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Marek’s disease (MD) is a lymphoproliferative disease of chickens caused by an oncogenic herpesvirus, Marek’s disease virus (MDV). Despite the availability of MD vaccines, little is known about the underlying immunological mechanisms that mediate vaccine-induced immunity. The objective of this research was to elucidate these mechanisms. To characterize host responses in the lungs, chickens were vaccinated with herpesvirus of turkeys (HVT) and infected with MDV-RB1B. Vaccinated MDV-infected chickens had a higher accumulation of viral genome in the lungs, associated with T cell infiltration in lung tissue and an up-regulation of interferon (IFN) - and interleukin (IL) -10. This finding led us to conclude that IFN-γ has a role in immunity; hence, we further investigated the role of this cytokine. The hypothesis tested was that the protective efficacy of HVT against MDV-RB1B would be enhanced when combined with recombinant chicken IFN-γ (rChIFN-). Chicken IFN-γ coding sequence was cloned into an expression plasmid, and the bioactivity of rChIFN- was confirmed. Administration of this plasmid led to a significant reduction in tumour occurrence in HVT vaccinated MDV-infected chickens, suggesting enhanced vaccine-induced immunity. To shed more light on the relevance of IFN-γ to immunity against MD, studies were designed to down-regulate the expression of IFN-γ in chicken tissues. Three small interfering (si)RNAs specific for chicken IFN- were selected which significantly inhibited expression of IFN-γ by up to 80% in cultured cells. These three siRNAs and a non-target control were cloned and expressed as short hairpin RNA (shRNA) using an avian adeno-associated virus (rAAAV) vector system. An MDV challenge trial was conducted once shRNA expression by the rAAAV was confirmed in vitro. It was reasoned that down-regulation of IFN- would lead to abrogation of immunity conferred by HVT. There was an increase in the number of chickens with tumours that received HVT and rAAAV + IFN-γ shRNA compared to the control group, though not statistically significant. However, no difference in MDV genome load in feathers was detected between vaccinated MDV-infected birds with or without rAAAV. In summary, we have demonstrated here that cytokines are induced in the course of vaccination against Marek’s disease and that IFN-γ plays a role in vaccine-induced immunity against MD.

Protein-protein interactions play an important role in nearly all processes of the living cells and the function of many proteins is dependent on their specific interactions with other biomolecules. A reliable tool to modulate these interactions would be invaluable for the development of molecules suitable for diagnostics, medicine, and biotechnology. In this work, we aimed to study the specificity of interactions in the model system of Interferon gamma receptor 1 (IFNgR1) and its natural ligand Interferon gamma (IFNg), important in innate immunity. We searched for mutations within the interferon receptor molecule IFNgR1 to modulate (increase as well as decrease) its affinity to IFNg by in silico analysis of the existing crystal structures of the complex between IFNgR1 and IFNg. We modeled amino acid substitutions and gauged how they influenced the interaction using empirical force field implemented in software FoldX. All selected promising IFNgR1 variants were expressed in Escherichia coli, purified to homogeneity, characterized, and kinetics of their interactions with IFNg was measured by Surface Plasmon Resonance (SPR). The first set of IFNgR1 variants included mutations on the interface of the IFNg/IFNgR1 complex. According to our SPR measurements, the affinity of most of these receptor...

The human Class Ib gene, HLA-E inhibits both Natural Killer Cells and a subset of CD8+ cytotoxic T lymphocytes by engaging the CD94/NKG2A inhibitory receptor. IFN-γ induces the expression of HLA-E as well as Class Ia molecules, which are required for the killing of target cells. Since HLA-E has negative effects on immune killing of target cells, we have sought to identify locus specific mechanisms of IFN-γ induction in order to identify molecular targets for selective activation of Class Ia genes, but not HLA-E. We have previously identified a unique upstream IFN-γ response region in the HLA-E promoter and showed that GATA-1 is required for its function in the K562 leukemic cell line. We have now examined the effect of GATA family members on IFN-γ induction of HLA-E in other cell types. HLA-E CAT reporter gene assays demonstrate that tumor cells that express GATA factors as determined by western blot and quantitative PCR, mediate a 2.4 to 4.0 fold enhanced response to IFN-γ stimulation. Functional constructs containing mutations of the core nucleotides in the GATA binding site had a 4.8 fold decreased response to IFN-γ in A2780 cells and a 8.5 to 14.0 fold decreased response to IFN-γ in SKOV3 cells. Knockdown of GATA-6 using siRNA resulted in a 40% decrease in HLA-E induction in Seg1 cells and a 30% decrease in HLA-E induction in HCT116 cells. Tetracycline regulated shRNA knockdown of GATA-6 expression in the SKOV3 cell line revealed a 3 fold decrease in the IFN-γ response of HLA-E reporter driven constructs. Additionally we observed a decreased IFN-γ response in SKOV3 cells transfected with siRNA specific for CBP and IRF-9. We conclude that GATA factors play a tissue specific role in regulation of IFN-γ mediated HLA-E expression and that IRF-9 may be a target for the differential manipulation of classical MHC and HLA-E.

Leishmaniasis is a spectrum of diseases caused by several species of protozoan parasites belonging to the genus, Leishmania. The disease, which is transmitted by Sandflies, ranges from self-healing cutaneous lesions to the life-threatening visceral leishmaniasis. Cutaneous leishmaniasis, caused by L. major, is the most common form of the disease. With no vaccine available for use in humans, cutaneous leishmaniasis remains a global public health problem. Since understanding the factors that regulate effective immunity to cutaneous leishmaniasis is critical for the development of an affective vaccine and treatment strategies, the overall aim of my thesis was to decipher the host and pathogen factors that regulate immunity in cutaneous leishmaniasis. Firstly, I show that parasite dose affects the expansion of different T cell subsets following L. major infection; with low dose infection inducing more CD8+ T cells while high dose infection induced more CD4+ T cells. However, although CD8+ T cells were important for optimal primary immunity following low dose infection, they where dispensable during secondary immunity. Secondly, I found that blockade of LIGHT, (lymphotoxin like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes) significantly impaired DC maturation, expression of co-stimulatory molecules, and early cytokine production (IL-12 and IFN-γ) following L. major infection. Interestingly, LIGHT was completely dispensable during secondary immunity in wild type mice but was critical for effective secondary immunity in CD40 deficient mice. Thirdly,I compared disease progression and immune response in CD40 and CD40L deficient mice infected with L. major under identical experimental conditions. I found significant differences in disease progression and immune response between CD40KO and CD40L KO mice infected with virulent L. major and treated with recombinant IL-12. My data revealed a novel pathway (CD40L-Mac-1 interaction) for IL-12 production and resistance to Leishmania major. Collectively, this thesis provides novel insights into the mechanisms involved in the development and maintenance of protective immunity against cutaneous leishmaniasis, which could lead to the development of a more efficient and effective immunotherapeutic and/or vaccination strategies against the disease. / October 2015

Severe influenza virus infection, including human infection with highly pathogenic H5N1 viruses is characterised by massive pulmonary inflammation, immunopathology and excessive cytokine production, a process in which macrophages may play a vital role. The aim of this project was to investigate the hypothesis that inhibition of inflammatory responses from infected macrophages, using either alternatively activated bone marrow derived macrophages (BMDMf), or IFNg receptor deficient (IFNgR-/-) mice may ameliorate the devastating immunopathology and inflammation routinely observed in highly pathogenic influenza virus infections. Infection of alternatively activated BMDMf resulted in enhanced positivity for viral proteins, compared with classically activated, inflammatory BMDMf. However, neither subset propagated the infection indicating that while infection is abortive in both classical and alternatively activated BMDMf, the latter may prove more efficient at removing infectious virus from the site of infection due to enhanced infectivity. However, influenza virus was capable of driving expression of proinflammatory mediators such as iNOS and TNFa from classical and alternatively activated BMDMf even in the absence of IFNg signalling. IFNgR-/- BMDMf demonstrated a reduced inflammatory response to infection compared to Sv129 counterparts, suggesting a potentially impaired inflammatory response in vivo. This was investigated by infection of IFNgR-/- mice, which resulted in ameliorated disease, lower viral titres and mild immunopathology, demonstrating that inhibition of IFNg signalling limits the severity of disease. Additionally, mRNA expression for key inflammatory mediators was reduced, demonstrating that inhibition of the overwhelming inflammatory response to influenza virus infection is beneficial to the host, resulting in protection from immunopathology and improved prognosis, without impairing viral clearance.

Mammalian cell culture is typically operated at the physiological temperature of 37°C. Low temperature cell culture at 30-33°C, in particular for CHO cells, increased the specific productivity of many recombinant proteins amongst many other benefits. However, the cell density is lower, thus reducing the total protein yield. Of the 17 mammalian cold-stress genes reported to be up- or down-regulated at low temperature, CIRP shows potential as a gene target for improving recombinant protein production, as its expression levels were reported to affect both growth and specific productivity. In this study, it was shown that over-expression of the cold-stress gene CIRP did not cause growth arrest in CHO cells, in contrast to previous reports. However, over-expression of CIRP successfully improved the specific productivity and total yield of a recombinant interferon-γ CHO cell-line at 37°C by 25%. / Singapore-MIT Alliance (SMA)

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No description available.