HIV infection is now the fastest-growing serious health hazard in the United Kingdom (UK), with an estimated 53,000 infected adults at the end of 2003. Despite a recent increase in heterosexually acquired infections, the most prevalent clade of virus within the country remains subtype B, from the main group of HIV-1, which is mainly transmitted through sex between men. To date, very little is known about how subtype B successfully invaded the British population, and how the virus has subsequently spread and evolved. Given that molecular data on HIV-1 is becoming increasingly available since the introduction of routine gene sequencing for drug-resistance monitoring, the present thesis proposes to assess the reliability of the HIV-1 pol gene for molecular analyses of epidemiological relevance. Identification of transmission networks by phylogenetic means were primarily conducted, with the further goal to investigate the dynamics of HIV-1 transmission at both individual and population level in the UK. Evolutionary and epidemiological approaches were then combined in order to assess the correlates of transmission within a population of primary HIV-1 infected individuals within a localised risk group, exploiting both molecular and clinical data. Finally, the epidemic history of HIV-1 subtype B in the UK was reconstructed from sampled HIV-1 pol gene sequences, providing new insights into the complexity of HIV- 1 epidemics that must be considered when developing monitoring and prevention initiatives. The analyses presented in these pages emphasizes the advantage of combining state-of-the-art epidemiological studies to phylogenetic frameworks when investigating the dynamics of a viral epidemic as complex as HIV-1.

Several diseases including cancer and pathogen infection are mediated by protease activity. In HIV infection, the viral protease plays a central role in the virus lifecycle, which has made it a clear therapeutic target. The dominant approach for the treatment of HIV is heavily dependent on inhibitors of this enzyme, but no new drugs have reached the market since 2006. There is thus a need for new design principles for the development of anti-retroviral therapies. Traditional methods of HIV detection are also limited in their use at point-of-care in resource-limited settings due to their reliance on highly trained laboratory personnel, cold-chain transport and expensive reagents. This thesis examines the role of peptide-protein interactions for the inhibition and detection of HIV-1 protease. Phage display is used to isolate heptameric peptide sequences which interact specifically with the enzyme. These peptides are then utilised as sensors for the detection of the enzyme through Forster Resonance Energy Transfer (FRET). The inhibitory properties of the peptides, both in isolation and through multivalent conjugates are also investigated. Finally, insights into the nature of these peptide-protein interactions are explored through molecular docking and all-atom classical molecular dynamics simulations. The expression of recombinant HIV-1 protease in E. coli is also discussed. The peptide based systems described here are expected to be more stable to environmental effects than protein based therapies and it is hoped that this work will provide new pathways for the design of peptide-based therapeutics and diagnostics for protease related diseases which do not rely on traditional methods.

Men's uptake of HIV-testing and counselling services across sub-Saharan Africa is inadequate relative to universal access targets. A better understanding of the effectiveness of available interventions to increase men’s HIV-testing and of men’s HIV-testing behaviours is required to inform the development of strategies to increase men’s levels and frequency of HIV-testing. My thesis aims to fill this gap. To achieve this, I combine a systematic review of randomised trials of interventions to increase men’s uptake of HIV-testing in sub-Saharan Africa with analyses of two population-based surveys from Zambia, through which I investigate the levels of and factors associated with HIV-testing behaviours. I also conduct an integrated analyses to explore whether the scale-up of voluntary medical male circumcision (VMMC) services between 2009 and 2013 contributed to increasing men’s population levels of HIV-testing. In the systematic review I find that strategies to increase men's HIV-testing are available. Health facility-based strategies, including reaching men through their pregnant partners, reach a high proportion of men attending facilities, however, they have a low reach overall. Community-based mobile HIV-testing is effective at reaching a high proportion of men, reaching 44% of men in Tanzania and 53% in Zimbabwe compared to 9% and 5% in clinic-based communities, respectively. In the population-based surveys, HIV-testing increased with time: 52% of men evertested in 2011/12 compared to 61% in 2013. Less than one-third of men reported a recent-test in both surveys and 35% multiple lifetime HIV-tests. Having a spouse who ever-tested and markers of socioeconomic position were associated with HIV-testing outcomes and a history of TB with ever-testing. The scale-up of VMMC provided men who opt for circumcision with access to HIV-testing services: 86% of circumcised men ever-tested for HIV compared to 59% of uncircumcised men. However, there was little evidence that VMMC services contributed to increasing HIV-testing among men in this rural Zambian setting. Existing strategies to increase men’s uptake of HIV-testing are effective. Over half the men in two population-based surveys reported ever-testing for HIV in rural Zambia. Nonetheless, some 40% of men never-tested. Men’s frequency of HIV-testing was low relative to recommendations that individuals with continued risk of HIV-infection retest annually for HIV. Innovative strategies are required to provide never-testers with access to available services and to increase men’s frequency of HIV-testing.

This thesis describes an investigation of the mechanisms by which DC-SIGN mediates enhancement of HIV-1 infection. Mannose-binding C-type lectin receptors, expressed on Langerhans cells and subepithelial dendritic cells (DCs), play an important role in HIV-1capture and subsequent dissemination to lymph nodes. DC-SIGN mediates both productive infection of DCs and trans-infection of CD4+ T cells that occurs in the absence of replication. The molecular events involved in transmission have not been fully defined. In this study, surface plasmon resonance analyses demonstrated that DC-SIGN, but not langerin, increases the binding affinity of trimeric gp140 envelope glycoproteins to CD4. In vitro infection experiments demonstrated significantly lower enhancement of a CD4-independent compared with a CD4-dependent strain. DC-SIGN increased the relative rate of infection of the CD4-dependent strain but had no effect on the CD4- independent strain. These findings are consistent with a mechanism in which DC-SIGN binding to glycans of the HIV envelope protein increases exposure of the CD4 binding site to enhance infection. To investigate the specificity and nature of glycan binding required for enhancement of HIV infection, a chimaeric C-type lectin was prepared where the carbohydrate recognition domain (CRD) of DC-SIGN was substituted by the CRD of langerin. Enhancement of HIV infection was then determined in TZM-bl cells expressing DCSIGN, langerin or the chimaeric DC-SIGN/langerin lectin. All lectins significantly enhanced infection compared with TZM-bl cells (in the absence of lectin). These data suggest that the increased flexibility of the CRD associated with DC-SIGN does not contribute significantly to C-type lectin-mediated enhancement of infection. To determine whether DC-SIGN binding to specific glycans of the envelope protein is associated with enhanced infection, mutagenised HIV-1 virions with specific glycan deletions (at residues 386 and 397) were compared with the parental strain using an in vitro model of DC-SIGN mediated enhancement of infection. Deletion of the N-Linked glycan at residue 386 reduced the level of enhancement. These studies also provided some evidence that DC-SIGN binding to the HIV envelope protein may induce further perturbation in the structure leading to increased exposure of the co-receptor binding site.

Background: Peanut allergy (PA) is responsible for life-threatening allergic reactions. Household peanut consumption (HPC), used as an indirect measure of environmental peanut exposure (EPE), is associated with PA especially when compared against atopic controls. Aims: To determine the association between EPE, peanut sensitization (PS), PA and explore the modifying effect of an impaired skin barrier. To assess the route of PS using peanut specific immune responses in skin versus gut derived T-helper (Th) cells. Methods: Peanut antigen in dust was assessed using ELISA, Mass Spectrometry (MS) and basophil activation test (BAT). HPC was compared to peanut-dust levels and airborne peanut. The impact of EPE on PS was determined in three cohorts with genotypic and phenotypic skin barrier function measures. Recall responses to peanut in skin versus gut-homing memory Th-cells were assessed using gene expression profiles and intracellular cytokine staining. Results: HPC was the most important factor for peanut-dust in the infants’ environment. BAT confirmed biological activity of peanut in dust; MS confirmed whole sequences of major peanut allergens. Airborne peanut was only transiently above peanuts being shelled. Early EPE increased the risk of PS; this was augmented by atopy and markers of skin barrier impairment. Th2 gene expression was not increased in skin versus gut-homing memory CD4+Th cells from peanut allergic children. IL9 was the most accurate classifier for PA versus PS and atopic non-peanut allergic (NA) children. Conclusions: EPE is a risk factor for PS in atopic children, especially when skin barrier is impaired; this supports the concept of epicutaneous peanut sensitization. Peanut is unlikely to be sufficiently airborne to induce inhalational sensitization. Although, there was no differential expression of Th2 cytokines in skin versus gut-homing Th cells, longitudinal assessment as children progress from PS to PA may show Th2 cytokines initiate in skin-homing Th cells then spread to gut-homing Th cells. IL9 may be a useful biomarker for peanut allergy. The role of IL9 in mast cell activation, trafficking and proliferation also provides a compelling explanation for the immunobiology underlying epicutaneous sensitization and elicitation of allergic reactions.

Improving maternal reproductive health, combating HIV/AIDS, and preventing sexually transmitted infections (STIs) are major global health priorities specifically highlighted under the UN Millennium Development Goals. Often, the risks associated with these clinical indications are inter-related. For example, genital HSV-2 infection in women is associated with a three-fold increased risk of HIV acquisition. Unfortunately, these individual health goals have not yet been achieved. A preferred strategy would involve a single pharmaceutical product/device that addresses at least two, and preferably three, of these clinical indications. A matrix-type VR, developed by the Queen’s University Belfast (QUB) and the International Partnership for Microbicides (IPM) and providing controlled release of the antiretroviral compound dapivirine (DAP), is presently being tested in a Phase III study as a potential microbicide for HIV prevention. Second generation VRs, comprising either two HIV microbicides (DAP and maraviroc, MVC) or a combination of DAP and levonorgestrel (LNG), are also in or about to enter early stage clinical development. The primary objective of this PhD project was to develop a multiple core, reservoir-type vaginal ring for delivery and maintenance of potentially clinically effective drug concentrations of anti-HIV drug dapivirine (DAP), progestin (levonorgestrel) and anti-HSV drug famciclovir (FMV). DAP-only and LNG-only vaginal rings have already been tested clinically, and there is much consensus on their clinical effective levels. Given than FMV has not previously been tested for vaginal administration, we developed different strategies to control and maximise the release of the drugs from the rings. In addition to FMV, two more experimental anti-HSV drugs pritelivir (PVR), its salt form pritelivir mesylate (PVR-M) and second drug north-methanocarbathymidine (N-MCT) have been tested for vaginal ring formulations in this research project.

This thesis explores the relationship between the use of antiretroviral therapy (ARVs) and drug resistance emergence in HIV-1 infected individuals. It also describes the combined impact of treatment use and resistance mutations on virological and immunological response in individuals who are under follow-up in one of four trials: MaxCminl, MaxCmin2, COLATE or SMART and three observational studies: EuroSIDA, the UK CHIC study and the UK drug resistance database. The emergence of resistance to an ARV that an individual is receiving may influence viral replication rates, which could increase the risk of CD4+T cell count deterioration, clinical progression and death, unless changes are made to the treatment regimen. Individuals may exhaust all treatment options if large amounts of resistance mutations are detected in their viral sub-populations. In the current era, new ARVs are still arriving on the market, but resistance to these ARVs is only partially understood. Understanding what mutations emerge in individuals who are failing treatment and the impact of specific mutations and combinations of mutations on the likelihood of responding to future regimens is still essential for being able to administer long-term therapy successfully. Research for this thesis started just after the introduction of ritonavir boosted protease inhibitors (Pl/rs). Drug resistance emergence among individuals who experienced virological failure on a Pl/r containing regimen is described in detail, and the relationship between resistance at baseline and virological response is also quantified. Other aspects of drug resistance are investigated, including: the potential benefit of harbouring the M184IA/ mutation, the impact of resistance on immunological response and the relationship between resistance and viral re-suppression rates amongst patients who interrupt an NNRTI containing regimen. This thesis outlines the benefits of resistance testing and highlights some of the key issues with interpreting resistance data. Resistance tests are generally performed on a selective group of individuals so caution needs to be used when extending the results to all HIV-1 infected individuals. Further, consensus sequences are useful for indicating resistance that is present in the predominant virus however, more minor, archived, species are not usually identified through this method and these may also be an important determinant of therapy response.

No description available.

No description available.

Natural Killer (NK) cells are innate cells of the immune system and constitute 10% of lung lymphocytes. Increasing evidence implicates a role for innate immunity in the pathogenesis of asthma and although there is evidence of a role for NK cells in the development of allergic inflammation, the mechanisms by which NK cells contribute to allergy is not known. To characterise the NK cell response and determine the phenotype of NK cells in allergic pulmonary inflammation we employed a model in which mice are dosed intranasally 3 times a week for 3 weeks with house dust mite (HDM) extract. Numbers of NK cells in the bronchoalveolar lavage (BAL) increased over the time course of HDM challenge and followed a similar trend to eosinophils and Th2 cells. Airway NK cells were activated and expressed NKG2D and granzyme B. In addition, expression of the NKG2D ligand (MULT-1) was upregulated in the lungs of mice treated with HDM. To determine the importance of the NKG2D receptor in allergic inflammation, the HDM model was tested on NKG2D knock out (KO) mice. There was a dramatic reduction in the extent of the inflammatory response in the absence of this receptor, including a reduction in BAL eosinophilia, Th2 responses and serum IgE. Adoptive transfer of wild type (WT) NK cells into NKG2D KO mice restored allergic inflammatory responses to HDM, whereas transfer of granzyme B-/- NK cells did not, demonstrating the requirement for NK cell expression of NKG2D and granzyme B. Detailed phenotypic analysis of NKp46+ cells in the HDM model showed that NKp46+ cells in BAL and lung consisted of RORγt+ and RORγt- subsets. NKp46+RORγt- cells resembled conventional NK cells as they express NKG2D and granzyme B, however NKp46+RORγt+ had similar phenotype to type-3 innate lymphoid cells (ILC3) cells and produced Th2 cytokines upon HDM challenge. We have shown for the first time that NK cells promote allergic lung inflammation via NKG2D and granzyme B production. We have also described for the first time the presence of NKp46+RORγt+ cells in the airways and lung which identifies potential novel therapeutic targets.