Tuberculosis (TB) is an important communicable disease affecting the human population world-wide. Despite the efforts of the scientific community, national governments and WHO in controlling the disease, TB still remains a major killer in resource poor settings. New rapid assays and techniques that are simple and cost-effective are urgently needed to identify, treat and understand pathogenesis including the geographical distribution of the disease. The aim of the thesis is to develop a novel genomic mapping tool using Insertion Element, IS6110 that could aid in epidemiological studies of Mycobacterium tuberculosis complex (MTBC) in low and middle income countries. IS6110, a bacterial transposon, plays an essential role in changing the physical and biochemical traits of MTBC. Due to their transposition in TB genomes, they are used as epidemiological markers for differentiation of TB organisms and the mapping of these elements could also shed light on the putative altered function of adjacent genes. In the era of Whole Genome Sequencing (WGS) where repeat elements are difficult to sequence with short read technologies, a rapid and simple method of insertion site mapping using IS6110 FAFLP PCR was developed. This work is aimed at developing a rapid, cost-effective and robust genomic tool box exploiting the IS6110 FAFLP PCR assay that can both identify and characterise the TB genotypes / genetic lineages in any geographical location. For the first time using the assay above, TB samples from Nepal were categorised into different genetic lineages. Fifty-five percent of the samples analysed belong to Principal Genetic Group 1 (PGG1), Beijing and Central Asian strains. Also, new primers were designed targeting the Beijing and the T- groups using the FAFLP derived data that gave rise to the development of rapid lineage specific PCR assays. In addition, it was noticed that 3.9% of the Nepalese strains tested in this research work were likely multi-drug resistant (MDR-TB) using PCR targeting the Rifampicin-resistance-determining region (RRDR) of the rpoB region. It is demonstrated here that IS6110 FAFLP methodology could easily characterise the TB samples into different genetic lineages provided they have more than four IS6110 copies. In addition, lineage specific PCR does not need any expensive instruments or reagents except for PCR blocks and gel visualisers, and could be very effective in the rapid identification of different TB genotypes within hours. These data also add to knowledge about the circulating strains of TB in Nepal, currently a poorly characterised region of the world in this regard, and could help in contact tracing studies by epidemiologists. The IS6110 FAFLP technique thus can be employed in any geographical location to map TB genetic lineages where there is little or no information available on the prevailing TB strains.

The age-related decline in the immune system, known as immunosenescence, predisposes old individuals to increased mortality and morbidity from infectious diseases (such as shingles, caused by reactivation of varicella zoster virus, VZV) and is associated with impaired vaccine responses. While multiple age-related changes have been identified in circulating memory T cells, little is known about the effects of ageing on memory T cells within peripheral tissues. T cells within human skin were characterised, and surprisingly neither their numbers, differentiation markers or effector functions were altered during ageing. VZV-specific CD4+ T cells were more frequent in the skin than in the blood, and their frequency in the skin did not decline with age. Increased PD-1 expression on T cells and an increased frequency of regulatory T cells in ageing skin consolidated evidence that old skin represents an inhibitory microenvironment. Vaccination of old individuals with the shingles vaccine Zostavax® did not alter the total number of T cells, or frequency of VZV-specific T cells, in the skin but did boost the frequency of circulating VZV-specific T cells. This was associated with a heightened delayed type hypersensitivity (DTH) response to VZV antigen, and with an upregulation of genes involved in T cell migration and activation. It is proposed here that Zostavax® prevents shingles by enhancing the recruitment of circulating VZV-specific memory T cells to sensory nerves during episodes of silent VZV reactivation. Increased early expression of p38 MAPK-associated pro-inflammatory genes has been observed in VZV- or saline-challenged old skin and was associated with poor DTH responses to VZV antigen. In an experimental medicine study, pre-treatment of ageing individuals with the p38 MAPK inhibitor losmapimod effectively restored robust VZV-specific DTH responses. Ageing of the population necessitates improved vaccination strategies, and p38 MAPK inhibition prior to vaccine administration presents a potential therapeutic opportunity to achieve this.

The MAP 3-kinase TPL-2 is required for activation of ERK1/2 MAP kinases in macrophages after stimulation of Toll-like receptors and the receptors for TNF and IL-1β. TPL-2 drives inflammation in a number of autoimmune and inflammatory disease models, and consequently is considered a potential anti-inflammatory drug target. Recent evidence from the Ley laboratory has indicated that TPL-2 regulates the production of the critical pro-inflammatory cytokine TNFα in an ERK1/2-independent manner. The aim of this study was to use mass spectrometry-based phosphoproteomics to investigate the signalling pathways regulated by TPL-2 in an unbiased manner to more fully understand how TPL-2 regulates innate immunity. A method for SILAC labelling of bone marrow-derived macrophages was developed and used to quantify phosphorylation changes after genetic and pharmacological perturbation of TPL-2 signalling. Across 11 pair-wise comparisons, 31,457 unique phosphosites were identified, from which a shortlist of 37 potentially TPL-2-dependent, ERK1/2-independent phosphorylations was generated. The biological consequences of the majority of these TPL-2 regulated sites remain to be determined, although bioinformatic analysis suggest that they may have roles in regulation of gene expression and the cytoskeleton. The activating residues of the MAP 2-kinases, MKK3 and MKK6 were validated as novel TPL-2 targets in LPS-stimulated macrophages and MKK6 was shown to be a direct substrate by in vitro kinase assays using recombinant proteins. It was also demonstrated that TPL-2 controlled the phosphorylation of the activation loop of the MAP kinase p38γ, which is a direct target of MKK3/6. These data identified a novel signalling pathway downstream of TPL-2, increasing our understanding of how TPL-2 controls inflammatory responses.

Mycobacterium tuberculosis is an intracellular pathogen that arrests phagosome maturation to survive within macrophages and cause disease. The process of phagosome maturation is important for the innate immunity against intracellular pathogens as well as the adaptive immunity and antigen processing. Activation by cytokines such as IFN-γ can modulate immune responses to infection. Several Rab GTPases regulate M. tuberculosis phagosome maturation, highlighting the importance of intracellular trafficking in this process. There is compelling evidence that M. tuberculosis can colonise several niches within macrophages, both in heterogeneous membrane-bound compartments as well as in the cytosol. In this thesis, the control of M. tuberculosis infection by IFN-γ through modulation of intracellular trafficking and thereby the intracellular niche occupied by this pathogen was investigated. First, a novel IFN-γ/Rab20 dependent pathway that targets pathogens into spacious phagosomes for elimination in macrophages is described. IFN-γ induced Rab20 expression and association with phagosomes. The analysis of spatiotemporal dynamics of M. tuberculosis phagosomes showed that in contrast to Rab5, PI3P or Rab7; EGFP-Rab20 was retained on M. tuberculosis phagosomes for up to 24 hours, which was comparable to endogenous Rab20 association with M. tuberculosis phagosomes in IFN-γ activated macrophages. IFN-γ increased interaction of M. tuberculosis phagosomes with the endosomal network via Rab20, targeting mycobacteria to spacious, proteolytic compartments resulting in inhibition of M. tuberculosis growth. Furthermore, this targeting of M. tuberculosis to spacious phagosomes reduced phagosomal damage and access of mycobacteria to the cytosol. In resting macrophages, M. tuberculosis was able to subvert this targeting to spacious compartments in a manner that was dependent on its ESX-1 secretion system. Altogether, this work reports a cell-autonomous, IFN-γ/Rab20 dependent pathway that results in elimination of M. tuberculosis by retention of mycobacteria in membrane-bound spacious compartments.

Airway epithelia are the first targets of influenza A virus (IAV) infection and the first cells to respond, contributing to immunity, pathology and recovery. The magnitude and quality of epithelial responses may depend on exacerbations like bacterial superinfections and be determined by the host genetic background. We therefore use a primary mouse tracheal epithelial cell (mTECs) cultures to identify epithelial determinants of susceptibility and protection. We assessed mTEC responses to IAV-bacterial co-exposure and found that the massive in vivo cytokine and chemokine response to co-infection is reflected in vitro by strongly increased epithelial responses to a combined viral-bacterial stimulus compared to single stimuli. The antiviral transcriptional responses dominate the overall response in vivo and in vitro. We also compared the epithelial response to IAV between high interferon producing, susceptible 129S8 mice, and IAV-resistant moderate interferon producers, C57BL/6 mice. We found that 129S8-derived mTECs do not produce more interferons in response to IAV but respond more strongly to interferons and IAV by induction of cytokines and interferon-stimulated genes. 129S8-derived epithelia also proliferate and differentiate less well than C57BL/6 mTECs, suggesting reduced repair potential. The initial epithelial cell-intrinsic antiviral response and control of IAV depends on type I and/or III interferons (IFNαβ or IFNλ). When IFNα and IFNλ influenza treatments were compared, IFNα stimulated both innate immune cells and mTECs, increasing immunopathology and mortality. IFNλ treatment only induced antiviral epithelial responses but not the immune-mediated pathology triggered by IFNα. Therefore, IFNλ treatment helps control IAV and protects without inducing immunopathology. During recovery, lung epithelial stem cells must proliferate and differentiate to repair infection-induced epithelial damage. We found that IFN impaired epithelial regeneration by reducing stem cell proliferation and differentiation, likely through IFN-induced blockade of epidermal growth factor signalling. Thus antiviral immune responses, if mistimed or excessive, may impede post-infection lung repair. Together, mTECs help identify determinants of susceptibility and protection.

Most of our understanding of bacterial chemotaxis comes from studies of Escherichia coli. However, recent evidence suggests significant departures from the E. coli paradigm in other bacterial species. In the first part of this work, we argue that the observed departures may stem from different species inhabiting distinct environments and thus adapting differently to specific environmental pressures. We therefore study the performance of various chemotactic strate¬gies under a range of stochastic time- and space-varying attractant distributions in silico. We describe a novel type of response in which the bacterium tumbles more when attractant concen¬tration is increasing, in contrast to the “adaptive” response of E. coli, and demonstrate how this response explains the behavior of aerobically-grown Rhodobacter sphaeroides. In this “specu¬lator” response, bacteria compare the current attractant concentration to the long-term average. By tumbling persistently when the current concentration is higher than the average, bacteria maintain their position in regions of high attractant concentration. If the current concentration is lower than the average, or is declining, bacteria swim away in search of more favorable con¬ditions. When the attractant distribution is spatially complex but slowly-changing, this response is as effective as that of E. coli. In the latter part of this work, we show that optimal response sensitivity is high for both adaptive and speculator responses. We argue that response sensi¬tivity would increase over long evolutionary timescales and show that increases in response sensitivity could drive the evolution of adaptive and speculator responses.

Adoptive transfer of T cell receptor (TCR) gene-modified T cells is a promising field of tumour immunology. However whilst these cells can potently control tumour growth, this occurs in only a subset of patients. In order to devise new strategies to improve the anti-tumour response we need a clear understanding of the mechanisms by which transferred T cells are activated to kill tumours. Whilst dendritic cells (DC) are required to activate and control T cell function in adaptive immune responses it is not known whether control of tumours by adoptively transferred T cells depends on similar interactions with endogenous DC. To address this the CD11c.DTR model was used to transiently deplete DC in mice with established B16.F10 sub-cutaneous tumours after having been treated with TCR-transduced T cells. Unexpectedly we found that depletion of CD11c+ cells facilitates enhanced expansion and effector-phenotype differentiation of the transferred T cells. This appears to be mediated by depletion of a DC population with regulatory capabilities. However depletion of DC in the closely related CD11c.DOG model fails to promote accumulation of TCR-transduced T cells. Indeed, in this model DC depletion leads to less T cell infiltration into tumours. These contrasting results following depletion likely reflect the heterogeneous CD11c+/DC compartment in the tumour, where different populations contribute pro- or anti-inflammatory roles. Nonetheless these data suggest that TCR-transduced T cells interact with the endogenous immune system, although the exact nature of this interaction requires further investigation.

HSV-1 and HIV-1 co-infection of human macrophages represents a clinically relevant model with which to investigate the host-pathogen interactions between macrophages and viruses. In this thesis, I demonstrate that HSV-1 productively infects human monocyte derived macrophages, with associated cell death and type I IFN responses, and additionally, that a proportion of macrophages support latent HSV-1 infection. I de ne latency as the absence of lytic gene transcription, virion production and cell death, in the presence of persistent expression of the HSV-1 latency associated transcript (LAT). I also demonstrate that HSV-1 super-infection increases HIV-1 transcription, and that latent HSV-1 can be reactivated by HIV-1. HSV-1 latent infection of neurons is well established, but this is the rst report, to my knowledge, of HSV-1 latent infection of myeloid lineage cells. The potential for macrophage reservoirs of latent HSV-1 may be an important factor for the clinical management of persistent reactive HSV-1 disease. Furthermore, HIV-1 infection in vivo is known to increase the frequency of HSV-1 reactivation in the host through the indirect mechanism of immune system suppression. However, a direct interaction between cells latently infected with HSV and HIV-1 has not previously been observed. Reactivation of latent HSV by HIV-1 therefore provides a novel mechanism for the well-established clinical synergy between these viruses.

Adoptive transfer of antigen specific regulatory T (Treg) cells was shown to be beneficial for the suppression of autoimmunity. In this study, we engineered antigen-specific Treg-like cells that recognise mouse MHC-class II molecules. The restricted expression of MHCII to professional antigen presenting cells and to inflammatory sites, allow the employment of MHCII specific Treg for suppression of unwanted immune inflammation. A mouse MHCII-specific Chimeric Antigen Receptor (CAR) was used to redirect the specificity of T cells via retroviral transduction, while Foxp3 gene transfer into purified CD4+ cells leads to the acquisition of a Treg-like phenotype. Incorporation of a suicide mechanism within the engineered Treg-like cells for their in vivo selective deletion will prevent long-term immune suppression. The functionality of the generated CAR and the specificity of responses elicited by CAR bearing T cells were validated in vitro. In preliminary in vivo experiments, intravenous injection of C57BL/6 mice with syngeneic mouse MHCII-specific CD4+ T cells, led to GVHD-like toxicity, while no signs of toxicity were observed when Treg-like cells of the same specificity were transferred alone or in a 1:1 mix with mouse MHCII specific CD4+ T cells. These data suggest the suppressive potential of the engineered Treg-like cells.

The success of ART has led to HIV infected individuals having normal life expectancies compared to the general population. However ART can only control the virus and not cure it and HIV persistence in long-lived cells represents one of the major barriers to HIV eradication. Moreover, an aging infected population shows an increase in cancer incidence. The classical successful strategy to treat cancer is chemotherapy by agents that can be either cytotoxic or immunomodulatory (IMiDs) to immune cells. Cytotoxic agents kill immune cells and therefore have the potential to constrict or shift the viral population via the activation of a new pool of latently infected CD4+ T-cells. IMiDs cause cellular activation and have the potential to clonally expand or shift the viral population. Using quantification of viral nucleic acids and single genome sequencing of longitudinally obtained samples from HIV infected individuals with comorbid neoplasms we demonstrate the effect of chemotherapy on HIV population dynamics. In individuals not on ART a change to viral population size and structure was seen in 1) a single case of Tcell ablation and disruption of viral control followed by a tropism switch and rebound of CCR5 using virus in an elite controller following myeloablative chemotherapy, and 2) a shift in viral population in an individual undergoing intensive chemotherapy. In individuals on suppressive ART we demonstrate that in the absence of strong effects on T-cells no change to HIV population size or structure was detected in 1) individuals undergoing treatment with a single cytotoxic agent, or 2) a cohort undergoing treatment with the IMiD pomalidomide. These results are important in the context of longer survival of HIV infected individuals, and therefore subsequent increased cancer risk and exposure to cancer drugs. This work shows that in the absence of ART chemotherapy agents with a detectable effect on CD4+ and CD8+ T- cells can alter the viral reservoir structure, reinforcing the need for ART alongside chemotherapy in the context cancer. This work demonstrates that these agents are unlikely to be of benefit in cure strategies but offer a new insight into a potential utility of CCR5 antagonists during future functional cure protocols.