With 429 000 deaths per year, malaria remains the most devastating parasitic disease for humans. It is caused by Plasmodium parasites and transmitted by Anopheles mosquitoes. The efficiency of artemisinin-based combination therapies (ACTs), the spearhead of malarial treatments, is now threatened by the appearance and spreading of artemisinin-resistant parasites. Moreover, the development of control strategies to block parasite transmission is a priority of WHO. In this project, we propose to study the mode of action of two antimalarial drugs, primaquine (PQ) and plasmodione (PD) that are active against gametocytes, the parasite stages responsible for human to mosquito transmission. PQ is the only available antimalarial medicine with established activity against mature gametocytes and is currently under intense clinical validation for widespread use in combination with ACTs. PD and derivatives are new early leads displaying fast-acting antimalarial activity and potent transmission-blocking properties. These drugs kill parasites most likely through pleiotropic redox-mediated mechanisms that remain poorly understood. While having distinct bioactivations, some of their modes of action seem to share common features. Indeed, while PQ is transformed by human cytochromes cytP450 into presumably highly active hydroxylated metabolites, the antimalarial activity of PD comes largely from its specific bioactivation within the infected erythrocytes and subsequent redox cycling properties. The existence of putative targets proteins for these compounds also remains an opened question. To decipher their complex mode of action, we propose to set up a multidisciplinary approach combining different expertise in organic synthesis, chemical proteomics, biochemistry, cell biology, and genetics, and to use the yeast as a model in parallel to Plasmodium studies. Our project has three main objectives: 1) to monitor the oxidative damages caused by PQ/PD, 2) to identify target proteins and redox enzymes controlling drug sensitivity and resistance in yeast and parasites, 3) to confirm the contribution of these candidate genes to drug sensitivity and resistance in Plasmodium. We expect that this proposal will allow us to shed light and understand the biochemical pathways and genes implicated in the modes of action of these antimalarials, and will allow their drug targeting as essential components in asexual and sexual parasites, responsible for malaria physiopathology and transmission, respectively.

To date, globally one out of four children is stunted and the current best- of -practice treatments are not able to correct for more than a third of the observed growth delays. The development of complementary, innovative interventions are therefore of utmost importance. In the Afribiota study we showed that stunting was associated with small intestinal bacterial overgrowth dominated by bacteria that normally reside in the oropharyngeal cavity and are associated with small intestinal inflammation and decrease lipid absorption. The oral-hygienic approach emerges as an entirely new preventive approach to tackle stunting. Therefore, our primary objective is to demonstrate by a specifically-designed clinical trial among 720 infants and their families in Bangui (Central-African Republic (CAR), that educating children and their families to implement strict and sustained rules of oral and nasopharyngeal hygiene will significantly prevent or reverse stunting; Our second objective is to investigate the oral cavity as a microbiological hub whose qualitative and quantitative alterations may impact on the future of child’s health. This will encompass fine description and monitoring of the oral microbiome assembly by and dynamics from birth, including key parameters influencing its ecological successions, like maternal and child hygiene, antibiotic use, and nutrition (including breastfeeding); Our third objective is to nucleate the conditions for the development of a global program of education to oral health in the CAR. Beyond the crucial question on how oral microbial communities expand to the gut resulting in the establishment of dysbiosis and stunting, this study will thus have a direct clinical benefit regarding both oral health and the treatment of undernutrition in Africa. The planned intervention on oral health will also allow us to assess the role of oral hygiene on other comorbidities, an area of much interest, as there are no data available on oral health in Africa.

Postnatal diagnosis of congenital toxoplamosis infection, caused by the parasite Toxoplasma gondii is imperative to ensure optimal medical care. Thus, the early identification of specific antibodies developed by the newborn is of crucial importance. This is a challenge because of the joint presence with maternal immunoglobulin G (IgG) in the serum of the newborn. The present proposal aims to identify newborn IgG that are specific for the pathogen. To achieve this goal, we will explore the individual signatures carried by the heavy chain of IgG and resulting from polymorphisms of several amino acids. We will use affinity purification to select specific IgG, adapted device to miniaturize these protocols and middle-down mass spectrometry for the proteogenomic characterization of these IgGs . Mapping of glycosylation of IgG will also be performed. Many scientific challenges will be overcome: (1) we will propose a workflow for the purification of pathogen-specific IgGs; (2) we will develop a proteolytic system to release the shortest discriminant sequences to identify peptide variants; (3) we will integrate the most recent updates of genomic information available in the public repository with dedicated proteomics and intermediary approaches in a proteogenomic strategy; (4) we will analyze the glycosylation profile in order to study a potential correlation with the specificity of IgG and their maternal or infant origin. We will use key samples that are already well characterized to validate our approach, and then apply it to paired samples of mothers (peripheral blood) and newborns (cord blood) derived from two existing cohorts in France and Benin focused on toxoplasmosis. The samples of interest are those taken in cases where the mother had a toxoplasmosis primary infection inducing seroconversion during pregnancy and where the newborn is suspected of having contracted the infection in utero. To be successful, the strategy will be optimized to achieve the sensitivity required to be directly compatible with the small amount of peripheral blood that can be collected from a neonate (maximum total serum volume 100 µL). This project will benefit from the expertise of complementary teams in immunological studies of congenital diseases and middle-down proteomics. It will be accompanied by three particularly adapted structures: the IRD, which coordinates the establishment of cohorts of interest, the ESPCI proteomics platform and in particular a high-resolution UVPD MS/MS mass spectrometer with a regional funding obtained in 2018, and the IPGG Labex for microfluidics. The two partners have been working together since 2011, which has been formalized by a collaborative research program between IRD and ESPCI. In the long term, this technological breakthrough will pave the way for many applications such as the diagnosis and monitoring of other congenital parasitic diseases (mainly Chagas disease), or more generally other infections of bacterial or viral origin, as well as other pathologies associated with autoimmune disorders such as insulin-dependent type 1 diabetes.

In the current perspective of global elimination of malaria, the search for more sensitive diagnostic tools is a key element in the fight against the disease. Alternative automated microscopic diagnostic techniques, based on deep learning image analysis algorithms are emerging, and a recent promising avenue is the development of diagnostic applications on smartphones. However, current solutions achieve a tool sensitivity equivalent to that achieved by an experimented microscopist. The original idea of this study is also based on a deep learning approach, but we propose a new learning paradigm allowing to considerably increase the sensitivity of the tool. Preliminary funding allowed us to establish the proof of concept by testing our hypothesis on field data from Bénin. The objectives of the study are now to increase the performance of our algorithm, then to implement the tool in a smartphone application which will be tested and validated in the field in Benin.