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.

Although Epstein–Barr virus (EBV) usually establishes a harmless infection in human memory B cells, it is implicated in the development of germinal centre (GC) B-cell-derived malignancies, including Hodgkin’s lymphoma (HL). I have shown using gene expression profiling that lymphoblastoid cell lines derived from GC B cells are a useful model for studying early EBV-associated changes contributing to the pathogenesis of HL. EBV infection of GC B cells is followed by the up-regulation of the DNA methyltransferases, DNMT3A, and the down-regulation of DNMT1 and DNMT3B, a pattern of expression which is re-capitulated in HL cell lines. I have also shown that the major EBV oncogene, LMP1, is responsible for the down-regulation in GC B cells of DNMT1, and that DNMT3A binds to the EBV promoter, Wp which is silenced by DNA methylation. Genome-wide promoter arrays revealed that EBV infection of GC B cells is followed by methylation changes in a substantial number of cellular genes. These changes were not randomly distributed across the genome but clustered at certain chromosomal locations and were strongly associated with the CpG content of gene promoters. Finally, I have shown that EBV also modulates the expression of another set of epigenetic regulators which control arginine methylation.

Measles is a leading cause of mortality in infants in countries with suboptimal vaccination coverage. This disease is caused by a negative-strand RNA virus, measles virus (MeV). Wild-type strains of the virus use two cellular receptors to invade cells and establish infection: the signalling lymphocyte activation molecule f1 (SLAMF1), which is present on certain immune cells, and nectin-4, which is expressed in the lung epithelium. During infection, MeV can spread through the release of virions or by inducing cell-cell fusion. The aim of this thesis is to determine the molecular mechanism underlying viral entry and exit. Herein, I observed that, upon attachment to SLAMF1+ cells, MeV particles induce extensive but transient membrane blebbing and cytoskeleton contraction. MeV entry occurred simultaneously with fluid-uptake and was sensitive to inhibitors of macropinocytosis and cytoskeleton dynamics. In contrast, the cortical actin network restricted the early stages of MeV-induced cell-cell fusion, in RhoGTPases, ezrin and moesin dependent manner. By resolving the proteome of infected cells, conserved phosphorylated residues in the viral haemagglutinin were also shown to impact on dimerization and cell-cell fusion. These results suggest the manipulation of several cellular components and pathways during entry and exit of MeV.

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.

Hepatitis C virus is an infectious disease affecting millions of people worldwide and causing chronic liver disease. The current standard of care is not only long but causes numerous side effects. Due to incomplete virological response and poor tolerability, only 50% of the patients are cured, with variability by genotype. Despite the development of non-enzymatic viral protein inhibitors, new therapies target mainly enzymes responsible for viral replication or translation. Being commonly used for antiviral and anticancer therapy, nucleosides analogues have played an important role as anti-HCV agents. Despite their potency and selectivity, nucleoside analogues appear to be poor substrates for metabolic enzymes. In particular, the first essential phosphorylation step is often rate-limiting thus, resulting in poor bioactivation to the active triphosphate form. Hence, monophosphate prodrug strategies have been applied to efficiently deliver intracellularly the key monophosphate derivatives. Such strategies have been successfully used for anti-HCV therapy and the phosphoramidate ProTide INX-08189, discovered in our lab, is one such example. Aiming at developing back-up molecules of INX-08189, we report in the present work, the synthetic strategies to obtain several modified β-2’-C-methyl-6-O-methyl guanosine and other modified β-2’-C-methyl purine nucleoside analogues. The phosphoramidate ProTide approach and the phosphorodiamidate approach were applied to these modified nucleosides. In-vitro, and sometimes in-vivo evaluation against HCV replication is reported, and the mechanism of bioactivation to their corresponding monophosphate species is discussed. Enzymatic experiments using carboxypeptidase Y and Huh-7 cell lysates were carried out to investigate the release of the monophosphate forms. We also investigated the hydrolysis of the 6-O-methyl group at the nucleoside level with adenosine deaminase enzyme, and at the monophosphate level using molecular docking in adenosine deaminase like protein-1. Eventually, the intracellular putative mechanism of activation of the ProTides was studied using molecular modeling with cathepsin A enzyme and human Hint-1 phosphoramidase.

Classical Hodgkin lymphoma (cHL) is one of the commonest lymphomas in the developed world, frequently affecting young adults. The majority of patients will be cured of their disease, but the toxicities of the therapy required to achieve this can lead to long-term morbidity in survivors. In addition, whilst most patients are cured, there remains approximately 15% -20% of patients who will not respond to primary therapy and may ultimately die of their disease. Approximately one-third of cases of cHL in the developed world are associated with the Epstein-Barr virus (EBV), where this association is believed to be causal. A ubiquitous herpesvirus, persistently infecting more than 95% of the world’s population, precisely how this virus causes malignant disease in a minority of immune competent hosts remains ill-understood. Epidemiological evidence points to inherited genetic factors. Long-recognised to have an association with the class I human leukocyte antigen (HLA) system, recent studies have confirmed that risk of EBV-associated cHL is related to an individual’s HLA-A* allotype, with HLA-A*01:01 being associated with increased risk of disease and HLA-A*02:01 being protective. Heterozygotes are observed to have an intermediate risk. HLA plays a central role in the recognition and cell killing of virally-infected or malignant cells by the cytotoxic T lymphocytes (CTLs) of the cell-mediated immune system. The exact mechanism whereby HLA-A* exerts its effect on risk of cHL unknown, but CTL responses to EBV in this context are hypothesised to be crucial. The CTL response to EBV is well-studied. Immunodominant epitopes restricted through common class I alleles have been described, many directed towards peptides derived from proteins expressed in the lytic cycle of viral infection. In spite of intensive study, no confirmed HLA-A*01:01-restricted EBV-specific CTL responses have been described, raising the possibility that absent or weak CTL responses specifically to EBV might lead to elevated risk of disease. However, particularly given the intermediate risk of disease seen in HLA-A*01:01 heterozygotes, it remains a possibility the HLA-A*01:01-associated risk might be due to qualitative or inhibitory changes to the EBV-specific immune response. The work of this thesis set out to address a number of specific questions regarding the role of HLA class I in the aetiology and clinical outcome of cHL. Firstly, whether an HLA-A*01:01 allele could modify the magnitude of the CTL response to HLA-A*02:01-restricted epitopes was examined. In a study of healthy adults examining CTL responses using interferon-γ ELISPOT, overall HLA-A phenotype did not significantly affect the EBV-specific CTL response restricted through HLA-A*02:01. However, exploratory analysis of cytokine levels in response to stimulation with EBV peptides demonstrated significantly higher secretion of IL-10 (with a nearly 10-fold difference), IL-17 and IL-5 in response to stimulation with EBV peptides in HLA-A*02:01/A*01:01 heterozygotes, compared to other HLA-A*02:01 phenotype groups. This suggests a possible effect of HLA-A*01:01 in HLA-A*02:01/A*01:01 heterozygotes which might begin to explain some of the HLA-associated differences in risk of developing EBV+ve cHL. Secondly, again in a study of healthy EBV-seropositive adults, and using sensitive methodologies, HLA-A*01:01-restricted EBV-specific CTL responses were sought, and, in an exploratory analysis, cytokine responses were examined. No HLA-A*01:01-restricted CTL responses to EBV were detected in this study, however, exploratory analysis demonstrated statistically significant differences in cytokine levels following simulation with EBV, with HLA-A*01:01 homozygotes generating much higher levels of IL-6. Lastly, given the importance of class I HLA in determining risk of developing EBV+ve cHL, a study of 424 patients with cHL was performed to determine if HLA-A*01:01 and A*02:01 alleles are a factor in determining clinical outcome. In this study, HLA-A*02:01 was associated with inferior overall survival (OS) and disease-specific survival (DSS) in EBV+ve cHL, and was independently prognostic in an adjusted analysis. Given the extremely poor outcomes seen in this study in HLA-A*02:01 carriers with EBV+ve disease (61.7% 10-year OS), it is possible that this group of patients is not currently being well-served by standard first-line therapy and may benefit from novel therapies.

Hepatitis C virus (HCV) infection is one of the major causes of liver morbidity and mortality worldwide. While effective therapies are now available, if eradication of this virus is to be achieved globally, an effective vaccine is still necessary. During hepatitis C virus (HCV) infection, broadly neutralizing antibody (bNAb) responses targeting E1E2 envelope glycoproteins are generated in many individuals. It is unclear if these antibodies play a protective or a pathogenic role during chronic infection or if they could prevent infection or reinfection with the virus. I investigated the presence and clinical associations of bNAb responses in three cohorts of individuals infected with or exposed to HCV infection. One with chronic HCV infection at differing disease states, one with chronic HCV infection at an early disease state and one group of individuals at high risk of HCV exposure who remained uninfected by conventional testing. I also studied bNAb responses in an individual from a HCV-HIV co-infected cohort who experienced spontaneous clearance of HCV after a post-therapy relapse (‘secondary spontaneous clearance’). I found a proportion of individuals when exposed to or infected with HCV produce a polyclonal bNAb response which may contribute to viral clearance in some cases. Host genetics and the ability to target multiple neutralising epitopes on the envelope protein are associated with such responses, although resistance mutations to bNAbs do exist in vivo. The presence of bNAbs is associated with lower levels of liver fibrosis. Using next generation sequencing technology in the study of B cell receptors in HCV infection revealed subtle changes in the B cell repertoire on HCV infection, this technology may be used in future to gain insight into the generation of bNAb responses.

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