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HIV-2 is a contemporary human retrovirus with the majority of infections localised to West Africa. Both HIV-1 and HIV-2 are able to cause AIDS; however, in contrast to HIV-1 infection, a common outcome following HIV-2 infection (∼ 37% of patients in this study cohort) is long-term non-progression (LTNP), where patients remain aviraemic and asymptomatic in the absence of treatment, often for decades. HIV-1 and HIV-2 both arose following zoonotic transmission of SIVs from non-human primates at around the beginning of the 20^th century and when patients develop AIDS caused by HIV-2 infection, it is clinically indistinguishable from AIDS following HIV-1 infection. Whilst the estimated number of HIV-2 infections remains small in the context of the global HIV pandemic (HIV-2 ∼ 2 million, HIV-1 group M ∼75 million), the differences in pathogenicity between these two viruses has been a source of great interest, particularly the features of LTNPs that allow control of viral replication in the absence of anti-retroviral treatment. The studies described in this thesis were carried out using samples collected from a well-characterised longitudinal community cohort in Caió, Guinea-Bissau. Chapter 3 of this thesis presents an investigation into the variation and evolution present in the HIV-2 specific accessory gene vpx. The data showed significantly increased signals of positive selection pressure in vpx in viraemic when compared to non-viraemic patients and also allowed the identification of novel variations at high frequencies (up to 22%) in this cohort that were previously un-described. Chapters 4 and 5 present a novel application of shotgun RNA sequencing (RNA- Seq) to HIV ex vitro and ex vivo samples. Chapter 4 demonstrates the divergence seen in a cultured viral isolate at the level of the whole genome, in the absence of many of the biases typically involved in sequencing of RNA viruses. Chapter 5 further extends this method to show the applicability of using RNA-Seq on primary patient HIV samples for the first time. Analysis of diversity estimates over the whole genome in the context of a low bias sequencing method show a high level of diversity in HIV-2 pol and low diversity in vpx. The aim of this work was to combine traditional and novel sequencing methods to facilitate assessment of the variation and evolution acting on vpx and to generate an accurate picture of the genetic diversity over the whole genome of HIV-2.

Hepatitis C virus (HCV) is emerging as one of the leading causes of morbidity and mortality in individuals infected with HIV and has overtaken AIDS-defining illnesses as a cause of death in HIV patient populations who have access to highly active antiretroviral therapy. For many years, the clonal analysis was the reference method for investigating viral diversity. In this thesis, a next generation sequencing (NGS) approach was developed using 454 pyrosequencing and Illumina-based technology. A sequencing pipeline was developed using two different NGS approaches, nested PCR, and metagenomics. The pipeline was used to study the viral populations in the sera of HCV-infected patients from a unique cohort of 160 HIV-positive patients with early HCV infection. These pipelines resulted in an improved understanding of HCV quasispecies dynamics, especially regarding studying response to treatment. Low viral diversity at baseline correlated with sustained virological response (SVR) while high viral diversity at baseline was associated with treatment failure. The emergence of new viral strains following treatment failure was most commonly associated with emerging dominance of pre-existing minority variants rather than re-infection. In the new era of direct-acting antivirals, next generation sequencing technologies are the most promising tool for identifying minority variants present in the HCV quasispecies populations at baseline. In this cohort, several mutations conferring resistance were detected in genotype 1a treatment-naïve patients. Further research into the impact of baseline HCV variants on SVR rates should be carried out in this population. A clearer understanding of the properties of viral quasispecies would enable clinicians to make improved treatment choices for their patients.

Background: The idiopathic inflammatory myopathies (IIM) are a heterogeneous group of rare autoimmune diseases comprising of polymyositis (PM), dermatomyositis (DM), and inclusion body myositis (IBM). They are characterised primarily by muscle weakness, and can present with extramuscular manifestations such as skin rashes, interstitial lung disease and malignancy. Aims: This aim of this study was to identify novel genetic risk factors in IIM and to further elucidate the relationship between genotype and serotype. Methods: 2,566 IIM samples were collected from 14 countries through the Myositis Genetics Consortium (MYOGEN) and genotyped on the Immunochip, a custom array covering 186 established autoimmune susceptibility loci. SNP2HLA was used to impute classical HLA alleles and constituent amino acids. Results: In a combined IIM analysis, the HLA region and PTPN22 reached genome-wide significance (p<5x10-8). A further nine regions reached suggestive significance (p<2.25x10-5) including UBE2L3, STAT4 and CD28 that have been implicated in autoimmune disease previously. Independent effects were seen within the STAT4 region. In a PM subgroup analysis (n=931), the HLA region and PTPN22 reached genome-wide significance. A further seven regions reached suggestive significance including SLC26A1/IDUA and RGS1. In an adult and juvenile DM analysis (n=1,360), only the HLA region reached genome wide significance. Three loci reached suggestive significance including GSDMB. In the IBM analysis (n=252), only the HLA region reached genome wide significance and 3 loci reached suggestive significance, including the CCR2 locus. Identification of exonic and eQTL SNPs has localised association signals to several potential causal variants. HLA imputation on the combined dataset confirmed that alleles of the 8.1 ancestral haplotype (AH) are most strongly associated with IIM. The cohort was stratified in to clinical subgroups. In PM the strongest effect was found with HLA-DRB1*03:01 with an independent effect with HLA-B*08:01. Amino acid position 74 lies within the peptide binding groove and may explain the risk in HLA-DRB1. HLA-B*08:01 was the most associated variant in both DM and JDM, with independent effects of amino acid position 57 of HLA-DQB1 in DM and HLA-C*02:02 in JDM. In IBM, the strongest associations were with amino acids positions 26 and 11 of HLA-DRB1.HLA imputation was conducted on antibody subgroups. The most associated variant for anti-Jo-1 and anti-PM/Scl antibodies was with amino acid 74 of HLA-DRB1. Alleles of the 8.1 AH were most associated with anti-TIF1-γ, anti-SAE and anti-cN1A antibodies. Alleles independent of the 8.1 AH were replicated such as anti-Mi-2 antibodies and HLA-DRB1*07:01, and anti-HMGCR antibodies and HLA-DRB1*11. Conclusions: This represents the largest study to date in IIM and has considerably expanded our knowledge about the genetic architecture of this rare disease. This study has identified novel disease susceptibility genes for IIM and independent associations with PM and DM, IBM and antibody subgroups that show that stratifying patients in to more homogenous cohorts is important to expand our knowledge of IIM. Ongoing sample collection is required to identify additional genes and environmental risk factors that lead to the development of IIM, and to expand our limited understanding of the pathogenesis of this disease.

With increasing age, aspects of the innate and adaptive immune systems show functional decline. In the skin this is associated with an increased incidence of epidermal malignancies and infections, a decreased incidence of contact allergy, and the development of autoimmunity. The mechanisms underlying these clinical effects in aged skin are poorly understood. Langerhans’ cells (LCs), which are members of the wider family of dendritic cells (DCs), reside in the epidermis where they act as sentinels of the immune system by processing and presenting antigen and inducing T cell responses. Previous investigations have suggested that the number of epidermal LCs is reduced, and that the motility of LCs is impaired in aged skin. A series of investigations was performed to characterise the mechanistic basis for the reduced frequency and restricted mobility of epidermal LCs in the skin of the elderly. Initially LC-like cells were cultured from circulating monocyte precursors and characterised using flow cytometry. The ability of precursors to differentiate into LC-like cells was not impaired in the aged; furthermore there were no age-associated differences in expression of markers of LC activation at baseline or upon stimulation. The phenotype of epidermal LCs was assessed using flow cytometric analysis of epidermal cell suspensions and did not appear altered in aged individuals. In addition, using the same techniques with dermal cell suspensions the dermal DC population was not altered with age. Langerhans’ cell migration from epidermal explants prepared from the skin of aged individuals was impaired but could be restored with exogenous interleukin (IL)-1β. There was no age-related reduction in the epidermal levels of IL-1β or caspase-1 (IL-1β converting enzyme which converts pro-IL-1β to the active form) or the expression of the IL-1 receptor I (IL-1RI), to account for this observation. However, the amount of IL-1 receptor antagonist was reduced in aged skin suggesting a change in the overall local cytokine balance. Based on previous reports that topical retinoic acid (RA) can increase cutaneous IL-1 production, a 4-day patch test assay was performed using 0.025% all-trans RA cream to explore whether this could restore LC migration in the aged. There was no effect on LC migration from epidermal explants prepared after treatment with RA in the aged.These data demonstrate that changes in LC function in the elderly may not be associated with changes in systemic DC biology. Age related changes in the cutaneous microenvironment are likely to be more relevant.

HIV-1 chronically infected individuals represent a large disease burden, making the development of a therapeutic vaccine for use in chronic infection a priority. However, therapeutic vaccination has not been successful to date. Most approaches have employed viral vectored vaccines encoding full length viral proteins, which aimed to boost pre-existing CD8&plus; T cell responses by mimicking natural HIV-1 infection. Simply boosting these pre-existing CD8&plus; T cell responses which have previously failed to control the virus may be insufficient. Although HIV-1 has a huge capacity to diversify, certain regions are less tolerant of mutations due to structural and functional constraints. We therefore hypothesised that it would be necessary to redirect the immune response to more vulnerable regions of the proteome, such as conserved regions. HIVconsv is a rationally designed conserved region vaccine. Vaccination of 19 chronically HIV-1 infected HAART treated patients with MVA.HIVconsv was safe and well tolerated. There was a significant increase in the magnitude of HIVconsv-specific T cell response following vaccination (p = 0.001), as measured by IFN-&gamma; Elispot assay, but changes observed in vaccinees did not reach statistical significance when compared with placebo recipients (p = 0.48). The capacity of CD8&plus; T cells to inhibit HIV-1 replication in vitro is highly predictive of virus control in vivo and is thus a possible surrogate of vaccine efficacy. There was a trend towards increased CD8&plus; T cell mediated inhibition following vaccination with 2x10⁸pfu MVA.HIVconsv (17&percnt; inhibition pre- vs 54&percnt; inhibition post-vaccination, p = 0.06). However, measurement of the latent HIV-1 reservoir by quantification of total HIV-1 DNA in circulating CD4&plus; T cells by droplet digital PCR showed no reduction in size upon vaccination, indicating CD8&plus; T cells induced by vaccination with MVA.HIVconsv were not of sufficient potency to impact the reservoir. In a second cohort of HIV-1 infected individuals, antiviral inhibitory activity was measured in 36 HIV-1-infected STEP and Phambili trial participants. Sustained potent CD8&plus; T cell antiviral inhibitory responses were rare but were strongly correlated with IFN-&gamma; responses to so-called ‘beneficial’ low entropy regions in HIV-1 Gag and Pol, that had been reported previously to be associated with HIV-1 control, (r = 0.69, p = 0.0001). This correlation was still significant after controlling for protective HLA alleles, whereas responses to conserved elements were only weakly correlated with viral inhibition (r = 0.41, p = 0.04). These data indicate that immunogens that are based on the selection of regions within the viral proteome by conservation score alone may not induce the most effective HIV-1-specific T cell responses and they highlight the importance of systematically selecting specific regions associated with HIV-1 control, together with exclusion of immunodominant decoy epitopes.

Regulatory T cells (Treg) are critical to the maintenance of immune tolerance, partly by controlling the unwanted activation of effector T cells (Teff) and thereby enhancing the resolution of autoimmune and allergic inflammation. Recent data suggest that Treg can specialize to better control different types of inflammation by using transcriptional machinery which controls differentiation and function of Teff. This thesis addresses questions related to the efficacious use of Treg, notably their ability to adopt distinct phenotypic profiles under different inflammatory contexts and their need to recognize antigen in the inflamed organ. Two differentially mediated mouse disease models were used in this project, namely Th1/Th17-mediated experimental autoimmune encephalomyelitis (EAE) as a model of multiple sclerosis and Th2-mediated allergic airways inflammation (AAI) as a model of asthma. A new model of rMOG-induced AAI was developed to specifically answer the questions on the importance of cell phenotype versus antigen-reactivity for the effective Treg-mediated suppression. It was demonstrated that Treg from the inflamed CNS in EAE had an upregulated expression of Th1 master regulator T-bet and Th1-associated chemokine receptor CXCR3, whereas Treg derived from the inflamed lung in AAI had an increased expression of Th2 master regulator GATA-3, lacked expression of T-bet and displayed decreased levels of CXCR3. This specialized and activated phenotype was restricted to tissue-derived Treg. The importance of appropriate Treg phenotype for effective suppression was suggested by the observed inability of CNS-derived Treg to inhibit AAI. A different Treg subset, TGF-β-induced Treg (iTreg), was shown to express high levels of T-bet and CXCR3, but not GATA-3 upon induction in vitro. iTreg effectively suppressed both Th1 and Th2 types of inflammation and the antigenreactivity was key to this. This thesis demonstrates that Treg are capable of acquiring a distinct phenotype corresponding with a CD4+ T cell response driving inflammatory disease and identifies antigen-reactivity as key to the efficacious suppression of inflammation. It also highlights substantial phenotypic differences between iTreg and naturally-occurring Treg which could be associated with different modes of suppression.

Regulatory T cells expressing the transcription factor Foxp3 have a critical role in the maintenance of tolerance to both self and innocuous exogenous antigens. Humans and mice die from overwhelming autoimmunity in the absence of Foxp3+ Treg whilst administration of regulatory T cells has shown promise therapeutically in ameliorating autoimmunity in several animal models. Regulatory T cells arise naturally in the thymus (nTreg) but may also be induced from naïve Foxp3- cells in the presence of TGF-β (iTreg), both in vitro and in vivo. This thesis focuses on in vitro generated mouse iTreg, testing the hypothesis that they are able to effect bystander suppression; iTreg activated by a given antigen are able to suppress other responding cells with different antigen reactivities. Chapter 3 details an in vitro assay system using iTreg and responder cells recognising different antigens (TCR transgenic cells). Evidence for bystander suppression is presented and that did not require the presence of iTreg-relevant antigen but did require iTreg-relevant MHC Class II. The kinetics of iTreg suppression are discussed, with evidence presented that iTreg exert their effects early in co-culture. Chapter 4 identifies the production of three pro-inflammatory cytokines by iTreg - IFN-γ, GM-CSF, and TNF. These were not involved in the in vitro suppressive mechanism, but early abrogation of TGF-β signalling did inhibit suppression. Chapter 5 describes the in vivo function of iTreg under various experimental protocols. iTreg did not limit initial proliferation of naïve T cells in response to antigen but did limit the development of effector cells producing pro-inflammatory cytokines. Exposure to a pro-inflammatory environment in vivo led to iTreg producing IFN-γ and TNF, but not GM-CSF. This could be replicated in vitro by exposure to IL-6, IL-12 or IL-27. Finally, evidence for bystander suppression by iTreg in vivo is presented, with a reduction in effector cells producing pro-inflammatory cytokines shown in an allergic airways diease model.

The applicant's research has led to a substantial body of published work and 17 pieces from this work are submitted here. Of these, eight are in peer refereed journals testifying to the importance of the body of work submitted. The work has attracted external funding of £73,000 from North West Thames Regional Health Authority which attests further to the quality of the work undertaken. In addition the applicant's expertise in the area has been recognised internationally by her appointments as World Health Organisation Advisor (Guidelines on Counselling of HIV Infected and AIDS Patients; Intravenous Drug Use and Risk of HIV Infection) and as UK representative to the Commission of the European Communities (Prevention of AIDS for Intravenous Drug Users) and she has presented evidence to a Home Office Working Party (Advisory Council on the Misuse of Drugs) and acted as academic referee for several academic journals including AIDS, AIDS Care, and Addiction. The submitted research publications are based upon five studies drawing upon 623 injecting drug users (idus) surveyed between 1985 and October 1992. The demographic characteristics of the clients included in each of the studies did not vary substantially between studies. Three of the studies involved evaluation of interventions for harm minimisation and two of those included designs of innovative interventions. The remaining two were aimed at identifying trends in risk - related behaviours and risk reduction. The body of work with its regular data collection over a seven year period from one location in the UK charts the behavioural changes and service responses from a point in time when AIDS awareness among idus was virtually non-existent through the response to the awareness of risks of sharing injecting equipment, followed by the introduction of needle exchange schemes and their evaluation, awareness of sexual transmission risks and need for sexual counselling, provides a unique perspective. The first and the final study had longitudinal components but the over all behavioural and attitudinal trends are identified from cross-sectional data. The approach taken by the research was to place risk-related behaviours within a context of the idus' social lifestyles rather than isolating behaviours from the contexts in which they occur. This approach contrasts with the individualistic social-cognitive models that have been used by others rather unsuccessfully to try to account for health related risk behaviours. The aims of the research were to obtain accurate information about the behaviours of idus with specific reference to HIV transmission related behaviours especially injecting practices and sexual behaviours by (1) identifying the characteristics of idus attending a drug dependency unit and/or syringe exchange unit in Central London (2) examining the sexual and drug-related behaviours of clients attending those services and their risks for HIV infections.

Genome Wide Association Studies (GWAS) have uncovered many genetic regions which are associated with autoimmune disease risk. In this thesis, I present methods which I have developed to build upon these studies and enable the analysis of the causal variants of these diseases. Colocalization methods disentangle whether potential causal variants are shared or distinct in related diseases, and enable the discovery of novel associations below the single-trait significance threshold. However, existing approaches require independent datasets to accomplish this. I extended two methods to allow for the shared-control design; one of these extensions also enables fine mapping in the case of shared variants. My analysis of four autoimmune diseases identified 90 regions associated with at least one disease, 33 of which were associated with 2 or more disorders; 14 of these had evidence of distinct causal variants. Once associated variants have been identified, we may wish to test some aggregate property, such as enrichment within an annotation of interest. However, the null distribution of GWAS signals showing association with a trait and preserving expected correlation due to linkage disequilibrium is complicated. I present an algorithm which computes the expected output of a GWAS, given any arbitrary definition of "null", and hence can be used to simulate the null distribution required for such a test. Commonly, GWAS report only summary data, and determining which genetic variants are causal is more difficult; the strongest signal may merely be correlated with the true causal variant. I have developed a statistical method for fine mapping a region, requiring only GWAS p-values and publicly available reference datasets. I sample from the space of potential causal models, rejecting those leading to expected summary data excessively different from that observed. This removes the need for the assumption of a single causal variant. In contrast to other summary statistic methods which allow for multiple causal variants, it does not depend upon availability of effect size estimates, or the allelic direction of effect and it can infer whether the pattern of association is likely caused by a non-genotyped SNP without requiring imputation. I discuss the effect of choice of reference dataset, and the implications for other summary statistics techniques.