Whole genome sequencing (WGS) has transformed molecular infectious diseases epidemiology in the last five years, and represents a high resolution means by which to catalogue the genetic content and variation in bacterial pathogens. This thesis utilises WGS to enhance our understanding of antimicrobial resistance in two clinically important members of the Enterobacteriaceae family of bacteria, namely Escherichia coli and Klebsiella pneumoniae. These organisms cause a range of clinical infections globally, and are increasing in incidence. The rapid emergence of multi-drug resistance in association with infections caused by them represents a major threat to the effective management of a range of clinical conditions. The reliability of sequencing and bioinformatic methods in the analysis of E. coli and K. pneumoniae sequence data is assessed in chapter 4, and provides a context for the subsequent study chapters, investigating resistance genotype prediction, outbreak epidemiology in two different contexts, and population structure of an important global drug-resistant E. coli lineage, ST131 (5-8). In these, the advantages (and limitations) of short-read, high-throughput, WGS in defining resistance gene content, associated mobile genetic elements and host bacterial strains, and the relationships between them, are discussed. The overarching conclusion is that the dynamic between all the components of the genetic hierarchy involved in the transmission of important antimicrobial resistance elements is extremely complicated, and encompasses almost every imaginable scenario. Complete/near-complete assessment of the genetic content of both chromosomal and episomal components will be a prerequisite to understanding the evolution and spread of antimicrobial resistance in these organisms.

Long-term non-progressors (LTPNs) in HIV infection target the structural protein Gag more frequently than individuals who progress to disease. However, the targeting of Gag per se does not always distinguish these two groups. Various factors have been put forth as likely explanations for this discrepancy including differences in the breadth and magnitude of observed responses, the HLA type of the host, the nature of the individual epitopes targeted and the ability of the virus to mutate these antigenic regions. The purpose of this thesis was to examine, using PBMCs isolated from HIV-2 infected LTNPs and CTL clones established in vitro, the clonotypic architecture and quality of an immunodominant HIV-2 Gag-specific response directed towards the HLA-B*3501-restricted epitope NPVPVGNIY (NY9: Gag245-253). The data presented in this thesis show that in spite of the expression of multiple inhibitory receptors on the surface of NY9-specific CD8+ T-cells, the NY9-response, which is a clonotypically 'private' response, bears a signature characterised by an increased cytotoxic sensitivity and the production of an array of cytokines, most notably IFN-γ and MIP-1β. Moreover, the results of this thesis indicate that the NY9-specific CD8+ T-cells are able to cross-recognise and lyse target B-cells pulsed with the corresponding HIV epitope PY9 and its variants at functional avidities (EC50) that are close to those exhibited by PY9-specific T-cells. However, not all mobilised TCR clonotypes are equally sensitive or equally cross-reactive. When individual CTL clones were studied it emerged that dominant clonotypes within the NY9-specific CD8+ T-cell memory pool possessed a higher avidity for tetramer and sensitivity for antigen than subdominant ones and demonstrated a better cross-reactive potential towards variants of the HIV-2 epitope. Hence, future HIV vaccine strategies may benefit from the inclusion of epitopes like NY9, the presentation of which appears to mobilise CD8+ T-cells with superior functional profiles.

Diagnoses of tuberculosis, 'TB,' currently rely upon non-specific techniques such as X-ray exams and acid-fast microscopy. Improved diagnostics would preferably consider specific bacterial processes to provide real-time readouts of disease burden and response to chemotherapy. This dissertation presents the cell-wall incorporation of trehalose analogues (fluorescent and radioactive) by the mycobacterial antigen 85 enzymes as a novel method to label the causative bacteria of TB, Mycobacterium tuberculosis (Mtb). The trehalose mycolyltransesterase enzymes (antigens 85A, B, and C (Ag85)) serve as essential mediators of cell envelope function and biogenesis in Mtb. We show that the Ag85 enzymes display activities so broad that they allow added non-natural carbohydrate probes to be incorporated into Mtb growing in vitro and within macrophages. Design and synthesis of a library of structurally-diverse analogs of the sugar trehalose (Tre) revealed that Ag85-enzymes catalyze esterification of a wide variety of non-natural Tre structures, even stereoisomers and those appended with charged or bulky groups (Chapter 2). A novel mass-spectrometry based Ag85 enzyme assay was developed and employed to screen the library of compounds against all three isoforms of Ag85 (Chapter 3). This screen revealed that the Ag85 enzymes exhibit preference for dissacharides over monosaccharides and a broad tolerance for most modified trehalose compounds. This activity assay also afforded full kinetic analysis and the discovery of a novel, covalent inhibitor of the Ag85 enzymes. The Ag85 activity assay informed the design of a fluorescent trehalose-based compound (FITC-Tre), which is the first, non-toxic, selective, small molecule probe for mycobacterial infection. FITC-Tre was acylated with mycolyl esters by growing mycobacteria, anchoring the probe in the cell envelope resulting in fluorescent bacteria (Chapter 4). Adding FITC-Tre to Mtb-infected macrophages allowed selective, fluorescent tagging of Mtb in vivo (Chapter 5). Colocalization studies with antibodies against a variety of phagosomal associated components have hinted at the possibility of FITC-Tre as readout of cellular trafficking of bacteria. ¹⁸F-trehalose, biotin-trehalose and rhodamine-trehalose are also substrates of Ag85. ¹⁸F-trehalose shows promise as Mtb selective PET probe in an infected rabbit model of tuberculosis. Future work with these probes may allow for fluorescent tracking of the Mtb during the macrophage infection process, as well as the ability to label Mtb in infected tissue. The functional differences between the three isoforms of Ag85, A, B and C, are not well understood and may have implications for the survival and persistence of mycobacteria within humans. The differences in substrate specificity and catalytic activity between the Ag85 isoforms (discussed in Chapter 3) has been further investigated (Chapter 6). Mutation of three secondary site amino acids from Ag85C into Ag85B afforded nearly a twenty-fold gain in enzyme activity. Mutation of the equivalent Ag85B residues into Ag85C triggered nearly a twenty-fold loss in activity. Dissection of the roles of these three amino acids helps to explain the previously reported large differences in catalytic activity between Ag85A, B and C. Overexpression of Ag85A, B and C under tetracycline regulation revealed that these enzymes differentially modulate incorporation of mycolates into the cell wall. The Ag85 enzymes are not functionally redundant, and instead serve unique purposes in cell wall biosynthesis. In summary, this research has demonstrated that the broad substrate tolerance of Ag85 enzymes, coupled with their extracellular location, opens the door to probes of mycobacterial infection using many imaging modalities.

During the course of infection with chronic pathogens such as Hepatitis C virus (HCV), Hepatitis B virus (HBV) and HIV, virus-specific CD8^+ T cells differentiate into heterogeneous dysfunctional subpopulations. Advances in multi-parameter flow cytometry have allowed these subpopulations to be further classified into classes of exhausted T cells, primarily by their expression of multiple inhibitory receptors. However, the exact phenotype of CD8^+ T cells during exhaustion is an area of great interest as many inhibitory receptors are also expressed on functional CD8^+ T cells. Discovering novel and specific markers of T cell exhaustion is fundamental in developing strategies to restore CD8^+ T cell function in chronic viral infections. Recently, genome wide expression profiles have identified broad molecular phenotypes in exhausted T cells that could not have been discovered by flow cytometry alone. I show how similar genomic approaches identify and further characterise the ectonucleotidase CD39 as a novel marker of CD8^+ T cell exhaustion in chronic viral infection. I show that CD39 is highly expressed in HCV and HIV-specific CD8^+ T cells and that CD39^+ CD8^+ T cells are enriched with gene signatures of exhaustion. CD39 is highly co-expressed with multiple inhibitory receptors including PD-1, enzymatically active on CD8^+ T cells in HCV infection and positively correlated with viral load in both HCV and HIV. I also demonstrate the discovery of a novel CD39^High population of cells in the mouse model of chronic Lymphocytic Choriomenigitis Virus (LCMV) infection, which express the highest degrees of PD-1, LAG3 and 2B4 in the CD39^+ fraction. Thus, CD39 is a novel and specific marker of severe CD8^+ T cell exhaustion in human and animal models of chronic viral infection.

Norovirus is the commonest cause of viral gastroenteritis, affecting all age groups worldwide. Outbreaks frequently occur in semi-closed communities such as schools, cruise ships, prisons and hospitals. Within the healthcare environment, the economic and logistical burdens and the inconvenience caused by norovirus is significant, since ward closure remains central to infection control. The aim of this study was to investigate norovirus transmission dynamics during hospital outbreaks. The ultimate goal was to provide information that could, in future, lead to the development of novel, less disruptive approaches to curtailing the spread of infection. The study explored the application of 'next generation' high throughput DNA sequencing technologies to the determination of large numbers of norovirus genomes. Whole genome sequences provide the highest possible level of discrimination among viruses, information which is essential to the identification of linked and independent cases of infection. The approach exploits the high norovirus mutation rate, which is typical of RNA viruses. Consequently, viruses within a single ward which differ by more than a few SNVs can be considered to represent independent introductions, rather than a single outbreak. Whole genome sequence data (determined for noroviruses collected between 2009 and 2013) were combined with epidemiological data, providing further insights into transmission dynamics. These data identified multiple independent virus introductions during single ward outbreaks. The possible origin of such outbreaks in Oxfordshire hospitals were investigated using viruses originating in the local community, and in other healthcare environments distributed throughout the UK. Whole genome sequences of noroviruses from consecutive years were genetically divergent, confirming the rapid evolution of the virus over time and excluding the possibility of prolonged environmental contamination as a reservoir of infection. Such detailed information on norovirus transmission within the healthcare environment could inform alternative future approaches to optimising infection control within the healthcare setting.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several tumours and hyperproliferative disorders. Hypoxia plays an important role in KSHV lifecycle, as hypoxia-inducible factors (HIFs) are involved in the latent/lytic switch and affect other KSHV genes, and as KSHV infection can in turn enhance cellular levels of HIFs. Two KSHV-associated tumours tend to develop in settings of relative hypoxia; Kaposi's sarcoma (KS) often occurs in the lower extremities and primary effusion lymphoma (PEL) exists in pleural effusions. A better knowledge of the pathways that regulate KSHV infection in hypoxia is therefore essential for an improved understanding of viral infection and pathogenesis. MicroRNAs (miRNAs) have been shown to play important roles in regulating the expression of genes in oncogenesis, and herpesviruses, including KSHV, encode for miRNAs. This thesis describes a multidisciplinary approach toward understanding the mechanisms behind the hypoxia-regulated miRNA-mRNA networks in the context of KSHV infection. The question of miRNA and mRNA regulation through hypoxia, KSHV or both is addressed in this thesis by deep sequencing and gene expression assays as well as various transfection and functional assays. In chronically infected cells compared to uninfected controls, it is demonstrated that the majority of cellular miRNAs whose expression is affected are substantially down-regulated. A third of this down-regulation can be attributed to a single genomic region, 14q32 cluster, where miRNAs are lowly expressed in infected cells. In hypoxia, hsa-miR-210 is the only miRNA to be consistently up-regulated in the KSHVinfected cell lines subjected to deep sequencing in this study. Computational approaches additionally allowed for the investigation of mRNA targets. Inversely correlated miRNAmRNA target pairs were identified and distributed into canonical pathways and biological networks. Taken together, these results suggest that miRNAs affected by hypoxic stress and/or viral infection are implicated in the pathogenesis of KSHV-related diseases. It is expected that the outcomes of these studies will change our understanding of how KSHV uses the host RNA silencing machinery to its advantage and how this intersects with the use of the cell's response to hypoxia.

HIV-1 has claimed the lives of millions of people globally and continues to spread despite development of highly active antiretroviral therapy. In 2013, 2.1 million new infections occurred and over 35 million people were living with HIV-1 infection. A prophylactic HIV-1 vaccine that can prevent infection or reduce viremia and subsequent transmission will always be an important part of the solution to bring this epidemic under control. In this thesis, the first HIV-1 vaccine candidate to focus on conserved regions of the virus (HIVconsv) was assessed in a phase I clinical trial conducted in healthy HIV-1/2 negative volunteers in Oxford. The HIVconsv T-cell immunogen was delivered using three leading vaccine modalities (DNA (D), modified vaccinia virus Ankara (M) and chimpanzee adenovirus serotype 63 (C)), in several novel heterologous prime-boost regimens. The frequency of T cells elicited through HIVconsv vaccination in the CM and DDDCM regimens surpassed that of previous HIV-1 cell-mediated vaccines. A large proportion of these T cells produced multiple cytokines and proliferated in response to recall peptides. The breadth of T-cell responses were also greater than the non-efficacious STEP study vaccine, with an average of 10 T-cell epitopes per vaccine recipient recognised across CM and DDDCM regimens. In vitro HIV-1 control mediated by CD8⁺ T cells was demonstrated for all vaccinees receiving the CM regimen, mainly against clade A (U455) and clade B (IIIB) isolates. Two vaccinees, demonstrated superior control of 6/8 and 7/8 viruses from the panel. The CM regimen induced significantly higher magnitudes of viral inhibition compared to the DDDCM or DDDMC regimens, with this regimen showing potential to overcome the disadvantage for subjects of carrying non-protective HLA alleles. Investigation of T-cell specificities revealed that the frequencies of T cells specific for conserved Gag but more so Pol regions significantly correlated with in vitro virus control. Direct examination of peptide expanded T-cell lines showed that all Pol pool- and limited Gag pool-specific cell lines reduced HIV-1 replication in vitro. In most individuals, targeting multiple HIV-1 epitopes concomitantly resulted in higher levels of virus inhibition than targeting a single viral epitope and two T-cell specificities showed enhanced control of HIV-1; the first within Pol (TAFTIPSI) and second from Gag (TERQANFL). These data support further development of the conserved region strategy for T-cell vaccines against HIV-1.

A knowledge about infectious diseases and vaccination is very important in this time. The questionnaire, which is part of this thesis, is designated for 1st to 4th grade high school students and the upper grade grammar school pupils. The questionnaire was used to analyse pupils' knowledge and attitudes about infectious diseases and vaccination. Pupils' knowledge and attitudes are quite good and students are able to voice their opinion. Females have better knowledge than males and older students are better too. The thesis is further focusing on how are the infectious diseases included in the National Curriculum and examine the prevention programmes for schools. The other purpose is to follow up the threat of pandemic diseases and present state of knowledge and awareness about this topic.

Hepatitis C virus (HCV) is a major global pathogen estimated to infect over 170 million people worldwide. A recent study has shown that vaccination with adenoviral vectors, based on rare human and simian serotypes encoding the non-structural (NS) proteins of HCV, induces highly potent, multi-specific and durable T cell responses in healthy human volunteers. In this thesis I assess the safety and immunogenicity of these vaccines (ChAd3–NSmut and Ad6-NSmut), for the first time in HCV infected patients. This work also explores whether vaccine-induced T cell responses target in vivo circulating HCV antigens and common naturally occurring epitope variants. Patients with treatment naive chronic genotype 1 HCV infection were vaccinated (i.m.) with ChAd3-NSmut and Ad6-NSmut in a heterologous prime boost schedule, either with or without current IFN and ribavirin (IFN/RBV). Epitope-specific T cell responses were defined by fine mapping using HCV peptides. Circulating viral genomic sequence was determined in vaccinated patients at baseline and at any point of viral relapse. Cross-reactivity of vaccine-induced T cell responses was determined in T cell assays, using peptides corresponding to both circulating host virus and common population HCV epitope variants. An in vitro dendritic cell /T cell priming model was used to identify possible candidates for a cross-reactive vaccine immunogen at the most immunodominant epitope, NS3₁₄₀₆. 33 patients were vaccinated. Vaccination was well tolerated. At the highest vaccine dose (2.5 x 10¹⁰vp) vaccine-induced T cell responses were detectable in 11/20 patients receiving concurrent IFN/RBV and 2/4 patients receiving vaccination alone. In total 14 antigenic targets were identified, 2 of which have not previously been described. However, T cell responses were of lower magnitude and more narrowly focused than those observed in healthy volunteers vaccinated with the same regimen. Analysis of viral sequence showed that in many cases vaccine-induced T cells did not target the circulating virus. At the most immunodominant epitope (NS3₁₄₀₆), T cells induced by vaccination failed to target common circulating genotype 1 HCV variants. An in vitro model suggested that in order to target all genotype 1 sequences at this epitope, it would be necessary to insert both a genotype 1a and 1b version of this epitope into a vaccine immunogen. Vaccination with adenoviral vectors induces T cell responses in patients with chronic HCV infection, however immune responses are attenuated compared with healthy volunteers. Ultimately a successful therapeutic or prophylactic vaccine strategy will rely on inducing responses that target conserved or cross-reactive epitopes.

Une partie de ce travail de thèse a consisté à appliquer les méthodes de FISH pour l’étude de trois bactéries pathogènes intracellulaires. La viabilité de Bartonella henselae a été évaluée à partir de ganglions de patients atteints de la maladie des griffes du chat (CSD). Le faible taux d’ARN détecté par biologie moléculaire, la stérilité des cultures, l'absence de détection par analyses histologiques et FISH confirment que B. henselae n'est pas ou rarement viable dans les ganglions de patients atteints de CSD. Tropheryma whipplei, l’agent de la maladie de Whipple, a été identifié et localisé par FISH, dans les macrophages d’un ganglion et d’une biopsie pulmonaire, confirmant le diagnostic infectieux. Deux méthodes de FISH ont été testées pour détecter Coxiella burnetii dans des cas d’endocardites et d’infections vasculaires en utilisant des sondes oligonucléotidiques et des sondes PNA. Les résultats ont confirmé une meilleure efficacité des sondes PNA et démontré que les techniques de FISH sont plus sensibles que l’immunohistochimie pour le diagnostic des endocardites et des infections vasculaires à C. burnetii. Nous avons également évalué les stratégies moléculaires mises en place pour le diagnostic syndromique. Bien que la PCR conventionnelle à large spectre permette l'identification de micro-organismes fastidieux et anaérobies, la PCR spécifique en temps réel révèle une supériorité significative dans le diagnostic syndromique. En conclusion, ce travail a permis de démontrer l’efficacité et l’applicabilité de la FISH pour la détection bactérienne. Cette méthode peut être utilisée comme un outil complémentaire afin d'améliorer le diagnostic de microbiologie clinique. / We applied FISH methods to the study of three intracellular pathogenic bacteria. The viability of Bartonella henselae was evaluated in a large series of lymph nodes from patients with cat scratch disease (CSD). The results obtained, associated with sterile cultures and negative histological analyzes and FISH, as well as the low level of RNA detected by molecular biology, provide evidence that B. henselae are not or are rarely viable in the lymph nodes of patients with CSD. Tropheryma whipplei has been identified by FISH in macrophages from one lymph node and for the first time in a pulmonary biopsy, confirming the diagnosis of infection. Two methods of FISH have been tested to detect Coxiella burnetii in cases of endocarditis and vascular infections using oligonucleotide and PNA probes. The results attested to the greater efficiency of PNA probes, and demonstrated that FISH were applicable for the diagnosis of C. burnetii endocarditis. We also evaluated the molecular strategies used for syndrome-driven diagnosis of infectious diseases. Although conventional broad-spectrum PCR allows for the identification of fastidious and anaerobic microorganisms, real-time specific PCR reveals a significant superiority in syndrome-driven diagnosis. The addition of specific PCRs in real time PCR would improve our molecular strategies, for example, in the case of the detection of Staphylococcus aureus for the diagnosis of lymphadenopathy. In conclusion, this work demonstrates the effectiveness and applicability of FISH for the identification of intracellular bacteria. This method can be used as an important complementary tool to the improvement of clinical microbiological diagnosis.