Apicomplexan parasites are widespread protozoan parasites of animals, which include major pathogens of humans, domestic animals and livestock. Many existing measures against these parasites remain insufficient for disease control, and new strategies for prophylaxis, treatment and control of transmission are urgently needed. Sporogony is defined as the production of sporozoites by repeated divisions of a zygote. It is an essential part of the life cycles of many apicomplexan parasites including Plasmodium, the causative agent of malaria, where sporogony takes place in the mosquito within extracellular encysted forms named oocysts. In Plasmodium, sporogony is critically dependent on a unique organelle named the crystalloid, which is exclusively found in the ookinete (a motile form of the zygote) and young oocyst stages. Many of the crystalloid proteins thus far identified in Plasmodium are conserved in and unique to the Apicomplexa, supporting the hypothesis that the molecular processes underlying sporogony are at least partly conserved across the phylum. In this proposal, we will use an integrated cross-disciplinary approach to study the crystalloids of Plasmodium berghei aimed at increasing our fundamental knowledge of the essential cellular and molecular processes underlying sporogony. This will be achieved via the following specific objectives: Objective 1: Study crystalloid loss in the oocyst by microscopy. Using various parasites lines expressing different crystalloid proteins tagged with fluorescent protein as markers of the crystalloid organelle, combined with superresolution confocal microscopy, the destination of the crystalloid and its protein cargo will be investigated in live oocysts during sporogony. This will help elucidate the processes involved in loss of the organelle at the subcellular level, and our understanding of crystalloid function. Objective 2: Refine the crystalloid protein interactome. Three transgenic parasite lines expressing different crystalloid proteins (LAP3, NTH and TPM2) each fused to a twin fluorescent protein/affinity tag (GFP/Strep) will be generated and used to harvest protein complexes from ookinetes by Strep-based affinity purification, followed by quantitative mass spectrometry-based protein identification. These analyses will produce a high confidence crystalloid protein interaction network (interactome). Objective 3: Determine the crystalloid proteome. Parasite lines expressing the GFP/Strep-tagged crystalloid membrane proteins NTH and TPM2 will be used to purify intact organelles from mechanically lysed ookinetes by Strep-based affinity purification, followed by quantitative mass spectrometry-based protein identification. This analysis will help determine the entire crystalloid protein repertoire (proteome) irrespective of protein interactions. Objective 4: Establish the crystalloid metabolome. Whole ookinetes and Strep-based affinity-purified crystalloid organelles from parasites expressing GFP/Strep-tagged NTH (generating NADPH), and a parasite line expressing an enzymatically inactive NTH version, will be subject to mass spectrometry-based global metabolite analysis. These analyses will allow differences in the metabolite repertoire (metabolome) of NTH-deficient parasites to be identified, which will help inform which NADPH-dependent biosynthetic processes are taking place in the organelle. Objective 5. Validate newly identified crystalloid proteins. Select proteins identified from the above objectives will be validated by GFP tagging and gene knockout studies in transgenic parasite lines to make sure they are genuinely linked with crystalloid function and sporogony. The collective results will provide new basic knowledge of the essential functions of the crystalloid in sporogony, and help to identify new molecules and molecular pathways for rational intervention strategies against development and transmission of malaria and related apicomplexan parasites

Fever is the leading reason to seek healthcare globally, with over 1 billion cases of febrile illness occurring in children annually. The vast majority of infections are uncomplicated and self-limited and can be treated conservatively. A few (<1%) are life-threatening but are often challenging to identify early in the course of illness. Our project challenges the current status quo and inefficiencies of triaging practices. We propose to develop and validate a rapid triaging tool to determine, objectively, quantitatively and with high precision those patients at risk of dying, so as to prioritize their care. The breakthrough solution lies in the ENDOTHELIUM, a newly recognized in vivo biosensor, which plays a critical role in our defense against pathogens. Endothelial cell activation and subsequent loss of integrity is a common pathway of injury in several life-threatening infections, including sepsis, malaria, or even COVID-19. Measuring specific mediators of this pathway (sTREM-1, Ang2, etc.) at first clinical presentation can reliably identify individuals at risk of dying, irrespective of the disease causing the fever, and more robustly than previously known predictors such as clinical algorithms or ""classic"" biomarkers. Importantly, these pathways are also ""druggable"", thus allowing the testing or re-purposing of specific interventions to improve outcome. We will design and produce a RTT (glucometer-like) that quantitatively measures the two markers with best predictive performance (sTREM-1 and Ang2). We will then incorporate it into 2 clinical trials (to be done in Mozambique, Gabon and Ethiopia) to 1) verify the improved performance of the proposed PoC RTT in risk-stratifying and predicting mortality among paediatric patients when compared to standard of care, and 2) test whether a specific intervention (nutritional supplementation of L-Citrulline), guided by biomarker results, improves (vs. placebo) long-term outcomes and survival after hospital discharge.

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A healthily maintained livestock is essential for the economy and prosperity of the UK. Additionally some infected livestock are the source of human diseases, particularly through foodborne infections. Historically, vaccines have been the most successful and effective intervention to reduce the burden of infectious diseases in humans. By contrast, the application of vaccines in veterinary medicine is rudimentary, mainly due to the economic necessity for reduced costs to vaccinate animals and because our knowledge of the pathogens that cause animal diseases lags behind that of human counterparts. A defining characteristic of a successful vaccine is the ability to evoke long-lasting protective immunity with minimal side effects. Many of the most successful human vaccines are glycoconjugates, a combination of a protein coupled to a glycan, which induces both a T-cell dependent and independent immune response generating a protective and lasting immunity. Examples of currently licensed human glycoconjugate vaccines include those against Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae, in which glycans (lipopolysaccharides or capsular polysaccharides) are chemically coupled to immunogenic carrier proteins. However, the production of these vaccines requires multistep procedures that are often complex and expensive, and can exhibit batch-to-batch variation. We recently developed Protein Glycan Coupling Technology (PGCT) that can overcome the complex procedures required for chemically synthesising glycoconjugate vaccines by expressing the vaccine in an Escherichia coli cell in a single-step procedure. The advantages of applying PGCT to veterinary vaccines are (i) glycoconjugate vaccines can be produced at low cost, (ii) the flexibility of coupling "any glycan" with "any protein" facilitates the production of vaccine combinations providing the opportunity to evaluate a greater variety of vaccine candidates, and (iii) combination vaccines against more than one disease can be produced, further reducing cost and obviating the need to administer multiple vaccines (or antibiotics). In this study we will use PGCT to produce inexpensive triple combination poultry vaccines to reduce infection from E. coli, Salmonella, Campylobacter jejuni/coli and C. perfringens. This will not only protect poultry flocks from severe disease but would also protect the human population from the most common foodborne infections including those caused by Salmonella and Campylobacter. In addition we will construct and evaluate a dual Coxiella/C. perfringens vaccine to protect cattle, sheep and goats against severe disease. This vaccine would also prevent the spread of Q-fever to humans, which is caused by the highly infectious Coxiella burnetii pathogen. The principles developed in this proposal could subsequently be widely applied to produce inexpensive efficacious vaccines against most animal species and promise to break new ground in veterinary vaccine production.