Tripartite motif (TRIM) proteins are numerous in the human proteome, and a number of these molecules are known to restrict retroviral replication. TRIM5α (T5α) is one such factor. It targets the viral capsid and imposes a block to infection between entry and reverse transcription. Capsid recognition is mediated by the C-terminal B30.2 domain, which contains surface-exposed loops of high amino acid variability. Restriction is then effected via proteasome recruitment and the induction of innate immune cascades. Although T5α is well-characterised in this respect, other factors – such as the highly divergent TRIM1 (T1) – remain poorly understood. To further characterise the T1 restriction phenotype, chimeras of this protein and its non-restricting paralogue, T18, were generated by overlapping PCR. The restriction activities of the resulting molecules were then measured using an established flow cytometry assay. These experiments revealed that T1 also binds capsid via the B30.2 domain, although the majority of this region can be functionally replaced. Other aspects of T1 biology addressed in this work include the contribution of N-terminal components to restriction potency, and the relationship between protein expression level and restriction activity. Following a number of attempts to generate a functional chimera of T1 and 5α, the latter half of this thesis explores how the spacing between capsid-binding and effector domains can influence restriction activity. To this end, a panel of mutations were made in the linker 2 (L2) region of T5α, and their effects on restriction measured. These experiments revealed that even small changes in interdomain spacing can have profound phenotypic consequences. Collectively, this work reinforces the notion that TRIM family members share a common overall design, allowing individual components to be shuffled between them. At the same time, each molecule has been shaped by unique evolutionary pressures, which can render them sensitive even to relatively minor modifications.

Clostridium difficile continues to be a leading cause of healthcare-associated infections in the developed world. Increased detection of C. difficile infection (CDI) and development of typing schemes to differentiate between strains is primarily due to the recognition of global outbreaks of a single strain, BI/NAP1/027 which is characterised by three common typing techniques; restriction endonuclease analysis (REA), pulsed-field gel electrophoresis (PFGE) and PCR ribotyping. Phylogenetic analysis using multilocus sequence typing (MLST) divides C. difficile into five phylogenetic lineages which align the well-known PCR ribotypes; 027, 023, 017, 078 and a lineage containing diverse PCR ribotypes. MLST data in this thesis confirmed the five phylogenetic lineages were maintained after testing a larger collection of isolates from varied sources with further micro-diversity within the individual lineages. MLST investigation did not identify a lineage exclusive to nonhuman strains or any correlation between sequence type and geographical location. Data in this thesis also supports the notion that PCR ribotyping and REA do not correspond as well as previously considered. This may result in phylogenetically similar strains being designated as a different type or variant. The toxin A-B+ PCR ribotype 017 strain that forms a predominant lineage is little investigated. Through whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis, a historical clone of PCR ribotype 017 was identified from a London hospital ward. Although no phenotype exclusive to the clonal strain was characterised, this is the first report in the UK investigating the phylohistory of isolates from hospitalised patients with CDI due to PCR ribotype 017. Further investigation of PCR ribotype 017 with a larger and global collection of strains revealed two distinct sub-lineages containing multiple independent clonal expansions, antimicrobial resistant SNP determinants, deletions and insertions which were well distributed geographically and temporally. The data suggests transmission between humans and animals and findings support a USA origin with multiple, global transmission events. The key findings of this thesis are that C. difficile as a species is continually evolving with the appearance of divergent sub-lineages. WGS is superior to routine typing methodologies for tracking this evolution and will have significant impacts for outbreak investigation, understanding the phylohistory and phylogeography of C. difficile and other pathogens that are a threat to human health.

Neisseria meningitidis is a pervasive bacterial coloniser of the human nasopharynx with up to 40% carriage in the population. Paradoxically, invasive disease occurs in only ~1 in 100,000 individuals. Sophisticated immune evasion mechanisms allow the bacterium to persist in the host, yet these adaptations may also enable its transition from a harmless commensal to an invasive pathogen. Dendritic cells (DC) are principal controllers of mucosal immunity, and bacteria can exploit their maturation process to evade the immune system. In this study, it is reported for the first time that live serogroup B meningococcus (NmB) impedes DC maturation by disrupting STAT1 through dephosphorylation of tyrosine 701 (Y701). DC were therefore refractory to interferon stimulation—which is essential for DC function—and had low levels of maturation markers and other STAT1 dependent genes. Interestingly, infected DC retained the activity of other major signalling pathways, including the MAP kinases, which induced PD L1hi DC that were CD4+ T-cell suppressive. Disruption of STAT1 also had the major effect of dampening SOCS1 and CISH expression, which are critical for homeostatic control of DC inflammatory cytokine production, and correlated with enhanced inflammatory cytokine production. Restoration of STAT1-Y701 phosphorylation reprogrammed DC towards a typical CD86hi maturation state, normalised cytokine profiles and T-cell responses. These data establish an unconventional inflammatory pathway for the meningococcus, but also link immune evasion and pathogenesis via a shared mechanism. These findings elucidate how NmB may undermine immunity within the normal mucosa but also inflict damage in the context of septicaemia due to uncontrolled inflammation.

Interleukin-10 (IL-10) is an immunoregulatory cytokine that has a vital role in maintaining a balanced and appropriate immune response. CD4+ T helper (Th) cells are important in regulating an effective immune response, and are a dominant source of IL-10. Despite the different signalling pathways that result in the polarisation of each Th subset and lead to the expression of their hallmark cytokines, IL-10 is expressed by all of the different Th subsets. We show that Th1 cells cultured with IL-12 produce IFNγ and small amounts of IL-10, while Th1 cells cultured with IL-12 and IL-27 produce large amounts of IL-10 and IFNγ. Furthermore, we show that Th17 cells can produce IL-10, or not, depending on the presence of IL-2. However, these Th cell populations are phenotypically heterogeneous, particularly with respect production of IL-10 protein. Less than half of each of these in vitro cultured Th cell populations expresses IL-10 or the hallmark cytokine, and co-expression of IL-10 and the hallmark cytokine is also heterogeneous. Therefore we devised and implemented an innovative new technique that enables RNA-Seq analysis of different intracellular cytokine producing cell subpopulations from within Th subsets. We find that it is possible to extract high quality mRNA from Th samples, even though they have been fixed and stained for intracellular cytokines, and that the data from these samples is replicable. Using this technique we have identified potential molecules and pathways by which (I) IL-27 drives IFNγ and IL-10 production by Th1 cells, and (II) IL-2 drives IL-10 production by Th17 cells. Furthermore, by separating different intracellular cytokine producing subpopulations within different Th1 and Th17 cell subsets we have found that cytokine producing and non-cytokine producing subpopulations within heterogeneous Th subsets have significantly different transcriptional profiles and that some pathways and molecules are enriched in IL-10 producing cells.

Solid organ transplant (SOT) patients are at risk of end-organ diseases such as pneumonitis, hepatitis or enteritis caused by HCMV. HCMV infection can occur via primary infection of a seronegative recipient or upon reinfection or reactivation in a seropositive transplant recipient. Seronegative recipients have the greatest risk of viraemia and disease, showing that pre-existing natural immunity provides substantial protection. This, in turn, underpins vaccination as a viable strategy to control HCMV in the transplant setting. To test this, a clinical trial with a vaccine based on HCMV glycoprotein B (gB) antigen plus MF59 adjuvant was performed in SOT awaiting transplantation. The study showed that antibody titres against the gB antigen were significantly increased in both seropositive and seronegative recipients of the vaccine in comparison to the patients who received placebo, and importantly, higher titres correlated directly with reduced viraemia post-transplant. The aim of my thesis was to identify the component of the specific humoral response responsible for this effect. In comparison with placebo recipients, I could find no evidence for the protection being due to induction of antibodies that mediate neutralisation, antibody dependent cellular cytotoxicity, or prevent cell to cell spread of virus in culture. In contrast, analysis of antigenic domains of gB bound by the antibodies revealed that vaccination of seropositive individuals enhanced antibody responses against antigenic domain 2 and that these correlated with reduced viraemia post-transplant. Antibodies against three other antigenic domains were induced by the vaccine, but did not correlate with protection. These results suggest that antigenic domain 2 should be an important component of future HCMV vaccines to boost pre-existing immune responses that protect from HCMV infection. The protection afforded to seronegatives remains unidentified, but could be explained if another antigenic domain on gB remains to be discovered.

Interferon-inducible transmembrane (IFITM) proteins are host cell derived restriction factors. Mammalian IFITM proteins have been shown to confer antiviral resistance when challenged with a diverse range of both enveloped and non-enveloped viruses. Little characterisation has been undertaken to date with the specific aim of elucidating their function and the antiviral properties of the chicken IFITM (chIFITM) gene family. The chIFITM gene family contains four genes located within a 17kb region on Gallus gallus chromosome 5. Currently there is little information available about the sequence diversity of these genes, their expression profiles or the role that they may play in restricting avian viral pathogens. Data presented in this thesis outlines a novel DNA pull-down sequencing technique which has allowed for the generation of a high quality contiguous reference sequence, alongside targeted sequencing of chicken cell lines and ex vivo cell cultures. Studies in this thesis have established that the chIFITMs are interferon stimulated and have characterized their upregulation in response to viral challenge with influenza A virus (IAV) in ovo, in vivo and in vitro, alongside other avian viruses. Stably-overexpressing DF-1 (immortalized chick embryo fibroblast) cells that express chIFITM1, 2, 3 and 3MUT (C71/71A) have been generated. These cell lines have been challenged with avian viruses including diverse strains of IAV and infectious bronchitis virus (IBV) and this data demonstrates that the chIFITMs are able to restrict avian viruses in vitro. Moreover, novel interactions have been identified which may help to uncover a possible mechanism of action. Global food security and protection of livestock from infectious agents remains a key priority, both in the United Kingdom and Internationally. This study examines the role of chIFITM proteins during viral infections and highlights one potential method of safeguarding the poultry industry, ensuring continuity of global food security.

Background Cardiovascular disease is increased in HIV-infected adults but the underlying aetiology is incompletely understood. Pre-clinical changes in cardiovascular structure and arterial stiffness in HIV-infected children are described in mainly middle to high-income countries with minimal longitudinal data available. Limited cross-sectional data is available from Africa in settings where 90% of HIV-infected children live. Methods ART-naïve and ART-experienced (on d4T+3TC+NNRTI for >2years, virologically suppressed at enrolment) HIV-infected children underwent serial assessment within the CHAPAS-3 trial (evaluating d4T vs ZDV vs ABC-based first line ART in Zambia/Uganda): extensive cardiovascular assessment of structure (carotid intimal medial thickness [cIMT] and arterial stiffness (pulse wave velocity [PWV]). Additionally markers of inflammation, disordered thrombogenesis, vascular damage and immune activation were measured. Age-matched HIV-uninfected controls had a single assessment. Baseline differences between ART-naïve/experienced children vs controls, and longitudinal changes in HIV-infected children were assessed. Results In 208 ART-naïve children with median age 2.9y (IQR 1.7–4.4), median CD4% 18% (11-23) and 209 HIV-uninfected controls median age 3.0y (2.1–4.1), mean(sd) cIMT was 0.46(0.04) v 0.44(0.04) mm respectively (p < 0.001); PWV was 5.85(0.8) vs 5.67(0.74)m/sec respectively (p=0.05). Among 74 ART-experienced children on ART for a mean of 3.7y with a median age of 6.9y (5.9–8.50), median CD4% 33% (27-39) and 75 uninfected controls with median age 6.7y (5.6-8.6), the mean(sd) cIMT was 0.46(0.05) vs 0.45(0.04)mm respectively (p=0.09); PWV was 5.63(0.61) vs 5.69(0.68)m/s respectively (p=0.57). In ART naïve children IMT and PWV significantly decreased from baseline (ART initiation) to week 96 mean(sd) cIMT -0.02(0.04)mm (p < 0.001), PWV -0.37(0.82)m/s (p < 0.001). In contrast whereas cIMT had significantly reduced by mean -0.2(0.06)mm (p=0.01) at week 96 in the ART experienced group PWV increased by 0.34(0.62)m/s (p < 0.001). There was no evidence that the changes in IMT or PWV over 96 weeks differed by randomisation ART in either ART naïve or ART experienced children (p≥0.27). Significant differences in a panel of 19 biomarkers and immunophenotyping (markers of activation and proliferation) were demonstrated between HIV infected ART naïve children and healthy age matched HIV uninfected children. No significant relationship between any of the biomarkers, immunophenotyping markers and IMT or PWV was demonstrated. Conclusion In this large study of arterial structural and stiffness in HIV-infected children in Africa, ART-naïve HIV-infected children had significantly poorer IMT and PWV than age-matched controls, with significant improvement seen after 96 weeks of ART. After a mean 3.7 years on ART, HIV-infected children had cIMT and PWV comparable to uninfected age-matched controls. IMT continued to improve after a further 96 weeks on ART. These findings illustrate that ART can reverse some of the structural/stiffness changes caused by HIV, strengthening the argument for early diagnosis and treatment of HIV-infected infants.

Genetic conflict between viruses and their hosts has driven an ‘arms race’, forcing the evolution of both immune defencesdefenses and multiple viral strategies to counteract and evade them. In the case of HIV many of these processes are well characterised – the virus carries with it a set of accessory proteins which target specific host restriction factors. Of these accessory proteins Vpr is the least well understood, with no described role that adequately explains its conservation across all known primate lentiviruses. Unpublished data from our lab indicate that Vpr is able to rescue infection in macrophages from addition of cGAMP, a second messenger protein produced by the cytosolic DNA sensor cGAS which activates antiviral immune signalling pathways. This study first sought to test the hypothesis that Vpr has evolved to counteract cGAS/STING mediated cytosolic DNA sensing using a co-transfection assay to test Vpr proteins from all groups of primate lentiviruses. Initial observations appeared to demonstrate specific degradation of innate immune signalling proteins. It was subsequently shown that HIV-1 M Vpr antagonises expression from all tested co-transfected plasmids. This phenotype was demonstrated to be species specific, and to correlate with both the history of zoonotic transmission and localisation of Vpr to the nuclear rim. Additionally, it was shown that Vpr antagonises NFB signalling activated by TNF, independent of an effect on expression from transfected plasmids, but with the same dependence on nuclear localisation, putatively by the same mechanism. Next, this study characterised an observation that the Vpr from the lentivirus infecting a mona monkey (SIVmon) stimulates NFB signalling. It was hypothesised that the SIVmon Vpr might have molecular binding partners in common with the HIV-1 M Vpr and conditions were optimised for proteomics studies to determine these binding partners. This study provides insights into the role of Vpr in antagonising innate immune sensing. Additionally, overexpression assays have been used widely in the literature describing Vpr. The data presented here indicate that observations using these assays, apparently demonstrating specific degradation of host cellular proteins, should be interpreted cautiously.

Epstein-Barr virus is one of the most common viral infections in humans and, once acquired, persists within its host throughout their life. EBV therefore represents an ex- tremely successful virus, having evolved complex strategies to evade the host’s innate and adaptive immune response during both initial and persistent stages of infection. While infection is mostly harmless in the majority of cases, EBV has the ability to be oncogenic in some individuals, and is associated with a wide range of malignancies as well as non-cancerous diseases. To generate new and useful insights into the evolution of EBV interactions with its host, a hybridization-based target enrichment methodology was optimised to enable whole genome sequencing of EBV directly from clinical samples. This allowed the gen- eration of whole genome sequences of EBV directly from blood for the first time. This methodology was subsequently applied to a number of distinct EBV sample col- lections and the resulting data used to investigate the intra- and inter-host variation in various clinical settings, such as infectious mononucleosis and immunosuppression with chronic EBV infection. Additionally, the number of available whole genomes from East Asia is expanded by eleven (unique) novel genomes from primary infection from a NPC- non-endemic area. These sequences were used for a comparative analysis between NPC- and non-NPC-derived EBV genomes and a number of sites were determined differenti- ating these two groups. Finally, comparative genomic analyses of world-wide EBV strain diversity were per- formed using genome sequences generated here in conjunction with a large number of publicly available EBV genome sequences. The comprehensive data sets generated, which included measures of diversity, selection, and linkage, were used to identify poten- tial targets of T cell immunity. In addition, the population structure of EBV was analysed to better understand the forces that have shaped the evolution of EBV.

Cell-mediated immunity (CMI) is central to the host response to intracellular pathogens such as Mycobacterium tuberculosis (Mtb). The function of CMI can be modulated by human immunodeficiency virus (HIV)-1 via its pleiotropic effects on the immune response, including modulation of macrophages, which are parasitized by both HIV-1 and Mtb. HIV-1 infection is associated with increased risk of tuberculosis (TB), and so in this thesis I sought to explore the host/pathogen interactions through which HIV-1 dysregulates CMI, and thus changes the natural history of TB. Using an in vitro model of human monocyte-derived macrophages (MDMs), I characterise a phenotype wherein HIV-1 specifically attenuates production of the immunoregulatory cytokine interleukin (IL)-10 in response to Mtb and other innate immune stimuli. I show that this phenotype requires HIV-1 integration and gene expression, and may result from a function of the HIV-1 accessory proteins. I identify that the phosphoinositide 3-kinase (PI3K) pathway specifically regulates IL-10 production in human MDMs, and thus may be a target for HIV-1 to mediate IL-10 attenuation. I show that HIV-1 may attenuate IL-10 to maximise its own replication, and identify potential consequences of IL-10 attenuation for CMI. By using the tuberculin skin test (TST) as a human challenge model, I evaluate HIV-1 modulation of CMI in vivo in active TB patients, and demonstrate IL-10 attenuation in this context. I identify a role for type I inteferons (IFNs) in HIV-1 anergy, and observe exaggerated T helper 2 responses associated with the immune reconstitution inflammatory syndrome (IRIS). To fully explore CMI in vivo by transcriptional profiling, I utilize the transcriptional heterogeneity of stimulated macrophages to develop a modular analysis strategy for transcriptional profiles, and apply this in the TST model. My results delineate novel modulatory effects of HIV-1 on the function of CMI, and thus provide insights into immunopathogenesis in HIV-1/TB co-infection.