I investigated the relationship between in-host ecology and transmission using the rodent malaria parasite <i>Plasmodium chabaudi</i> as an experimental laboratory model. This required the initial development of a novel quantitative reverse transcription PCR technique (qRT-PCR) to measure the in-host density of <i>P. chabaudi</i> transmission stages and provide better estimates of transmission investment. My doctoral research revealed that the majority of total parasite and transmission stage production occurs within the initial phase of infection, however, many individuals developing very low level long lasting chronic infections which are transmissible to vectors, confirming current findings in the field. During conspecific competition, a small degree of transmission investment plasticity was observed, but did not affect the competitive outcome. Competition success was greatly influenced by drug treatment when drug resistant and sensitive genotypes co-infected the same host. In this case, it was observed that strong drug dosage completely cleared the drug sensitive clone, releasing the resistant clone from competition and then resulted in its enhanced growth and increased transmission potential. Reduced drug dosage which did not completely clear the sensitive genotype also released the resistant clone from competition, but prevented its enhanced growth. These findings suggest that low drug treatment, which just alleviates host clinical symptoms but does not completely clear parasites, will preserve within host competition and many reduce the rate at which drug resistance spreads. This contradicts current medical thinking that incomplete drug treatment regimes will accelerate drug resistance evolution.

Plasmids were constructed to encode C3d/PfEMP-1 fusions and expression of recombinant protein in mammalian cells in culture was assayed. Eukaryotic expression of <i>P. falciparum </i>proteins proved to be problematic and a re-condoning approach was adopted to address this. The production of polyclonal anti-PfEMP-1 antibodies in mice was assessed in immunofluorescence assays and in immunoblots with <i>P. falciparum</i>-infected erythrocytes. The data question the suitability of a DNA vaccine approach in the development of a PfEMP-1-based vaccine using C3d. In order to raise specific antibodies to PfEMP-1 and to develop a suitable assay to assess immunogenicity of this antigen, research efforts became focussed on the production of recombinant PfEMP-1 protein, with a view to immunising mice. A recombinant PfEMP-1 domain was expressed in mammalian cells and characterised, demonstrating it to be the ligand involved in binding uninfected erythrocytes. Its reactivity with immune sera and, therefore, its suitability as a malaria vaccine candidate was assessed. Findings highlight the need for further work on the development of methods to produce functionally active recombinant protein. They also show the necessity of improving methods of detecting surface expression of PfEMP-1. The suitability of the <i>Saimiri </i>monkey model for C3d-based vaccination and <i>P. falciparum</i> challenge experiments was assessed. In other species, the receptor for C3d is CR2, expressed on B cells. <i>Saimiri</i> B cells were characterised and their capacity to bind human C3d was demonstrated, indicating that <i>Saimiri</i> is potentially a suitable model for pre-clinical vaccination studies using human C3d-based immunoprotentiation.

It is now widely accepted that clinical symptoms develop as a result of an excessive inflammatory response to malaria antigen. I hypothesise that inflammation is primarily due to a "Th-1-like" response and that the development of clinical immunity is associated with the downregulation of pro-inflammatory cytokines. I further hypothesise that immune responses of naïve and clinically immune individuals are mediated by different populations of mononuclear cells. In order to test these hypotheses, I first developed specific and sensitive methods to measure cytokine production. I then compared proliferative responses and IFN-g-production of peripheral blood mononuclear cells (PBMCs) to <i>P. falciparum </i>schizont extract (PfSE) from malaria naive, malaria-exposed (but not clinically immune) and malaria-immune individuals. In order to determine how PfSE-induced IFN-g production is regulated, I have also measured IL-12 p40, IL-12 p70 and IL-10 from PfSE-stimulated PBMCs and investigated the role of neutralising antibody to IL-12 in modulating IFN-g production. Finally, a combination of cell surface staining and intracellular cytokine staining was used in order to determine the phenotypes of cells responding to PfSE. Cells from all individuals proliferated vigorously in response to PfSE, but there were no significant differences between any of the groups. Cells from naïve individuals produced moderate levels of IFN-g which were mainly IL-12-dependent. Intracellular cytokine staining analysis indicated that IFN-g was primarily produced by ab+ T cells, although significant proportions of gd+ T cells and NK cells also produced IFN-g. IFN-g levels were significantly higher in PBMC cultures of exposed individuals than in cell cultures from naïve individuals and were only partially IL-12-dependent. In contrast, minimal levels of IFN-g were produced from PfSE-stimulated cells of immune individuals and were significantly lower than those found in either naïve or exposed populations.

Introduction and Objectives: Nontyphoidal Salmonellae (NTS) are a major cause of bacteraemia in Sub-Saharan Africa. Most cases of NTS bacteraemia in this region occur in children under five years of age and HIV-infected adults. The objectives of this work were to investigate the role of antibody in immunity against NTS in Africans and to explore the potential of measuring anti-Salmonella antibody as the basis of a diagnostic assay. Materials and Methods: We obtained blood from healthy Malawians of all ages and NTS bacteraemic children. Invasive strains of NTS were from bacteraemic Malawian children. We measured anti-Salmonella antibody levels by flow cytometry and we assessed the ability of serum to kill Salmonella in a serum bactericidal assay. Phagocytic cell function of peripheral blood cells in relation to NTS was examined by flow cytometric oxidative burst and phagocytosis assays and a blood cell killing assay. Serum cytokine levels were measured using a cytokine bead array assay. Results: We developed and optimised a flow cytometric assay that measured antiSalmonella antibody titres over 4 log-decades of fluorescence. Using this assay in a crosssectional study of healthy Malawians, we found that anti-Salmonella antibody titres increased with age with no reduction in old age. Oxidative burst by peripheral blood cells in African children under two years in response to stimulation with NTS was impaired compared with older children and the oxidative burst activity correlated with anti-Salmonella IgG titres. Preopsonising NTS with immune serum overcame this deficit in oxidative burst activity. We found that anti-Salmonella antibody and complement were required for phagocytosis, oxidative burst and blood cell killing of NTS. High levels of IL-6, TNF-a, IFN-y and IL-10 cytokines were secreted by peripheral blood cells following stimulation with NTS, but antibody was not required for this. In children with NTS bacteraemia, titres of antiSalmonella antibodies were increased compared with healthy age-matched individuals. These antibodies were not bactericidal or protective against further episodes of NTS bacteraemia some children. Using our antibody assay as the basis of a potential diagnostic tool for NTS bacteraemia, the sensitivity was low using sera from the acute phase of infection (around 50%) and higher using convalescent sera (greater than 80%). Conclusion: Anti-Salmonella antibody together with complement are required for both cellindependent and phagocyte cell-dependent killing of NTS in peripheral blood of Africans, the latter modality of immunity involving phagocytosis and oxidative burst function. Specific antibody is not a clear requirement for cytokine production in response to NTS.Protective anti-Salmonella antibody develops naturally with age in healthy Africans and high titres of antibody are produced in response to NTS bacteraemia, but are not always protective. The low sensitivity found when using our antibody assay for diagnosis of NTS bacteraemia during acute illness requires further work to establish whether this can be used as the basis of a rapid diagnostic test for this condition. Our findings indicate that a vaccine against NTS for Africa should induce production of protective bactericidal and opsonic antibodies in young African children.

Schistosomiasis is one of the most prevalent diseases in the world and a major cause of morbidity in Africa. Accurate determination of the geographical distribution of schistosomiasis in Africa along with the number of people affected is difficult, since reliable prevalence data are often not available for most of the African continent. Effective schistosomiasis control programmes rely on accurate statistics regarding the geographical distribution of disease, the population at risk, and the intensity of disease transmission. These estimates can be obtained using a number of statistical methods which relate prevalence and intensity of disease to risk factors, measured at the individual level and at the population level. Schistosoma mansoni is largely a climatedriven parasite, which relies on the availability of a suitable snail host. The survival of parasitic infection depends on climatic variables, such as temperature, rainfall and vegetation. Statistical models which incorporate spatial or individual heterogeneity are highly complex and require large numbers of parameters. Until recently, the most common approach was to use regression modelling to identify risk factors for disease transmission. However, this method has a number of limitations. In particular, it gives no information on the dynamics of transmission, e. g. will the disease reach an endemic state under a certain set of conditions or be subject to a periodic cycle? The aim of this thesis was to a) develop mechanistic transmission models to study how schistosomiasis disease dynamics change with water temperature change and to parameterise these models to provide better estimates for a specific host-parasite combination; b) explore how the efficacy of control programmes changes with changing water temperature; c) produce continent-wide maps of schistosomiasis prevalence in Africa, using a combination of geospatial models and environmental data; d) to quantify the impact of climate change over the next 50 years on the prevalence and intensity of disease. A mechanistic model describing the transmission dynamics of schistosomiasis at a range of water temperatures was developed and showed that as the long-term mean temperature increases up to 29°C, the mean worm burden increases. At 34°C, the mean worm burden starts to taper, as the thermal limits of both the snail and the parasite are reached. Adding complexity to the models, such as snail density-dependence and adult parasite density-dependence, had no significant impact on the overall transmission patterns. However, a sensitivity analysis revealed subtle changes in the relative importance of certain parameters. The most detailed model showed that the parameters describing the transmission of schistosomes from snail to man were the most sensitive to change and therefore, provided a useful target point for control strategies. The effects of various control programmes were modelled using discrete time series models and manipulation of the individual parameters. The most effective control programme was repeated mass chemotherapy, although reducing contact with contaminated water also proved highly effective. Producing maps of geo-referenced point prevalence data highlighted the areas in which no data currently exist. This provides an invaluable tool for determining which regions need further study. Four separate geospatial models were developed to predict the distribution of schistosomiasis over Africa, and each was validated using existing data. The ordinary kriging model provided the best estimates for prevalence data and the indicator kriging model provided the best estimates for the probability of infection within a population. These models are useful for determining high-risk populations and locating areas in which control efforts should be focussed. Two types of regression models were used to investigate associations between climatic variables and prevalence of disease. Monthly rainfall and mean annual temperature were shown to have important roles in defining the limits of schistosomiasis transmission. Using these data, it is possible to define a threshold, outside which schistosomiasis transmission is unlikely to occur. These models were used to predict how the distribution of schistosomiasis would change with climate change. It was shown that over the next 50 years, there will be an increase in the number of areas able to support the intermediate vector. Without socio-economic development or intervention strategies, this will almost certainly be followed by an increase in disease transmission. The use of mathematical and geospatial models can greatly enhance our understanding of schistosome epidemiology and are an essential tool in the planning stages of any intervention strategy.

No description available.

Malaria is a major threat to human health. It is caused by Plasmodium s-pp., a protozoan parasite that belongs to the phylum Apicomplexa. Malaria parasites have a complicated life cycle and clinical symptoms occur during replication of the parasite in erythrocytes. Plasmodium falciparum subtilisin-like serine protease 1 (PfSUB1) belongs to the subtilisin-like family of serine proteases (subtilases). It is encoded by a single copy gene and is translated as a protein with a molecular mass of about 78 kDa. Attempts to disrupt the gene have not been successful, indicating an essential role in the intraerythrocytic life cycle of the parasite. Recent studies have shown that PfSUB1 is discharged into the parasitophorous vacuole (PV) in the final stages of schizont maturation to mediate proteolytic maturation of an abundant PV protein called SERA5. In this project, an examination of the SERA5 processing sites along with the use of a small library of internally quenched fluorogenic peptides, revealed a consensus PfSUB1 substrate recognition motif that showed a striking resemblance to all known primary processing sites in merozoite surface proteins. The role of PfSUB1 in these processing events was therefore examined. The results showed that PfSUB1 is responsible for the proteolytic processing of merozoite surface proteins 1, 3, 6 and 7 that takes place in the final stages of schizont maturation. Different strategies to obtain a knock-down of the pfsub1 gene by means of reverse genetics were also attempted. Mutagenesis studies were performed to study the structural characteristics of PfSUB1 that are responsible for its substrate specificity, and in an attempt to optimise the expression of soluble recombinant PfSUB1 for future crystallographic studies. The results obtained identify PfSUB1 as a multifunctional processing protease of the malarial PV that plays a key role in both egress and proteolytic remodelling of the developing merozoite in preparation for its release from the confines of the infected cell.

No description available.

No description available.

No description available.