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TPL-2 functions as a MEK1/2 kinase that is an essential component of an ERK1/2 MAP kinase pathway activated by Toll-like receptors and members of the IL-1 and TNF receptor families. TPL-2 is required for production of the pro-inflammatory cytokine TNF by macrophages after lipopolysaccharide stimulation. Recent work from the Ley laboratory has demonstrated that TPL-2 induces TNF secretion by macrophages independently of MEK1/2 and ERK1/2 phosphorylation. However, the target protein that TPL-2 phosphorylates to control TNF production is not known. The aim of this project was to determine the primary amino acid sequence specificity of TPL-2 by peptide library screening to allow bioinformatic identification of novel TPL-2 substrates. To do this, a strategy was developed to purify TPL-2, complexed with its cellular binding partners ABIN-2 and NF-κB1 p105, from lysates of transiently transfected HEK293 cells. This method generated large amounts of highly purified TPL-2 complex, which was soluble, stable, and catalytically active. Interestingly, mass spectrometric analysis of highly purified TPL-2 complex identified several novel binding partners, including mTORC2, IRAK1, and TRIM25, which may be functionally linked to the TPL-2/ABIN-2/NF-κB1 p105 complex. Screening of a positional scanning peptide library demonstrated that TPL-2 complex had an unusual amino acid specificity, in which negatively charged amino acids were preferred at all five positions N-terminal to the phosphorylation site. A synthetic peptide based on this optimal motif was specifically phosphorylated by the TPL-2 complex much more efficiently than a positive control MEK peptide, strongly suggesting that the screen had worked. A Scansite search of protein sequence databases with the positional-specific scoring matrix for TPL-2 identified a number of potential novel substrates, including several proteins involved in intracellular membrane trafficking. A strategy for future validation of potential TPL-2 substrates and the possibility of using an optimal TPL-2 substrate peptide for high throughput inhibitor screening are discussed.

Streptococus pneumoniae is an important human pathogen, yet in most individuals it establishes only transient nasopharyngeal colonisation without causing disease. Using murine models, this thesis explores the hypothesis that colonisation induces acquired immune responses which protect against subsequent pneumonia. Colonisation models with wild-type (WT) and mutant S. pneumoniae were established in outbred CD1 mice. Mutants lacked either capsule or lipoproteins, or were auxotrophs unable to replicate in vivo. WT colonisation protected against subsequent pneumonia. Mutants were cleared more rapidly than WT, were not immunogenic and did not protect. When the auxotroph was supplemented, colonisation, immunogenicity and protection were improved, suggesting duration of a colonisation event is an important factor in determining immunogenicity. This may be one factor explaining the poor immunogenicity of the other mutants. The mechanism by which previous colonisation protected against subsequent lethal pneumonia was then defined in a series of studies in inbred CBA/Ca mice. Colonisation induced both mucosal and systemic antibody responses to bacterial surface antigens but not capsule. There was also evidence of more robust cytokine production during subsequent pneumonia, including systemic and mucosal IL-17 responses dependant on the presence of CD4-cells. Protection was primarily against systemic invasion following pneumonia. Passive transfer studies and experiments using genetically modified mice demonstrated that systemic antibody was both necessary and sufficient to protect, and in vitro and in vivo models showed this to be via opsonophagocytosis and bloodstream clearance of bacteria. Antigenic protein targets of protective serum were defined using Western blotting and multiplex bead immunoassay techniques. Overall this thesis demonstrates that nasopharyngeal colonisation can protect against lethal pneumonia in mice via opsonophagocytic antibody against surface proteins thus preventing bacteraemia.

The immunosuppressive function of regulatory B cells (Bregs) has now been confirmed in several murine models of chronic inflammation, including collagen induced arthritis (CIA), inflammatory bowel disease, and experimental autoimmune encephalomyelitis (EAE). In particular, our group has previously been reported that IL10+ regulatory B cells, known to play an important role in controlling autoimmunity and inflammatory disorders, are contained within the Transitional-2 immature (T2) B cell pool (T2Bregs). In this thesis I first of all characterize regulatory B cells in the MRL/lpr mouse model of systemic lupus erythematosus (SLE) and report that agonistic anti-CD40 specifically targets T2 B cells and enriches T2 Bregs upon short term in vitro culture. Whilst transfer of unmanipulated T2 B cells, isolated from mice with established lupus, failed to confer protection to diseased mice, transfer of in vitro anti-CD40- generated T2 B cells (T2-like-Bregs) significantly improved renal disease and survival by an IL-10-dependent mechanism. T2-like-Bregs readily accumulated in the spleen after transfer, suppressed Th1 responses, and induced the differentiation of IL-10+CD4+T cells conveying regulatory effect to CD4+T cells. In addition, in this thesis I demonstrate that freshly isolated human CD19+CD24hiCD38hi B cells contain a population with potent regulatory capacity. Immature CD19+CD24hiCD38hi Bregs suppressed the differentiation of Th1 cells via the provision of IL-10, but not TGFβ, and their suppressive capacity was abrogated by the addition of anti-CD80. In addition, although they comprise a greater percentage of the B cells in the peripheral blood of SLE patients, CD19+CD24hiCD38hi SLE Bregs were found to be refractory to further CD40 stimulation, produced less IL- 10, lacked the functional suppressive capacity of their healthy counterparts. Altered cellular function within this compartment may impact effector immune responses in SLE and other autoimmune disorders. Therapeutic strategies facilitating the enrichment, or enhancement of the suppressive activity, of human Bregs offer new treatment possibilities.

The thesis is premised on the fact that India‟s National AIDS Control Organization (NACO) employs the typology of female sex work in outreach and other components of the HIV programme in order to identify high-risk female sex workers (FSWs). However, the current typology – distinguishing between FSWs based on their main place of solicitation – may not adequately reflect the variation in HIV risk. Using data from integrated biological and behavioural assessment surveys among FSWs from three south Indian states, I propose a method for devising evidence-based typologies of sex work which prioritizes place of solicitation and explores other factors potentially helpful for targeted interventions by indicating which FSWs are at high risk. For Karnataka state, the analysis suggests that the typology should distinguish between women based on the main place of solicitation and the main place of sex; this typology identifies street to lodge and brothel to brothel FSWs as being at highest risk for HIV. The strongest HIV/STI risk factor among FSWs from Andhra Pradesh is marital status, while among Tamil Nadu FSWs it is marital status and alcohol consumption respectively. Of the three states, Karnataka typology has the highest outreach applicability, as the main place of sex is linked to a geographical location. In addition, a qualitative study was conducted in Belgaum district, Karnataka to understand what it is about the mode of operation in different sex work settings that may help explain why some FSW categories are at higher risk for HIV compared to others. The qualitative data identified a number of factors which might help explain why brothel to brothel, lodge to lodge, street to lodge, dhaba to dhaba and highway to highway FSWs are likely at highest HIV risk, with different vulnerability factors applying to different modes.

Lentiviral‐based vectors are effective and promising tools for the generation of cell mediated immunity. Multiple studies have demonstrated that subcutaneous injection of lentivectors encoding tumour antigens results in induction of strong CTL responses and often in tumour killing. However, integration of lentivectors into human genomic DNA poses a risk of insertional mutagenesis. Indeed, this possibility has been highlighted by gene therapy trials that resulted in the development of T cell leukaemia in several patients. For this reason, non‐integrating lentiviral vectors (NILVs) have been developed as a safer alternative for gene delivery. The first part of this thesis demonstrates that lentivectors carrying multiple mutations preventing integration are effective vaccines. Subcutaneous injection of these vectors resulted in induction of systemic dose‐dependant CD8+ T‐cell responses to the encoded antigen. The duration of the persistence of antigen presentation was measured using transfer of OT1 transgenic T cells into previously immunized mice. Measuring expansion of those cells revealed that the antigen was present and presented for at least 30 days. CD8+ T‐cell responses were further enhanced by addition of dendritic cell (DC) stimulators: p38 MAP kinase and NF‐κB stimulators. These activators led to a more rapid response peaking at day 7. Finally, NILVs expressing the antigen and DC activators were tested in a tumour therapy model and were found to be effective. The second part of this thesis focused on altering DC‐T cell interactions to enhance responses to immunization by lentivector‐mediated knockdown of PDL1 on DCs. The analysis of DCs infected with anti‐PDL1 shRNA showed that knocking down this molecule drives DCs towards a mature phenotype. The influence of PDL1 knockdown was assessed on co‐cultured T cells. The absence of PDL1 enhanced their proliferation and reduced antigenic stimulation induced TCR complex degradation. DCs transduced with lentivectors expressing PDL1 shRNA were also tested in vaccination and tumour therapy.

Background: Recent advances in biomedical HIV prevention highlight the importance of successfully engaging male participants, yet previous participant engagement methods have had mixed results. Participatory research and collaborative community development methods of engagement focus on the importance of culture and community and have been successful for engaging participants in research. However, these methods have not been used to engage male participants in biomedical HIV prevention intervention studies in the global south. Methods: The Impilo Yamadoda: Men’s Health Study is presented as a case study to explore use of a “strategic community engagement method” based on theories of participatory research and collaborative community development to engage young Zulu-speaking men in a multi-phase HIV prevention intervention in rural KwaZulu-Natal, South Africa. This engagement method included the identification, recruitment, and training of local volunteers (Research Partners). Research Partners were responsible for: recruitment and implementation of a brief community based men’s health survey; collaborative planning of experimental phase recruitment (N=200) with researchers. Qualitative interviews with Research Partners (N=6) and participants (N=83; 8 focus groups, 20 interviews) were explored alongside analysis of the design, methods, and results of the Impilo Yamadoda study. Results: Research Partners were expected to collect ~300 (7/day) surveys; N=735 (12.9/day) were returned. Analysis of questionnaires confirmed data quality. Similar recruitment methods were used in the biomedical phase; 95.3% (223/234) of participants completed enrolment, including a behavioural questionnaire, blood sample, and randomisation. Discussion: Research Partners discussed the importance of community-level incentives to participants’ decisions to engage in research during their training programme, which was confirmed through analysis of qualitative interviews and focus groups with participants. These findings suggest that methods of participant engagement such as the strategic community engagement method used in the Impilo Yamadoda study could be used to successfully engage participants in future biomedical HIV prevention studies in the global south.

Regulatory T cells (Tregs) have shown considerable potential in the treatment of murine models of immuno-pathology. Whilst poly-clonal Tregs are able to suppress immuno-pathology in a number of models, the superiority of Ag-specific Treg treatment has been demonstrated using Tregs from T cell receptor (TCR)- transgenic animals. Translation of these promising results to the clinic has been hampered by difficulties in isolating or enriching the rare Ag-specific Tregs from the polyclonal population. Here I describe two distinct approaches to generate Ag-specific T cells with regulatory ability: firstly, TCR gene transfer into purified CD4+CD25+ T cells was used to redirect the specificity of naturally occurring Tregs. Secondly, co-transfer of FoxP3 and TCR genes served to convert conventional CD4+ T cells into Ag-specific ‘Treg-like’ cells. Both approaches generated T cells that suppressed in vitro and engrafted efficiently, retaining TCR and FoxP3 expression, when adoptively transferred into recipient mice. Using an established arthritis model, I demonstrate Ag-driven accumulation of the gene modified T cells at the site of joint inflammation, which resulted in a reduction of joint swelling. In animals treated with TCR-transferred natural Tregs this was accompanied by a local reduction in the number of inflammatory Th17 cells and a significant decrease in arthritic bone destruction. Together, I have described a strategy to rapidly generate Ag-specific Tregs capable of antigen-dependent amelioration of autoimmune damage in the absence of general immune suppression. These approaches could practicably be translated into the clinic in order to treat numerous different immuno-pathologies.

Lentiviral vectors, derived from HIV-1, are promising tools for gene therapy. Recent clinical trials have demonstrated the translation of their effectiveness in laboratory studies to clinical trials. However there are still limitations, relating to vector safety and efficiency of production, that could confine their future use. I investigated the ability of lentiviral vectors to perturb cellular gene expression by insertional mutagenesis (IM), using an in vitro model that detects aberrant splicing from lentiviral vectors to the growth hormone receptor gene (Ghr). The lentiviral vector pHV with full long terminal repeats (LTRs) and an internal spleen focus forming virus promoter (SFFV), was previously found to activate Ghr expression by a fusion mRNA transcript initiated in the HIV LTR (46). I extended this discovery to show that the SFFV promoter was enhancing expression from the HIV LTR, leading to IM (269). Application of our in vitro IM assay to potential clinical lentiviral vectors revealed that the novel ‘UCOE’ (ubiquitously acting chromatin opening element) promoter, within a selfinactivating (SIN) lentiviral vector, could drive UCOE-Ghr mRNA transcripts, causing IM. Mutation of splice donor sites in UCOE alone was insufficient in abrogating IM, however internal deletions around these splice donor sites were more successful. In other work, I made a packaging cell line for lentiviral vectors by stably expressing rev and a modified RD114 env (RDpro) in a cell line expressing HIV gag-pol. This led to the isolation of 57R10E, a cell line that made a titer of over 104 infectious units per ml when a SIN lentiviral vector was transiently or stably expressed. Taken together, this work will broaden the application of lentiviral vectors in clinical gene therapy by reducing both the chances of adverse events and the costs associated with vector production.

Infection with Human Immunodeficiency Virus, type 1 (HIV-1) is associated with a gradual progressive decline in the number of CD4+ T lymphocytes. Effective treatment suppresses viral replication and is accompanied by a concomitant increase in the number of CD4+ T cells. Immune reconstitution of CD4+ T cells in children following treatment is characterised by a sustained increase of naïve cells, a pattern that differs from that seen in adults. The aim of this thesis was to explore how these changes occur. CD4+ T cells in blood samples from HIV-1 infected children were identified, divided into sub-populations and analysed for apoptosis, proliferation, activation and differentiation by flow cytometry. Four CD4+ T cell sub-populations, with varying contributions to the total CD4+ T cell pool were thus identified: (i) recent thymic emigrants (RTEs) made up the largest population yet maintained very low levels of proliferation despite increased viral replication and cellular activation, and were consistently greater in children with undetectable viraemia; (ii) central naïve cells, which were fairly constant in HIV-1 infected children of all ages regardless of CD4 count; (iii) CD31- memory cells that increased as CD4 count fell and (iv) CD31+ memory cells that despite their high level of activation and proliferation remained a small population across age, viral load and CD4 count. Treatment interruption and the resulting increased viraemia and decreased CD4 count were associated with only transient changes to the percentage contribution of each subset, which supports the existence of a setpoint for each subpopulation. This thesis infers the importance of thymic output in maintaining the CD4 count and hence the potential for using RTEs in monitoring response to treatment and sheds light on the role and origin of CD31+ memory cells as a small but highly activated population that may be important in disease pathogenesis.