Dengue viruses (DENV) are mosquito-borne flaviviruses that cause a severe febrile illness, and sometimes a potentially lethal syndrome called dengue haemorrhagic fever (DHF). Despite decades of effort, the global resurgence of dengue is testament to the inadequacy of current control measures. Dengue has become an immense international public health concern: WHO estimates that there are 50-100 million dengue infections and 500,000 cases of DHF hospitalised each year. Thus dengue has a major economic impact in the developing world through loss of healthy life and utilisation of constrained health resources. Global dengue control is likely to require a combined approach based on the development of successful strategies for immunization and antiviral drugs, as well as vector control. Better understanding of DENV pathogenesis presents new opportunities for design of rationally attenuated vaccine candidates and antiviral therapies. This thesis focuses on understanding a critical step in DENV pathogenesis: evasion of human innate immune responses mediated by interferon. A lentiviral vector system was developed to express dengue non-structural (NS) proteins in human cells, and we used this to show that expression of dengue NS5 alone inhibited IFN-α, but not IFN-γ, signalling. The IFN-α signalling cascade is blocked downstream of Tyk2 phosphorylation: NS5 binds to the transcription factor STAT2 and inhibits its phosphorylation. The polymerase domain of NS5 is sufficient to block IFN-α induced signal transduction, and inhibition does not require NS5 nuclear translocation. We finally tested several hypotheses to explain why STAT2 degradation occurs in both DENV-infected and replicon-containing cells, but not when NS5 is expressed alone. The most important conclusion from this work is that DENV NS5 is a potent and specific type I IFN antagonist. The results of this study are an important step in defining the molecular pathogenesis of dengue, and provide clues to potential new approaches to combat this disease.

The E6 protein from high-risk Human Papillomaviruses (HPVs) has previously been shown to be necessary for the persistence of viral episomes in cells, however, the mechanism for this remains unclear. High-risk E6 proteins have many activities including the ability to degrade p53 and the ability to bind to and degrade PDZ proteins. In this study I aimed to further elucidate the role of E6 in the persistence of viral episomes. I used two HPV16 mutant genomes with mutations in the E6 open-reading frame; one that is unable to degrade p53 (16E6p53m), and one that lacks the PDZ-binding motif (16E6PDZ). I found that both are unable to persist episomally in cells thereby implicating these two activities of E6 in HPV episomal persistence. Upon closer investigation of the two mutant genomes, I found that the 16E6p53m genome does not replicate as efficiently as the wild-type genome. This result suggests a function for p53-degradation in genome replication, and consequently in genome persistence. Furthermore, by carrying out a more detailed analysis of the relationship between E6 and the PDZ protein hScrib, I showed that the wild-type E6 protein is stabilised by virtue of the PDZ-binding motif, present on its C-terminus. On the other hand, the mutant E6 protein that lacks the PDZ-binding motif (E6PDZ) is more susceptible to proteasomal degradation. These findings provide evidence for a previously unknown outcome of the E6-PDZ protein interaction, in stabilising wild-type E6 protein. In addition to the implications of this stabilisation in the persistence of viral episomes, it is also significant when considering the activities and properties of E6 that contribute to the development of neoplasia. Finally, I have also found that wild-type HPV16 genomes cannot persist in cells that constitutively express E6 protein, suggesting that the correct regulation of E6 expression is crucial.

This PhD investigated the role of human cytomegalovirus (HCMV) in inflammatory disease associated with common variable immunodeficiency (CVID) by examining the functional competence of HCMV specific CD8+ T cell responses. The project was based on the hypothesis that HCMV is a major factor driving the expansion of CD8+ T cells that contribute to the inflammatory pathology. HCMV specific CD8+ T cell frequencies were significantly elevated in inflammatory patients compared to non-inflammatory patients or healthy subjects. The frequency of EBV (GLC) epitope specific CD8+ T cells did not differ between patient groups. HCMV CD8+ T cells from inflammatory patients displayed a distinct cytokine expression profile with the majority of cells producing IFN-γ only or IFN-γ and TNF-α in response to antigen stimulation. These cells did not show evidence of exhaustion, with low PD-1 expression; rather, they showed high functionality, high TCR avidity and high proliferative potential. CD8+ T cells from inflammatory patients but not non-inflammatory patients or healthy donors expressed high levels of Ki-67 and proliferated in response to antigen stimulation in vitro without co-stimulation. Further phenotypic analysis revealed striking correlations between the frequencies of HCMV specific CD8+ T cells expressing PD-1 or granzyme B and the overall frequency of CD8+ CD27-CD28- T cells. Consistent with their hypothesized role in the inflammatory disease, the CD8+ T cells from inflammatory patients expressed reduced levels of the anti-inflammatory marker CD73. Further support for the involvement of HCMV in driving the inflammatory pathology came from collaborative work in which viral antigen was detected at the sites of inflammation. The results support the hypothesis that HCMV and HCMV-specific T cells are key factors in CVID associated inflammation. They explain previously reported T cell ‘abnormalities’ seen in CVID and provide an evidence base for clinical trials of anti-TNF therapy and/or antiviral therapy in these patients.

The Wilms tumour antigen is a promising target for T cell-based tumour immunotherapies. Vaccines against WT1 peptides tested in cancer patients showed immunological and molecular responses. However, the clinical responses observed were partial and it is currently not known whether physiological levels of WT1 expression in some healthy tissues results in the deletion or tolerance induction of WT1-specific T cells. In this PhD project, TCR gene transfer into purified haematopoietic stem cells (HSCs) was used to study the thymic development of WT1-specific T cells and their fate in the periphery. Lentiviral constructs containing the genes for an HLAA2 allorestricted, murinised WT1 TCR or the genes for a control, viral peptide-specific, LMP2 TCR, were generated. The conditions for lentivirally-transduced HSC transplants were optimised. The results obtained from WT1 TCR tranduced HSC transplants in HLA-A2Kb transgenic mice demonstrated that thymocytes expressing this high-avidity WT1 TCR were positively selected into CD8 T cells and emerged in the recipient’s periphery. WT1-specific T cells exhibited a memory, CD44hi phenotype correlating with rapid antigen specific killing, proliferation and cytokine secretion of WT1-specific T cells in the absence of vaccination. LMP2-specific T cells exhibited a naive-like, CD44low phenotype without any antigen specific function. WT1-specific T cells persistent long-term in the periphery of transplanted mice, and no autoimmunity was noted. The results presented in this thesis show for the first time that T cell specificity for a tumour-associated, self-antigen did not result in tolerance induction, but instead mediated the spontaneous generation of functionally competent, memory phenotype T cells.

Unlike many parts of the world, both HIV-1 and HIV-2 are endemic in West Africa. The work in this thesis was based on a clinic cohort at the Medical Research Council Laboratories in the Gambia and a community cohort based in Caió, a rural village in Guinea-Bissau. Although HIV-2 was the dominant infection in these countries two decades ago, HIV-1 has increased rapidly in recent years. Chapter 3 demonstrates that although HIV-1 CRF02_AG is responsible for most HIV-1 infections in both cohorts, the subtype distribution varies considerably – including the presence of a novel circulating recombinant form (CRF49_cpx) in the Gambia, accounting for approximately 15% of all cases. Chapter 4 applies modern phylogenetic and phylodynamic techniques to viral sequences from rural Guinea-Bissau to characterise the molecular epidemiology and population dynamics of HIV-1 and HIV-2 in this region. Viruses from HIV-2 elite controllers and progressors can share a most recent common ancestor, suggesting that host factors are responsible for the dichotomous outcomes observed in HIV-2 infection. Chapter 5 explores whether humoral immunity plays a role in HIV-2 non-progression. Neutralising antibody responses of remarkable magnitude and breadth are seen in the plasma of HIV-2 infected subjects, yet there is no direct relationship to viral control. Chapter 6 demonstrates that polyfunctional HIV-2 Gag-specific CD8+ T-cell responses are, however, associated with control of HIV-2. The phenotype of these CD8+ T-cells varies from what is thought to be important in HIV-1 control. In contrast to HIV-1, escape from both humoral and cellular immune responses may be limited in HIV-2. The aim of this work was to help understand the spread and transmission of HIV-1 and HIV-2 in West Africa, as well as to provide insight into why most HIV-2 infected individuals behave as long term non-progressors, whereas others progress to AIDS like their HIV-1 infected counterparts.

Alloreactive immune responses directed against malignant cells in recipients of allogeneic haematopoietic stem cell transplants are able to cure patients with haematological cancers. However, such immune responses may cause severe morbidity when directed against healthy recipient tissue, resulting in graft-versus-host disease (GvHD). Naturally occurring regulatory T cells (Tregs) are CD4+ T cells characterized by their expression of the transcription factor Foxp3. Whilst adoptively transferred polyclonal Tregs suppress GvHD in several murine models, their lack of specificity may compromise beneficial immunity against malignancy or infection. The generation of MHC class I-restricted, alloantigen-specific Tregs would allow them to recognize antigen presented directly on GvHD target tissues, concentrating their sites of activation at these tissues and potentially reducing non-specific immune suppression. I have generated ‘converted’ Tregs through retroviral transfer of genes encoding Foxp3 and specific MHC class I-restricted T cell receptors (TCRs) into conventional CD4+ T cells. I used the 2C-TCR, which recognizes the MHC class I molecule H-2Ld, expressed in Balb/c and other H-2d mice, in complex with the ubiquitously expressed peptide p2Ca; and the MH TCR, which recognizes the MHC class I molecule H-2Db, expressed in B6 and other H-2b mice, in complex with the male peptide WMHHNMDLI. In vitro, Foxp3 2C-TCR-transduced B6 CD4+ T cells are hyporesponsive to stimulation and are able to suppress the alloreactive proliferative response of B6 CD4+ and CD8+ T cells to Balb/c splenocytes, consistent with the acquisition of regulatory function. When adoptively transferred to lethally irradiated Balb/c recipients of MHC-mismatched B6 bone marrow and conventional T cells, Foxp3 2C-TCR-transduced B6 CD4+ T cells reduce early proliferation of donor T cells, weight loss and GvHD score in the recipients. Similarly, CD4+ T cells transduced with Foxp3 and the MH-TCR are able to suppress allogeneic responses both in vitro and in vivo. However, while both the 2C-TCR and the MH TCR confer specificity to their cognate antigens in vitro, antigen specificity of suppression is not evident in these in vivo models. In this thesis I show that the endogenous TCR of transduced CD4+ T cells contributes to this lack of specificity, a finding that has important implications for the use of class I-restricted TCRs alongside Foxp3 in CD4+ T cells to direct regulatory activity.

The Drosophila cytokine Spätzle (Spz) triggers the Toll signalling cascade in flies upon infection with Gram-­‐positive bacteria and fungi. It is an analogue to the mammalian IL-­‐1 and is produced by the fat body as a precursor protein (Spz-­‐ precursor). Active Spz is produced from its precursor by a tightly regulated cascade of serine proteases. However, the transcriptional regulation of Spz in Drosophila in response to bacterial or fungal infection is still poorly deciphered. Therefore, the aim of the project was to interrogate the regulation of Spz transcription, with specific interest in hemocytes, which are equivalent to mammalian monocytes/macrophages. For this purpose, we have generated and characterised a transgenic Spz-­‐neGFP reporter that allows the visualisation of Spz transcription in individual cells and tissues during different stages of development. We have identified populations of cells that transcribe the Spz gene in the steady state throughout development. We have infected the transgenic reporter flies with the fungus Candida albicans and examined the transcription of Spz after immune challenge. In future, these strains and results will help develop an in vivo genome wide assay, employing Drosophila RNAi libraries, in order to gain more knowledge about the transcriptional regulation of Spz and to identify novel regulatory genes and pathways for Spz transcription.

Immunity declines with ageing resulting in an increase in incidence and severity of infections such as shingles. We used the delayed type hypersensitivity response as a model for a memory T cell response to investigate differences in the response to VZV antigen administered into the skin between old and young individuals. We found that there is a reduced clinical response, which is associated with a lack of T cell accumulation in the old subjects. The earliest differences between the two groups include reduced activation of dermal endothelium and a reduced infiltration of CD11c+ dendritic cells in the skin of the old individuals after VZV antigen injection. This may reflect a defect in the ability of VZV specific T cells and / or innate immune cells resident in the skin to condition the cutaneous environment adequately to induce an effective adaptive immune response. There is an increased level of Tregs in the normal skin of old individuals and these Tregs may suppress the activation of skin resident cells, thereby reducing early recruitment of memory T cells from the circulation as part of the immune response. Without adequate recruitment and activation of a few VZV specific T cells, we propose there is failure to produce additional mediators needed to amplify the memory response.

CD4 T cell lineage choice epitomises the ability of the immune system to become tailored to a specific threat and provides a framework for understanding the mechanisms behind cell specification. The differentiation of T effectors from naïve cells gives rise to pro-inflammatory lineages including T helper 1 (Th1) and Th2 and anti-inflammatory regulatory T cells (Treg). An additional lineage of Treg also exits the thymus in parallel to naïve T cells and together these Treg are required for prevention of autoimmunity. These T cell lineages are distinct in terms of their cytokine production and functional effects but also through their differences in gene expression and its regulation, which are orchestrated by the presence of lineage-specifying transcription factors specific for each lineage. In addition, post-translational modification of histones also provide insights into this transcriptional regulation and more recently the pervasive and tissue-specific transcription of multiple classes of RNA species without protein coding capacity, non-coding RNA (ncRNA), has been found to play a role in cell differentiation and function. In this thesis I identify several ncRNAs with differential expression different T cell lineages. This includes ncRNAs upregulated Treg compared to T responders. The characterisation of these, including their expression in the autoimmune context of systemic lupus erythematosus (SLE), is presented and their possible biological functions are examined. The relevance of histone modifications for influencing Treg identity in SLE is also investigated. An additional class of ncRNAs that originate from gene enhancer regions, eRNA, is also investigated in the context of Th1 versus Th2 lineage choice. This enhancer transcription is increased genome-wide in Th1 cells at enhancers with high density T-bet binding in, termed ‘super-enhancers’. The functional relevance of these eRNAs, including at the super-enhancer upstream of the Th1 signature cytokine gene, IFNG, is also investigated in knockdown experiments.

This thesis examines the evolution of HIV-1 subtype C multiple linked multi-class antiretroviral resistance mutations in the viral protease (PR) and reverse transcriptase (RT) genes of vertically infected children. Emergence of PI resistance on the backdrop of pre-existing non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance could compromise long-term treatment options in such children. We characterised multi-class drug resistance using single genome sequencing (SGS) in children with viraemia while receiving PI-based ART. We applied SGS of HIV-1 protease (PR) and reverse transcriptase (RT) to longitudinal samples from a cohort of the Children with HIV Early Antiretroviral Therapy (CHER) trial with viral loads >1000c/ml after 40 weeks of early ART. Bulk sequencing revealed NVP-selected resistance in 50% of these children while SGS revealed NVP-selected resistance in 70%. Two children had baseline NRTI and PI mutations, suggesting previous maternal ART. Linked multi-class drug resistance following PI-based ART was detected by SGS in 2/10 children. In one child, the majority species contained M184V in RT linked to L10F, M46I/L, I54V and V82A in PR and a triple-class drug resistant variant with these mutations linked to the NNRTI mutation V108I. In the second child, the majority species contained M184V and V82A linked within viral genomes. I correlated nucleotide variation of PR-RT with the number of single genomes obtained at each time point and ART status and used maximum likelihood trees, recombination analysis, positive selection analysis and co-evolution analysis to describe the evolution of PR-RT of the viral populations. Six children who received early ART for 40/96 weeks only or received continuous ART for the duration of the CHER trial had clusters of identical sequences from baseline and week 40 of ART. These sequences did not harbour known drug resistance mutations. Therefore one could hypothesize viral replication from a persisting viral reservoir that was established from infection that occurred prior to the initiation of ART. The rooted ML trees of 2 children who developed drug resistance during ART had clusters of identical sequences harbouring common drug resistance mutations from multiple time points which is characteristic of the selection of drug resistant viral populations that cause virological failure during ART. When drug resistant viral populations developed during treatment failure, M184V single mutated viruses were selected from multiple wildtype viral populations but only one population became the major contributor to drug resistant viraemia in both children. Triple-class drug resistant sequences that had common DRMs (M184V, V108I in RT and M46I in PR) did not cluster together. I found no evidence of recombination or coevolving sites in PR-RT for any of these children. I used a luciferase based single replication cycle assay to examine drug susceptibility and replication capacity (RC) conferred by multi-class drug resistant PR-RT from the 2 children who developed such drug resistant variants. I tested the susceptibility of pseudoviruses to the components of early ART (AZT, 3TC and LPV), the components of second-line therapy for these children (Abacavir (ABC), Didanosine (ddI), Efavirenz (EFV) and (NVP)), the PIs Nelfinavir (NFV) and Saquinavir (SQV), which are also approved for use in children and Darunavir (DRV), which has been identified as a PI option needed in paediatric co-formulation. Pseudoviruses with known PI resistance conferring mutations showed reduced susceptibility to all PIs except DRV. Those with known NNRTI resistance conferring mutations showed reduced susceptibility to EFV and NVP. M184V mutated pseudoviruses conferred high-level resistance to 3TC. In one child, a combination or one of the RT mutations V35T, E36D, T39R, S48T, T165I, K173A, D177E, T200A, Q207D, R211K, V245Q, E248N, D250N, A272P, K277R, E291D, I293V, T296N may be associated with high-level ABC and ddI resistance when genetically linked with M184V. Population sequence analysis was used to characterize the viral gag genes that encoded matrix, capsid, nucleocapsid, p6, and spacer peptides 1 and 2 along with PR-RT as a single amplicon. I determined the presence of compensatory PI-resistance mutations in gag, drug resistance mutations in PR and RT and other amino acid changes that occurred during ART. To determining the polymorphic nature of these sites, I compared them to a position-specific scoring matrix for gag that was derived from HIV-1 subtype C sequences from children from Sub-Saharan Africa. P453L in the p1/p6 cleavage site of Gag emerged in the viral population of one child during PI-based ART. It was the only amino acid change in Gag that emerged among all children in the study cohort that has been characterised as a compensatory mutation that is selected by and enhances PI-resistance. This project is the first to identify multi-class drug resistance mutations in PR and RT that were linked on the same genome as well as characterise their development during early PI-based ART in children. Triple class drug resistant viruses detected in the minority species of the viral population of one child demonstrated significant levels of resistance to LPV, SQV, NFV, 3TC and NVP, and established that such variants could compromise future ART regimes if they became the dominant species of the viral population. I note that the small convenience sample (n = 10) chosen for this project limited the power of this study so that findings could not be generalized.