Newborns born to mothers with Plasmodium falciparum (Pf) placental malaria (PM) have a greater susceptibility to all infections in infancy. Recent studies, including our data, showed that immune cells of those newborns exhibited a dysregulated innate immune response at birth and at 18 months of age. Of note, in those studies, neonatal immune cell responses varied from attenuated to enhanced depending on the timing of in utero Pf-infection. The underlying mechanisms of PM-induced immunomodulation of neonates and the effect of the timing/duration of PM on this modulation remain unknown. Trained innate immunity is defined by epigenetic reprogramming of innate cell precursors that leads to an attenuated or enhanced innate response lasting for months or years in their progeny. The capability of innate cells to respond depends on the nature, the magnitude and the duration of exposure to components responsible for the first stimulation of the precursor. In Myeloid-PM, we hypothesize that PM induces such modifications in neonatal monocyte precursors, which leads to long-lasting susceptibility to all infections in infancy. Pf-infected erythrocyte sequestration in intervillous causing PM modifies the local environment into a pro-inflammatory state. Molecules from the Pf-infected placental microenvironment are very important to contextualize our findings and identify probable effectors at play from the hosts and the parasite in the modulation of neonatal immunity. State-of-the-art technologies, already implemented in the lab during a previous pilot study, will depict precisely the Pf-infected placental environment and its contribution to neonatal immunomodulation. Benin is an endemic area for malaria. In the project Myeloid-PM, we will enroll 210 pregnant women and their newborns in a one-year longitudinal study. Timing/duration of PM will be considered in the selection of women and four clinical groups will be constituted based on presence/absence of Pf-parasites and of hemozoin-loaded maternal immune cells (marker for persistent infection) in the placenta: (i) not infected, (ii) recent and active PM, (iii) chronic and active PM and (iv) past PM. Using epigenetic, transcriptomic and functional approaches on mononuclear cells collected at birth (from cord blood) and at 12 months of age from naturally exposed-individuals, we propose to clarify the mechanisms of neonatal and infant innate cell modulations following in-utero priming during PM. Single-cell ATAQseq/RNAseq will be performed on freshly collected cells. The same approach will be implemented on CD34+ cells to determine whether epigenetic modifications on myeloid precursors may explain the long-lasting susceptibility to infections. In parallel, we will deploy a systemic and integrative approach, at organ-, cell-, protein- and metabolite-levels to shed light on the modifications in the placental environment. Specifically, 12 placentae (3 per clinical groups) will be selected for spatial transcriptomic and scATAcseq/RNAseq on dissociated placental cells. Plasma from maternal blood and cord blood will be characterized using Somalogic technology (~7000 human proteins), mass spectrometry and metabolomics in order to identify soluble factors that can induce directly or indirectly epigenetic modification in neonatal immune cells. Finally, candidate molecules will be tested in vitro on naturally exposed cells to reverse the modulation through functional experiments. Myeloid-PM will likely uncover new aspects of trained immunity and placental malaria. Because epigenetic modifications are plastic and reversible, our project will provide the proof-of-concept, on naturally exposed individual cells, that PM-induced neonatal immunomodulation can be reverted and will offer new directions for future therapies targeting metabolism and epigenetic reprogramming.

French research is internationally recognized in the field of public health and in the traditional scientific disciplines of epidemiology, biostatistics, health economics, and clinical research. The Institut de Recherche pour le Développement (IRD), which is primarily interested in public health to improve the health of Southern populations, is a world leader in this regard. Thus, French scientists are renowned for producing evidence on the effectiveness of life-saving public health interventions. This is especially true of the researchers of UMR 2016 “MERIT” IRD-Paris Descartes, who are working on women’s and children’s health (reproductive health). However, this expertise is most often concerned with interventions organized in experimental and controlled contexts (efficacy). Yet this information is not sufficient, because for an intervention to be reproducible, generalizable, and effective on a large scale, we also need to understand why it works, how, for whom, and in what contexts (effectiveness). Implementation science in public health is still in the emergence phase of its development, and very few French scientists have developed this type of expertise to study the organization of complex health interventions in real life situations. For example, no French organization was present at the largest international conference on this topic related to Southern healthcare systems held in Peking in 2012 (Global Symposium on Health Systems Research). Following a World Health Organization status report in 2013, the first implementation science training guide has just been published in 2014, without any involvement of French experts. The principal candidate in this project, Valéry Ridde, is a Canadian international expert in the science of implementing public health interventions, an expertise he has been able to develop in Africa. The overall objective of the project is to develop and reinforce in France the implementation science for complex reproductive health interventions in the South by conceptually and empirically developing this field of knowledge, while inserting France into the existing global network and strengthening its national capacities. To achieve this objective, the scientific challenges to be tackled have mainly to do with implementing an interdisciplinary and applied research program in reproductive health, with the active involvement of researchers from UMR 216 “MERIT”, which would be the candidate’s home base, and maintaining a very close working relationship with national and international decision-makers. This project will enjoy exceptionally good conditions for achieving these objectives because of its complementarity with the current activities of UMR 216 “MERIT”, its implementations in Africa and Asia, and its future prospects for development through the planned creation of a new joint international unit entitled “Quality of care, vulnerability, and risk management in global health” (QUARISM), whose research capacities will be greatly reinforced by the principal candidate. The work plan for the project’s 48 months is structured around the following activities: using implementation science to conduct complex reproductive health and equity projects in Africa; developing conceptually the implementation science for reproductive health interventions; producing scientific results and applying knowledge with decision-makers and stakeholders; creating university courses and professional training programs; and training doctoral candidates and young researchers. This project will have many positive spinoffs: supplementary research grants; development of a new interdisciplinary conceptual framework; 20 scientific articles and one collective book; 15 policy briefs and a website, training of 3 doctoral candidates and 2 young postdoctoral researchers—all of this while contributing to improving the efficiency of French public assistance to maternal and child health in the South.

Background and Rationale In sub-Saharan Africa, preventive drug strategies against malaria in pregnancy are recommended from the 2nd trimester of pregnancy onwards. Consequently, pregnant women remain insufficiently or not protected during the first trimester of pregnancy. However, this period may be at particular high risk for the fetus since pregnancy-associated parasites that accumulate into the developing placenta may alter fetal development and growth. The consequences of malaria in early pregnancy have been poorly explored so far. Although studies have indicated that malaria before 4 months of pregnancy was associated with poor maternal and child outcomes, they were not specifically designed to address this issue and both selection and confounding biases cannot be ruled out. Our project will provide considerable value-added findings compared with existing studies for four main reasons: (i) pre-conception recruitment of women (allowing the detection of the earliest malarial infections during pregnancy), (ii) intrauterine growth retardation as primary endpoint, (iii) assessment of both women’s nutritional status before and during pregnancy and gestational hypertension as potential confounders, and (iv) detection of submicroscopic malarial infections. Project objectives, design, analysis plan, and expected outcomes Our goal is to assess the consequences of malaria in early pregnancy in both the mother and her fetus by answering the following questions : (1) What is the effect of malaria in the first 3 months of pregnancy on intrauterine growth restriction (IUGR), duration of pregnancy, birth weight and maternal anemia? (2) What are the burden and the consequences of submicroscopic malarial infections in the first trimester of pregnancy for the mother and the fetus? (3) Does women’s nutritional status modify the relation between malaria in early pregnancy and child outcomes? The project will last 42 months. Three tasks will be dedicated to the recruitment and follow-up of 500 pregnant women. For this purpose, 2,000 women of childbearing age living in the area of Abomey-Calavi (South Benin) will be followed-up monthly until they become pregnant. The first 500 to be pregnant will be then followed-up monthly at the facility-level for, specifically, malaria (microscopy and RT-qPCR), gestational hypertension and nutrition (anthropometrics and feeding practices). Five doppler ultrasound scans will be scheduled during pregnancy with the first one between 8 and 14 weeks of gestation to date the pregnancy accurately. The 4th task will use data collected before and during pregnancy to assess the effect of malaria in the first trimester on maternal and child outcomes, using both hierarchical mixed models and path analysis. The main outcome will be IUGR defined as small-fetal/birth weight-for-gestational-age or reduction in growth according to the new WHO charts. The 5th task will be dedicated to project management. In terms of outcomes, our findings will help to introduce new interventions or to revise existing ones, for the prevention of malaria in pregnancy. New strategies are urgently needed, as the short-term efficacy of present guidelines is now questioned. Organizational capacity Valérie Briand (UMR216 IRD/UPD), the coordinator, has a 8-year expertise in the field of epidemiology and prevention of malaria in pregnancy and a great experience in conducting field-studies. She has been the principal investigator of a large clinical trial for the prevention of malaria in Benin. The project’s team involves scientists specialized in epidemiology, biostatistics, anthropology and biology belonging to UMR216, as well as two French collaborators (UMR204-IRD and US953-Inserm) specialized in nutrition and perinatal health and two Beninese collaborators from the Université d’Abomey-Calavi.

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.

Small molecule fluorescent probes are indispensable tools in most sectors of biological and biomedical research. Besides strong, bright and stable fluorescence, organic fluorescent dyes need to possess additional properties to meet ever-growing requirements. Among these, photoactivability, covalent capture and fluorogenesis (PCCF) constitute invaluable characteristics in modern chemical biology for interactomic and bioimaging applications. While the integration of the PCCF triad in single molecular elements ("tags") is very desirable, this association of properties is extremely rare and difficult to generalise conceptually. Currently, only the azidoprofluorophores (APF), photoactivated in bio-incorporated aminofluorophores, possess general PCCF properties. APF show however important limitations, such as irreversible photoactivation (with destruction of the tag in absence of biological interactome) or difficulty to chemically conjugate with the studied entities of interest. We propose a unique modification of most categories of fluorescent dyes commonly used in research under a form called "OXO-Tags", to confer them the desired PCCF triad. We show by a great body of preliminary proofs-of-principle, both experimental and in silico, that these novel tags indeed possess integrated PCCF properties. These initially non-fluorescent tags can thus be photoactivated to yield covalently-bioincorporated fluorescent photoadducts, demonstrated in fluorimetry, SDS-PAGE and cell imaging. The OXO-Tags can be easily conjugated to various entities of interest (e. g., bioactive small-molecules, proteins, RNA) by use of self-immolative spacers, offering a control of the photoreactivity of the tag and capable of releasing the two components separately in a programmed manner post-photoactivation ("catch and release"). These tags would constitute precious reporters for the identification of targets of drugs and natural products, macromolecular interactomic studies, as well as cell imaging. The objectives of this ANR project are to synthesize, functionally then biologically validate an optimized generation of OXO-Tags in reference interactomic paradigms, involving known associations of small molecule (drug)-target and macromolecule (protein or RNA)-partner. Further, OXO-Tags will be used to explore the targets and interactomes of other entities of interest in these two molecular categories.