This thesis presents the development and optimization of a high-throughput fluorescence microbead based approach for multiplexed, large scale medical diagnostics of biological fluids. Specifically, different sizes of semiconductor nanocrystals, called quantum dots, are infused into polystyrene microspheres, yielding a set of spectrally unique optical barcodes. The surface of these barcodes are then used for sandwich assays with target molecules and fluorophore-conjugated detection antibodies, changing the optical spectra of beads that have associated with (or captured) biomolecular targets. These assayed microbeads are analyzed at a single bead level in a high-throughput manner using an electrokinetic microfluidic system and laser induced fluorescence. Optical signals collected by solid state photodetectors are then processed using novel signal processing algorithms. This document will discuss developments made in each area of the platform as well as optimization of the platform for improved future performance.

Public health planning for influenza is based on morbidity and mortality estimates derived from statistical models. Lower than anticipated 2009 H1N1 pandemic death estimates have raised questions about the method. Examining the statistical method is important for future policy and program development. We compared the main methods of estimating influenza burden through a systematic literature review and by comparing statistical estimates of influenza-attributable burden at the Ottawa Hospital (TOH) to clinical estimates validated through chart review. We identified heterogeneity in methods used to estimate influenza-attributable mortality in the literature which resulted in within-season estimate variation by study. We found statistical estimates of influenza burden at TOH to be 4-8 times greater than clinically validated data. We also found no significant association between the outcomes examined and epidemic periods at TOH. The findings of this study suggest discordance between model estimates by model approach and between model estimates and validated findings. Examining reasons for these discordances should be pursued.

New and emerging diseases in human and animal populations appear to be predominately associated with generalist pathogens that are able to infect multiple hosts. Carnivores are susceptible to a wide range of these pathogens and can act as effective samplers of their vertebrate prey, which are important reservoirs of many emerging diseases. This thesis evaluates the utility of carnivores as sentinels for pathogens present in their prey by exploration of four selected pathogen-prey-sentinel combinations in three rural study sites of varying habitat in northern England and Scotland over a twenty-two month period (2007-2009). Selected pathogens were Coxiella burnetii, Leptospira spp., Encephalitozoon cuniculi, and rabbit haemorrhagic disease virus (RHDV), selected prey species were wild rodents and rabbits, and selected carnivores were foxes, domestic cats and corvids. Seroprevalence to C.burnetii, Leptospira spp and E.cuniculi was assessed using adapted or novel test methodologies to enable their use for multiple mammalian species, however these were not applicable to corvids. RHDV seroprevalence was not assessed due to low acquisition of rabbit samples. Overall, seroprevalence to all three pathogens was significantly higher in predators than prey, at 24.2% and 12.4 % for C.burnetii, 22.73% and 1.95% for Leptospira spp and 39.06% and 5.31% for E.cuniculi in predator and prey species respectively. A similar pattern was found in all study areas and was consistent irrespective of individual prey or predator species, although serological evidence of exposure to E.cuniculi was not detected in domestic cats in any area. A semi-quantitative assessment of the time and financial costs of the study approach and application to hypothetical examples indicates that sampling carnivores is a much more costeffective approach to pathogen detection than sampling prey. The results indicate that carnivores can act as useful sentinels for broad-scale detection of pathogen presence and relative levels of prevalence in prey and predator populations. Careful selection of predator species and methods of sample acquisition are necessary to maximise their utility, and issues associated with diagnostic test performance and validation must also be acknowledged. Suggestions are made as to how this principle might be applied to future surveillance programmes. In addition, the study is the first report on the seroprevalence of C.burnetii, Leptospira spp and E.cuniculi in multiple wildlife species (field voles, bank voles, wood mice, foxes), the first detection of antibodies to C. burnetii in wildlife and cats, the first detection of antibodies to L mini, L hardjo prajitno and L hardjo bovis in wild rodents, and to L mini in cats, and the first detection of antibodies to E.cuniculi in wild rodents and foxes in the UK.

In this thesis, I examined the expression of CD1d on respiratory epithelial cells (REC) in human and explored its potential role in mucosal immunity in the lungs. Hitherto, there have been no published reports of CD1d expression on REC though it has been observed on other epithelial surface (notably intestinal epithelial cells). This observation, and work in my supervisor’s laboratory demonstrating CD1d-restricted natural killer T cells (iNKT) cells as early players in the lungs of influenza A virus (IAV)–infected mice prompted my interest in this area. I hypothesized that CD1d is expressed on REC and that it contributes to activation of iNKT cells in the lungs via presentation of endogenous or pathogenic glycolipids. I asked following questions – i) is CD1d expressed on REC ii) can this expression be regulated and iii) does CD1d expression on REC have a function. This thesis provides the first evidence for CD1d expression on human RECs (in cell lines and primary RECs) and also presence of alternatively spliced variants. CD1d expression was inducible by viral-associated signals in vitro and despite being non-professional antigen presenting cells, RECs can present glycolipid (α–GC) to, and activate iNKT cells in a CD1d-dependent process resulting in production of both Th1 and Th2 cytokines. Using whole genome expression profiling, I then showed that iNKT cells expressed a distinct profile of genes while in direct contact with α–GC-bound CD1d on RECs compared to cells separated by transwell membrane. Here early biological pathways were dominated by cytokine and chemokine related genes (JAK-STAT signaling pathways, cytokine-responsive elements and cytokine/chemokine genes) and apoptosis-related genes. This suggested that glycolipid-bound CD1d on REC was capable of inducing a programme of immune activation in iNKT cells. I concluded my work by examining if CD1d expression on RECs influenced its active role in immunity. Using wild type and CD1d-deficient transgenic mice challenged with IAV, I showed that CD1d expression is induced on REC in vivo after viral challenge, and in the absence of CD1d, mice showed worse outcome. RECs isolated from CD1d-deficient mice had a much stronger gene expression profile for pro-inflammatory genes. This suggested that CD1d expression on REC could have a bi-directional effect – on the RECs that expressed CD1d (preventing excessive immune-related genes activation) and on the iNKT cells that it engaged (activation, with pro-immunity effects). The thesis concludes with discussion of the potential implications of these findings and future work to examine hypotheses generated from this work.

The development of an efficacious P. falciparum malaria vaccine remains a top priority. Pre-erythrocytic vaccine efforts have traditionally focussed on two well- known antigens, CSP and TRAP, yet thousands of antigens are expressed throughout the liver-stage. The work described in this thesis aimed to assess the ability of other pre-erythrocytic antigens to induce an immune response and provide protective efficacy against transgenic parasites in a mouse model. Research undertaken in our laboratory has demonstrated the ability of prime-boost viral vectored sub-unit vaccination regimens to elicit high levels of antigen-specific T cells. Eight candidate antigens were therefore expressed individually in the viral vectors ChAd63 and MVA. Two antigens, PfLSA1 and PfLSAP2, were identified that confer greater protective efficacy in inbred mice than either CSP or TRAP. PfLSA1 was also able to induce almost complete sterile efficacy in outbred mice, suggesting this vaccine should be assessed in a clinical trial. Immune responses to the candidate antigens were also assessed in human volunteers following their first exposure to controlled malaria infection. The antigen TRAP was further characterised by epitope mapping in volunteers vaccinated with ChAd63-MVA ME-TRAP. However, no functional T cell assay exists to measure inhibition of P. falciparum liver-stage parasites. An improved murine in vitro T cell killing assay was developed, and preliminary experiments were conducted that demonstrate the potential and promise of a P. falciparum T cell killing assay. Such assays will not only allow mechanistic studies to be undertaken, but could also change the way we screen pre-clinical liver-stage vaccines.

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M.tuberculosis, the causative agent of human tuberculosis, is able to achieve long-term persistence within host organism macrophages. This persistence is achieved via the ability of the mycobacterium to prevent phagosomal-lysosomal fusion. The mechanisms by which fusion is inhibited remain incompletely understood. Here we provide evidence supporting a mechanistic link between infection with pathogenic mycobacteria and the cellular pathway defective in the rare lysosomal storage disorder Niemann-Pick disease type C (NPC). We observed that NPC phenotypes, including lipid storage and reduced lysosomal calcium release, can be induced in wild-type murine and human macrophages by infection with pathogenic mycobacteria. This phenotype induction did not occur following infection with the non-pathogenic M.smegmatis. Phenotype induction could be achieved in the absence of the mycobacteria using lipids from the mycobacterial cell walls. The importance of mycobacterial cell wall lipids to mycobacterial virulence has been well-documented. This lipid-mediated inhibition likely occurs through the NPC1 protein. Susceptibility to phenotype induction was inversely proportional to levels of functional NPC1, whilst a pre-existing dysfunction in the NPC pathway (either stemming from mutation or pharmacological inhibition) rendered cells less able to clear non-pathogenic mycobacteria. Finally, we demonstrate that therapies for NPC, particularly curcumin, are able to promote clearance of mycobacteria from infected macrophages. NPC therapies may hold promise for a new approach to the treatment of tuberculosis.

The human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte (CTL) response is important in the control of HIV-1 infection. Due to the virus having a high rate of mutation, immune pressure can select for variants that are no longer recognised by CTLs to dominate the viral quasispecies. This is similar to how antiretroviral resistance emerges. HIV-1 is therefore adapting to both human leukocyte antigen (HLA)-restricted immune responses and antiretroviral therapy. This thesis initially focused on the natural CTL response to an HLA-B*51-restricted epitope in integrase. This HLA class I allele is associated with slow progression to AIDS; however, as no CTL-driven escape mutation has been fully defined within this integrase epitope, we cannot determine what contributes to the association of HLA-B*51 and natural control of infection. By longitudinally studying a cohort of early HIV-infected individuals, we observed the emergence of polymorphisms that abrogate a CTL response to this epitope. CTL escape may also prove to be the downfall of current immunotherapy strategies attempting to combat HIV infection. T cell receptors (TCRs) have been genetically modified to enhance their binding affinity to an HLA-A*02-peptide complex and transduced into CD8+ cells to create an HIV adoptive therapy. We demonstrate through in vitro selection pressure assays that escape from these cells may be a difficult task for the virus given that the TCR is able to recognise the majority of variants of this epitope. Antigen processing mutations may represent the only option for escape. How this may translate clinically will only be determined through in vivo studies, which must be meticulously monitored. Finally, when this high affinity TCR was fused to an anti-CD3 single chain variable fragment to create proteins capable of redirecting non-HIV-specific CTLs to HIV-infected cells, we found that the result was specific lysis. These proteins may supersede the use of TCR-transduced cells when used in synergy with antiretroviral therapy.

Consistent efficacy in human clinical trials has been achieved by two leading malaria vaccine candidates encoding either the P. falciparum circumsporozoite (CSP) or thrombospondin-related adhesion (TRAP) proteins. However, protection in humans relies on high antibody (Ab) titers or potent T cells, respectively, when these antigens are used individually. Therefore, a concurrent induction of both anti-CSP antibodies and TRAP-specific T cells may further improve the protective efficacy associated with these immunogens. This thesis investigates the protective potential associated with CSP and TRAP combination vaccines by employing a pre-clinical Plasmodium berghei (P. berghei) mouse malaria challenge model. Depletion of CD4⁺ and CD8⁺ T cells indicated that protection by the CSP antigen relied solely on antibodies while TRAP protected through CD8⁺ T cell responses. Moreover, the administration of relatively small amount of anti-CSP monoclonal antibodies (mAb) substantially enhanced the sub-optimal protection elicited by TRAP. A way to maximize both, anti-CSP antibodies by adenovirus/protein (Ad-P) prime-boost vaccinations and anti-TRAP T cell responses using adenovirus/modified vaccinia strain Ankara (Ad-M), was explored. Combination of the two approaches stimulated optimal humoral and cellular responses that afforded sterile protection in the C57BL/6 animal model. Additional analyses of the effector functions of Abs indicated that the efficacy of this vaccine relied on a two-stage attack against the parasite: anti-CS antibodies reduced the number of sporozoites reaching the liver, thus decreasing the number of infected hepatocytes required to be eliminated by the dual action of natural killer (NK) cells and CD8⁺ T cells. These proof-of-concept studies using a wild-type (wt) P. berghei challenge model allowed progression towards the development of human malaria Plasmodium vivax (P. vivax) vaccines using PvCSP and PvTRAP antigens. The immunogenicity of PvCSP and PvTRAP-based vaccine candidates was assessed in multiple mouse strains. Additionally, transgenic (tg) P. berghei parasites expressing P. vivax genes were generated and used to demonstrate the protective efficacy of Ad-P PvCSP and Ad-M PvTRAP vaccines.

Despite 30 years of research, a prophylactic vaccine against HIV-1 is still lacking and is urgently needed in order to control the global AIDS pandemic. The discovery of broadly neutralising antibodies (BNAbs) was an important step for HIV-1 research but no vaccine candidate tested so far has been able to reproduce responses containing such antibodies, and it remains unclear how this could be achieved via immunisation. In this thesis, I attempted to explore this gap of knowledge in two ways. First, certain features (‘signatures’) of the Env protein that were associated with a broadly neutralising response were identified through machine learning. Further characterisation of these signatures revealed several ways by which these naturally-occurring mutations might alter the immunogenicity of the Env protein that could result in the elicitation of a broadly neutralising response. The incorporation of such signatures in future vaccine design could be useful as the Env protein might adopt a conformation that encourages the elicitation of a broadly neutralising response. Second, 3 novel vaccination approaches were proposed aiming to induce a BNAb antibody response. The development of 2 approaches proved to be difficult and was not continued. For the third approach, non-neutralising immunogen-derived antibodies were used to mask immunodominant epitopes on the Env protein (i.e. ‘antibody-shielding’), thus allowing the antibody response to be focused to the highly conserved CD4 binding site (CD4bs). Subsequent immunisation of the antibody-shielded gp120 proteins in mice and rabbits demonstrated that antibody-shielding was able to significantly dampen the V3-specific antibody response while retaining the CD4bs-specific response. However, the antibody response to the V1/V2 loop was enhanced upon V3-dampening which indicates that further optimisation of the antibody-shield is needed in order to eliminate any antibody response towards the immunodominant regions. In conclusion, these results are the first description of a number of novel vaccination ideas and provide valuable insights into how these approaches could be optimised to become effective HIV-1 vaccines that can lead to the elicitation of a broadly neutralising antibody response.