The premise of this project was to engineer a novel viral platform with the capacity to enhance antitumour immunity. To this effect, the N1L gene was disrupted in a Lister strain vaccinia viral backbone that had previously been engineered to be tumour selective (VVL15ΔN1L) and armed with transgenes encoding murine and human versions of GMCSF and IL12. In vitro, they retained potency for infecting, replicating in and killing a panel of murine, Syrian hamster and human cancer cells; all viruses were able to express their transgenes to detectable levels upon infection of every tumour cell line. In comparison to the parental virus (VVL15), VVL15ΔN1L administration into immune competent in vivo tumour models (of pancreatic and lung cancer) led to enhanced intratumour (IT) infiltration of neutrophils as well as markedly elevated circulating numbers of natural killer (NK) cells. VVL15ΔN1L also enhanced the tumour infiltration of CD8+ cells. Functional immunoassays and flow cytometric analysis of T cells provided evidence of enhanced tumour specific adaptive immunity. In comparison to VVL15, IT VVL15ΔN1L significantly reduced the growth of subcutaneously implanted syngeneic pancreatic tumours. This effect was predominantly due to cytotoxic lymphocytes, evidenced by the complete abrogation of efficacy upon repeating the experiment in mice that had been depleted of CD8+ cells. A similar treatment schedule reduced the formation of lung metastases from a primary spontaneously metastasising syngeneic lung cancer model; and translated into prolonged short-term post-operative survival when used as neoadjuvant to surgical resection. Efficacy in this context was contrastingly, due to an elevation in systemic NK cells; concurrent depletion of NK cells (but not CD4+ or CD8+ cells) completely abrogated the survival advantage. The IL12 transgene armed recombinant was the most effective antitumour therapeutic. Its IT administration into pancreatic tumours led to complete tumour eradication in over 80% of tumour bearing mice and was effective in slowing the growth of other aggressive flank tumours. Neoadjuvant administration of VVL15ΔN1L-mIL12 into metastatic lung cancers dramatically prolonged long-term post-surgical survival, with apparent cure of 88% of mice.

Over the past few decades, it has become increasingly apparent that host-associated microbial communities play an integral role in the development, physiology, and health of their host organisms. All hosts have evolved mechanisms to filter the microbial taxa that comprise their resident intestinal microbial community, or gut microbiota. Utilizing the zebrafish as a model host organism, we documented the development of the gut microbiota through time, and found a significant shift in the composition of the gut microbiota after the onset of adaptive immunity. This led us to hypothesize that adaptive immunity is an important determinant of gut microbiota composition. We tested this hypothesis using wild type and rag1-/- zebrafish, which lack a functional adaptive immune system. Additionally we tested the robustness of the effects of adaptive immunity to dispersal of microbes between immune-compromised and immune-competent genotypes. We found that adaptive immunity had less of an effect on the composition of the gut microbiota than we expected, although there were intriguing differences in the nature of selection imposed when adaptive immunity was present than when it was absent. Because “westernization”, or market-integration, has been associated with significant changes in the human microbiota and certain health risks, we used similar analyses to those we applied to the zebrafish system to determine whether market-integration alters the filtering effects of inflammation and intestinal helminth parasites on the intestinal microbial community. We found that market-integration increased inter-subject dissimilarity and reduced inter-subject dispersal. Even small changes in the inflammation marker, CRP, were associated with differences in the gut microbiota, but these effects were reduced in the presence of helminth infection, which has been hypothesized to affect the microbiota by reducing inflammation. In total, this dissertation provides evidence for the nature and importance of host filters of the gut microbiota across two vertebrate species, as well as providing a framework for future studies of the effects of such filters on the assembly of the gut microbiota. This dissertation includes previously published, and unpublished, co-authored material.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Immunity and metabolism have been viewed as separate fields, however recent evidence show that these two systems are intimately integrated, share resources and cross-regulate each other. Activated immune cells have to alter their metabolism in order to support effector functions. On the other hand, viruses are obligatory parasites that counter and exploit host pathways, including metabolism, to effectively propagate. Like immune cells, viruses have to alter the metabolic profile of infected cells in order to propagate. The regulation of metabolism in immune cells or virally infected cells has been well studied. However, the precise metabolic regulation that ensues when both immune system and viral infection in immune cells interact and compete for the limited resources and metabolic pathways available is not clear. In this thesis, I have sought to investigate the integrative process by studying the metabolic programming of macrophages infected with murine cytomegalovirus (MCMV) The central hypothesis of this thesis is that productive infection of macrophages by MCMV takes advantage of the early inflammatory metabolomic reprogramming of activated macrophages to establish infection, and modulates metabolism at late stages of infection towards fatty acid (FA) production to promote viral progeny. To study this interaction, I have analysed the temporal profile of the transcriptome and metabolome of bone marrow derived macrophages (BMDM) infected with productive (WT) and non-productive (attenuated) (MCMV) strains. This aimed to unravel the host-directed versus virus-driven metabolic alterations. I show evidence indicating that during early times of productive and non-productive MCMV infection glycolysis is, in infected BMDM, markedly increased. Furthermore, pharmacological and siRNA mediated inhibition of glycolysis resulted in attenuation of viral growth demonstrating the dependency of MCMV on this pathway. Additionally, using interferon receptor A (IFNAR) and interferon receptor A (IFNB) deficient BMDM showed that type-I interferon (IFN) signalling is essential for the early upregulation of glycolysis that was observed. In addition to the changes in glycolysis, MCMV infection alters the tricarboxylic acid (TCA) cycle in infected BMDM. Metabolomic and transcriptomic data revealed a shift from catabolic to anabolic function for the TCA to promote production of TCA intermediates. Finally, the urea cycle is also altered both on transcriptional and metabolomic level, consistent with the support of Nitric oxide (NO) production which is a hallmark metabolite in classically activated macrophages. These changes observed in the TCA cycle and glycolysis are consistent with supporting the FA elongation pathway during late time points of productive infection. Only productive MCMV infection upregulates this pathway. At the same time, pharmacological and siRNA mediated inhibition of FA elongation pathway greatly attenuates viral growth. This indicates that MCMV growth is dependenton FA elongation. The effect was very specific for the elongation and not the de novo synthesis pathway indicating that MCMV remodels FA that already in the cells. It is argued, that in agreement to known literature, MCMV uses these FA for the formation of its lipid membrane. To further investigate the dependency of MCMV on FA elongation pathways I studied additional lipids pathway associated with the former. I found that MCMV infection also upregulates the triacylglycerol formation and membrane remodelling pathways, which are dependent on FA biosynthesis and elongation. The inhibition of triacylglycerol formation and membrane remodelling pathway also attenuated MCMV growth. This indicates that apart from the formation of its lipid membrane MCMV requires FA to remodel the cellular environment. I have also explored the effects of infection on regulating lipid mediators, in particular eicosanoids. Eicosanoids are lipid signalling molecules that can act as potent inflammation modulators. Here I demonstrated that productive MCMV infection specifically increases PGE2 production in infected BMDM. Moreover, addition of PGE2 increased viral replication in infected fibroblasts in comparison to non-treated cells, while pharmacological blocking of EP4 (PGE2 receptor) rescued the phenotype. These studies reveal how MCMV advantageously use inflammatory lipid pathways to promote growth In conclusion, the data presented in this thesis support my hypothesis and provide an insight in the role of metabolism during viral infection. Evidence is provided to show that MCMV co-ops the early alterations that metabolic pathways undergo in activated macrophage, including but not limited to glycolysis, TCA cycle and urea cycle. These early changes in metabolism appear to be coupled with upregulation of FA elongation pathways and remodeling of lipids in infected cells. Finally, MCMV co-ops the function of regulatory lipids, in particular PGE2, to promote viral growth. It is further argued that MCMV productive infection dictates these fatty acid metabolism alterations in order to remodel the host cell's environment, regulate the immune system response and provide resources for its lipid membrane.

The hepatitis C virus (HCV) currently infects 3% of the world???s population, with chronic infection in 50-80% of exposed individuals. A small subset of individuals who are exposed to HCV do not develop anti-HCV antibodies, persistent viraemia or chronic hepatitis despite generating HCV-specific CD4+ and CD8+ T cells. These individuals are believed to develop an immune response which rapidly clears viraemia prior to the induction of an antibody response. Circumstantial evidence supports the likelihood that some of these individuals may generate these same responses and outcomes on repeated occasions of HCV infection. HCV-specific cellular immune responses in seronegative subjects have been the subject of only limited prior study, in part due to the lack of appropriate recombinant antigens and assay systems. Therefore, this thesis described the development and validation of an interferon-? (IFN-?) ELISPOT assay using overlapping peptides (n=441). Using this assay, HCV-specific cellular immune responses were detected in 5/10 (50%) of chronically infected subjects. Responses were identified more frequently, and were directed against more regions of the HCV genome, than with traditional assay systems. This IFN-? ELISPOT assay, a comparable interleukin (IL)-2 ELISPOT assay, and a multiplex in vitro cytokine production assay were then used to evaluate HCV-specific cellular immune responses in three cohorts of seronegative subjects at high-risk of exposure to HCV ??? babies born to infected mothers, multiply-transfused subjects with thalassaemia, and high risk injecting drug users. Cellular immune responses were evaluated in 23 infants born to HCV-antibody positive women. Responses were not detected in infants born to HCV-PCR negative mothers. IFN-? production was detected in 1/11 infants born to viraemic mothers using the ELISPOT assay, with cytokine production observed in an additional 3/5 infants studied using the in vitro cytokine production assay. HCV-specific cellular immune responses were assessed in a cohort of multiply transfused subjects with thalassaemia using assays for cytotoxic T lymphocyte activity, IFN-? and IL-2 ELISPOT, as well as lymphocyte proliferation and in vitro cytokine production. Responses were detected in 6/13 chronically infected subjects (46%), 4/7 subjects who had cleared infection (71%), and 14/17 seronegative subjects (82%). The seronegative subjects had responses which were broader and higher in magnitude than those with chronic HCV infection, although lower and narrower than in subjects who had cleared prior HCV infection. IFN-? and IL-2 ELISPOT assays, in additional to in vitro cytokine production assays, were performed on 41 injecting drug users (IDUs), with responses detected in 6 (15%). Seronegative IDUs with HCV-specific cellular immune responses had been injecting for a mean of 7.7 years, and reported multiple risk factors for exposure to HCV. The combined data from these three cohorts indicate that the HCV-specific cellular immune responses detected in seronegative subjects were generally broad in specificity. Cytokine production was generally Th1-biased, a pattern which has previously been associated with an increased likelihood of clearance in primary infection. The findings also suggest that responses can be maintained for decades after exposure, and may provide protection against repeated exposures. In summary, cellular immunity against HCV is evident in some seronegative high risk subjects, suggesting that the cellular immune responses may efficiently facilitate viral clearance. Understanding the mechanisms of this immune response pattern will allow better understanding of the host response to HCV and may provide key insights into vaccine design.

Les moustiques anophèles sont les vecteurs du parasite Plasmodium l’agent du paludisme. Le parasite subit des pertes massives pendant son cycle de développement chez l’anophèle, ce qui suggère que les moustiques sont capables de développer une réaction immunitaire efficace contre le parasite. La connaissance de l’immunité et de la résistance des moustiques au genre Plasmodium provient principalement de systèmes de laboratoire qui utilisent des espèces de parasites de rongeurs ou d’oiseaux comme modèles du paludisme humain. Les observations présentées dans cette thèse suggèrent que certains gènes comme Tep1 et LRIM1 sont des médiateurs de réponses antiparsitiques contre différents Plasmodiums dans différents vecteurs. Cependant, le degré d'efficacité avec laquelle un moustique est capable de réduire le nombre de parasites peut être variable surtout entre combinaison de souche de moustique et de souche de parasite différentes, selon que la paire soit hautement compatible ou non.

Plants have evolved sophisticated defence systems against pathogen infection. Initiation of induced defence signalling often involves specific recognition of invading pathogens by the products of specialized host Resistance (R) genes. Consequently, the pathogen is stopped at the site of infection. A unique dominant mutant in Arabidopsis thaliana, snc1, constitutively expresses pathogensis-related (PR) genes and exhibits enhanced resistance to bacterial and oomycete pathogens. SNC1 encodes an R-gene – a single amino acid change renders this protein constitutively active without interaction with pathogens. snc1 displays a stunted phenotype that may be caused by both the accumulation of toxic compounds and energy squandered on unnecessary defence instead of normal growth. The distinctive morphological phenotype of snc1 is intimately associated with the other resistance phenotypes, and provides a robust genetic tool for dissecting the signalling events downstream of snc1. To identify genes important for defence signalling, we carried out a suppressor screen to identify modifier of snc1 (mos) mutants that restore the wild type size and morphology in the snc1 background. Furthermore, in most cases, a loss of sneakiness in mos mutants correlated with a reduced or abolished constitutive PR gene expression, SA accumulation and pathogen resistance in snc1 plants. These loss of function mutants represent defects in positive regulators of the snc1 pathway. I cloned and characterized two mos mutants, and showed that they both have roles in Arabidopsis innate immunity as well. mos6 partially suppresses snc1 and exhibits enhanced disease susceptibility (EDS) to an oomycete pathogen. MOS6, identified by map-based cloning, encodes an alpha-importin subunit, one of 8 found in Arabidopsis, and has a demonstrated role in nucleocytoplasmic partitioning (protein import). Two other genes cloned by others from this screen, MOS3 and MOS7, encode components of the nuclear pore complex, implicating nuclear trafficking as a key regulator in plant innate immunity. mos4 exhibits EDS to virulent and avirulent bacterial and oomycete pathogens. There is evidence that MOS4-mediated resistance is independent of the signalling protein NPR1. MOS4 encodes a protein with homology to human Breast Cancer Amplified Sequence 2 and with predicted protein-protein interaction domains. Subcellular localization of MOS4-GFP shows that MOS4 is localized to the nucleus. To illuminate the biochemical function of MOS4, a yeast-2-hybrid screen was conducted. One MOS4-interactor was a putative myb transcription factor, MOS4-Associated Complex Protein 1 (MAC1), also known at AtCDC5. MAC1 interacts directly with MOS4 in vitro and in planta. mac1 insertional mutants exhibit defects in immune responses to pathogens similar to that of mos4. In addition, mac1 also partially suppressed snc1 morphology and enhanced resistance. Both MOS4 and MAC1 have homologs in humans and fission yeast that are members of a discrete protein complex that has been implicated in several different biological processes including RNA splicing, apoptosis and protein degradation. Using proteomics data from yeast and human, we found genes with homology to additional components of the orthologous complex in Arabidopsis, and isolated insertion mutants in these. Mutations in PRL1, which encodes a WD protein, display similar disease phenotypes to that of mos4 and mac1. AtCDC5 has DNA binding activity, suggesting that this complex may regulate defence responses through transcriptional control. Since the complex components along with their interactions are highly conserved from fission yeast to Arabidopsis and human, they may also have a yet-to-be identified function in mammalian innate immunity.

Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. While the discrimination of host and microbial DNA is presumed to reflect TLR9-mediated recognition of CpG motifs, little information is available to verify this hypothesis. Cell stimulation experiments demonstrate preferential activation of TLR9 by CpG-containing nucleic acids, however direct binding investigations have reached contradictory conclusions with respect to the ability of TLR9 to bind nucleic acids in a sequence-specific fashion. Here we report expression of the soluble, ectodomain of human TLR9 with characterization of its ligand-binding properties. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity is mediated specifically by the absolute concentration of nucleic acids in a sequence-independent manner.<p>The bovine hsp70A promoter was used to direct the heat-regulated synthesis of the ectodomain of human TLR9 in transfected cultured bovine cells. The protein was efficiently secreted from transfected cells in a temperature-dependent manner and the recombinant receptor produced was found to be relatively pure. A stably transfected cell line with regulated expression of the protein was obtained and repeated thermal cycling of the cultures enabled high-yield production of the receptor in an active ligand-binding form. Using this recombinant receptor to study the ligand binding properties of TLR9, a model of positive cooperativity is proposed in which the sensitivity of TLR9 ligand binding is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion, while activation of TLR9 is highly dependent on DNA sequence. That is to say that TLR9 is primed for activation by interaction with non-activating sequences but activation itself occurs in a sequence-specific fashion.

Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. While the discrimination of host and microbial DNA is presumed to reflect TLR9-mediated recognition of CpG motifs, little information is available to verify this hypothesis. Cell stimulation experiments demonstrate preferential activation of TLR9 by CpG-containing nucleic acids, however direct binding investigations have reached contradictory conclusions with respect to the ability of TLR9 to bind nucleic acids in a sequence-specific fashion. Here we report expression of the soluble, ectodomain of human TLR9 with characterization of its ligand-binding properties. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity is mediated specifically by the absolute concentration of nucleic acids in a sequence-independent manner.<p>The bovine hsp70A promoter was used to direct the heat-regulated synthesis of the ectodomain of human TLR9 in transfected cultured bovine cells. The protein was efficiently secreted from transfected cells in a temperature-dependent manner and the recombinant receptor produced was found to be relatively pure. A stably transfected cell line with regulated expression of the protein was obtained and repeated thermal cycling of the cultures enabled high-yield production of the receptor in an active ligand-binding form. Using this recombinant receptor to study the ligand binding properties of TLR9, a model of positive cooperativity is proposed in which the sensitivity of TLR9 ligand binding is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion, while activation of TLR9 is highly dependent on DNA sequence. That is to say that TLR9 is primed for activation by interaction with non-activating sequences but activation itself occurs in a sequence-specific fashion.

<p>Recently, the study of invertebrate innate immunity has garnered considerable attention after the discovery that mammalian homologues of the <italic>Drosophila melanogaster</italic> </p><p>Toll pathway play a role in mammalian innate immunity. One invertebrate model system that has begun to be intensely studied is the nematode <italic>Caenorhabditis elegans</italic>. Immunity in <italic>C. elegans</italic> has been shown to be inducible in that it responds uniquely to different pathogens. These changes in gene expression require transcription factors in order for certain genes to be transcribed. We utilized an RNA interference screen of potential transcription factors to identify the GATA transcription factor ELT-2 as a possible transcription factor involved in immunity. We then demonstrated that ELT-2 was required for resistance to a wide range of pathogens and was responsible for regulating expression of the C-type lectin <italic>clec-67</italic>, a marker of immunity. </p><p>We also studied another transcription factor known to play a role in C. elegans immune function, the FOXO transcription factor DAF-16. We specifically focused in on the role of DAF-16 in germline-deficient mutants, and we demonstrated that such mutants are resistant to many different pathogens. This led to further investigation of the germline-deficient mutant glp-4, which should also show broad range resistance to pathogens but fails to do so. Through whole genome sequencing, we identified mutations that may be responsible for the glp-4 phenotype. We also demonstrated that DAF-16 was active in glp-4 mutants, leading to us proposing a model where glp-4 plays a role in influencing <italic>C. elegans</italic> immunity besides its involvement in germline development.</p> / Dissertation