Background. Current empirical understanding of adherence to antiretroviral therapy (ART) is limited and the majority of current research is cross-sectional. Diary methodology provides an opportunity to capture the fluctuating nature of variables influencing adherence, close to the time that they occur. A limited number of diary studies have been conducted with people living with HIV (PLHIV) and few have investigated patterns of treatment adherence and psychological variables simultaneously. Aims. To investigate the day-to-day factors associated with adherence to ART in PLHIV. To investigate the feasibility of diary methodology used to investigate adherence to ART in PLHIV. Method. A mixed methods design comprising 2 parts was used: a diary study, using multilevel diary methodology (study 1) and qualitative semi-structured interviews (study 2). Study 1: participants (n = 18) completed an initial questionnaire, and a 14-day diary, the diary recorded adherence to ART and day-to-day variables (such as mood and stressors). Study 2: interviews were conducted with HIV service users (n = 4) and HIV healthcare staff (n = 5). The interviews explored factors influencing the general feasibility of diary methodology in HIV populations. Results. Full adherence (100%) was reported in terms of taking tablets, however, there was variability in reported adherence in terms of taking medication at the correct time (‘adherence timing’). Anticipatory negative affect associated with non-adherence and attitude (positive) towards treatment regime were the only two daily variables (measured in the diary) associated with optimal adherence timing. The relationships between adherence timing and the daily variables were moderated by a number of variables, including: illness perceptions, perceived stigma and beliefs about medications. The majority of participants who completed the diaries were male (88.9%) and white (72.2%), females and ethnic minority groups were under-represented. The interviews resulted in the identification of multiple barriers and facilitators of diary methodology in HIV populations. In particular, barriers to study uptake and completion were identified. Conclusions. A number of variables were found to be associated with optimal adherence timing. However, conclusions are limited due to the small sample size and under- representation of certain groups in the HIV population. Diary methodology may be more feasible within certain subgroups of the HIV population. Future research and clinical implications have also been considered.

Human Immunodeficiency Virus (HIV) infection is a global public health priority. An effective HIV-1 vaccine strategy must overcome the enormous genetic diversity generated by the virus while inducing potent, long lasting immune responses. In this thesis, virus-specific cellular immune responses directed towards HIV conserved regions were evaluated using a validated IFN-γ enzyme-linked immunospot (ELISpot) assay and two sets of peptide panels based upon HIV-1 vaccine candidates. One panel was based on the 14 of the most highly conserved (HIVconsv – 806 amino acids) regions of the HIV-1 proteome and was designed to provide extensive coverage of subtypes A, B, C and D. The second panel represents Gag, Reverse Transcriptase (RT), Integrase (INT), Nef and Envelope sequences derived from a consensus subtype A reference (GRIN/Env). The purpose of this study was to examine the extent of T cell responses to HIV-1 epitopes elicited at different stages of HIV-1 infection and in vaccinees. Individuals infected with HIV-1 subtypes (A, B, C and D) from the UK, Rwanda, Uganda, and Zambia representing a wide range of genetic backgrounds were tested using a HIVconsv peptide matrix and GRIN pools. CD8 responses were identified across Gag, RT and INT and there was no apparent subtype specific bias in terms of recognition of both peptide panels (HIVconsv and GRIN/Env). GRIN appeared to be more antigenic because GRIN incorporated both conserved and variable epitopes compared to HIVconsv panel that only comprised conserved peptide sequences. Furthermore, there was no association between IFN-γ T cell responses and control of viraemia in early infection between viraemic controllers (VC) and viraemic progressors (VP). However, VC appeared to be slightly more polyfunctional than VP. Viral inhibition assay (VIA) employed to assess the vaccine specific responses demonstrated that conserved regions of the HIV-1 proteome could result in an increased ability to inhibit a panel of HIV-1 isolates in vitro. Although detection of a CD8+ T cell response against a particular epitope does not necessarily translate to protection, the conserved nature of HIV-1 epitopes suggests that these domains are immunogenic and might be important for viral function. Thus, these data support the inclusion of conserved sequences in any future vaccine candidate.

Since the introduction of combination antiretroviral therapy (ART), there has been a dramatic improvement in the prognosis of people living with HIV. Indeed, data from numerous cohorts now show that for people commenced on ART at high CD4 counts and who are retained in care, life expectancy is similar to matched HIV-negative people. Consequently, the number of years an individual diagnosed with HIV can expect to be treated with ART is set to increase markedly. With the emergence of an older population, polypharmacy and drug-drug interactions are now more prominent clinical problems in the management of HIV. Furthermore, as the number of years an individual spends on ART increases, treatment fatigue leading to suboptimal adherence and antiretroviral resistance are frequently observed. As such, the development of long-acting parenteral antiretroviral drugs with low propensity for drug-drug interactions, low toxicity and high genetic barriers to resistance is highly desirable. Fusion inhibitors are a class of antiretroviral drugs which display limited systemic toxicities and few drug-drug interactions. They are rarely used, however, owing to injection site reactions associated with their delivery. C34-PEG4-Chol is a novel fusion inhibitor derived from the lead molecule, C34, but modified with the addition of polyethylene glycol (PEG) and cholesterol (Chol). With addition of cholesterol, potency of the drug is enhanced by concentrating it in cell membrane domains where viral fusion occurs. Moreover, the addition of cholesterol has been shown to markedly enhance its circulatory half-life in rodents such that it may potentially act as a long-acting antiretroviral drug in humans with infrequent subcutaneous injections. In this thesis, I assess the antiretroviral resistance pattern of C34-PEG4-Chol in vitro. An adaptive clinical trial design was simulated to examine options of optimising the output of useful pharmacokinetic data after administration of single doses of C34-PEG4-Chol to HIV-positive men. Finally, I undertook a ‘first-in-man’ study to examine the pharmacokinetic profile of C34-PEG4-Chol and potential of this agent as a long-acting antiretroviral drug. My work demonstrates the emergence of mutations to C34-PEG4-Chol via novel pathways within the heptad repeat (HR)-1 domains of gp41. Compensatory mutations were also observed within the HR-2 domain. The phenotypic effect of a HR-1 mutation was demonstrated following generation of mutant viral clones and assessing viral entry into cells in the presence of drug. An adaptive clinical trial design has shown, through simulations, to lead to more efficient allocation of trial participants across potentially therapeutic doses of the drug. Following a ‘first-in-man’ study, a promising pharmacokinetic profile was characterised which showed an extended drug half-life several fold above the 90% inhibitory concentration of the drug for over 4-days. This is the first time a cholesterol-endowed antiviral peptide has been entered into clinical trials in humans and has demonstrated the potential value of cholesterol-endowed peptides drugs in the field of various infectious diseases such as HIV, influenza and paraomyxoviruses.

HIV is a master of immune evasion, utilising a range of different techniques to not only survive the human immune system, but also mediate its eventual catastrophic decline. Understanding how HIV evades the adaptive immune response is paramount to developing effective treatments and vaccines. This thesis aimed to investigate three key ways in which HIV-1 and HIV-2 mediate immune evasion in the context of clinically latent infection. Chapter three summarises a study into selection pressure and mutation in the gp120 envelope gene in a narrow-source HIV-1 cohort of former plasma donors (FPDs) from China. This study further characterised the cohort, and identified specific mutations in the gene consistent with antibody and CTL-driven selection pressure. Chapter four describes an investigation into the downregulation of HLA-I mediated by primary isolates of HIV-1 and HIV-2 Nef. Nef-mediated HLA-I downregulation contributes to the evasion of CTL responses. In stark contrast to previous reports, no evidence for differential downregulation of HLA-A and HLA-B was detected, but primary isolates invariably showed reduced activity relative to laboratory-adapted and consensus variants. In performing this study, a number of limitations came to light regarding how bifurcate analyses are used to interpret flow cytometric data collected in studies of receptor modulation. A novel technique - SWARM - was therefore developed to address these limitations, and is described in chapter five. Chapter six aimed to address why CTL responses against HIV-2 Nef are rare, despite HIV-1 Nef being highly immunogenic. A series of in vitro (immuno)proteasomal processing assays revealed HIV-2 Nef is more extensively digested than HIV-1 Nef, but further experimentation is required to explain the difference in response. Finally, chapter seven briefly summarises a successful collaborative attempt to resolve the first ever crystal structure of HIV-2 Nef.

Human Immunodeficiency virus (HIV -1) uses various mechanisms to evade immune recognition by cytotoxic T cells. Down-regulation of Human Leucocyte Antigen class I (HLA-I) by the viral protein Nef is one such important mechanism. HLA-A and Bare believed to be down-regulated by Nef while HLA-C and E are not. The differences in downregulation were due to sequence differences between alleles. I evaluated the functional significance of polymorphisms in the cytoplasmic domains of HLA-A and B in Nef mediated HIV -1 pathogenesis. HLA -B cytoplasmic alleles resisted down-regulation mediated by nef compared to HLA-A cytoplasmic alle1es and these differences were seen to play a crucial role in maintaining CTL response as shown by live virus ELISPOT assays, viral suppression assays and CD107a secretion. Therefore, we propose that this relative resistance to Nef mediated down-regulation by HLA-B cytoplasmic allele contributes to the high efficacy and better control of HI V infection by HLA-B restricted CTLs. The partial resistance of HLA-B cytoplasmic domain was maintained in HIV -2 Nef alleles and sub group 0 HIV -1 viruses. The variability of the HLA-A and B susceptibility appeared to depend on the Nefallele while resistance ofHLA-C was global. Substitution Of315DR316 to GG and truncation of C terminal amino acids play a role in mediating resistance to Nef. Possibly these amino acid changes lead to different molecular trafficking profiles as observed in wet lab methods, and changes to secondary protein structures as predicted by in silico methods resulting in partial resistance. In addition, the functional significance of CD4 down-regulation by Nef, another important effect was also explored in this thesis. Nef was found to be ineffective in down-regulating CD4 molecules truncated at cytoplasmic domain and the viruses which were produced in cell lines expressing truncated CD4, were found to be defective in terms of replication and irtfection. Evaluating the potential of these CD4 molecules in gene therapy is highlighted in my in vitro studies. In summary this thesis provides insight into important but unexplored areas ofNef mediated

Type I food allergies are adverse reactions to food, mediated by immunoglobulin E (1gB) antibody, generated by the gut immune system towards food allergens. There is no known therapy at this time. The aim ofmy work was to dissect the role of dendritic cell-T cell interaction in the development of food allergy. Previous work showed that T cell-mediated apoptosis of dendritic cells (DCs) was impaired in a mouse model of food allergy but the biological relevance of this event is undetermined. T cell-mediated apoptosis was investigated in two mouse strains, with C3H1HeJ showing higher degree of susceptibility than Balb/c mice. In parallel, I aimed to compare the susceptibility of intestinal (Peyer's patch)- and systemic (spleen)-derived DC to T cell-mediated apoptosis in physiological condition (i.e non-sensitised). The latter study showed greater susceptibility of gut Peyer's Patch-derived DC (PP-DC) to antigen-specific T cell-mediated cell death (apoptosis) than spleen-derived DC. Also, similar levels of T cell-mediated apoptosis of DC were observed, irrespective of mouse strain, suggesting increased resistance to T cell-mediated apoptosis alone is not sufficient in triggering full 19B-mediated allergic reactions. However, significant alteration was observed in studies of DC capacity to secrete lymphokine Interleukin-12 (IL-12), which promotes T-helper 1 (Thl) responses. Interleukin-4 (IL-4)-mediated production of IL-12 was impaired in PP-DC of sensitised C3H1HeJ mice but not splenic, non-sensitised or in DC derived from Balb/c mice. This event was not dependent on IL-4 receptor availability upon DC but was linked to reciprocal control ofSTAT-6 and STAT-4 pathways leading IL-12 production by DC. My data indicates that the development of food allergy is linked to the simultaneous failure of at least two different immuno-regulatory mechanisms. A possible hypothesis from this suggests that restoring levels ofIL-12 in the gut may be a novel and effective strategy for food allergy therapy.

It is now more than 30 years since the Human Immunodeficiency Virus (HIV) became a pandemic, and now it is still a major global pathogen. Although several classes of antiretovirals are currently available for therapy, resistance and toxicity remain the biggest threats to the successful control and treatment of HIV. Therefore it is important to increase the range of targets to help reduce the chance of treatment failure. Nef serves as a pathogencity factor, and is closely associated with disease progression to Acquired ImmunoDeficiency Syndrome (AIDS) . Using structure based drug design (SBDD) methods, a range of compounds were identified to bind Nef and prevent CD4 down-modulation by Nef. Both cellular and in vitro assays were utilised to screen molecules for inhibitory activity. A set of 11 compounds were selected for the 1st generation screen. This approach identified two 'hits' with 50% inhibition of Nef function at 10 IlM. The lead molecule was selected for structural refinement, which lead to the identification of 9 analogues for use in the 2nd generation screenings. This screen identified 3 'hits' and activity against Nef function was titrated to nanomolar concentrations. Each subsequent generation utilised a re-iterative improvement/modification process, and resulted in 3 distinct compound groups, the 3rd being used to study ' differences in activity amongst similar structures.

Abstinence-based programs fall into two categories: abstinence-only interventions present sexual abstinence as the exclusive means ofHIV prevention; abstinence-plus interventions emphasize abstinence as the most effective prevention strategy, but also actively promote condom use and safer sex. This thesis examines the effects of abstinence-based programs in two systematic reviews of randomized controlled trials. Both reviews were conducted under the auspices of the Cochrane Collaboration. The review of abstinence-only interventions identified 13 trials, which consistently found no significant program effects on biological or behavioral outcomes compared to various controls. The review of abstinence-plus programs contains 39 trials, of which 24 found a protective biological or behavioral intervention effect compared to various conu::ols. Next, this thesis investigates data related to program implementation in primary trials: that is, the design of active and control programs, their delivery by trial staff, their uptake by trial participants, and the contextual factors that affect implementation. These data can affect heterogeneity and comparability oftrial evidence in a systematic review. Furthermore, implementation information is critical for practitioners seeking to use evidence in practice. However, current guidelines do not prepare reviewers to identify, appraise, or use implementation data in analyses. This thesis relied on consensus methods and a literature search to generate a conceptual framework of program implementation with relevance to systematic reviews. This model is the first to incorporate implementation ofcontrol-group interventions, contextual factors, vulnerability of implementation data to bias, and variations in intended treatment design across trials. The framework has been received favorably by systematic reviewers, methodological experts, and review users. Finally, the thesis assesses the relevance of the framework to the systematic review of abstinence-programs. This application suggests ways that implementation data can inform reviewers' judgments about grouping trials, assessing heterogeneity, interpreting the generalizability of evidence, and suggesting directions for research and practice.

Allergy is a hypersensitive immune response to innocuous environmental antigens, characterised mainly by inflammation. The development of allergy is often attributed to atopy, a genetic predisposition to produce elevated levels of immunoglobulin (lg) E. Allergic conditions such as atopic dermatitis, allergic asthma and allergic rhinitis can be severely detrimental to quality of life, and over the last few decades, their prevalence has increased considerably in the developed world. However, current therapies often have limited efficacy. There is substantial evidence to suggest that the histamine IL receptor (ILR) is associated with allergic inflammation. ILR is a seven transmembrane domain G protein-coupled receptor (GPCR), predominantly expressed on leukocytes, with putative roles in chemotaxis, Th2 cell polarisation, and cytokine/chemokine production. For these reasons, ILR represents a new therapeutic target to treat allergic inflammation. Little is known about the functional impact of single nucleotide polymorphisms (SNPs) in the ILR-encoding gene (HRH4) and its relationship with atopy, or whether changes in expression are associated with atopic phenotypes. The aims of this thesis were to attempt to: (i) assess the impact of the common Ala138Val and His206Arg mutations upon ILR function; (ii) analyse the association of HRH4 with atopy susceptibility in a Caucasian population; (iii) provide a detailed mRNA expression profile of full length IL~390) and its non-functional splice variants IL~302) and IL~67) in the human airway and periphery; and (iv) evaluate the effects of atopy and grass pollen season upon the expression of ILR isoform mRNA, in peripheral blood leukocytes ex vivo. Non-synonymous protein-coding HRH4 SNPs rs11665084 (AlaI38Val) and rs11662595 (His206Arg) were confirmed in a Caucasian cohort (n=42) with a minor allele frequency of 12%. A much rarer, previously novel SNP, rs58154316 (Ser284Cys), was identified and validated in a larger Caucasian cohort (n=181), giving a minor allele frequency of 0.8%. Predictive modelling based on GPCR sequence conservation suggested that these mutations would have no detrimental effect on receptor function. This prediction could not be tested physically in a recombinant expression system due to technical problems with transfection and the poor specificity of anti-Hale antibodies used for analysis of ILR protein expression.

Systemic Lupus Erythematosus is an autoimmune disease of complex aetiology. It presents several clinical and immunological features, amongst them the presence of autoantibodies and lymphopenia. It is known that genetic susceptibility plays a part in developing the diseases, although few specific genes have been identified. As disturbances of apoptosis play a part in the pathogenesis of SLE, genes that code for proteins leading to, or belonging to this pathway are good candidates for the polygenic background predisposing to the disease. Eight known genes were selected as candidates based on a literature review. Markers in the candidate genes were genotyped on 388 Caucasian trios, predominantly by mass spectroscopy with the Sequenom platform. Association was evaluated with the transmission disequilibrium test. A ninth gene was selected based on positional cloning ofthe SLE subphenotype ofB cell levels. Evidence for association to SLE was found for: a haplotype spanning the second and third exons ofFAS, a haplotype in the 3' end ofFASL, SNPs in the 5' region of TP53, a haplotype spanning PARP, SNPs in the promoter region of CRH, a 3' haplotype of PDCDl and a SNP in the 3' untranslated region of SMAC/DIABLO. Genotypes for the same SMAC marker also showed significant differences in B cell levels. No assoc~ation was found between BCL2 and SLE. Haplotype and linkage patterns were established for all the candidate genes in the UK Caucasian cohort used. This study strengthened the hypothesis of involvement ofapoptosis genes in lupus pathogenesis and delimited genomic regions ofinterest for further genetic screening.