Protein-carbohydrate (PC) interactions play a key role in various biological processes. In particular, PC interactions govern infected erythrocyte (IE) adhesion on placental cells during placental malaria (PM) leading to severe pathological conditions. Experimental description of PC interfaces remains very challenging. The main goal of SugarPred is the development of structure- and sequence-based carbohydrate binding site prediction tools through implementation of the most recent machine learning approaches on the basis of the available structural data. We will apply the developed tools to VAR2CSA, the protein responsible for IE adhesion during PM, and will verify our predictions in direct experiment. This interdisciplinary approach will allow identification of VAR2CSA sugar-binding residues, and thus fill an important knowledge gap currently limiting the improvement of PM vaccines. A set of machine learning tools for the carbohydrate binding site prediction will be made available to the scientific community.

Despite a significant decrease in number of Plasmodium falciparum-related deaths in recent years, malaria remains a major public health problem. Resistance of parasites to anti-malarial drugs could soon lead to a resurgence of the disease and calls for the need of developing new interventional strategies. We recently conceptualized a novel immunotherapeutic approach aiming at redirecting a pre-existing polyclonal antibody response against Epstein-Barr virus (EBV), which chronically infects over 95% of the population, towards defined target cells to mediate their clearance by immune effectors. As a proof-of-concept, we established that this strategy is efficient against cancer cells. We will here generate bi-modular fusion proteins (BMFPs) able to recruit polyclonal endogenous high-affinity antibodies (anti-EBV) towards P. falciparum-infected erythrocytes. BMFPs will be designed based on an EBV antigen coupled to nanobody-derived binding moieties targeting P. falciparum antigens specifically expressed at the surface of cells infected by asexual or sexual parasite forms. Following generation and screening of nanobody libraries specifically targeting parasite-derived proteins, best binders will be fused to the EBV antigen. The efficacy of the resulting BMFP candidates to recruit immune effector mechanisms and mediate infected erythrocytes’ clearance will be evaluated both in vitro and in vivo. The development of BMFP therapeutics, interfering with parasite pathogenicity and/or transmission, could offer new malaria control alternatives and allow a broader therapy access to individuals infected by P. falciparum in sub-Saharan Africa.

Impaired splenic function (hyposplenism) affects more than 0.4% of the French population, half of them splenectomised, portends a serious risk of complications, including severe infections, thromboembolism and leukemia. While many splenectomised patients are fully asplenic, loss of function is partial when the spleen has been damaged by acquired or inherited diseases like sickle cell disease (SCD). In SCD, hyposplenism emerges during infancy but spleen function may persist in adults, and correlate with disease severity. The spleen clears altered red blood cells (RBC) from the circulation. Best markers of hyposplenism thus assess this ability to retain stiff RBC and “pit” those that contain rigid bodies. RBC that have not been pitted in absence of a functional spleen contain nucleic acid-positive bodies (Howell-Jolly bodies, HJB) or empty vacuoles (Pocked RBC). The proportion of these RBC in circulation is highly discriminating. Pocked RBC are 30 times more abundant than HJB and predict hyposplenism with high sensitivity and specificity. Only pocked RBC counts closely correlate with splenic scintigraphy, the reference measure of spleen function. Pocked RBC counts are the most relevant marker of hyposplenism, but current quantification methods are suboptimal. Molecular markers of their vacuoles remain to be identified, and current counting is observer-dependent and slow. Our objective is to improve the relevance and applicability of splenic function markers, as a prerequisite for clinical studies to validate their prognostic & theranostic value. Specific aims are (i) to assess the correlations between residual splenic function and outcome in hyposplenism, and (ii) to establish reliable, observer-independent methods for measuring splenic function by quantifying RBC deformability with new microsphere-based and microfluidic techniques, or set-up counting of Pocked RBC by cytometry or imaging coupled with machine learning. The main barriers to be lifted are the poor adaptation of existing markers of spleen function to the clinical lab workflow, and their elusive validation as predictors of disease severity. The final product is a routine biological test that quantifies splenic function like serum creatinine levels quantify renal function. Important knowledge and medical gaps remain to be filled. Complex physical challenges on RBC as they cross splenic slits are poorly described. The removal of rigid bodies or vacuoles from RBC without cell lysis (the pitting process) is even more complex and can be fully observed experimentally only with highly specific microfluidic devices. On the medical side, there is a pressing need for biomarkers in SCD that accurately inform key treatment decisions, such as the best timing for treatment intensification, before irreversible organ damage occurs. Because the spleen is damaged early in SCD, spleen function markers are potentially informative. Impaired spleen function may altogether reflect past severity and portend a risk of further degradation, because pathogenic subpopulations of RBC are less efficiently cleared from the circulation. In some patients splenectomised for trauma, spleen function reappears due to regrowth of splenic nodules (splenosis). Results from markers will induce a beneficial simplification of follow-up in these patients. Conversely, if hyposplenism is confirmed, patient compliance to vaccination and antibio-prophylaxis will be enhanced with positive impact on prognosis. Our consortium brings together complementary inputs from physicians, physicists, pathophysiologists, and AI specialists. This shapes a cohesive translational task force mastering medical practice and sophisticated in silico and in vitro tools, including the only microfluidic chip with spleen-mimetic slits narrower than 1 micron currently available. We will undertake 6 tasks to find either the first specific markers of pocked RBC or to count them without label. We will also assess their predictive value.

Sickle cell disease (SCD) is a genetic recessive inherited disorder caused by a Glu-to-Val substitution in the β globin protein resulting in abnormal hemoglobin (HbS) that polymerizes under hypoxia driving red cell sickling and reduced half-life. SCD is a severe multisystem disease characterized by hemolytic anemia, high susceptibility to infections, inflammation, recurrent painful vaso-occlusive crises and organ failure. SCD is also characterized by stress erythropoiesis, with abnormalities during terminal erythroid differentiation, suggesting that anemia could also be impacted by defects of central origin. We recently demonstrated the occurrence of ineffective erythropoiesis in the bone marrow of SCD patients and characterized the molecular mechanism as involving the cytoplasmic trapping of HSP70 chaperon protein by HbS polymers under partial hypoxia. Although SCD has been investigated for decades, there is still an urgent need for more studies to understand the complexity of its molecular and cellular defects and to develop new treatments in a personalized medicine perspective. The main treatments in SCD target the causative defect, i.e. HbS, either by adding normal hemoglobin to the circulation, through chronic blood transfusion or allogeneic hematopoietic stem cell transplantation, or by inducing endogenous fetal hemoglobin (HbF) using hydroxycarbamide (HC). Surprisingly, although HC was first tested in SCD patients more than 35 years ago, the molecular mechanisms underlying its mediated induction of HbF are still poorly understood. HbF expression is a known modulator of disease severity in SCD as it inhibits HbS polymerization, prolonging the lifespan of the red cells in the circulation. Importantly, we have recently revealed a new anti-apoptotic role for HbF during terminal erythroid differentiation in SCD by showing that it rescues erythroblasts from cell death at the polychromatic and orthochromatic stages. We have designed an ambitious project to address the unknown molecular mechanisms involved in ineffective erythropoiesis in SCD and to test the effect of known and novel therapeutic strategies on erythroid differentiation. In particular, our IRIS project will investigate the (i) molecular bases of ineffective erythropoiesis in SCD related to the auto-oxidation of HbS and to the impaired α and sickle β chain coupling, and (ii) the impact of erythroblasts’ death on the erythroid niche, particularly on the central macrophage of the erythroblastic island. We will also develop innovative therapeutic strategies based on gene and base editing and assess their effect on ineffective erythropoiesis, together with the effect of other therapeutical molecules such as HC. Our study will be conducted in vitro and in vivo, using a wide panel of tools and human material comprising patient hematopoietic primary cells and cell lines engineered for the project purposes, as well as the humanized Townes SCD mouse model. The IRIS proposal tackles precedingly unexplored areas of the SCD pathophysiology, both on the fundamental and translational research levels. It will reveal new and important biological aspects of SCD that may explain the large variability in disease severity and degree of anemia. It will also offer new molecular therapeutical strategies that can improve patient life expectancy and quality of life and likely reduce the treatment costs.

Atopic diseases cause major morbidity and economic loss. Although rare immune cells, basophils are implicated in the pathogenesis of allergic diseases of respiratory system and skin (including rhinitis, asthma and atopic dermatitis), and also autoimmune and other inflammatory diseases like bullous pemphigoid, chronic urticaria and eosinophilic oesophagitis. Considering the impact of dysregulated basophil functions on the pathogenesis of various diseases, it is conceivable that basophils are the potential therapeutic targets. However, currently, there are no specific approaches to target them. In this fundamental-translational project (“BASIN”), we design and investigate an innovative immunotherapeutic approach to inhibit basophil activation in pathological conditions. As IL-3 cytokine, secreted mainly by T cells is the most potent activator of basophils and also a pre-requisite for IgE-mediated degranulation, we hypothesize that interfering with basophil-IL-3 axis will suppress basophil activation and hence benefit allergic and inflammatory diseases. To address this hypothesis, three objectives are planned in BASIN project by combining the complementary expertise of 4 partners in human immunology (Dr. J Bayry), patient tissues (Dr. K Boniface and Pr. J Seneschal), in vivo experimental models (Dr. M Li ), and bioinformatics (Dr. A G De Brevern). Objective 1 is aimed at cross-talk between human basophils and T cells, with a particular focus on regulatory T cells (Tregs), based on our newly published report showing that Tregs induce activation of basophils by IL-3 and STAT5-dependent mechanisms. This task involves both in vitro studies and in situ using allergic patients’ biopsies. Objective 2 is aimed at exploring the IL-3 signal axis in the recruitment and activation of basophils in the inflamed skin tissue, and the mechanisms underlying the loop regulation between basophils and T cells, by using novel genetically modified mouse tools (Il3-reporter mouse line, and Il3-conditional knockout), and atopic dermatitis models. Objective 3 aims at using bioinformatics approach for identification and validation of inhibitors to interfere IL-3 axis, thus to inhibit inflammatory basophil responses in atopic diseases. BASIN project is at the boundary of fundamental and clinical research and aims at translating fundamental findings for the benefit of the individuals and the society. We expect to achieve a better understanding of the expression and function of IL-3 in basophil-mediated inflammation, from cellular cross-talk to molecular signaling both in human and mice. If successful, this translational research brings promising new biotechnological innovations for the therapy of allergy and other inflammatory diseases where basophils and IL-3 are pathogenic. Hence our project has dramatic repercussions for the quality of life of the patients and in the long term reduces societal costs.