MALaria ARTemisinins RESistance – MALARTRES

The aim of this work is to better understand the mechanism of resistance developed by Plasmodium falciparum, the malaria agent, facing the molecules of the family of artemisinins (ARTs).
It is now demonstrated that the resistance is related to the ART parasite's ability to enter quiescent, very original resistance phenomenon, never before observed in Plasmodium.
The ART-F32 strain, is the only stable P. falciparum strain highly resistant to ART and cultivable in vitro. It will be the common basis of studies conducted in this research project.
Specific staining of this strain F32-ART and its twin-sensitive strain F32-TEM by insertion of an expression cassette GFP-Luciferase, will be a facilitator tool for the study of these parasites, especially in terms of identification, selection and quantification.
We will further define the parasitic stages capable of entering into quiescence, doses and exposure sequences of ART for entry or exit of quiescence.
The comparative analysis of genomes of strains resistant F32-ART and sensitive F32-TEM highlighted potentially involved in quiescence mutations. Genetic modification of F32 and F32-ART-TEM stem insertion / deletion candidate genes (mutated / wild) will confirm their involvement in the phenomenon of quiescence. Thus, the project will permit to offer a molecular test for identifying resistant strains easily used for epidemiological monitoring in the field.
A final objective will be to select molecules or new drug combinations, active on resistant parasites to ART. Compounds known to regulate the cycle of eukaryotic cells will also be evaluated. This should provide a pharmacological response to problems of resistance to ARTs and therapeutic impasse to come.

Among the objectives of this project, the task 5 «Investigation of the genotype malaria quiescence« aimed to analyze the genotypic basis of resistance to ARTs in Plasmodium.
The parasite gene «K13« of Plasmodium falciparum has been identified by a scientific consortium with partner 1 and partner 2 of this ANR project, as a molecular marker of resistance of malaria to artemisinin derivatives. The discovery of this marker can permit to better understand how the parasite resistant to artemisinin derivatives, to significantly improve the monitoring of the spread of resistant forms and to quickly adapt effective regimens to fight against this scourge. This work has been published in the journal Nature.
It was then necessary to demonstrate the central responsibility of K13 mutations in the resistant lines currently observed in Cambodia. With a fine genetic engineering technique, an international consortium, whose partner 1 and partner 3 of this ANR project, replaced the K13 gene mutations present in strains resistant to artemisinin with their wild equivalent and observed that these strains were becoming susceptible to malaria. Conversely, introduction of various mutations into sensitive strains confers the ability to resist artemisinins. This work, published in the journal Science, provides formal evidence that the K13 gene is the major determinant of resistance to artemisinin in Cambodia parasite strains.
This major discovery provides a powerful tool to detect resistant forms of malaria and map their distribution. Thus, since 2014, this gene entered the WHO definition of resistance of Plasmodium to Artemisinin-based combination therapies .

Comparative Whole genome sequencing of F32-ART and its twin sensitive strain F32-TEM permitted to identify the molecular marker associated with resistance to ART allowing rapid detection of strains with reduced sensitivity to ART in the field.
The strain F32-ART is an ideal model to study quiescence in Plasmodium. To date, the phenomena involved in quiescence such as parasites survival mechanisms, pathways involved in inducing, maintaining and output quiescent still extremely unclear. The analysis of this mode of resistance is a difficult scientific challenge because quiescence concerns only a small fraction of the total parasite population. F32-ART parasites of GFP-luciferase tagged will be a major tool to meet this goal.
This project has already identified the essential molecular marker for monitoring resistance to ART. Our work is now focused on understanding the new parasitic resistance mechanism that is quiescence to find active therapeutic solutions against ARTs resistant parasites.

Our work has helped to demonstrate that the K13 gene is the major determinant of Plasmodium falciparum parasite resistance to artemisinin.
These results led to:
- 1 article in the journal Nature in January 2014
- 1 article in the journal Science in January 2015
- The update of the WHO criteria for malaria resistance to ACTs (Status report on artemisinin resistance)
- The discovery of K13 gene linked to resistance to ART has been listed by the NIH among the 20 greatest scientific advances in 2014. www.niaid.nih.gov/about/pages/2014.aspx

PS The article published in Nature is a part of the task 5. This work was done and published after the funding application filing but before obtaining this funding ANR; that's why this grant is not indicated in this article.

Malaria is a major health problem in tropical and subtropical areas with an unacceptable toll of more than 1 million deaths each year and represents a significant risk to millions of travelers. In 2001, WHO recommended to use artemisinin derivatives (ARTs) in combination with a partner drug as first-line for Plasmodium falciparum treatment. To date almost all malaria-endemic countries have since changed their treatment policy accordingly leading to a 31% reduction in global malaria deaths for the last 10 years. However, since 2004, a major threat has emerged in Western Cambodia where clinical efficacy of ARTs has markedly decreased, with a delayed parasite clearance rate and high recrudescence rates in the following weeks. Artemisinin-resistant Plasmodium falciparum malaria parasites are now reported in all the Greater Mekong region (Cambodia, Vietnam, Thailand & Myanmar). Resistance to ARTs emerges at a time when no in vitro methodology or molecular or biochemical predictive markers exist to detect and study it. Moreover, there is currently no group of drugs that can replace artemisinins.
The general objective of this proposal is to better understand P. falciparum resistance to ARTs and to develop new diagnostic and therapeutic tools to fight ARTs-resistant parasites.
This innovative and ambitious project is proposing to characterize the in vitro quiescence phenotype induced by artemisinin resistance (Objective 1). This work is based on a major tool: the strain F32-ART, the only reported stable and highly artemisinin-resistant strain of P. falciparum that can be cultured in vitro, and able to survive 7000-fold the dose of ARTs that kills sensitive parasite strains. This strain permitted to Partners 1 & 2 to make a breakthrough by demonstrating that artemisinin resistance involves young stages which survive toxic effect of the drug through temporary growth arrest. Very recently, we have participated to show that quiescence is not only an in vitro phenomenon but also the mechanism of resistance to ARTs of malaria isolates from patients in Asia. F32-ART strain is thus a perfect model to study quiescence in the malaria parasite. Virtually nothing is known to date about this quiescence phenomenon in P. falciparum such as the mechanisms by which F32-ART parasites survive, and pathways involved in induction, maintenance and exit from quiescence. Analysis of ART-resistance phenomenon is an essential scientific challenge as quiescence affects a subset of young stages parasites which to date cannot be isolated from the rest of the parasite population. Technology of labeled parasites F32-ART GFP-luciferase developed by Partner 3 will be a major asset to meet this goal.
The lack of understanding the molecular basis of artemisinin resistance is a major roadblock for identification and monitoring of isolates with decreased sensitivity to ARTs in the field. Construction by Partner 3 of parasites transfected by targeted genes selected by sequencing F32-ART/F32-Tanzania whole genome should fulfill this gap. The success of this project should deliver tools for epidemiological monitoring of artemisinin-resistant parasites (Objective 2).
Moreover it is urgent to find therapeutic solutions to circumscribe these resistant parasites with selection of compounds or drug combinations active against ART-resistant parasites (Objective 3).
From a fundamental point of view, this project should help to better understand this new mechanism of parasite resistance, namely quiescence. From a practical point of view, it should determine molecular makers for monitoring artemisinin resistance and find active molecules against ART-resistant parasites.