T cell activation, differentiation and expansion are induced by signals generated upon engagement of TCR by agonist peptide:MHC. At the site of T cell interaction with an antigen presenting cell, TCR, accessory proteins such as CD28 and LFA-1, and the Src family kinases (SFK) Lck and Fyn, accumulate and form the immunological synapse. Lck is the key signaling molecule, initiating TCR signals by phosphorylating the ITAM in the CD3/ subunits resulting in the recruitment and activation of Zap-70 and further propagation of the signaling cascade. Fyn is a positive mediator of T cell signaling, sharing specific substrates in common with Lck, and also acts as a negative regulator by phosphorylating the adaptor protein, PAG. Phosphorylated PAG binds Csk and its recently identified binding partner, the protein phosphatase PEP, recruiting them to the plasma membrane where they can inhibit the SFK. The differential localisation of the kinases, with Fyn compartmentalised to lipid rafts in mature T cells and the majority of Lck being non-lipid raft associated could explain the unique functions attributed to each kinase. In order to examine the interactions between SFK and their negative regulators in T cell activation, CD8+ T cells from mice expressing the class I MHC-restricted TCR, F5, were investigated. In naïve T cells Lck, Fyn, Csk and PEP were shown to co-localise to the site of activation-induced tyrosine phosphorylation following early TCR crosslinking. In contrast, in memory T cells Csk, PEP and Fyn not only co-localised with the site of TCR activation but also distributed at the distal end of the cell, suggesting differential redistribution of key negative regulators away from the site of TCR engagement in these cells. In the absence of Fyn there was no change in Csk localisation despite the loss of PAG phosphorylation, suggesting another adaptor protein can recruit Csk to the plasma membrane. Caveolin-1, a cholesterol-rich membrane protein was investigated as a possible candidate for recruiting Csk and was shown to be present in CD8+ T cells. Moreover, caveolin-1 was shown to be phosphorylated by Lck, Fyn and Abl kinases upon TCR engagement and to redistribute and polarise to the cSMAC, co-localising with Csk. In summary we have shown that there are alterations in the distribution of the negative regulators Csk, PEP and Fyn, once T cells have encountered antigen, and that this could be associated to the more efficient responses of memory cells upon re-exposure to antigen. Furthermore, its appears there is redundancy of both signaling molecules and adaptor proteins, which may contribute to the fine tuning of the TCR signaling pathway, such that signals derived from the TCR can still modulate an appropriate immune response even in the absence of key regulators.

Immune disorders such as allergy and autoimmunity are becoming increasingly common in developed countries. Self-reactive T cells exist in both healthy and autoimmune individuals. It is generally understood that hyperimmune disorders are caused by insufficient regulation, namely loss of activity of regulatory T cells. Whilst regulatory T cells exist naturally it is also possible to induce them both in vitro and in vivo. Immunotherapeutic techniques aim to provide noninflammatory exposure of antigen to the immune system with the aim of inducing antigen-specific regulatory T cells. Interleukin-10 (IL-10) is a cytokine with well known immunosuppressive qualities. It inhibits both the migration and the antigen-presenting ability of dendritic cells. It also has direct effects on T cells. Indeed, IL-10-secreting TR1 regulatory T cells were identified almost 15 years ago; their in vitro generation being dependent on exposure to IL-10. Particle-mediated DNA delivery (PMDD) is a promising method of immunisation and is especially suited to vaccines intended to have greater control over the response they induce. One of the main reasons for this is the possibility of including genes encoding immunomodulatory molecules alongside the antigen gene. This study utilises a mouse model involving the adoptive transfer of TCR-transgenic CD4+ T cells and establishes the response of these cells to PMDD immunisation. The model was then used to examine the effect of coadministration of the IL-10 gene. Its inclusion in the vaccine suppressed the response to antigen. This effect was maximal when the IL-10 gene was expressed in the same cell as the antigen gene. Using sequential immunisations the model was extended in order to study long-term effects, namely tolerance and the induction of regulatory T cells. Finally a mouse model of allergic asthma was used to examine any tolerogenic/therapeutic effects of the antigen-IL-10 vaccine. No significant longterm tolerance to antigen was identified. These results demonstrate that whilst the presence of IL-10 clearly inhibits the T cell response to antigen it does not necessarily confer tolerogenic properties on these cells. This brings into question whether IL-10 in the periphery, supplied, for example, by TR1 cells, generates fresh regulatory T cells or merely inhibits the response to a particular antigenic challenge.

The study of human papillomavirus (HPV) utilises human keratinocytes as host cells that are co-cultured with lethally irradiated mouse fibroblasts. Although this co-culture system has been effective in recapitulating HPV’s life cycle, it resembles studies whereby radiation-induced bystander effect (RIBE) has been identified. We considered the possibility that Normal Immortalised Keratinocytes (NIKs) that we used as host cell for HPV16 may be in receipt of DNA damaging agents from heavily irradiated J2-3T3 fibroblasts. We report that NIKs (+/ - HPV16) cells respond to the presence of feeders by increasing DNA damage response leading to H2AX phosphorylation. NIKs receive DNA damaging agents from irradiated feeder cells primarily through direct cell-to-cell contact and secondarily through the culture media. Although ATM, ATR, and DNA-PK were activated by RIBE in NIKs, we found that feeders inhibited ATM activation in NIKs (+HPV16). Moreover, we found that expression of HPV16 E6 and E7 oncoproteins activated ATM, ATR, and DNA-PK. HPV16 also activated H2AX, 53BP1, RPA32, and NBS-1. We considered that both feeder cells and HPV16 could induce DNA damage response in NIKs. This would change the interpretations of HPV16’s interactions with host cell’s DNA damage surveillance system in NIKs (+ HPV16) cells co-cultured with feeders. We proceeded to enhance our understanding of how feeder cells and/ or HPV16 changed the way NIKs cells respond to direct DNA damage. We found that NIKs cells (+feeders or +HPV16) respond to direct gamma radiation in a broadly similar way, whereby activation of ATM was inhibited, increase in ATR was extremely small and delayed, while activation of DNA-PK was still generally responsive. We report that presence of HPV16 sensitised NIKs cells to direct gamma radiation and as such, the implications of these observations could be considered for radiotherapy of cancers with HPV. Our initial belief that feeder cells were essential in ensuring stable autonomous replication of HPV16 in NIKs cells was proven otherwise, as NIKs maintained HPV16 DNA episomal copies in the absence of feeders. Contrary to current accepted publication, ATM was found be unnecessary for maintenance of HPV16 DNA episomes and viral genome amplification.

The human malaria parasite, P. falciparum, replicates within a membrane-bound intraerythrocytic parasitophorous vacuole (PV). The resulting daughter merozoites actively escape from the host cell in a process called egress. There is convincing evidence that proteases are key players in this step. These proteases could serve as excellent targets for the development of new antimalarial drugs. P. falciparum Serine Repeat Antigens (SERAs) form a family of 9 proteins all containing a central papain-like domain that identifies them as putative cysteine proteases. They are highly conserved throughout all Plasmodium species, and there is strong genetic evidence that they may play a role in egress. P. falciparum SERA6 is one of the most highly-expressed SERAs in asexual erythrocyte stages. In this study biochemical fractionation and indirect immunofluorescence analysis were used to confirm localisation of SERA6 to the PV. It was shown that SERA6 is a substrate for PfSUB1, a subtilisin-like protease which is crucial for egress and which is released into the PV just prior to egress. SERA6 is cleaved by PfSUB1 at 3 positions, releasing the papain-like domain. Processing of SERA6 by PfSUB1 is partially indispensable in in vitro P. falciparum parasites, as some mutations in SERA6 that block cleavage are not tolerated in the parasite. In addition, the putative catalytic Cys of SERA6 cannot be replaced with a non-catalytic Ala residue in the parasite, indicating SERA6 is an indispensable cysteine protease. Finally it was demonstrated that PfSUB1-mediated processing of the P. berghei orthologue of SERA6 converts it to an active cysteine protease. It can be proposed that SERA6 is a key player in a proteolytic cascade that leads to egress of the blood-stage malaria parasite.

Classically, the emergence of resistance to protease inhibitors (PIs) in HIV-1 requires the stepwise accumulation of primary and compensatory mutations in the viral protease (PR). In addition, it was demonstrated that mutations occurring on one of the natural substrates of the PR, Gag, could behave as compensatory mutations in the presence of certain primary PR mutations. Furthermore, mutations on the Gag could account for resistance to PIs when appeared in isolation. So far, most studies assessing the effect of Gag mutations on resistance to PIs have focused on two cleavage sites (CS), namely P7/P1 and P1/P6. However, data on the remaining CS and non-cleavage sites is scarce. In my PhD studies, I developed and optimized an assay for the amplification and sequencing of HIV-1 Gag and PR genes in order to characterize mutations occurring in patients failing PI-based therapy. Initially, I performed a cross-sectional analysis by comparing the Gag and protease sequences from PI-experienced patients and PI-naïve subjects. A number of Gag mutations associated with PI-selective pressure were determined, which were not restricted to P7/P1 and P1/P6 CSs, but present throughout the Gag. Subsequently, I conducted a longitudinal analysis of patients failing a PI-based regimen, which confirmed that under PI-selective pressure the entire Gag evolved along with the PR and that changes were most prominent at P2/P7, P7/P1 and P1/P6 CSs and in the P17 protein outside CSs. Finally, I performed phenotypic characterization of PI susceptibility and replicative capacity studies on patient’s viruses and side-directed mutants. As a result of these investigations I found that the evolution of Gag in patients on unsuccessful PI therapy led to increased levels of PI resistance and improved viral replicative capacity. Specifically, I characterized two novel CS mutations (P17/P24: Y132F, and P2/P7: T375A) that conferred resistance in the context of a wild type backbone.

Erythrocyte invasion is a key step in the Plasmodium life cycle. This process is tightly regulated, involving the sequential release of specialised apical secretory organelles – the micronemes, rhoptries and dense granules. These organelles contain proteins required for invasion and establishment of the parasitophorous vacuole, but most of the proteins remain uncharacterised. The aim of this project was to uncover novel proteins with a role in invasion by the human malaria parasite Plasmodium falciparum merozoites. I identified proteins using the following selection criteria: a) expression in the schizont/merozoite form of the parasite; b) conservation across the genus; c) the presence of a signal peptide and d) one or more transmembrane (TM) domains. A list of 64 proteins was identified, and filtered further based on novelty, presence in the merozoite proteome, expression in other life cycle stages, and difficulty of study. Five proteins were selected, and I produced recombinant protein and raised antibodies against three, which I used to identify the sub-cellular location of the protein within the parasite. The proteins appear to reside in either the rhoptries or the endoplasmic reticulum of the merozoite. Attempts were made to epitope-tag and delete all 3 genes, with a focus on one protein, the type IV Hsp40, PF11_0443. This protein contains two TM domains and is expressed during schizogony. By immunofluorescence it is present in the ER of early schizonts, before accumulating at the apex of merozoites in a rhoptry location. Immunoprecipitation experiments indicated that the protein binds known rhoptry proteins and other chaperones. The protein has been epitope-tagged but attempts to delete the gene by genetic recombination were unsuccessful. The gene is conserved in Plasmodium spp. and there are orthologues in higher eukaryotes, but it is absent from other Apicomplexa. Current studies are focused on the role of this protein in erythrocyte invasion.

Introduction: Tuberculosis (TB) is a major cause of morbidity and mortality worldwide. The immune response during TB is complex and incompletely characterized, hindering the development of new diagnostics, treatments and vaccines. Studies performed in different disease settings (intermediate versus high burden) have sometimes yielded divergent results, limiting advances in our understanding of TB. We used microarray based approaches to obtain an unbiased comprehensive survey of the host response to TB in both the UK and South Africa. Methods: Whole blood was collected before treatment. RNA was extracted and used for whole genome expression studies using Illumina HT-12 microarrays. This was complemented by multiplexed cytokine analysis using the MILLIPLEX™ Multi-Analyte Profiling system. Biological data was integrated with comprehensive clinical data including radiology. Data mining was performed using Genespring GX 7.3 and Ingenuity® Pathways Analysis software in combination with a novel Genomic Modular Analysis Framework. A subset of patients was assessed at 2 and 12 months post treatment. Results: We identified a robust blood transcriptional signature for Active TB in both intermediate and high burden settings, independent of ethnicity, age and gender. Transcriptional profiles appeared to reflect radiographic extent of disease. Longitudinal analysis revealed that the signature of Active TB disappears during successful treatment. Analysis of blood leucocyte counts and serum cytokines, along with interrogation of gene expression data using pathway and Modular analysis suggests that this signature reflects changes in cellular composition and altered cytokine gene expression, with a key part of the signature being composed of interferon-inducible transcripts. This included several transcripts from the tripartite motif (TRIM) protein family, which have not previously been implicated in TB pathogenesis. Conclusions: This is the first whole genome expression profiling study in human TB. Our findings have implications for understanding disease pathogenesis, and could yield biomarkers for diagnosis and treatment monitoring.

Vav1 is a haematopoietic-specific guanine nucleotide exchange factor for Rho-family GTPases that acts downstream of antigen receptors and regulates events such as lymphocyte proliferation, cytoskeletal reorganization, calcium flux and the activation and clustering of integrins. It is well established that localization of signalling molecules is important for their function. Here, we investigated the subcellular distribution of Vav1 in primary CD4+ and CD8+ T cells following T cell receptor (TCR) stimulation using the combination of two imaging methods: confocal and total internal reflection microscopy. This study also aimed to examine the relationship between the localization of Vav1 and its function. We found that Vav1 polarizes to the immunological synapse (IS) and localizes to TCR-induced microclusters. Analysis of Vav1 mutants showed that loss-of-function mutations in the DH, PH and N-terminal SH3 domain did not affect TCR-induced localization. However a point mutation in the SH2 domain (R696A) completely abrogates polarization of Vav1 to the IS and its localization to microclusters. Furthermore, this mutant fails to undergo TCRinduced phosphorylation and does not rescue Vav1-deficient T cells from impaired calcium flux. Interestingly, the C-terminal SH3 domain mutant exhibited a nuclear localization. This mutant also showed slightly decreased ability to undergo movement toward IS, diminished TCR-induced phosphorylation and could only partially rescue Vav1-deficient T cells from impaired calcium flux. Taken together, these data show that the SH2 domain of Vav1 is required for its correct localization at the IS, phosphorylation and function. Furthermore, the C-terminal SH3 domain of Vav1 seems to play a role in the retention of Vav1 in the cytoplasm.

Signal transduction through the antigen receptor of B cells (BCR) is crucial in controlling their development and maintenance, and is also needed for B cell immune responses. Syk, a protein tyrosine kinase, has already been implicated in signalling downstream of the BCR. Mice deficient for Syk show a developmental block at the immature to mature B cell stage such that no mature B cells are generated. Thus it has not been possible to use these targeted mice to study the role of Syk in the activation of mature B cells or in antigen-driven B cell responses. To get around this, I used mice with an inducible conditional knock out (CoKO) of Syk to generate mature B cells lacking Syk. This has allowed me to study the role of Syk in mature B cell activation and its involvement in the immune response. In this work I show that B cells from Syk CoKO mice are unable to respond to BCR stimulation. I also addressed the requirement in Syk for T-dependent immune responses, looking at both primary and secondary responses. The primary response was Syk-dependent, while, surprisingly, the secondary response seems to be Syk-independent. Finally, I investigated the role of Syk downstream of TLR receptors. Unexpectedly, Syk CoKO B cells were unresponsive to TLR4 stimulation. This defect is B cell autonomous and may be due to reduced signaling through the Akt/Gsk-3/cMyc pathway, downstream of the BCR.

Papillomaviruses cause a wide spectrum of benign and neoplastic diseases in humans and animals. They may also cause infections that are characterised by the absence of clinical signs of disease and some of these may represent viral latency. Many viruses have a latent stage of their life cycle and papillomaviruses appear to be no different. For example, some low-risk human papillomavirus types such as HPV-11 and HPV-6 can cause infections that resemble latency. It has recently been shown that rabbit oral papillomavirus (ROPV) is an appropriate model of these virus types. Infection of the tongue mucosa with ROPV leads to the formation of benign papillomas that form and regress within a matter of weeks. In this thesis, we show that ROPV is a suitable model system for the study of latent papillomavirus infections. The regression of ROPV papillomas is followed by the persistence of viral DNA and RNA in the absence of clinical signs of disease. Persistence of ROPV DNA is generally restricted to basal epithelial cells at sites of previous infection. Low copy numbers of viral DNA in the basal layer are compatible with infection remaining in only a subset of these cells (possibly epithelial stem cells). Typically there is no amplification of viral DNA in the upper layers of the epithelium. ROPV proteins are undetectable during latency suggesting that the productive stages of the life cycle are not completed. Low levels of ROPV early transcripts are detectable and it is possible that early proteins are necessary to allow stable maintenance of viral episomes in basal epithelial cells. We attempt to demonstrate the ability of latent ROPV infection to reactivate to form clinical disease. Evidence of spontaneous reactivation was seen on one occasion, but efforts to initiate reactivation by immunosuppressing rabbits were hampered by the toxicity of the drugs used. However, our preliminary data suggest that immunosuppression of rabbits can cause reactivation of latent ROPV.