Comparison and Optimization of Different Methods for the In Vitro Production of Plasmodium falciparum Gametocytes

The generation of sexually committed parasites (gametocytogenesis) is poorly understood in malaria. If the mechanisms regulating this process were elucidated, new opportunities for blocking malaria transmission could be revealed. Here we compare several methods described previously for the in vitro production of Plasmodium falciparum gametocytes. Our approach relies on the combination of several factors that we demonstrated as impacting on or being critical to gametocytogenesis. An improved method has been developed for the in vitro production of P. falciparum gametocytes as the first step toward obtaining adequate numbers of pure gametocytes for in vitro studies, such as, for example, the identification of transmission blocking drugs

Activity of clinically relevant antimalarial drugs on plasmodium falciparum mature gametocytes in an atp bioluminescence >transmission blocking> assay

Background: Current anti-malarial drugs have been selected on the basis of their activity against the symptom-causing asexual blood stage of the parasite. Which of these drugs also target gametocytes, in the sexual stage responsible for disease transmission, remains unknown. Blocking transmission is one of the main strategies in the eradication agenda and requires the identification of new molecules that are active against gametocytes. However, to date, the main limitation for measuring the effect of molecules against mature gametocytes on a large scale is the lack of a standardized and reliable method. Here we provide an efficient method to produce and purify mature gametocytes in vitro. Based on this new procedure, we developed a robust, affordable, and sensitive ATP bioluminescence-based assay. We then assessed the activity of 17 gold-standard anti-malarial drugs on Plasmodium late stage gametocytes. Methods and Findings: Difficulties in producing large amounts of gametocytes have limited progress in the development of malaria transmission blocking assays. We improved the method established by Ifediba and Vanderberg to obtain viable, mature gametocytes en masse, whatever the strain used. We designed an assay to determine the activity of antimalarial drugs based on the intracellular ATP content of purified stage IV-V gametocytes after 48 h of drug exposure in 96/384-well microplates. Measurements of drug activity on asexual stages and cytotoxicity on HepG2 cells were also obtained to estimate the specificity of the active drugs. Conclusions: The work described here represents another significant step towards determination of the activity of new molecules on mature gametocytes of any strain with an automated assay suitable for medium/high-throughput screening. Considering that the biology of the forms involved in the sexual and asexual stages is very different, a screen of our 2 million-compound library may allow us to discover novel anti-malarial drugs to target gametocyte-specific metabolic pathways. © 2012 Lelièvre et al.Peer Reviewe

A novel validated assay to support the discovery of new anti-malarial gametocytocidal agents

Additional file 1. Graphical representation of the expression of 12 selected genes throughout the 30 days of gametocytogenesis. Y-axis shows the gene expression represented as (Ctgene−Ct18S rRNA)Ttime × −(Ctgene−Ct18S rRNA)T0, considering the time 0 as the basal expression. Although only the gametocytogenesis during 30 days is presented, similar results were obtained from day 0 to day 15 in both assays

A Murine Model of falciparum-Malaria by In Vivo Selection of Competent Strains in Non-Myelodepleted Mice Engrafted with Human Erythrocytes

To counter the global threat caused by Plasmodium falciparum malaria, new drugs and vaccines are urgently needed. However, there are no practical animal models because P. falciparum infects human erythrocytes almost exclusively. Here we describe a reliable falciparum murine model of malaria by generating strains of P. falciparum in vivo that can infect immunodeficient mice engrafted with human erythrocytes. We infected NODscid/β2m−/− mice engrafted with human erythrocytes with P. falciparum obtained from in vitro cultures. After apparent clearance, we obtained isolates of P. falciparum able to grow in peripheral blood of engrafted NODscid/β2m−/− mice. Of the isolates obtained, we expanded in vivo and established the isolate Pf3D70087/N9 as a reference strain for model development. Pf3D70087/N9 caused productive persistent infections in 100% of engrafted mice infected intravenously. The infection caused a relative anemia due to selective elimination of human erythrocytes by a mechanism dependent on parasite density in peripheral blood. Using this model, we implemented and validated a reproducible assay of antimalarial activity useful for drug discovery. Thus, our results demonstrate that P. falciparum contains clones able to grow reproducibly in mice engrafted with human erythrocytes without the use of myeloablative methods

Hundreds of dual-stage antimalarial molecules discovered by a functional gametocyte screen.

Plasmodium falciparum stage V gametocytes are responsible for parasite transmission, and drugs targeting this stage are needed to support malaria elimination. We here screen the Tres Cantos Antimalarial Set (TCAMS) using the previously developed P. falciparum female gametocyte activation assay (Pf FGAA), which assesses stage V female gametocyte viability and functionality using Pfs25 expression. We identify over 400 compounds with activities <2 μM, chemically classified into 57 clusters and 33 singletons. Up to 68% of the hits are chemotypes described for the first time as late-stage gametocyte-targeting molecules. In addition, the biological profile of 90 compounds representing the chemical diversity is assessed. We confirm in vitro transmission-blocking activity of four of the six selected molecules belonging to three distinct scaffold clusters. Overall, this TCAMS gametocyte screen provides 276 promising antimalarial molecules with dual asexual/sexual activity, representing starting points for target identification and candidate selection

The antimalarial efficacy and mechanism of resistance of the novel chemotype DDD01034957.

New antimalarial therapeutics are needed to ensure that malaria cases continue to be driven down, as both emerging parasite resistance to frontline chemotherapies and mosquito resistance to current insecticides threaten control programmes. Plasmodium, the apicomplexan parasite responsible for malaria, causes disease pathology through repeated cycles of invasion and replication within host erythrocytes (the asexual cycle). Antimalarial drugs primarily target this cycle, seeking to reduce parasite burden within the host as fast as possible and to supress recrudescence for as long as possible. Intense phenotypic drug screening efforts have identified a number of promising new antimalarial molecules. Particularly important is the identification of compounds with new modes of action within the parasite to combat existing drug resistance and suitable for formulation of efficacious combination therapies. Here we detail the antimalarial properties of DDD01034957-a novel antimalarial molecule which is fast-acting and potent against drug resistant strains in vitro, shows activity in vivo, and possesses a resistance mechanism linked to the membrane transporter PfABCI3. These data support further medicinal chemistry lead-optimization of DDD01034957 as a novel antimalarial chemical class and provide new insights to further reduce in vivo metabolic clearance

The antimalarial efficacy and mechanism of resistance of the novel chemotype DDD01034957.

New antimalarial therapeutics are needed to ensure that malaria cases continue to be driven down, as both emerging parasite resistance to frontline chemotherapies and mosquito resistance to current insecticides threaten control programmes. Plasmodium, the apicomplexan parasite responsible for malaria, causes disease pathology through repeated cycles of invasion and replication within host erythrocytes (the asexual cycle). Antimalarial drugs primarily target this cycle, seeking to reduce parasite burden within the host as fast as possible and to supress recrudescence for as long as possible. Intense phenotypic drug screening efforts have identified a number of promising new antimalarial molecules. Particularly important is the identification of compounds with new modes of action within the parasite to combat existing drug resistance and suitable for formulation of efficacious combination therapies. Here we detail the antimalarial properties of DDD01034957-a novel antimalarial molecule which is fast-acting and potent against drug resistant strains in vitro, shows activity in vivo, and possesses a resistance mechanism linked to the membrane transporter PfABCI3. These data support further medicinal chemistry lead-optimization of DDD01034957 as a novel antimalarial chemical class and provide new insights to further reduce in vivo metabolic clearance

Effect of the haematocrit layer geometry on Plasmodium falciparum static thin-layer in vitro cultures

<p>Abstract</p> <p>Background</p> <p><it>In vitro </it>cultivation of <it>Plasmodium falciparum </it>is usually carried out through the continuous preservation of infected erythrocytes deposited in static thin layers of settled haematocrit. This technique, called the candle-jar method, was first achieved by Trager and Jensen in 1976 and has undergone slight modifications since then. However, no systematic studies concerning the geometry of the haematocrit layer have been carried out. In this work, a thorough investigation of the effects of the geometric culturing conditions on the parasite's development is presented.</p> <p>Methods</p> <p>Several experimental trials exploring different settings have been carried out, covering haematocrit layer depths that ranged from 6 mm to 3 mm and separation between the walls of the culturing device that ranged from 7.5 mm to 9 mm. The obtained results have been analysed and compared to different system-level models and to an Individual-Based Model.</p> <p>Conclusion</p> <p>In line with the results, a mechanism governing the propagation of the infection which limits it to the vicinity of the interface between the haematocrit layer and the culture medium is deduced, and the most appropriate configurations are proposed for further experimental assays.</p