Tuberculosis (TB) is an infectious bacterial disease mainly infecting the pulmonary system of the human body. It affects around 1.5 million people every year, most of whom live in developing countries. The incidence of TB has increased in line with the rise in incidences of Human Immunodeficiency Virus (HIV) infections and Acquired immune deficiency syndrome (AIDS). Due to the pressing concerns of TB, the World Health Organisation (WHO) came up with the Direct Observed Treatment (DOTS) programme. Unfortunately, the development of several resistant strains against first-line drugs and consequently second and third-line drugs have developed. As the current TB drug regimen is inadequate, a good screening strategy, discovery of newer drugs and identification of the mode of action would help in developing better treatment routines and determining bacterial pathways more clearly. Drug discovery follows two major routes, one leading from the drug to the target and the other from target to the drug. Both methods have been applied in this work in order to identify new drugs effective against mycobacteria. Screens performed against a drug library approved by the Food and Drug Administration (FDA) have resulted in some promising hits. Functional characterisation of a putative enoyl CoA hydratase EchA12, which was targeted by florfenicol, revealed a novel lipid chaperone functionality associated with cell wall lipid biosynthesis. Furthermore, a target based phenotypic drug screen of the GSK177 box set against Mtb-PrsA provided further evidence that this enzyme as a viable drug target (Ballell et. al., 2013).

Influenza A viruses (IAVs) are common infections of certain avian reservoir species, and they periodically transfer to mammalian hosts. These cross-species jumps are usually associated with sporadic outbreaks, and on rare occasions lead to the establishment of a lineage in the new host species. The immune pressure exerted by the new host on the emergent virus forces it to evolve and adopt strategies to evade immunity in order to survive in nature. Understanding the biological mechanisms that allow successful inter-species transmission and adaptation to mammals is crucial to develop the theoretical tools required to predict and/or control emergence of new viruses in humans and animals. H3N8 equine influenza virus (EIV) represents an interesting model to study the dynamic of within-host variation of an avian-origin IAV. Indeed, this virus has emerged from birds in 1963 and has circulated in horse populations for more than fifty years despite the availability of vaccines. Evidence of evolution of EIV virulence factor non-structural protein 1 (NS1) also exists. NS1 is the main viral antagonist of the host interferon (IFN) response, and it relies on different strategies for overcoming these responses, which varies depending on the viral strain. While some NS1 proteins effectively block the induction of IFN and IFN stimulated genes (ISGs), others block general gene expression at a post-transcriptional level, and therefore reduce the synthesis of IFN and ISGs indirectly. Importantly, little is known about the contribution of these NS1 functions to EIV infection phenotype and adaptation to horses. In this work, we characterised NS1 proteins spanning the entire EIV lineage and showed that NS1s from different time periods after EIV emergence counteract the IFN response using different and mutually exclusive mechanisms. While EIVs circulating in the early 1960s blocked general gene expression by a NS1-mediated blockade of the cleavage and polyadenylation specificity factor 30 (CPSF30), NS1s from contemporary EIVs specifically inhibit the induction of ISGs by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially during EIV evolution, E186K substitution and C-terminal truncation. These changes in NS1 allowed contemporary EIVs to replicate in the presence of high levels of IFN. The results shown here with EIV indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation.

Innate immunity is a branch of the immune system that is responsible for controlling the early events of pathogen infection. One of the key components of the innate immune systems arsenal are the interferon (IFN) cytokines. IFNs are small signalling proteins that are released by cells in response to invading pathogens, and viruses in particular. They are named for their ability to interfere with viral replication. The result of IFN signalling is the up-regulation of a diverse collection of genes termed interferon-stimulated genes (ISGs). These genes act in synchrony to limit the replication of viruses. The protein products of ISGs are involved in a multitude of cellular pathways that limit replication and additionally intercept viral proteins and nucleic acid directly. Some of these ISGs are mediators of an important cell-death response, apoptosis. Apoptosis is a vital component of innate immune signalling and controls viral replication by sacrificing the infected cell to limit further infection of neighbouring cells. The function of specific ISGs in mediating this response is poorly understood.

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Strong adaptive evolutionary forces shape the interactions between pathogens and their hosts and typically lead to a stable co-existence. In this process of co-evolution, mammals have developed restriction factors that limit retrovirus infectivity, replication or assembly and narrow the spectrum of potential host species. These restriction factors are either constitutively expressed, such as APOBEC3 proteins, cytidine deaminases that interfere with reverse transcription, or form part of the type I interferon-induced innate immunity, such as TRIM5, a member of the tripartite motif protein family that induces degradation of retroviral capsid, blocks reverse transcription, or tetherin (BST-2, CD317), which inhibits release of nascent viral particles from infected cells. Conversely, viruses have evolved antagonists of restriction factors or proteins that limit IFN-induced gene expression, thus evading immune surveillance. The interaction between host and viral components is delicately balanced and has a significant impact on disease outcome. Feline immunodeficiency virus (FIV), a lentivirus closely related to human immunodeficiency virus (HIV), is a recent introduction into domestic cats and causes an immunodeficiency syndrome analogous to human AIDS. Interestingly, non-domestic cats such as lion or pumas have co-existed with lentiviruses for prolonged periods of time and FIV infections are largely benign. Although plasma viral and proviral loads are high in both domestic and non-domestic cats, in vitro studies have shown that FIV infection of non-domestic cat T lymphocytes is significantly less efficient than that of domestic cat T cells. Thus, this thesis tests the hypothesis that the differential disease outcome of FIV infections in felids is caused by differences in lentiviral restriction factor activities or their sensitivities to FIV restriction factor antagonists. Data presented in this study show for the first time that feline APOBEC3 proteins are expressed in tissues and cell types relevant for FIV infection. The APOBEC3 proteins A3H and A3CH exhibited a high antiviral activity against FIV lacking the APOBEC3 antagonist Vif in single-cycle replication assays, with no difference in activity being detected between domestic and non-domestic cat proteins. However, domestic cat A3CH was significantly more sensitive to antagonism by FIV Vif than lion or puma A3CH, which would allow efficient viral replication in domestic cat T lymphocytes and subsequently lead to T cell loss and immunodeficiency. Furthermore, this thesis provides evidence that felid tetherins can prevent FIV particle release from producer cells in single-cycle replication assays; however, stable expression of domestic and non-domestic cat tetherins in feline cell lines did not abrogate FIV replication. Indeed, syncytium formation indicative of viral cell-to-cell spread was significantly enhanced in type I interferon-treated feline cells infected with CD134-independent strains of FIV which often arise in chronic (late) stages of FIV infections in vivo. Finally, this work reports the generation of a synthetic domestic cat TRIM5α-cyclophilin A fusion protein which was highly efficient at preventing FIV pseudotype and productive infection. This novel feline restriction factor represents a potent antiviral defence agent with very low potential for toxicity and could in future be used in gene therapy approaches to treat FIV-infected cats.

Human herpes virus 6 (HHV6) infects the majority of individuals in childhood, followed by a lifelong asymptomatic latent infection. However, in immunosuppressed individuals reactivation of HHV6 can cause significant clinical pathology. Recent successes with adoptive T cell therapy against other viral infections, notably the human herpes viruses Epstein-Barr virus (EBV) and Human cytomegalovirus (HCMV), suggest that this may be a useful therapeutic approach for HHV6-driven disease in immunosuppressed individuals. However, very few studies have been carried out analysing the immune response to HHV6 in any detail. This thesis was aimed at characterising the CD8+ T cell response to HHV6 in a group of healthy individuals, with the aim of mapping and characterising novel CD8+ T cell epitopes. Initial studies included four HHV6B antigens (U11, U39, U54 and U90), predicted to be immunogenic based on their HCMV homologues. Whole antigen peptide mixes (pepmixes) were used to stimulate peripheral blood mononuclear cells (PBMC) from healthy subjects. T cell responses were analysed by intracellular cytokine staining (ICS) after overnight stimulation and/or by interferon-γ (IFN-γ) ELISpot assay after 10 days of stimulation. For responses to U11 and U90, peptides libraries were used to map minimum CD8+ restricted epitopes. Further characterisation of HHV6B-specific T cells was carried out by identifying the HLA restriction elements and determining whether these T cells were capable of killing HHV6B-infected cells. PBMC from 30 healthy donors were stimulated with pepmixes corresponding to HHV-6B antigens U11, U39, U54 and U90. A weak CD8+ response (0.02-0.2%) to U90 and U54 was observed in a number of donors. Short-term in-vitro reactivations of PBMC (in 25 healthy donors) with HHV6B pepmixes followed by analysis of antigen and peptide specific response were performed by IFN-γ ELISpot assay. T cell responses to U54, U90, U11 and U39 were observed in 88%, 84%, 76% and 72% of the donors, respectively. Subsequently, the breadth of epitope specificity within U90 and U11 was screened for 9 healthy donors; with successful identification of 10 CD8+ T cells specific (9-mer) epitopes within these antigens. Seven of them were within U90 antigens and three of them were within U11 antigens. Allelic association of the U90 epitopes were; VEESIKEIL - B40 (60), FESLLFPEL - B40 (60), NLITAAKNI - A2, ITAAKNIGI - A2, LNIDPSESI - A1, PSKSKKIKL - A29, NHCFINHFV - B39. Allelic association of the 2 U11 epitopes were LKTQRRHKF - B37 and GILDFGVKL - A2; the HLA association for FNAVYSQRV was not identified. CD8+ T cell populations specific to some of these epitopes were also able to kill HHV6B infected cells. HHV6B T cells responses are detectable in healthy donors. Peptide specific responses against U11 and U90 have been mapped and characterised. These findings are relevant to the development of T cell mediated immunotherapy of HHV6-associated diseases.

Patients living with many cancers, including ovarian cancer (OC), often suffer from a lack of adequate treatment options. In the case of OC, primary debulking surgery followed by platinum and paclitaxel chemotherapy has led to a vast improvement in patient survival over the past few decades, however, rates of drug-resistant recurrence remain high. Research into new, experimental treatment options is therefore warranted for OC and other cancers. Oncolytic viruses (OVs) are replication-competent viruses that can selectively infect and destroy cancerous cell types, while leaving healthy cells unharmed. OVs do this by exploiting differences between cancer and normal cell phenotypes. Herpes simplex virus (HSV)-1, strain 1716 is one example of this type of virus that has shown selectivity for cancer cells in previous preclinical studies, as well as high levels of safety in humans. One prominent area of current OV study seeks to investigate the ability of OVs to induce immunogenic cell death (ICD) – this term describes multiple modes of programmed death pathways that culminate in release of proimmunogenic factors, which facilitate a modification of the host immune system. Two of the most prominent of these pathways are necroptosis and immunogenic apoptosis (IA). Here, I show that while many OV cell lines express the necessary components for necroptosis, they are unable to undergo classical necroptotic death (induced by TSZ). Despite this, HSV-1716 can infect and kill a range of OC lines successfully. I showed that HSV-1716-induced cell death displays two markers of IA yet does not seem to rely solely on apoptosis to kill cells. In addition, it appears not to rely on any components of the necrosome in order to kill cells, even in cells that are competent to typical necroptosis. However, when RIPK3 is overexpressed in HeLa cells, virus-induced cell death increases, as do markers of both necroptosis and IA. To investigate the role of ICP6 in HSV-1716-induced ICD, viral and cell mutants were made possessing various forms of the protein. Full-length ICP6 protein expressed in cell lines had the effect of blocking cellular response to TSZ, but constructs lacking a region known as the RHIM did not. A functionally similar mutation was produced within the RHIM of live HSV-1716 using CRISPR/Cas9 technology, which was shown to have the effect of disrupting ICP6/RIPK3 binding – thought to be the determinant of necroptotic cell death. Despite this, no changes in cell death signalling could be determined between the viruses at all. Interestingly, when cells were infected in combination with TNF-α, or TNF-α in addition to SMAC mimetic, the RHIM-modified virus produced significantly more death than HSV-1716. This suggests that while loss of RIPK3 inhibition is not sufficient to lead to increased necrosis alone, cells infected with this virus are more sensitive to further necrosis induction. This finding may prove to have great utility for producing the next generation of oncolytic viral therapeutics which can induce greater levels of proimmunogenic cell death. From this we can conclude that HSV-1716 is capable of inducing IA in OC cells. Death is not dependent on necroptosis, however additional RIPK3 seems to sensitise cells to death by other means. Cellular binding of viral ICP6 and RIPK3 can be disrupted by modification of the RHIM, although this change has no bearing on ICD signalling alone but can sensitise cells to TNF-α-induced death.

Immune responses towards Cytomegalovirus (CMV) often increase in magnitude with age; a phenomenon termed ‘memory inflation’. Elevated CMV-specific immunity is correlated with an increased mortality rate in elderly individuals and there is considerable interest in therapeutic approaches that may reverse this. Latent CMV infection is characterised by intermittent episodes of subclinical viral reactivation which may play a role in boosting CMV- specific immunity however, the relative importance of reactivation in the development of "memory inflation" is currently uncertain. In order to investigate these questions valaciclovir was administered as to aged mice with established murine CMV (MCMV) infection to block stochastic lytic reactivation from latency. Following 12 months of treatment there were highly significant reductions in the frequency of the MCMV-specific CD8\(^+\) T-lymphocytes and the residual MCMV-tetramer specific response exhibited a less differentiated phenotype. The accumulation of memory cells associated with untreated MCMV infection suppressed the proportion of naïve CD8\(^+\) T-cells by 60%, whereas antiviral treatment was able to completely restore this effect. Furthermore, valaciclovir treatment of MCMV reduced the elevated viral load that followed influenza virus challenge demonstrating that anti-MCMV treatment can lead to improved immunity to other pathogens in old age.

Feline immunodeficiency virus (FIV) is an important pathogen of domestic cats which in some cases can lead to feline AIDS. It shares many similarities with its human counterpart and is studied to understand correlates of immune-protection and mechanisms of disease progression in cats, both to improve the welfare of infected cats and as an animal model for the pathogenesis of HIV infection in humans. FIV is believed to evolve during the course of infection as a result of the error prone nature of reverse transcriptase and recombination between viral variants, but relatively little is known about this process in naturally occurring infection. Ultimately, it remains unknown why some infected cats remain healthy while others progress to AIDS rapidly. The studies reported in this thesis addressed this lack of knowledge by examining sequential blood samples obtained during the course of natural FIV infection in a population of 44 privately owned domestic cats. Employing Bayesian coalescent framework, it was demonstrated that the FIV env gene is relatively stable genetically. Although not necessary a prerequisite, this is likely to explain why many naturally infected cats can remain healthy and do not progress to AIDS. By determining the cell tropism of isolated viral variants, it was shown that sick cats were more likely to harbour viruses of the “late” phenotype than healthy animals, similar to the co-receptor switch observed during the progression of HIV infection. Intra-host diversity analyses highlighted a likely role for the leader region of the env gene in viral pathogenesis. Furthermore, recombination was demonstrated to be abundant in natural infection, indicating a requirement for the current phylogenetic classification of FIV to be revised. By assessing the strength and breadth of neutralising antibodies (NAbs), it was shown that NAbs did not appear to influence the course of natural FIV infection, arguing against a role in controlling infection and disease progression. Following an examination of samples collected from a group of privately owned Australian vaccinates, it was shown that the Fel-O-Vax FIV vaccine did not induce cross-reactive neutralising antibodies. Furthermore, in the country where commercial FIV vaccine is licenced, we identified and characterised the virus strain which was likely able to establish infection in vaccinated cat and raised concerns of vaccine’s efficacy. Overall this study broadens our understanding of natural FIV infection, and highlights that much can be learned, not from the similarities but rather by studying the differences between the feline and human lentiviruses. Such comparative studies are likely to contribute to design of highly desirable, safe and fully efficacious lentiviral vaccines.