Katharine R. Trenholme

A pilot randomised trial of induced blood-stage Plasmodium falciparum infections in healthy volunteers for testing efficacy of new antimalarial drugs.

Background Critical to the development of new drugs for treatment of malaria is the capacity to safely evaluate their activity in human subjects. The approach that has been most commonly used is testing in subjects with natural malaria infection, a methodology that may expose symptomatic subjects to the risk of ineffective treatment. Here we describe the development and pilot testing of a system to undertake experimental infection using blood stage Plasmodium falciparum parasites (BSP). The objectives of the study were to assess the feasibility and safety of induced BSP infection as a method for assessment of efficacy of new drug candidates for the treatment of P. falciparum infection. Methods and Findings A prospective, unblinded, Phase IIa trial was undertaken in 19 healthy, malaria-naïve, male adult volunteers who were infected with BSP and followed with careful clinical and laboratory observation, including a sensitive, quantitative malaria PCR assay. Volunteers were randomly allocated to treatment with either of two licensed antimalarial drug combinations, artemether–lumefantrine (A/L) or atovaquone-proguanil (A/P). In the first cohort (nu200a=u200a6) where volunteers received ∼360 BSP, none reached the target parasitemia of 1,000 before the day designated for antimalarial treatment (day 6). In the second and third cohorts, 13 volunteers received 1,800 BSP, with all reaching the target parasitemia before receiving treatment (A/L, nu200a=u200a6; A/P, nu200a=u200a7) The study demonstrated safety in the 19 volunteers tested, and a significant difference in the clearance kinetics of parasitemia between the drugs in the 13 evaluable subjects, with mean parasite reduction ratios of 759 for A/L and 17 for A/P (95% CI 120–4786 and 7–40 respectively; p<0.01). Conclusions This system offers a flexible and safe approach to testing the in vivo activity of novel antimalarials. Trial Registration: ClinicalTrials.gov NCT01055002

Effect of Antimalarial Drugs on Plasmodium falciparum Gametocytes

Gametocytes are the sexual stage of the malaria parasite and are essential for transmission to the mosquito. Antimalarial drugs have been reported to affect gametocyte production in vivo, which leads to a potential increase in transmission. We used transgenic Plasmodium falciparum parasites expressing a green fluorescent protein tag in a fluorescence-activated cell sorting-based assay to measure the effect of 8 antimalarial drugs on gametocyte production in vitro. Exposure to antimalarial drugs resulted in an increase in the number of gametocytes in test cultures. Although a dose-dependent reduction in late-stage gametocyte viability was observed, none of the drugs tested statistically significantly reduced gametocyte numbers.

Targeting of the Ring Exported Protein 1 to the Maurer’s Clefts is Mediated by a Two-Phase Process

Early development of Plasmodium falciparum within the erythrocyte is characterized by the large‐scale export of proteins to the host cell. In many cases, export is mediated by a short sequence called the Plasmodium export element (PEXEL) or vacuolar transport signal; however, a number of previously characterized exported proteins do not contain such an element. In this study, we investigated the mechanisms of export of the PEXEL‐negative ring exported protein 1 (REX1). This protein localizes to the Maurer’s clefts, parasite‐induced structures in the host‐cell cytosol. Transgenic parasites expressing green fluorescent protein–REX1 chimeras revealed that the single hydrophobic stretch plus an additional 10 amino acids mediate the export of REX1. Biochemical characterization of these chimeras indicated that REX1 was exported as a soluble protein. Inclusion of a sequence containing a predicted coiled‐coil motif led to the correct localization of REX1 at the Maurer’s clefts, suggesting that association with the clefts occurs at the final stage of protein export only. These results indicate that PEXEL‐negative exported proteins can be exported in a soluble state and that sequences without any apparent resemblance to a PEXEL motif can mediate export across the parasitophorous vacuole membrane.

Targeted mutagenesis of the ring-exported protein-1 of Plasmodium falciparum disrupts the architecture of Maurer's cleft organelles

Mature red blood cells have no internal trafficking machinery, so the intraerythrocytic malaria parasite, Plasmodium falciparum, establishes its own transport system to export virulence factors to the red blood cell surface. Maurers clefts are parasite‐derived membranous structures that form an important component of this exported secretory system. A protein with sequence similarity to a Golgi tethering protein, referred to as ring‐exported protein‐1 (REX1), is associated with Maurers clefts. A REX1–GFP chimera is trafficked to the Maurers clefts and preferentially associates with the edges of these structures, as well as with vesicle‐like structures and with stalk‐like extensions that are involved in tethering the Maurers clefts to other membranes. We have generated transfected P. falciparum expressing REX1 truncations or deletion. Electron microscopy reveals that the Maurers clefts of REX1 truncation mutants have stacked cisternae, while the 3D7 parent line has unstacked Maurers clefts. D10 parasites, which have lost the right end of chromosome 9, including the rex1 gene, also display Maurers clefts with stacked cisternae. Expression of full‐length REX1–GFP in D10 parasites restores the 3D7‐type unstacked Maurers cleft phenotype. These studies reveal the importance of the REX1 protein in determining the ultrastructure of the Maurers cleft system.

A high-throughput assay for the identification of drugs against late-stage Plasmodium falciparum gametocytes

Recent success in the global reduction campaign against malaria has resulted in the possibility that it may be feasible to drastically reduce or even eradicate malaria even without the introduction of a vaccine. However, while there has been significant effort to design the next generation of antimalarial drugs, one area that is underrepresented in the current antimalarial pharmacopeia is that of transmission blocking drugs directed at late-stage gametocytes. Here we describe the development of a robust and simple assay that is amenable to a high throughput format for the discovery of new antigametocyte drugs.

Anti-malarial drugs: how effective are they against Plasmodium falciparum gametocytes?

BackgroundRecent renewed emphasis on the eradication of malaria has highlighted the need for more tools with which to achieve this ambitious goal. One high priority area is the need to determine the gametocytocidal activity of both currently used anti-malarial drugs and those in the development pipeline. However, testing the activity of compounds against Plasmodium falciparum gametocytes is technically challenging both in vivo and in vitro.MethodsHere the use of a simple robust assay to screen a panel of currently used and experimental anti-malarial drugs against mature P. falciparum gametocytes is described.ResultsEight of 44 compounds tested reduced gametocyte viability by at least 50% and three showed IC50 values in nM range.ConclusionsThere is a need to identify new compounds with activity against late stage gametocytes and the information provided by this in vitro assay is a valuable first step, which can guide future clinical studies.

Antimalarial Asexual Stage-Specific and Gametocytocidal Activities of HIV Protease Inhibitors

ABSTRACT The stage-specific antimalarial activities of a panel of antiretroviral protease inhibitors (PIs), including two nonpeptidic PIs (tipranavir and darunavir), were tested in vitro against Plasmodium falciparum. While darunavir demonstrated limited antimalarial activity (effective concentration [EC50], >50 μM), tipranavir was active at clinically relevant concentrations (EC50, 12 to 21 μM). Saquinavir, lopinavir, and tipranavir preferentially inhibited the growth of mature asexual-stage parasites (24 h postinvasion). While all of the PIs tested inhibited gametocytogenesis, tipranavir was the only one to exhibit gametocytocidal activity.

CLAG 9 is located in the rhoptries of Plasmodium falciparum

Abstract Clagxa09, a gene located on chromosomexa09 of Plasmodium falciparum has previously been associated with the cytoadherence of parasitized erythrocytes to CD36. This gene is part of a multi-gene family found in all Plasmodium species studied to date. Using data from the Malaria Genome Sequencing Project, peptides specific for clagxa09 were designed, synthesized and used to immunize mice. This antisera was used in Western blotting and immunofluorescence experiments to determine the cellular localization of CLAGxa09 in the parasitized erythrocyte. Co-localization using immunofluorescence of wildtype and knockout parasites unequivocally shows that CLAGxa09 is localized to the rhoptry organelles of P. falciparum.

Enhanced Gametocyte Formation in Erythrocyte Progenitor Cells: A Site-Specific Adaptation by Plasmodium falciparum

Gametocytogenesis by Plasmodium falciparum is essential for transmission of the parasite from human to mosquito, yet developing gametocytes lack expression of surface proteins required for cytoadherence. Therefore, elimination from the circulation should occur unless they are sequestered in regions of low blood flow such as the extracellular spaces of the bone marrow. Our data indicate that gametocytogenesis is enhanced in the presence of erythroid progenitors found within the bone marrow. Furthermore, atomic force microscopy indicates that developing gametocytes undergo remarkable shifts in their erythrocyte membrane elasticity, which may allow them to be retained within the bone marrow until maturation.