Upon infection with a virus, a cellular innate immune response is rapidly initiated to contain the pathogen. A potent interferon (IFN)-α/β cytokine response underlies host defence by prompting the expression of antiviral genes and stimulating adaptive immunity. Pattern recognition receptors (PRRs) are integral components of this response, as they recognize molecules specific to pathogens, including viral nucleic acids and couple to IFN induction. For my studies, I pursued lines of investigation aimed at deepening our understanding of immune responses trigered by the sensing of viruses within the cell cytosol. Initially, I focused on the PRR RIG-I. This RNA helicase senses RNA present in the cytosol of mammalian cells infected with certain negative-sense RNA viruses. Although many molecules capable of activating RIG-I had been proposed, the precise nature and contribution of RIG-I agonists during the course of a viral infection was undefined. I was able to show with the help of two colleagues that viral genomic RNA, but not other RNA species, constitutes the prominent source for RIG-I activation in influenza and Sendai virus infected cells. Next, I focused on the identification of new regulators of the innate pathways dedicated to the sensing of cytosolic nucleic acids following infection. My first approach, involving the use of a vaccinia-encoded immunomodulator as bait for novel components of the cytosolic DNA sensing pathway, was unsuccessful. So I turned my attention to DDX60: an IFN-inducible superkiller-2- like RNA helicase that I fished-out from microarray data. I was able to generate a ddx60-knockout mouse and demonstrate that this helicase is dispensable for the induction of IFN-α/β in response to different PRR stimuli. These results shed light on the role of this poorly characterized helicase in antiviral immunity and suggest that DDX60 may function as a specific antiviral restriction factor rather than a component of the IFN-inducing PRR pathways.

TCR signalling is crucial to both T cell development and naive T cell homeostasis. Naïve T cell survival in the periphery is thought to depend on both cytokine signalling and constitutive TCR signalling. The nature of this TCR dependent survival signal remains controversial. The tyrosine kinase, Zap70, is essential for TCR signalling. The aim of this project is to investigate the role of Zap70 in the transduction of survival signals in naïve T cells. Using mice that conditionally express Zap70 by means of a tetracycline-inducible system, we found that Zap70 is absolutely required for the maintenance of naïve T cells in the periphery. Loss of Zap70 resulted in a dramatic and rapid reduction in mature naive CD8 T cells in the blood and peripheral organs consistent with a naïve T cell survival defect. This survival defect could not be accounted for by cell-intrinsic differences in IL-7Rα or Bcl-2 expression. Analysis of T cell survival in vitro revealed no differences in responses to IL-7 between F5 T cells with or without Zap70. Survival in vitro was found to be enhanced by the presence of CD1 1 c+ enriched splenocytes but not T or B cells. Survival of F5 T cells in these cultures was dependent on Zap70 expression and also required intact MEK signalling. In addition, we also tested the ability of previously described Zap70 mutants, Zap70 SKG and Zap70YYAA, to transduce homeostatic TCR survival signals in vivo. Both mutants were unable to support the development, survival or antigen-induced expansion of F5 T cells. Additionally, we found evidence that Zap70Y YAA had a dominant negative effect on T cell development and reconstitution in F5 TetZap70 mice. In conclusion, we find that Zap70 is essential for transmission of signals required for naive T cell survival and requires both full expression and functionality of Zap70.

Considerable interest has been focused on inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although small interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi to the central and peripheral nervous system remains a major challenge limiting its applications. This thesis describes the development of a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) using replication-defective herpes simplex viral (HSV-1) vectors by identifying and evaluating various approaches to induce RNAi, i.e. expression of individual short-hairpin RNAs (shRNAs), artificial microRNAs (miRNAs) and multiple tandem miRNAs. Following the development of these systems, HSV-mediated delivery of shRNA or miRNA against reporter genes was shown to result in highly effective and specific silencing in neuronal and non-neuronal cells in culture and in the DRG of mice in vivo, including in a transgenic mouse model. Proof of concept was established by demonstrating in vivo silencing of the endogenous trpv1 gene, thought to be involved in nociception, by assessing both mRNA and protein levels. These data are the first to show silencing in DRG neurons in vivo by vector-mediated delivery of shRNA and support the utility of HSV vectors for gene silencing in peripheral neurons and the potential application of this technology to the study of nociceptive processes and in pain gene target validation studies. Moreover, a disabled HSV-1 vector targeting p75, Lingo1 and NgR2, which are involved in myelin inhibition of axonal regeneration, was developed and evaluated for its ability to promote regeneration of sensory axons into the spinal cord, following injury of the dorsal roots. This is the first time such an appoach to silencing multiple genes has been employed. Although HSV-mediated delivery of multiple miRNAs resulted in highly effective silencing of these genes in dividing cells in culture, while highly effective silencing of p75 was achieved, only modest silencing of Lingo1 and NgR2 was observed in DRG neurons in vivo. Preliminary regeneration experiments, which were largely outside the scope of this thesis, were inconclusive and require more extensive study as a stand-alone project, if the in vivo potential of the approach developed for silencing multiple genes targeted at axonal regeneration is to be further explored.

Lentiviruses stably integrate their genome into the host genome. Although this feature can be advantageous for long term transgene expression, it also has the potential to cause mutagenesis and cell transformation. To address this problem, thereby improving the safety of lentiviral gene therapy, non-integrating lentiviral vectors (NILVs) were developed. NILVs were generated by mutating the cis-acting sequences that interact with integrase (att sites) or by mutating specific residues in integrase in different domains (catalysis - D64V, strand transfer – Q148A, K264R, K266R, K273R, DNA or chromatin binding - N120L, W235E). Relevant mutations were then combined in order to improve the safety of these vectors. It was shown that all mutant vectors were efficiently produced and mutations did not affect infectivity. In contrast to dividing cells, differentiated muscle cells infected with NILVs show stable transgene expression over time without degradation of episomal viral DNA. The vectors were also tested in vivo by intramuscular injection in neonate mice. Transgene expression from muscle cells was maintained for 8 months using both integrating and NILVs. The vectors were then tested in a haemophilia B disease model. It was shown that plasma levels of FIX produced by muscle cells infected with integrating lentiviral vectors were above the therapeutic threshold. However, expression from NILVs was lower. This was studied in detail and it was found that integrating lentiviral vectors are transcriptionally more active than NILVs. A comparison of expression levels revealed that integrated lentivectors express more transgene protein per vector copy than NILVs and AAV vectors, but both episomal vectors display similar levels of transgene expression per vector copy. In conclusion, NILVs have the potential to be used as tools for prolonged transgene expression in non-dividing muscle cells or transient expression in dividing cells. However, vectors may need to be optimised if high expression levels are required.

Following the discovery that porcine endogenous retrovirus (PERV) can infect human cells, the potential risk of a zoonotic infection by PERV has been a major obstacle in the xenotransplantation field. The aim of this thesis is to gain a better understanding of PERV biology, so as to help assess and reduce the risk of PERV zoonosis. PERV subgroup A can enter human cells through two human PERV-A receptors (huPAR-1 and -2). To determine critical regions in the receptor for PERV-A infection, chimeric receptors between huPAR-2 and the non functional murine PAR (muPAR) have been analysed. A single amino acid difference (amino acid 109) was found responsible for the inability of muPAR to mediate PERV-A binding and infection. These results were then applied to the evaluation of PERV infection of non-human primates (NHP). NHP could represent an ideal animal model for assessing the risk of zoonosis following long-term exposure to porcine material. However, PERV does not infect NHP cells with the same efficiency as it does human cells. The data presented in this thesis suggests that in some NHP species the poor infectivity is due to mutation of the same critical amino acid (a.a.109) described for muPAR. However, African green monkey cells express two functional receptors and other mechanisms are likely to be responsible for the low susceptibility to PERV-A infection. Secondly, I evaluated the effect of a release inhibitor as a possible strategy to reduce PERV dissemination from pig cells. Human tetherin can inhibit retrovirus production from cells. I showed that overexpression of human and newly cloned porcine tetherin in pig cells can reduce the release of PERV. My data suggests that tetherin-expressing transgenic pigs could represent a safer donor in xenotransplantation.

Dendritic cells play a key role in both innate and adaptive immunity. Recognition and presentation of antigens on the major histocompatibility complex (MHC) provide an efficient response against infections. However, this response strongly depends on antigen presentation. During inflammation several oxidative reactions occur that lead to the production of oxidants. Under these conditions protein antigens are exposed to high concentrations of hypochlorous acid (HOCl) and hydrogen peroxide (H2O2) that conduct changes in antigen structure and enhance the specific T cells response. However, the mechanism of presentation of oxidized antigens is presently unknown. In this thesis, we have focused on the mechanism involved in the enhancement of the T cell response to oxidized proteins. In particular, we have studied the uptake, processing and presentation of oxidized antigens in in vitro and in vivo mice models to MHC II‐restricted T cells; we have investigated the potential receptor‐mediated mechanism underlying the enhanced immunogenicity of oxidized antigens; and finally we have analyzed the connection between protein chemical modifications and the enhanced response to oxidized antigens.

Antigen recognition by B cells results in their activation followed by specific antibody production. These events are initiated by antigen binding to their surface B cell receptors (BCR) which triggers both signalling and internalization of the receptor bound antigen to the endosome. However B cells also express features of the innate immune system such as Toll like receptors (TLRs), that can be located either on the surface of the cell or intracellularly where they recognize bacterial and viral nucleic acids. Engagement of these receptors within B cells is associated with enhancement of humoral responses. The aim of my PhD project was to investigate how endosomal TLR ligands in a particulate form could gain access to their intracellular receptors in the B cell and which impact the subsequent TLR engagement had on B cell fate. To achieve this, I directly linked both antigen and TLR9 ligand to particulates. Immunisation of mice with those particulates resulted in enhanced specific antibody titers compared to stimulation with particulate antigen alone. To dissect the underlying mechanism, I employed transgenic B cells bearing BCR specificity for the same antigen and stimulated them with particulate antigen-TLR9 ligand conjugates. Particulate TLR9 ligand could not gain access to its receptor within B cells via unspecific macropinocytosis and instead depended on BCRmediated internalization. Subsequent engagement of intracellular TLR9 by its ligand present in the conjugates resulted in B cell activation and proliferation, followed by differentiation into plasma cells and antigen specific antibody secretion. The uptake of the antigen-TLR9 ligand particulates both in vitro and in vivo depended on the affinity of the antigen once a defined threshold required for internalization was surpassed. The extent of plasma cell differentiation however could be modulated by the amount of TLR9 ligand present on the particulates. Thus I observed that direct linking of antigen and TLR ligand resulted in PC differentiation through antigen specific BCR mediated internalization and subsequent TLR engagement. This reveals a mechanism that may operate during the initiation of a primary immune response.

Umbilical cord blood (CB) is recognised to be a valuable alternative to bone marrow (BM) as a source of hematopoietic stem cells (HSC). The occurrence of Graft vs. Host Disease (GvHD) after CB transplantation has been reported to be less severe in comparison to BM transplants. In addition to the naive state of immune cells, the action of immuno-suppressive cells such as regulatory T cells (Treg) may contribute to the positive aspects observed in CB transplants. This study investigated the phenotypic and functional characteristics of CB Treg and their potential for expansion in culture. In addition, the allogeneic response of CB and AB CD4+ cells was compared. Two main subsets of Treg have been described: resting (CD45RA+FOXP3low) and activated (CD45RA-FOXP3high). Results presented here showed that CB contained mostly resting Treg whereas AB contained mostly activated Treg. In addition, freshly isolated CB Treg were less capable of inhibiting the proliferation of responses in comparison to AB Treg. CB Treg acquired an activated Treg phenotype and potent suppressive activity after expansion, and expanded CD25+ cultures maintained Treg characteristics for longer in comparison to CD25+ cells expanded from AB. Importantly, unlike AB Treg, expanded CB Treg suppressed the proliferation of autologous and allogeneic responders equally. Finally, ‘putative Treg’ were induced from CB CD25- cells following allogeneic stimulation, the putative Treg were CD4+FOXP3+CD25+CTLA-4+ and were capable of suppressing the proliferation and cytokine responses to primary and subsequent allogeneic challenges. In conclusion, Treg subsets from CB display different phenotypic and functional properties to AB Treg. These properties of CB Treg may clarify the cellular interactions in clinical settings in which CB is currently used and highlight potential future uses.

Tuberculosis, caused by infection with the intracellular pathogen Mycobacterium tuberculosis, remains a major global threat to humanity. BCG remains the only prophylactic vaccine against tuberculosis but gives variable protection against pulmonary disease. While it is unclear what determines an effective immune response against Mycobacterium tuberculosis infection, the generation of host Th1 responses is accepted to be a major mechanism of protection; and promoting earlier and enhanced Th1 responses may increase the efficacy of tuberculosis vaccines. Early production of interleukin-17 in the lungs following Mycobacterium tuberculosis challenge of previously vaccinated mice has been shown to be required for efficient Th1 cell recruitment. Interleukin-10 is an immunosuppressive cytokine that regulates various processes involved in the generation of both Th1 and Th17 responses. The investigations within this thesis extend on previous studies showing interleukin-10 negatively regulates the immune response to primary Mycobacterium tuberculosis infection. Firstly, these investigations show that interleukin-10 signalling inhibits host control of infection with virulent and hypervirulent Mycobacterium tuberculosis strains, in both Mycobacterium tuberculosis-resistant C57BL/6 mice, and Mycobacterium tuberculosis-susceptible CBA/J mice. Furthermore, inhibition of interleukin-10 signalling specifically during BCG vaccination enhanced host-generated antigen-specific interferon-γ and interleukin-17 responses, and this regime gave significantly greater protection against aerogenic Mycobacterium tuberculosis challenge in both mouse strains. In susceptible CBA/J mice, antibody blockade of interleukin-10 receptor specifically during BCG vaccination resulted in an unprecedented 1-Log10 further protection against Mycobacterium tuberculosis challenge as compared to BCG-vaccination alone. This robust protection was sustained during the course of Mycobacterium tuberculosis infection, and correlated with superior lung Th1 and Th17 responses, and enhanced interferon-γ and interleukin-17 production from innate lymphoid cells. In summary, interleukin-10 inhibits both host-mediated protection against primary Mycobacterium tuberculosis infection, and optimal BCG-elicited protection. Antagonists of interleukin-10 may therefore be greatly beneficial as adjuncts to tuberculosis therapy, and as adjuvants in preventive vaccination against tuberculosis.

In chronic hepatitis B virus (HBV) infection, the loss of hepatitis B e antigen (HBeAg) and seroconversion to anti -HBe coincide with a decrease in viral load and reduction in clinical hepatitis. This thesis describes a programme of work undertaken to investigate viruses causing an unusually aggressive phenotype of anti-HBe positive chronic hepatitis with high viral load. The thesis is broadly divided into two sections. The first describes a genomi cs approach to investigating these viruses. A large multiple alignment of whole genome H BV sequences was generated using sequences retrieved from GenBank. This alignment was used for phylogenetic studies, as well as providing the reference set for developing a whole genome HBV next generation sequencing technique. A scheme for whole genome sequencing was developed using the SEQUENOM M assCLEAVE protocol, in which RNA transcribed PCR products are subjected to base-specific cleavage and MALDI-ToF mass spectrometry, followed by computer-aided pattern matching. Specific issues encountered with this technology will be described in depth. Phylogenetic and bioinformatic analysis of DNA sequencing information obtained for clinically significant viruses will also be described. The replication cycle of HBV remains poorly understood, partially due to the limitations of existing in vivo and in vitro models. The second half of the thesis describes the development of in vitro culture methods for investigating HBV replication. Mammalian expression vectors were designed and generated to express elements of the HBV. Transfection of individual vectors into human hepatoma cell lines was performed, and protein expression and viral DNA characterised using a number of techniques, including immunofluorescent microscopy, ELISA, western blotting and qPCR. This was followed by co-transfection experiments to demonstrate trans-complementation of the viral genome and restoration of viral replication. Once fully optimised, these techniques will allow comparisons of replication efficiency between viruses to be performed in a safe and flexible manner.