The malaria pre-erythrocytic stages have been identified as an ideal therapeutic target, but complex in vitro models for Plasmodium vivax and Plasmodium falciparum lack the efficiency needed for rapid screening and evaluation of new vaccines and drugs, especially targeting the P. vivax hypnozoite. To address this challenge, we employed a multi-parameter approach using “omics’” to identify pre-erythrocytic targets and biomarkers, guide phenotypic therapeutic screening, and study parasite functionality with innovative bioassays using highcontent screening. Herein, we discuss three novel bioassays formatted in 384-well plate systems with utilization of commercially-available materials and application of high-content imaging for rapid bio-image analysis. To refine functional assessment of pre-erythrocytic targets in early infection phases, we developed a real-time, ‘live’ sporozoite motility assay and a live sporozoite hepatocyte cell traversal assay to examine chemotherapeutic and immunoprophylactic interventions in biologically relevant environments. Furthermore, our 384-well primary hepatocyte culture system and methodology maintains stable hepatocyte physiology of cryopreserved primary human hepatocytes in addition to primary non-human primate hepatocytes for greater than 30 days, thus ideal for robust liver parasite development following infection with P. vivax, P. falciparum or P. cynomolgi sporozoites. We report antimalarial drug and vaccine studies performed in all bioassays with identification of novel anti-LS inhibition mechanisms. Additionally, this research discusses the discovery of potential sporozoite and liver stage targets identified through transcriptomic profiling of freshly isolated P. vivax and P. cynomolgi sporozoites using a candid approach of recapitulating the pivotal transition period from mosquito to human through microenvironment reconstruction and exposure to biological stimuli. We further characterize sporozoite invasive phenotypes through the application of the bioassays. Together, these novel functional assays enable us to rapidly evaluate potential preerythrocytic therapeutic candidates and analyze complex Plasmodium sporozoite phenotypes.

Malaria is a severe public health concern in certain regions, causing 445,000 deaths and over 200 million cases in 2016 (Ashley et al., 2018). The vast majority of these cases and deaths are located in warm climates where the Anopheles mosquito is present, especially in sub-Saharan Africa and southeast Asia. Current efforts aim to prevent the disease through vaccination, which has proven to be challenging. Plasmodium falciparum, the pathogen responsible for malaria, is transmitted between humans via the female vector Anopheles mosquito. This parasite has a complex life cycle that is not fully understood, making it difficult to treat the infection and even more difficult to inoculate a population through vaccination. P. falciparum is also capable of polymorphism, changing structure once a host antibody has identified a pathogenic antigen. For this reason, it is very technically challenging to develop a vaccine that is able to confer a high enough immune response through sufficient host antibody production. This thesis will begin with a review on the prevalence and severity of malaria and an overview of the principles of immunology and vaccine development. We will then discuss the parasitic life cycle and how it results in the pathogenesis of the disease. Current antimalarial treatments and resistance to those treatments will be analyzed. This thesis will conclude with an in-depth analysis of the current prophylactic vaccines against P. falciparum, focusing on their mechanism, efficacy, and probability of success.

The development of a malaria vaccine is necessary for disease eradication. Successful vaccine candidates to date have targeted the asymptomatic, pre-erythrocytic stage of the disease, however even the most efficacious vaccines are only partially protective. Research undertaken in our laboratory has demonstrated that one such regimen, using an 8 week prime-boost viral vector approach of ChAd63 ME-TRAP and MVA ME-TRAP, induces sterile efficacy in 21% of vaccinees, with a key role identified for TRAP-specific CD8⁺ T cells. The work described in this thesis explores the most immunogenic regimen by which to administer these two pre-erythrocytic malaria vaccines. A shortening of the prime-boost interval from 8 to 4 weeks, and the addition of an extra ChAd63 ME-TRAP priming vaccination, both demonstrated improved T cell immunogenicity over the standard 8 week regimen. Further to this, novel assays were developed to aid the evaluation of vaccine-induced immune responses. Adaptations of the existing methodology for ELISpot analysis and to whole blood flow cytometry techniques, enabled more detailed analyses of paediatric vaccine-induced T cell responses in The Gambia. This work also permitted the comparison of vaccine immunogenicity in this paediatric population, with malaria-naïve and malaria-exposed adult vaccinees. The results suggest that vaccine-induced T cell responses in infants of 8 weeks and older are comparable to that of adults. A second approach involved the development of a novel functional assay. This assay quantitatively measured the in vitro inhibition of intrahepatic Plasmodium parasite development using T cells from ChAd63.MVA ME-TRAP vaccinated volunteers. The assay demonstrated the ability of CD8⁺ T cells to inhibit parasite development in a TRAP-specific manner, and provides a platform with which to further explore pre-erythrocytic immune responses.

Plasmodien spp. sind obligat intrazellulär lebende Parasiten, welche einen evolutionär konservierten aktinabhängigen molekularen Motor für die Fortbewegung und den Wirtszellein- und -austritt nutzen. In dieser Arbeit werden die Aktinregulatoren Adenylyl- Zyklase- assoziierte Protein (C-CAP), Profilin sowie die Aktin depolymerizierenden Faktoren 1 und 2 (ADF1, ADF2) in Plasmodium berghei charakterisiert. Die Geninaktivierung von C-CAP besitzt keinen Einfluss auf die Entwicklung von pathogenen Blutstadien. C-cap(-) Ookineten bewegen sich jedoch deutlich langsamer, sind aber in der Lage den invertebraten Wirt zu infizieren. Defekte treten während der extrazellulären Replikationsphase im Mosquito auf und führen zu Abbruch des Lebenszykluses. Die erfolgreiche Komplementierung der Defekte mit dem orthologen Gen aus Cryptosporidium parvum CpC-CAP bestätigt die funktionale Redundanz zwischen beiden Proteinen. Profilin, als ein weiteres G-Aktin bindendes Protein, ist hingegen nicht in der Lage die Defekte des c-cap(-) Parasiten auszugleichen. Mittels transgener Parasiten welche ein C-CAPmCherry Fusionsprotein exprimieren, wird das C-CAP Protein im Zytoplasma lokalisiert. Erstmals wird mit dieser Arbeit ein G-Aktin bindendes Protein, C-CAP beschrieben, welches eine essentielle Funktion während der Oozystenreifung in Plasmodium berghei besitzt. Die Transkription der Aktinregulatoren Profilin, ADF1 und ADF2 wird in Sporozoiten drastisch herunterreguliert und Profilin kann als Protein nicht mehr nachgewiesen werden. Um die Funktion von C-CAP und Profilin zu überprüfen, wurden beide Proteine spezifisch in Sporozoiten überexprimiert. Diese Parasiten sind nicht in der Lage die Speicheldrüsen des Wirtes zu besiedeln, was zum Abbruch des Lebenszykluses führt. Anhand dieser Ergebnisse entwickele ich ein „minimalistisches“ Model zur Beschreibung der Aktinregulation in Sporozoiten in welchem das ADF1 als regulatorisches Protein im Mittelpunkt steht. / Plasmodium spp. are obligate intracellular parasites, which employ an conserved actin-dependent molecular motor machinery that facilitates their motility, host cell invasion and egress. In this work I report implications of the actin-regulators adenylyl cyclase-associated protein (C-CAP), profilin and actin depolymerization factor 1 and 2 (ADF1, ADF2) in distinct and previously unanticipated cellular processes during the life cycle of in the rodent malarial parasite Plasmodium berghei. Fluorescent tagging of the endogenous C-CAP genetic locus with mCherry revealed cytosolic distribution of the protein. Gene deletion demonstrates that the G-actin binding protein C-CAP is entirely dispensable for the pathogenic blood stages. Ookinetes show reduced motility, but are competent infecting the mosquito host. Defects emerging in the extracellular replication phase, leading to attenuation of oocyst maturation. Successful trans-species complementation with the C. parvum C-CAP ortholog, rescues the c-cap(-) phenotype and proves functional redundancy. The actin regulator profilin fails to rescue the defects of c-cap(-) parasites, despite sharing its actin sequestering activity with C-CAP. Taken together, C-CAP is the first G-actin sequestering protein of Plasmodium species that is not required for motility but performs essential functions during oocyst maturation. Characterization of the actin regulators profilin, ADF1 and ADF2 revealed dramatic transcriptional down-regulation and the absence of the profilin protein in sporozoites. To test whether G-actin binding proteins interfere with sporozoite functions, I ectopically overexpressed of profilin and C-CAP stage-specifically in sporozoites. This conducted to abolishment of salivary gland invasion and lifecycle arrest. Based on these unexpected findings and the available literature data, I developed a “minimalistic model” for actin regulation in sporozoites that predicts ADF1 as the main actin-turnover regulating factor.