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Plasmodium falciparum malaria is one of the leading causes of child mortality worldwide, and currently there is no vaccine which confers sterilising immunity against infection. One approach to reducing the morbidity and mortality associated with P. falciparum malaria would be the development of a vaccine which targets P. falciparum virulence phenotypes, thereby protecting against the severest forms of the disease. Rosetting, where erythrocytes parasitised with P. falciparum bind to uninfected erythrocytes, is a well-established virulence phenotype that could serve as such a target. Although the major family of parasite proteins responsible for rosetting, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), displays a staggering degree of antigenic polymorphism, there is some evidence to suggest that functionally related PfEMP1 subsets are relatively conserved. Therefore, it is possible that antigenic conservation within the rosette-mediating subset of this family exists. In this thesis, I aim to determine whether rosette-mediating PfEMP1 variants are sufficiently conserved to be considered viable vaccine targets. Firstly, I used rosetting Kenyan culture-adapted P. falciparum lines to characterise four novel rosette-mediating PfEMP1 variants via var gene expression profiling, and tested the ability of recombinant protein domains from these variants to bind to uninfected erythrocytes. I then examined the relationship of the var gene sequences encoding these new variants to those of known rosetting variants. Of the four newly characterised rosetting variants, three were not characteristic of rosetting variants. One was from a PfEMP1 subset which would be predicted to bind to endothelial protein C receptor, and two had a previously uncharacterised binding phenotype. This surprising discovery suggests that multiple different subsets of PfEMP1 can mediate rosetting, and some potentially have dual binding phenotypes strongly linked to severe malaria. Secondly, I investigated the existence of naturally acquired, broadly strain-transcending antibodies to rosetting parasitised erythrocytes in individuals living in malaria endemic countries. To do this, I used a method for the elution of intact IgG from human plasma from the surface of parasitised erythrocytes and transfer to heterologous parasite lines. I found that individuals do raise strain-transcending antibodies to rosetting parasite lines, suggesting the presence of conserved epitopes. Importantly, this strain-transcending recognition was seen with allopatric plasma-parasite pairs, implying that the conserved epitopes are present in geographically diverse regions. Lastly, I set out to determine whether rosetting clinical P. falciparum isolates from Uganda are recognised by polyclonal IgG raised in rabbits to rosetting PfEMP1 variants expressed by laboratory lines derived from Southeast Asia, Central America and Kenya. To do this, I undertook a study to collect P. falciparum isolates from children with malaria attending Mbale District Hospital in eastern Uganda, and then tested these isolates for recognition with the PfEMP1 antibodies by flow cytometry. I found that some of the IgG do cross-react with certain clinical isolates, suggesting the presence of conserved epitopes, but these were not universally present in all rosetting isolates. In summary I have found that, whilst rosette-mediating PfEMP1 variants may be diverse, individuals do raise strain-transcending antibodies to rosetting PEs, and conserved epitopes can be detected on PfEMP1 variants displayed by rosetting clinical isolates. These findings suggest that several distinct PfEMP1 subsets can mediate rosetting, and that rosetting PfEMP1 variant types linked to severe malaria merit further investigation as vaccine candidates.

For vector-borne parasites such as malaria, how within- and between-host processes interact to shape transmission is poorly understood. In the host, malaria parasites replicate asexually but for transmission via mosquitoes to occur, specialized sexual stages (gametocytes) must be produced. Once inside the mosquito vector, gametocytes immediately differentiate into male and female gametes, and motile male gametes must swim through the hostile environment of the bloodmeal to find and fertilise female gametes. Despite the central role that gametocytes play in disease transmission, explanations of why parasites adjust gametocyte production in response to in-host factors remain controversial. Furthermore, surprisingly little is known about the mating behaviour of malaria parasites once inside the mosquito. Developing drugs and/or vaccines that prevent transmission by disrupting sexual stages are major goals of biomedicine, but understanding variation in gametocyte investment and male gamete behaviour is key to the success of any intervention. First, I propose that the evolutionary theory developed to explain variation in reproductive effort in multicellular organisms provides a framework to understand gametocyte investment strategies in malaria parasites. I then demonstrate that parasites appear to change their reproductive strategies in response to environmental cues and in a manner consistent with our predictions. Next, I show how digital holographic microscopy can be used to characterise the morphology and motility of male gametes. I then provide evidence for non-random movement of male gametes and that gamete interactions with red blood cells appear to hinder mating success in a bloodmeal. Finally, I discuss the variation in gametocyte differentiation and fertilisation success when exposed to a number of factors implicated in gametocyte activation. The data presented here provides important information on the basic biology of malaria parasite reproductive stages and demonstrates considerable variation in parasite traits and behaviours in response environmental changes; both in the host and in the mosquito vector.

Malaria, one of the most deadly infectious diseases in the world, is caused by a protozoan parasite, Plasmodium. The malaria parasite has a very unique way of disposing of toxic free heme. Heme detoxification is achieved through heme polymerization, generating hemozoin (or malaria pigment). In vivo heme polymerization has been demonstrated in the presence of Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a 30 kDa protein composed of 35% histidines. In addition, chloroquine, a 4-aminoquinoline antimalarial drug, has been shown to inhibit PfHRP2-mediated heme polymerization. Investigation of PfHRP2 and its roles in heme detoxification has demonstrated PfHRP2 to be capable of binding 50 hemes per polypeptide of PfHRP2; furthermore, spectrosopic studies have revealed that 95% of the bound hemes are six-coordinate, low-spin, and bis-histidyl ligated. We propose that heme binding has the immediate effect of lowering the free heme concentration within the parasite thereby protecting the parasite against free heme-mediated toxicity; moreover, heme binding is the first step to PfRP2-mediated heme polymerization. Probing the mechanism of chloroquine inhibition of PfHRP2-mediated heme polymerization demonstrated that (i) chloroquine binds to the PfHRP2-heme complex only in the presence of heme, (ii) chloroquine does neither displace the heme bound to PfHRP2 nor inhibit heme binding to PfHRP2. Chloroquine binding brings about spectroscopic changes to the PfHRP2-heme complex, suggesting a close interaction between PfHRP2 and chloroquine. Based on our findings, we have proposed new models for the mechanism of action of chloroquine. As heme detoxification is both required for parasite survival and uniquely distinct from that of the human host, it makes for an attractive drug target. We have developed a quick and efficient microplate-based assay to screen for compounds that inhibit PfHRP2-mediated heme detoxification (by inhibiting heme binding to PfHRP2). Using this novel microplate heme-binding inhibition assay, we screened a library of potential antimalarial compounds. Our results reveal a good correlation between heme-binding inhibition and parasite growth inhibition. Hence, we have not only established the effectiveness of this novel screening method but have identified compounds with potent antiparasitic activity.