Natural exposure to Plasmodium falciparum’s asexual blood-stage results in protection against severe disease, but no vaccine using the widely-studied blood-stage antigens apical membrane antigen 1 (AMA1) or merozoite surface protein 1 (MSP1) has proven convincingly protective in clinical trials. Challenges include antigenic polymorphism, the apparent requirement for exceptionally high antibody concentrations for protection, and clinical-grade production of conformationally-accurate recombinant protein antigens followed by formulation with a human-compatible adjuvant. This thesis describes the generation of viral-vectored vaccines targeting ten less-studied blood-stage antigens, focusing upon antigens implicated in erythrocyte invasion. These vaccines were immunogenic in mice and rabbits. The rabbit antibodies raised were functionally active in the in vitro assay of parasite growth inhibitory activity (GIA). GIA with antibodies against one antigen, RH5, exceeded that achieved with antibodies against the ‘gold standard’ AMA1 or MSP1 antigens. This antigen’s amino acid sequence is relatively conserved between parasite strains. Importantly, and unlike anti-AMA1 and MSP1 antibodies, the GIA effects transcend genetically diverse strains. It was hypothesised that blockade of the interaction of RH5 with its receptor basigin was likely to be a mechanism of action of anti-RH5 antibodies. Vaccine-induced polyclonal anti-RH5 serum was found to be capable of blocking this interaction, as well as merozoite attachment to erythrocytes. A panel of RH5-specific monoclonal antibodies were raised: those which block the RH5-receptor interaction were capable of neutralising parasites. Minimal linear epitopes recognised by these antibodies were mapped, and are likely to be within or close to RH5’s receptor binding site. These data support prompt clinical testing of RH5-based vaccines, and shed light upon the mechanism of action of anti-RH5 antibodies. However substantial challenges remain in establishing whether this antigen, selected on the basis of the in vitro assay of GIA, will be capable of achieving in vivo protection against P. falciparum. Further work presented in this thesis addresses the use of quantitative PCR data to assess blood-stage vaccine efficacy in experimental human challenge with P. falciparum, and the use of surface plasmon resonance to establish more detailed characterisation of vaccine-induced antibody responses. Finally, the results of P. falciparum challenge of RH5-vaccinated Aotus nancymaae non-human primates are presented.

Background: Hepatocellular Carcinoma (HCC) is the third most common cause of cancer related death worldwide and is often diagnosed by measuring serum Alpha-fetoprotein (AFP); a stand-alone biomarker with limited diagnostic proficiency. To compensate for this, AFP is commonly used in conjunction with high performance imaging and radiological methods. However, as the burden of HCC is predominantly in the developing world where such technologies are not readily available, it is imperative that efforts are made to pursue the discovery of novel, high performance, easy to measure and robust biomarkers. With the aim of improving on the diagnostic ability of AFP, our project focuses on the study of plasma proteins as identified by Mass Spectrometry (MS) in order to investigate differences seen in the respective proteomes of controls and subjects with liver cirrhosis (LC) and HCC. Methods: Matrix Assisted Laser Desorption Ionization Time-of-Flight MS (MALDI-TOF MS) was first attempted on weak cation exchange (WCX) fractionated plasma in a pilot selection of forty subjects. On the main case-control group, quantitative MS analysis using liquid chromatography electro spray ionization quadrupole time-of-flight (LC-ESI Q-TOF) was conducted on 339 subjects using a pooled expression profiling approach. Enzyme-linked immunosorbent assays (ELISA) and 1 and 2Dimentional electrophoresis methods were performed to validate and detail candidate protein levels and modification patters in individual and pooled subjects. The human plasma used for the MS based protein discovery experiments was collected as part of a five year Liver Cancer Case-control Study (Gambia, West Africa). A smaller set of samples from subjects who formed a spectrum of non-liver disease controls, LC and HCC were obtained from the Jos University Teaching Hospital (JUTH) in Nigeria and ELISA and gel electrophoresis assays conducted on them to confirm the trends and differences seen in the Gambian subject set. Results: Bioinformatic evaluation of MALDI-TOF data highlighted peak masses 2444m/z, 2583m/z and 2559m/z to have high diagnostic abilities based on area under curve (AUC) statistics of >0.75. Of these polypeptide fragments, one was identified as the plasma glycoprotein, alpha chain fibrinogen. Results from the large-scale label free discovery experiments indicated twenty-six proteins to be differentially expressed between the three subject groups. These prospective markers include proteins previously linked to HCC as well as novel candidates, namely glutathione peroxidase 3, serum amyloid p, carboxypeptidase N and complement factors I and H which have not been implicated in the context of HCC diagnostics. Direct measurement of Hemopexin (HPX), alpha-1-antitrypsin (α1AT), apolipoprotein A1 (Apo A1) and complement component 3 (CC3) levels confirmed their change in abundance in LC and HCC versus control patients. Further biochemical characterization of glycosylated HPX isolated from glycoprotein enriched plasma sample pools showed evidence of isoelectric point shifts, indicating differential glycosylation patterns in high mannose structures of HPX which may be disease stage linked. The direct measurements of HPX, α1AT, Apo A1 & CC3 conducted on the independent Nigerian subject group also confirmed much of the trends reported from the Gambia Liver Cancer Study (GLCS) plasma. Conclusions: The independently validated, significant changes in the quantitative expression of ApoA1, α1AT, CC3 and HPX could be exploited for development into high-performance affordable assays, usable in the diagnosis and monitoring of HCC and LC patients. The unique signatures observed for most of these proteins, from liver disease free controls to LC and HCC suggest their involvement in independent pathways. As such, combining some or all of these four markers within a diagnostic panel could offer a much-needed boost in robustness and accuracy for AFP. The differences in the processing and molecular weight separation of these proteins also offers a novel inroad into biomarker identification. These suggested disease specific signatures could with further study offer highly specific biomarkers able to discern the key stages that predispose individuals to hepatocarcinogenesis. Impact: This is the first MS based discovery and extensive validation study on West African subjects whose primary cause of HCC are the Hepatitis B Virus (HBV) and fungal toxins.

Treatment of lower respiratory tract infection (pneumonia and pleural infection) relies on the use of empirical broad spectrum antibiotics, primarily because reliable pathogen identification occurs infrequently. Another consequence of poor rates of pathogen identification is that our understanding of the microbiology of these infections is incomplete. This thesis addresses some of these issues by combining the acquisition of high quality lower respiratory tract samples, free from nasooropharyngeal contamination, with novel molecular microbiological techniques in an attempt to increase rates of pathogen identification. Four main areas are examined: (i) The role of so-called ‘atypical pneumonia’ bacteria in causing pleural infection. These pathogens have been previously identified in the pleural space infrequently and routine culture usually fails to isolate such bacteria. High sensitivity nested polymerase chain reaction (PCR) is a culture-independent technique which is used to undertake a systematic evaluation for these pathogens in pleural infection samples. (ii) The role of Pneumocystis jirovecii in pleural infection, either as a co-infecting pathogen or in monomicrobial infection. This fungus causes severe pneumonia, particularly in the immunosuppressed, but is increasingly recognised as a co-pathogen in community-acquired pneumonia, and is frequently isolated in the upper and lower respiratory tract in health. A high sensitivity real-time PCR assay is used to examine for this fungus. (iii) Ultra-deep sequencing of the 16S rRNA gene is used to perform a comprehensive microbial survey in samples taken from the multi-centre MIST2 study of pleural infection. The techniques employed allow analysis of polymicrobial samples and give very high taxonomic resolution, whilst incorporating methods to control for potential contamination. Further, these techniques provide confirmation of the results from the ‘atypical’ bacteria nested PCR study. (iv) Bedside ultrasound-guided percutaneous transthoracic needle aspiration (TNA) of consolidated lung is undertaken in patients with pneumonia, as part of the PIPAP study. An evaluation is undertaken of the efficacy and acceptability of TNA. Aspirate samples acquired are also processed using ultra-deep sequencing of the 16S rRNA gene. Other samples obtained as part of the PIPAP study, such as ‘control’ lung aspirates and ‘control’ pleural fluid samples, are similarly processed to enable calculation of sensitivity and specificity of the sequencing methodology.

The Human immunodeficiency virus (HIV) pandemic has resulted in a heavy global disease burden, and clinically causes Acquired Immuno-Deficiency Syndrome (AIDS). The development of highly active antiretroviral therapy (HAART) has achieved remarkable control of the rapidly evolving HIV. However, HIV remains neither curable nor preventable by vaccine, and in the developing regions worst affected by HIV, HAART remains inaccessible to most patients. Furthermore, the change in both immunology and viral evolution during chronic HIV infection and its relation to AIDS pathogenesis remains unknown. Following the failure of recent HIV vaccines, it is believed that a better understanding of host-pathogen interaction is vital to advance therapeutic (vaccine and drug) design. In this thesis, I have performed an investigation of viral adaptation in response to different selection forces during advanced HIV infection and AIDS. The thesis first examined a case study that reveals the potential role of B cell-mediated neutralising antibody (NAb) in chronic HIV infection through the unexpected effect of B cell depletion agent, anti-CD20 (Rituximab). Here, longitudinal results have shown that viral load (VL), env gene diversity, and NAb sensitive strains increased during B cell and NAb depletion as a result of Rituximab administration, and reversed as B cells recovered. The study provides preliminary evidence to support the idea that NAb may be effective at suppressing HIV. The rest of the thesis focused on the cross-sectional cohort at Bloemfontein, South Africa (n=1491), a resource-limited region affected by the pandemic. Here, we used methods that include molecular and pretherapy drug resistance epidemiology, mathematical modelling, phylogenetically adjusted bioinformatics analysis and in vitro viral replication capacity (VRC) assay to study materials including cohort demography, plasma samples, CD4 cell count, VL, viral genetic sequences and host human leukocyte antigen (HLA) tissue types. Our analysis was further augmented by the additional data kindly contributed by our neighbouring Durban cohort collaborators (n=775), which also includes an IFN! ELISPOT assay that measures cytotoxic T lymphocyte (CTL) responses. Using the HIV pol sequencing data and phylogenetic analysis we confirmed that the local molecular epidemiology is similar to the circulating strains documented in the regional database. However, the pretherapy drug resistance mutation screening results have revealed an unexpected high incidence of drug-induced viral mutants in the AIDS patients with CD4 counts <100 cells/μl. According to mathematical modelling, this finding is attributable to additional sources of antiretroviral therapy exposure, which warrants public health caution. The investigation then focused on studying the changes in HLA class I mediated CTL selection and viral evolution as CD4 counts are reduced in AIDS. Interestingly we have noted evidence that suggest weakening CTL immune selection against gag during AIDS is associated with increased viral fitness (measured by VRC) and reversion of previous immune-escape mutations which conferred high fitness costs. In conclusion, this thesis compared different sources of host and drug mediated HIV selection and its implication for viral evolution. The identification of more bottleneck sites conferring high fitness costs to the selection of escape mutants is expected to be helpful in the design of future therapeutics (via vaccine, drug, immune therapy, or public health strategy). As we have learnt from the principle of combinational ARV, it would be desirable for a vaccine to select HIV at multiple sites of high escape-mutation fitness cost, hence offering protective effect.

Cette thèse évalue le risque d'émergence d'un pathogène via l'avifaune sauvage dans une région indemne en combinant deux approches : 1) L'étude de pathogènes partageant des caractéristiques éco-épidémiologiques communes avec le pathogène émergeant. 2) L'utilisation de données écologiques disponibles dans la région indemne. Le Chapitre 1 montre que l'étude de la circulation de pathogènes partageant des caractéristiques éco-épidémiologiques communes (Influenza Aviaire Faiblement Pathogène et Maladie de Newcastle) avec un pathogène émergeant (H5N1 HP) permet d'apporter des éléments d'information sur la circulation potentielle de ce pathogène en cas d'émergence. Les principales conclusions de ce chapitre nous permettent de construire les trois hypothèses testées aux chapitres suivants portant respectivement sur les étapes d'une émergence : introduction (Chapitre 2), circulation (Chapitre 3), et transmission à la faune domestique (Chapitre 4). Ces trois chapitres permettent une meilleure évaluation du risque d'émergence d'un pathogène (le H5N1 HP) dans une zone indemne (le DIN) à partir de méthodes basées sur les données écologiques disponibles dans cette zone indemne et obtenues à partir de techniques telles que le comptage aérien, la télémétrie satellitaire, ou la télédétection. Ils permettent notamment d'estimer que le risque d'émergence du H5N1 HP dans le DIN via l'avifaune sauvage est le plus élevé lors des mois de janvier à mars des années de faible crue, et que la Sarcelle d'été et le canard Pilet sont les deux espèces à surveiller en priorité. Cette thèse montre donc comment l'utilisation de données éco-épidémiologiques disponibles dans une zone indemne permettent une évaluation et un meilleur contrôle du risque d'émergence d'un pathogène. L'utilisation d'outils satellitaires permet l'obtention de larges plages de données ayant des résolutions spatiale et temporelle suffisante pour évaluer des dynamiques éco-épidémiologiques évoluant rapidement. / This thesis aims to evaluate the risk of emergence of a pathogen from wildlife in an uninfected area by combining two approaches:1) The study of pathogens sharing similar eco-epidemiological characteristics with the emerging pathogen2) The use of eco-epidemiological data available in the uninfected area Chapter 1 shows that studying pathogens sharing similar eco-epidemiological characteristics (Low Pathogenic Avian Influenza and Newcastle Disease) with an emerging pathogen (HPAI H5N1) enables to provide information on the potential circulation of this pathogen if it would emerge. The main conclusions of this chapter allow us to formulate the three hypothesis tested in the following chapters, each related to a stage of an emergence: introduction (Chapter 2), circulation (Chapter 3), and transmission to domestic poultry (Chapter 4). These three chapters enable a better evaluation of the risk of emergence of a pathogen (HPAI H5N1) in an uninfected area (IND) by using methods based on eco-epidemiological data available in this uninfected area, obtained from techniques like aerial census, satellite telemetry, and remote sensing. The risk of emergence of HPAI H5N1 from wildlife in the IND is evaluated to be the highest between the months of January and March during years with a low flood level. Garganeys and Pintails are identified as the two main species to be surveyed in priority. This thesis shows how eco-epidemiological data available in an uninfected area enable an evaluation and a better control of the risk of emergence of a pathogen. Satellite tools allow acquiring large dataset with temporal and spatial resolution compatible with eco-epidemiological dynamics evolving rapidly.

Enteric fever caused by Salmonella enterica serovars Typhi and Paratyphi A are a major public health concern in Kathmandu. The aim of this thesis was to identify and assess the population most at risk by investigating epidemiologic trends of enteric fever within a subset population of Kathmandu. Therefore,the burden and incidence of enteric fever within the study population and the seasonal and gender distribution of enteric fever was assessed. Considerable burden of enteric fever, unrelated to population density, correlating with the seasonal fluctuations in rainfall was observed. This thesis also aimed to improve the understanding of enteric fever transmission by identifying probable transmission routes,hence various water and food samples were analysed and the extent of faecal contamination in them was determined. S. Typhi isolates were sequenced and genotyped and combined with GPS data to longitudinally study the local distribution and infer transmission of this human restricted bacterial pathogen. Extensive clustering of typhoid independent of population size and density and existence of an extensive range of genotypes within typhoid clusters including individual households with multiple cases was observed. These observations predict that indirect transmission had an overwhelming contribution for disease persistence, potentially through contaminated water. Consistent with this hypothesis, S. Typhi and S. Paratyphi A were detected in water supplies and it was observed that typhoid was spatially associated with public water sources and low elevation. A concurrent case-control study was also conducted which allowed for the determination of risk factors in the population at risk. These studies imply that resources should be allocated toward controlling the most important vectors of enteric fever, including food sold by vendors, chlorination of drinking water, construction of proper water distribution and sewage networks,vaccination campaigns and hygiene education.

Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes (IEs) is a common property of field isolates associated with severe disease (Pain, Ferguson et al. 2001). Platelet receptors CD36 (Pain, Ferguson et al. 2001), P-Selectin (Wassmer, Taylor et al. 2008) and gC1qR (Biswas, Hafiz et al. 2007) mediate clumping. To characterize the molecular specificities of the clumping phenotype, I cloned clumping parasite line IT/C10 by limiting dilution. I characterized var gene expression in the IT/C10 clones using generic primers for the DBL tag region (Bull, Berriman et al. 2005). Clumping assays were conducted in the presence of specific reagents to delineate host factors hypothesized to contribute to development of the clumping phenotype. Finally, I conducted a clinical study with isolates from children with malaria in Kilifi, Kenya. This study shows that in parasite line IT/C10, platelet-mediated clumping is associated with Itvar30 suggesting a prominent role for the PfEMP-1 encoded by this var gene in development of platelet-mediated clumping. For IT/C10 parasites, platelet activation appears to be involved in platelet-mediated clumping. Platelet P-Selectin appears to mediate clumping using lectin-dependent interactions. To further elucidate the mechanisms that mediate clumping by host platelets, I have used a panel of platelet antagonists to delineate specific platelet activation pathways. Our results show that platelet activation plays an important role in platelet-mediated clumping. Finally, in this study, platelet-mediated clumping was associated with parasitaemia, but not with disease severity.

Deubiquitinating enzymes (DUBs) constitute a diverse protein family. The specificities and functions of the majority of DUBs are unknown, although their impact on many biological and pathological processes is widely appreciated. This dissertation entails a detailed characterization of otubain 1 (OTUB1), an ovarian tumor domain-containing DUB. The presented work describes OTUB1’s specificity, localization, protein interactions, importance in infection with Yersinia, and proposes a novel model of regulation of its enzymatic activity. I first discuss the structural and biochemical properties of OTUB1, demonstrating its selectivity towards ubiquitin and NEDD8. Moreover, I show that OTUB1 cleaves lys48- but not lys63-linked polyubiquitin, emphasizing its role in ubiquitin-mediated proteasomal degradation. Mass spectrometric identification of interaction partners and localization studies suggest possible involvement of OTUB1 in RNA processing and cell morphology. Furthermore, I demonstrate that invasion of the host cells by the enterobacteria Yersinia can be altered by changing OTUB1 expression. This effect is dependent on the catalytic activity of OTUB1 and its ability to stabilize RhoA-GTP prior to infection. YpkA and OTUB1 modulate RhoA-GTP stability in opposing ways, leading to cytoskeletal rearrangements that may be involved in bacterial invasion. Moreover, OTUB1 is post-translationally modified by phosphorylation that modulates its ability to stabilize RhoA-GTP and counteracts its effect on bacterial invasion. These findings provide a novel entry point for the manipulation of the host—pathogen interactions. Lastly, a kinase screen revealed that FER, an oncogenic kinase with a role in cell morphology, phosphorylates OTUB1, as demonstrated by overexpression, siRNA and in vitro studies. The phosphorylated site was mapped to tyr26 and the activity-based labeling revealed that this modification interferes with the deubiquitinating activity of OTUB1. In summary, the results presented in this thesis confirm that OTUB1 exerts properties of a “classical DUB” and uncover some of its physiological functions.

Replication-deficient adenovirus (Ad) and modified vaccinia virus Ankara (MVA) vectors expressing single Plasmodium falciparum antigens can induce potent T cell and antibody responses and have entered clinical testing using a heterologous prime-boost immunisation approach (Ad_MVA). This thesis describes a number of pre-clinical approaches aimed at enhancing the efficacy of these viral vectored vaccines targeting the blood-stage of malaria. First, the development of a highly efficacious malaria vaccine is likely to require a multi-antigen and/or multi-stage subunit vaccine. The utility of an Ad_MVA immunisation regime combining vaccines expressing the 42kDa C-terminus of the blood- stage antigen merozoite surface protein 1 (MSP142) and the pre-erythrocytic antigen circumsporozoite protein (CSP) in the P. yoelii mouse model was investigated. It was found that vaccine co- administration leads to maintained antibody responses and efficacy against blood-stage infection, but reduced secondary CD8+ T cell responses and efficacy against liver-stage infection. CD8+ T cell interference can be minimised by co-administering the MVA vaccines at separate sites, resulting in enhanced liver-stage efficacy. The mechanisms of CD8+ T cell interference were explored. Second, Ad_MVA regimes expressing blood-stage antigens that can protect against P. chabaudi and P. yoelii blood-stage infection were tested against P. berghei, but did not confer protection. Similarly, IgG from rabbits immunised against P. falciparum MSP1 (PfMSP1) could not protect mice from a chimeric P. berghei parasite expressing PfMSP1. Third, two molecular adjuvants, the C4bp α-chain oligomerisation domain (IMX108/313) and the Fc fragment of murine IgG2a, were tested for their ability to enhance immunogenicity of recombinant adenoviruses when fused at the C-terminus of a blood-stage antigen. IMX108/313 was found to adjuvant T cell responses of small (< 80kDa) antigens and this was associated with antigen oligomerisation. However, the Fc fragment did not adjuvant responses. Finally, it was found that using a strong early promoter to drive antigen expression enhances the immunogenicity of single administration MVA vaccines, but that this did not enhance post-boost immunogenicity in an Ad_MVA regime.

Human immunodeficiency virus (HIV) Rev and Hepatitis B virus (HBV) e-antigen are both viral proteins that have key functions in their respective viral replication cycles. Both have evaded crystallization for decades due to their tendency to aggregate and/or form higher-order species. In this thesis the structure determination of HIV Rev and HBV e-antigen is presented—achieved via complexing with monoclonal antibody Fab fragments—and their structures are analysed. HIV Rev is a small regulatory protein that mediates the nuclear export of viral mRNAs, an essential step in the HIV replication cycle. In this process, Rev cooperatively oligomerises onto a highly structured RNA motif, the Rev response element. The structure of Rev (complexed with Fab), determined to 2.3 Å resolution, reveals a molecular dimer where the ordered portion of each subunit (N-terminal domain; NTD; residues 9-65) contains two coplanar a-helices arranged in hairpin fashion. Rev subunits dimerise via interaction of identical hydrophobic patches that overlap to form a V-shaped assembly. Mating of hydrophobic patches on the outer surface of the dimer promotes higher order interactions. Cryo-electron microscopy and helical image reconstruction of in vitro assembled Rev filaments were performed to better understand higher-order Rev oligomerisation. Reconstructions of Rev filaments were determined to ~13 Å resolution, permitting docking of the Rev NTD structure. Conformational variability of the Rev dimer subunits and use of a third ligomerisation interface engender filaments that can expand and contract. Both characteristics were also observed in the crystal structures of Rev. Surface features of the Rev filaments are altered in different expansion states, which may have implications for the assembled forms that Rev adopts during nuclear export of RNA and subsequent re-import into the nucleus. Various models for Rev oligomerisation onto the viral RNA are proposed. Chronic Hepatitis B virus (HBV) infection afflicts millions worldwide with cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the protein (core antigen, HBcAg) that forms the building-blocks of capsids. HBeAg is not required for virion production, but is implicated in establishing immune tolerance and chronic infection. The crystal structure of HBeAg clarifies how the short N-terminal propeptide of HBeAg induces a radically altered mode of dimerisation relative to HBcAg (~140 rotation), which is locked into place through formation of intramolecular disulfide bridges. This structural switch precludes capsid assembly and engenders a distinct antigenic repertoire, explaining why the two antigens are cross-reactive at the T-cell level (through sequence identity) but not at the B-cell level (through conformation). The structure offers insight into how HBeAg may establish immune tolerance for HBcAg while evading its robust immunogenicity.