Human Immunode ciency Virus (HIV) has a high mutation rate which allows it to evolve rapidly in response to selective pressures. We explore how transmission may naturally select HIV virulence. Set-point viral load (SPVL), the level of stable viraemia in early asymptomatic infection, is linked with the severity of infection, and is used as a proxy for virulence. The rst part investigates the virulence-transmission trade-o , which has been identi ed in several host-pathogen systems. Previous work hypothesised that this trade-o favours HIV-1 viruses of intermediate SPVL which are optimal for transmission. We analysed a deterministic model incorporating host and viral contributions to SPVL, transmission rates and duration of infection. We estimate key parameters governing SPVL evolution and examine the rate of evolution towards intermediate virulence. This model was extended to incorporate stochasticity to investigate how the founding virulence a ected the probability of emergence into a new population. Strains of intermediate virulence were found to be more likely to emerge. The second part explored heritability of virulence. Several recent studies have estimated heritability of SPVL, mostly by comparing between individuals identi ed as transmission pairs, but one used phylogenetic comparative analysis instead, which is applicable to more datasets. Many methods are available for this approach, and we evaluate these using simulated and previously analysed data, and develop new methods. None were able to identify heritability below a substantial threshold, which was higher than many of the results in previous studies. The two most successful methods were applied to a new dataset from the Netherlands, with an ambiguous result suggesting borderline detectable heritability. This work explores the estimation of heritability using phylogenies and provides some further evidence for genuine heritability of SPVL. The models provide strong support for a previous hypothesis and a framework for investigating how HIV virulence evolves in populations.

In recent years there has been an increased focus on biomedical interventions as a means of Human immunodeficiency Virus (HIV) prevention and the use of antiretroviral therapy (ART) has been a particularly successful tool in prevention efforts, with evidence for treatment in reducing HIV transmission. This is dependent on several factors including the early identification of those infected with HIV. In this thesis I will explore current challenges to testing for HIV in the UK by systematically reviewing current national levels of testing and investigating demographic characteristics associated with timing of diagnosis, testing practices and routes to diagnosis among those recently diagnosed with HIV in West London in order to identify barriers to increased and repeat testing for HIV in the UK. My findings show that guideline recommended testing levels are poor in most clinical settings and this is reflected in overall low HIV test coverage in the UK. HIV diagnosis at an earlier point in infection remains significantly associated with men who have sex with men (MSM) and White ethnicity and both patient and provider barriers act as ongoing challenges in earlier identification of HIV in all groups. Current testing practices are not enough to achieve equitable access to early diagnosis for HIV. Testing practices of clinicians, along with system challenges play an important role in HIV testing and changing these may be the most effective method of increasing earlier identification of HIV positive individuals in the UK.

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Studying evolutionary dynamics is crucial for epidemiological containment and/or eradication of viral diseases. With recent advancements in genetic sequencing, the spread of viruses can be monitored and sequenced almost in real-time as epidemics unfold, and sophisticated statistical analytical methods allow for the inference of population genetic and evolutionary characteristics directly from epidemiologic and genetic data. In this thesis, I have investigated various evolutionary aspects of the human immunodeficiency virus, the causative agent of AIDS, in the context of (a) within-host evolution of the virus in the emergence of resistance to CCR5 antagonist anti-retroviral agents and (b) between-host global evolutionary dynamics of the virus including an investigation of closely related simian immunodeficiency virus endemic in simian hosts. The main outcomes of my work have been, firstly, the development of a novel computational predictor for determining viral tropism from genetic sequences and a study investigating the efficacy of utilising this approach, and other genotypic methods, for screening patients prior to therapy; secondly, the characterisation of complex within-host mutational pathways and population genetic dynamics that give rise to the emergence of a rare form of resistance to CCR5 antagonists, namely the ability for the virus to utilise antagonist-bound CCR5 coreceptor; thirdly, an investigation of the global spread of HIV-1 group O and a proposed reconciliation of previous attempts to characterise genetic diversity observed which were discordant; and fourthly, en exploratory study comparing differences in rates of intrinsic disorder in both HIV and species-specific SIV, as well as human host genes and homologous simian host genes, and investigating the hypothesis that HIV pathogenicity might be attributable to promiscuous protein-protein interactions facilitated through protein intrinsic disorder. These studies have all highlighted aspects that are collectively important for epidemiological and genomic surveillance of infectious disease, especially in characterising transmission dynamics, monitoring changes and adaptation of viruses. Ultimately, I hope that these contributions will asset the wealth of knowledge that exists in this field, which collectively guides research into the development of new drugs and treatment regimens, as well as guidance on public health measures for control of infectious disease.

This research examined past microbicide effectiveness trials to better understand how adherence and adherence assessment could be improved in future vaginal microbicide trial design. No product is currently available despite decades of clinical trials of candidate microbicides, yet the need for women to have a method to reduce their risk of sexual transmission of HIV that does not rely on male partner agreement remains urgent. Low product adherence and inaccurate adherence reporting has inhibited the ability of trials to accurately assess the biological efficacy of candidate products. Methods Three different studies were conducted to examine adherence as a clinical trial design issue. The comparative study examined how five trials measured, calculated, and reported microbicide adherence. The quantitative study used latent class and latent profile analysis and multinomial logistic regression to examine if patterns of adherence could be identified in four trials and, if so, what individual-level factors were associated with the patterns. The qualitative study sought opinions from former trial participants about how to improve adherence and adherence reporting in future microbicide trials through focus group discussion workshops in South Africa and Tanzania. Results There was diversity in methods used to collect and calculate adherence among the included trials. Two methods to calculate averages of overall adherencewere identified. Trial documentation and publications lacked clarity in exact methods used to calculate adherence estimates. Latent structure analysis identified different patterns of adherence in all included trials, and these patterns were similar. Multinomial logistic regression identified factors associated with adherence patterns in all trials. Women join and stay in microbicide trials for their own needs, which are not necessarily related to interest in using the investigational product. Key reasons for joining and staying in trials included access to health care and financial reimbursements. Fear of adverse effects from the investigational product was the most important reason why participants did not use the gel. Participants reported that male partners can act as barriers to gel use and the key reason for inaccurate reporting of gel use was fear of removal from the trial. This study demonstrated that trial teams and participants can work together to develop improved trial designs. Recommendations There are improvements to be made in how trialists plan, conduct, analyse and report results of microbicide trials. Trial teams can improve the clarity of their trial materials, and use analysis methods to identify patterns of adherence. To improve adherence and trial implementation, trials can test applicators for evidence of vaginal insertion and report results to participants, better engage male partners, develop a less watery gel, and create an atmosphere of transparency and respect between research teams and participants. Identifying HIV prevention products for women requires better understanding of the lives of women asked to join these trials, and application of that understanding to collaboratively develop innovative trial designs that meet both the needs of the research and the needs of participants.

CD4+ T cells play a central role in controlling the adaptive immune response by secreting cytokines to control the responses of different lymphocytes. Naïve CD4+ T cells differentiate into at least four subsets, Th1, Th2, Th17, and inducible regulatory T cells under antigen and corresponding cytokine microenvironment during an immune response. Therefore, each subset has unique functions for pathogen elimination. The differentiation of these subsets is induced in response to conditioned cytokine stimulation, which induces specific master regulator transcription factors. Multiple other transcription factors, both subset specific and shared, are also involved in promoting subset differentiation. Previously, our group has discovered that early growth response gene-2 (Egr-2) is important for the maintenance of T cell homeostasis and controls the development of autoimmune disease. However, the underlying mechanisms are unknown. In this study, using Egr-2 conditional knockout mice, a novel mechanism of Egr-2 in the control of Th17 differentiation has been discovered. Egr-2 is induced by TGFβ and IL-6 in naïve CD4+ T cells. The induced Egr-2 negatively regulates the expression of IL-17, the signature cytokine for Th17. In contrast, Egr-2 has less effect on the expression of IL-2 or IFN-γ in effector T cells. In the absence of Egr-2, CD4+ T cells produce high levels of Th17 cytokines. Deletion of Egr-2 in T cells renders mice susceptible to EAE induction; a model for Th17 mediated autoimmune diseases. T cells lacking Egr-2 show increased propensity for Th17, but not Th1 or Th2, differentiation in vitro. The key mechanism of Egr-2 in regulation of Th17 differentiation is to inhibit the function of Batf for transactivation of IL-17 expression and promotion of Th17 differentiation. Egr-2 interacts with Batf in CD4+ T cells and suppresses its interaction with DNA sequences derived from the IL-17 promoter, while the activation of STAT3 and expression of RORγt is unchanged in Th17 cells in the absence of Egr-2. Thus, Egr- 14 2 plays an important role to intrinsically control Th17 differentiation. We also found that CD4+ T cells from multiple sclerosis (MS) patients have reduced expression of Egr-2 and increased expression of IL-17 following stimulation with anti-CD3 in vitro. Collectively, our results demonstrate that Egr-2 is an intrinsic regulator that controls Th17 differentiation by inhibiting Batf activation which may be important for the control of inflammatory autoimmune diseases.

Background: Globally, the number of adolescents living with perinatally-acquired HIV continues to rise including in Malawi. To date, this group has received relatively less attention in the field of HIV care; yet they are increasingly surviving into adulthood. There is a growing need for the development of appropriate care and support services for this group; however their sexual and reproductive health (SRH) needs remain poorly addressed. Research Aim: This study aimed to explore perinatally-infected young women’s experiences of growing up with HIV in order to understand their SRH outcomes within their wider socio-cultural and structural context. Methods: A qualitative case study design was adopted whereby each ‘case’ comprised a female adolescent (15-19 years), a nominated caregiver and a service provider. Data was collected for 14 cases through in-depth interviews. The interviews with adolescents were based on an innovative visual method known as ‘my story book’. Results: The study found that young women endured multiple losses that negatively impacted upon their sense of self and belonging. Emotional, material and social support were essential in helping them to build a sense of identity, but their access to such resources was highly variable. Young women’s strategies to seek love, acceptance or support often led them to take sexual risks and left them with little control over their reproductive health. Both the service providers and caregivers often ‘turned a blind eye’ to young women’s sexual activities, leading to poor SRH outcomes. Lack of open discussion on SRH issues was related to cultural and religious norms hindering young women’s access to information and contraception. Conclusion: Addressing the complex needs of the young women poses a key challenge for Malawi’s HIV services. One way forward is to explore ways in which services could develop integrated models of care, offering a ‘one-stop shop’ to this vulnerable group.

Background: Phylogenetic characterisation of local HIV-1 epidemics can be used to understand emerging transmission networks. We analysed subtype-unassigned sequences in the UK HIV Drug Resistance Database (UK HIV DRD) to map the emergence and origin of genetically diverse recombinant strains using phylogenetic analyses, near full-length sequencing of plasma HIV-1 and CD4 cell count decline. Methods: 55,556 genotyped pol sequences (protease amino acids 1-99, RT amino acids 1-234) were analysed for evidence of recombination. Near full-length single genome sequencing of plasma HIV-1 was performed on stored specimens from six patients with a putative A1/D novel recombinant strain. Recombination and phylogenetic analyses were performed using RIP, PhyML, jpHMM, Simplot, SCUEAL and FastTree v2.3.1. Evolutionary analysis of putative novel recombinants was performed using Bayesian Evolutionary Analysis by Sampling Trees (BEAST). Geographic screening for additional instances of recombinant structures was performed using HIV BLAST and the Los Alamos National HIV database. Demographic data were available for 50/72 patients. CD4 cell count decline was assessed using linear regression. Results: The proportion of subtype-unassigned HIV-1 strains in the UK HIV DRD increased significantly during 1999-2008 (p=<0.01). 2,030 putative B-recombinant sequences were analysed for evidence of transmission of novel recombinant strains; 15 novel recombinant clusters comprising 94 individuals were identified. The proportion of intravenous drug users (IVDU), males and people of white ethnicity was significantly higher among novel recombinant clusters than among people infected with pure subtypes and recognised CRFs. Geographic screening showed co-circulation of novel strains in seven countries over three continents and import and export of novel strains from the UK was identified. Near full-length sequencing of six plasma specimens showed five patients sharing an identical A1/D strain; this was registered as CRF50_A1D and 67 further instances in the UK HIV DRD were identified. Geographic analysis showed close relation of component subtype A1 and D regions to East African strains; monophyletic clustering indicated a single introduction of this variant into the UK. Time scaled analysis showed a time to most recent common ancestor of approximately 1992. Demographic data showed the earliest CRF50 transmissions were confined to men who have sex with men (MSM), with subsequent transmissions to heterosexuals and IVDUs. Analysis of the sixth, complex, sequence demonstrated onward recombination of CRF50 with a subtype B strain (median divergence year 2000). CD4 cell count analysis suggested infections with CRF50 progressed in a similar manner to subtype B infections. Conclusions: Significant increasing genetic diversity of HIV-1 in the UK is linked to both UK and international transmissions among multiple exposure routes. These findings highlight the changing dynamics of HIV transmission in the UK and the converging of the two previously distinct MSM and heterosexual epidemics.

Nontyphoidal \(Salmonella\) is strongly associated with HIV infection, whereas there is a negative association between HIV and typhoid fever. To investigate this phenomenon, we explored humoral and cytokine responses in HIV-infected and uninfected blood and sera against \(S.\) Typhimurium and \(S.\) Typhi, finding that HIV-infected sera had significantly impaired bactericidal and opsonic activity against \(S.\) Typhi compared with HIV-uninfected sera. We observed dysregulated cytokine responses in blood from HIV-infected individuals after Salmonella stimulation, with RANTES levels being modulated by HIV status and \(Salmonella\) serovar. Most \(S.\) Typhi isolates express Vi capsule and O:9 O-antigen. The roles played by the Vi capsule and by anti- \(S.\) Typhi antibody in infection are unclear. Effective vaccines against typhoid exist, but the mechanisms of protection afforded by the antibody they elicit is poorly characterised. We investigated the role of Vi capsule and anti-S. Typhi antibody in the killing of \(Salmonella\). Using isogenic Vi+/- \(Salmonella\) we show that Vi-expression is associated with reduced antibody and complement deposition on \(Salmonella\) and increased resistance to serum and phagocyte killing. We characterised the bactericidal and opsonic activity of purified human anti-Vi and anti-O:9 antibodies against \(S.\) Typhi, finding that both antibodies kill \(Salmonella\) but anti-O:9 antibody has poor opsonic activity. Collectively our data suggest cytokine, but not antibody, dysregulation may underlie the dichotomy of \(Salmonella\) infection in the context of HIV. We demonstrate that both anti-capsular and anti-O-antigen antibodies elicit bactericidal activity against \(S.\) Typhi but anti-O:9 antibody is a poor opsonin. This has implications in the development of O:9 vaccines against O:9 –expressing \(Salmonellae\).