, which prevents the establishment of virus replication, but this can be overcome by increasing multiplicities of infection. Of those IFN-induced antiviral molecules, human MxA is an essential component of the IFN-induced antiviral state in blocking influenza virus genome import, as this block can be abolished by lentivirus-mediated knockdown of MxA. I also show that in cells constitutively expressing MxA the viral genome still manages to be transported into the nucleus, indicating that MxA might require an unidentified IFN-induced factor to block nuclear import of the influenza virus genome. These results reveal that IFN exerts its action at an early stage of virus infection by inducing MxA to interfere with the transport of viral genome into the nucleus, which is the factory for viral RNA production.