A Mosquito Pick-and-Place System for PfSPZ-based Malaria Vaccine Production

The treatment of malaria is a global health challenge that stands to benefit from the widespread introduction of a vaccine for the disease. A method has been developed to create a live organism vaccine using the sporozoites (SPZ) of the parasite Plasmodium falciparum (Pf), which are concentrated in the salivary glands of infected mosquitoes. Current manual dissection methods to obtain these PfSPZ are not optimally efficient for large-scale vaccine production. We propose an improved dissection procedure and a mechanical fixture that increases the rate of mosquito dissection and helps to deskill this stage of the production process. We further demonstrate the automation of a key step in this production process, the picking and placing of mosquitoes from a staging apparatus into a dissection assembly. This unit test of a robotic mosquito pick-and-place system is performed using a custom-designed micro-gripper attached to a four degree of freedom (4-DOF) robot under the guidance of a computer vision system. Mosquitoes are autonomously grasped and pulled to a pair of notched dissection blades to remove the head of the mosquito, allowing access to the salivary glands. Placement into these blades is adapted based on output from computer vision to accommodate for the unique anatomy and orientation of each grasped mosquito. In this pilot test of the system on 50 mosquitoes, we demonstrate a 100% grasping accuracy and a 90% accuracy in placing the mosquito with its neck within the blade notches such that the head can be removed. This is a promising result for this difficult and non-standard pick-and-place task.Comment: 12 pages, 11 figures, Manuscript submitted for Special Issue of IEEE CASE 2019 for IEEE T-AS

Ultrasensitive detection of toxocara canis excretory-secretory antigens by a nanobody electrochemical magnetosensor assay.

peer reviewedHuman Toxocariasis (HT) is a zoonotic disease caused by the migration of the larval stage of the roundworm Toxocara canis in the human host. Despite of being the most cosmopolitan helminthiasis worldwide, its diagnosis is elusive. Currently, the detection of specific immunoglobulins IgG against the Toxocara Excretory-Secretory Antigens (TES), combined with clinical and epidemiological criteria is the only strategy to diagnose HT. Cross-reactivity with other parasites and the inability to distinguish between past and active infections are the main limitations of this approach. Here, we present a sensitive and specific novel strategy to detect and quantify TES, aiming to identify active cases of HT. High specificity is achieved by making use of nanobodies (Nbs), recombinant single variable domain antibodies obtained from camelids, that due to their small molecular size (15kDa) can recognize hidden epitopes not accessible to conventional antibodies. High sensitivity is attained by the design of an electrochemical magnetosensor with an amperometric readout with all components of the assay mixed in one single step. Through this strategy, 10-fold higher sensitivity than a conventional sandwich ELISA was achieved. The assay reached a limit of detection of 2 and15 pg/ml in PBST20 0.05% or serum, spiked with TES, respectively. These limits of detection are sufficient to detect clinically relevant toxocaral infections. Furthermore, our nanobodies showed no cross-reactivity with antigens from Ascaris lumbricoides or Ascaris suum. This is to our knowledge, the most sensitive method to detect and quantify TES so far, and has great potential to significantly improve diagnosis of HT. Moreover, the characteristics of our electrochemical assay are promising for the development of point of care diagnostic systems using nanobodies as a versatile and innovative alternative to antibodies. The next step will be the validation of the assay in clinical and epidemiological contexts

Functional characterisation of the FIKK kinase family of Plasmodium falciparum

Key to P. falciparum virulence is its capacity to remodel the host erythrocyte. Infected erythrocytes become rigid and cytoadhere to the vascular endothelium leading to the disease symptoms and preventing their filtration by the spleen. Unlike other human- infecting Plasmodium species, P. falciparum exports a family of 18 FIKK kinases into the host cell. Here, a conditional knockout strategy based on the DiCre/LoxPint technology was used to study 4 FIKK kinases (FIKK4.1, FIKK7.1, FIKK10.1 and FIKK11) and identify their potential targets by quantitative phosphoproteome analysis. The deletion of FIKK4.1 led to a significant reduction in the phosphorylation of host cytoskeletal proteins and parasite proteins involved in remodelling. The characterisation of FIKK4.1 KO parasites confirmed its role both in the rigidification of the infected erythrocytes and in the trafficking of the adherence-mediating virulence factor PfEMP1 to the host cell surface. Additionally, recombinant versions of several FIKK kinase domains were used to identify potential pan-FIKK inhibitors. When tested in vitro, these compounds showed activity on both P. falciparum and P. knowlesi, raising concerns regarding their specificity. A whole genome sequencing on drug-resistant parasites did not allow to identify additional targets. Moreover, it was shown that the compounds were not active on the FIKK kinases in culture due to the high intra-erythrocytic ATP concentration. Using the recombinant FIKK kinase domains it was also shown that FIKK kinases possess distinct substrate specificity. Whereas most of them conserved the ancestral basophilicity, some evolved to phosphorylate preferentially acidic motifs. Strikingly, FIKK13 was found to be a tyrosine kinase, a feature supposed to be absent in Plasmodium. Finally, by studying the FIKK kinases from another Plasmodium species closely related to P. falciparum, it was shown that FIKK kinases substrate specificity is conserved across species of the Laverania clade.Open Acces

Human Challenge Studies in Endemic Settings

This open access book provides an extensive review of ethical and regulatory issues related to human infection challenge studies, with a particular focus on the expansion of this type of research into endemic settings and/or low- and middle-income countries (LMICs). Human challenge studies (HCS) involve the intentional infection of research participants, and this type of research is rapidly increasing in frequency worldwide. HCS are widely considered to be an especially promising approach to vaccine development, including for pathogens endemic to LMICs. However, challenge studies are sometimes controversial and raise complex ethical issues, some of which are especially salient in endemic and/or LMIC settings. Informed by qualitative interviews with experts in infectious diseases and bioethics, this book highlights areas of ethical consensus and controversy concerning this kind of research. As the first volume to focus on ethical issues associated with human challenge studies, it sets the agenda for further work in this important area of global health research; contributes to current debates in research ethics; and aims to inform regulatory policy and research practice. Insofar as it focuses on HCS in (endemic) settings where diseases are present and/or widespread, much of the analysis provided here is directly relevant to HCS involving pandemic diseases including COVID19

Human Challenge Studies in Endemic Settings

This open access book provides an extensive review of ethical and regulatory issues related to human infection challenge studies, with a particular focus on the expansion of this type of research into endemic settings and/or low- and middle-income countries (LMICs). Human challenge studies (HCS) involve the intentional infection of research participants, and this type of research is rapidly increasing in frequency worldwide. HCS are widely considered to be an especially promising approach to vaccine development, including for pathogens endemic to LMICs. However, challenge studies are sometimes controversial and raise complex ethical issues, some of which are especially salient in endemic and/or LMIC settings. Informed by qualitative interviews with experts in infectious diseases and bioethics, this book highlights areas of ethical consensus and controversy concerning this kind of research. As the first volume to focus on ethical issues associated with human challenge studies, it sets the agenda for further work in this important area of global health research; contributes to current debates in research ethics; and aims to inform regulatory policy and research practice. Insofar as it focuses on HCS in (endemic) settings where diseases are present and/or widespread, much of the analysis provided here is directly relevant to HCS involving pandemic diseases including COVID19

A novel role for EXP2 in invasion by Plasmodium sporozoites

Plasmodium parasites, the causative agents of malaria, possess a translocon that exports parasite proteins into the infected erythrocyte, beyond the confines of the parasite. The parasite pore-forming protein Exported Protein 2 (EXP2) is a component of this translocon and the pore through which proteins are exported. Although EXP2, and other translocon components, are also expressed during the mosquito and liver stages of infection, their function remains unexplored. To study the function of EXP2 during the liver stage of infection, we used a genetically engineered Plasmodium berghei parasite line, generated by our collaborators. In this parasite line, the EXP2 gene is silenced during the mosquito stage, as the 3’ untranslated region of the EXP2 gene is excised, generating EXP2 knock-out sporozoites. The sporozoite is the mosquito transmissible form of the parasite, that when deposited in the mammalian host, infects the liver, giving rise to the pre-erythrocytic stage of Plasmodium infection. Using this transgenic parasite line, we observed that invasion of the hepatocyte by EXP2 conditional knock-out parasites is impaired, both in C57Bl/6J mice and in the hepatoma cell line HepG2, suggesting that invasion is dependent on EXP2. Using a combination of fluorescence and electron microscopy techniques, we show that EXP2 is present in the hepatocyte-infectious sporozoite in vesicles, in the apical end of the parasite. These vesicles are important for the invasion process, as they are discharged by the sporozoite and contain proteins important for hepatocyte adhesion and entry in the host cell. Stimulating sporozoites with conditions that mimic the host cell milieu, we observed that EXP2 relocates to the surface of the mosquito-transmitted sporozoite and that it is secreted by the sporozoite. We also observed that invasion defect of the EXP2 conditional knock-out parasites can be rescued by the exogenous administration of recombinant EXP2 or recombinant α-hemolysin, another pore-forming protein, suggesting that the function of EXP2 is through its pore-forming ability on the membrane of the hepatocyte. Invasion of cells via pores has been documented for a variety of intracellular pathogens, like Trypanossoma cruzi, Listeria monocytogenes and adenovirus. Importantly, the pore does not allow the parasite to cross the membrane, rather it induces changes in the host cell that make it endocytose the wounded region of the membrane. We observed that recombinant EXP2 can recapitulate the major hallmarks of the membrane repair pathway. On one hand, treating cells with recombinant EXP2 leads to influx of calcium to HepG2 cells. Moreover, invasion of Plasmodium berghei sporozoites is dependent of acid Sphingomyelinase and finally, the invasion defect of the EXP2 conditional knock-out sporozoites can be rescued by exogenously adding acid sphingomyelinase. This suggest that indeed EXP2 induces hepatocyte membrane repair, which plays a key role in parasite invasion. Overall, while our data show that Plasmodium parasites uses EXP2 for divergent functions in its life cycle, it also highlights the convergent evolution of different intracellular pathogens, which have developed similar strategies to take advantage of cellular responses to membrane damage, hitchhiking their way into the host cell

Social lives and afterlives of a malaria vaccine trial: partnerships in practice

This thesis focuses on the development of a malaria vaccine as an avenue to explore global health partnerships. In the last twenty years, public-private partnerships have become a prominent organizational form in global health. Hundreds of large transnational collaborations and countless smaller collaborations between the public, private and non-profit sectors have been established. Partnerships have been supported by the large increase of donor funding for research and control of infectious diseases in impoverished countries and many aim to develop or provide vaccines, medicines or interventions. Analysts generally agree that partnerships are saving many lives and revolutionizing drug and vaccine development for infectious diseases. However, while partnership is a notion that connotes equity and mutuality, often global health partnerships operate in contexts that involve vast disparities in power and resources and there is little known about the impacts of partnerships on the places where they operate. This raises the questions: How do global health partnerships operate in practice? What are their impacts in the places where they operate? Addressing these questions, this thesis examines a partnership established to develop the most advanced malaria vaccine, named RTS,S. Based on 17 months of ethnographic research in Tanzania and interviews with representatives of partnering organizations in Belgium and the United States, I trace the development of the RTS,S vaccine from laboratories to its clinical trials across Africa. I explore the social relationships formed between private companies, philanthropic institutions and non-profit organizations in the North, and research institutions and communities in north-eastern Tanzania, where a malaria vaccine clinical trial was conducted. Analyzing the impacts of the malaria vaccine partnership, I focus on community development, construction of infrastructure, the building of human capacity, provision of health care and extraction of data. The focus on partnerships is intended to improve understanding about this ever-increasing social, political and economic formation in global health, and contributes to discussions and debates about how partnerships operate and their role in international development, global health governance and transnational medical research

Investigation of azithromycin analogues and proteasome-like inhibitors as quick-killing antimalarials

Malaria is caused by mosquito-borne parasites of the genus Plasmodium which were responsible for ~435,000 of deaths annually, with >90% caused by the deadliest species, P. falciparum. Over the last two decades, global implementation of vector control and artemisinin combination therapies have resulted in significant reductions in the global burden of malaria. Of current concern is the spread of multi-drug resistant parasites that have severely limited the efficacy of antimalarials, including front-line artemisinins, highlighting the urgent need to identify new antimalarials for use as treatments. The aim of this thesis was to investigate novel antimalarial development avenues and identify new chemotypes that could be used in the near future as treatments. The macrolide antibiotic azithromycin is known to target the malaria parasites remnant plastid organelle (the apicoplast’s) bacterial-like ribosome and causes slow-killing ‘delayed death’, where the parasite dies in the second replication cycle (4 days). Azithromycin has also been shown to inhibit invading merozoites and kill blood stages within the first replication cycle (2 days) via an unidentified mechanism, proposed to be independent of delayed death. Thus, we hypothesised that azithromycin could be redeveloped into an antimalarial with two different mechanisms of action against parasites: delayed death and quick-killing. Over 100 azithromycin analogues that featured a high proportion of different structural profiles were obtained, leading to improved quick-killing activities over azithromycin. Quick-killing was also confirmed to be completely unrelated to delayed death, as blood stage parasites lacking the apicoplast were equally susceptible to quick-killing of azithromycin and analogues. Two different avenues were also confirmed for azithromycin’s antimalarial re-development: delayed death and quick-killing or quick-killing only, which could be modulated depending on the location of added functional groups. Azithromycin and analogues were found to be active across blood stage development, with only short treatments required to kill parasites. The metabolomics signatures of parasites treated with azithromycin and analogues suggested that quick-killing acts multi-factorially, with the parasite’s food vacuole and mitochondria being likely targets. Finally, in vitro activities of two subtypes of tri-peptide proteasome-like inhibitors, vinyl sulfone and aldehydes, were addressed against P. falciparum and the zoonotic malaria parasite P. knowlesi. All compounds exhibited low-nanomolar activities against both Plasmodium spp. and showed excellent selectivity for parasites over human cells, suggesting these inhibitors provide viable chemical scaffolds for optimisation. There was no evidence of increased protein ubiquitination upon treating parasites with these compounds, suggesting they do not target the proteasome. We also investigated whether hypoxia inducible pro-drug proteasome-like inhibitors could be used to reduce host toxicity of antimalarials. However, these pro-drugs could be not activated in in vitro culture conditions and there was limited evidence suggesting this strategy would be applicable in malaria. These studies build on previous findings on the drug-killing efficacy, mechanism of action and possible application of redeveloping azithromycin analogues as new and improved antimalarials. I also identified new proteasome inhibitor-like scaffolds as starting points for further development. This body of work provides thorough biological characterisation of a panel of compounds that could lead to new avenues for antimalarial development.Thesis (Ph.D.) -- University of Adelaide, School of Biololgical Sciences, 202