RNA interference (RNAi) is a conserved sequence-specific, gene-silencing mechanism that is induced by double-stranded RNA (dsRNA). One of the functions of this pathway is the defense against parasitic nucleic acids: transposons and viruses. Previous results demonstrated that viral infections in Drosophila melanogaster are fought by an antiviral RNAi response and that components of the endocytic pathway are required for dsRNA entry to initiate the RNAi response. Recently we have shown that infected insect cells spread a systemic silencing signal that elicits a protective RNAi-dependent immunity throughout the organism. This suggests that the cell-autonomous RNAi response is insufficient to control a viral infection and that flies also rely on systemic immune response to fight against such infections. As a junior group leader, I will study the mechanisms that mediate the RNAi-based antiviral response in insects. By combining biochemical, cellular, molecular and genomic approaches, both in vivo and in cell culture, I will analyze the mechanisms underlying viral tropism, systemic propagation of the antiviral signal and the basis of the persistence of the antiviral state. Furthermore, I will examine whether the dsRNA-uptake pathway is conserved in mosquitoes and its relationship with viral immunity in that host. This comprehensive approach will tackle how this nucleic acid-based immunity works in insects to generate an anti-viral stage. A better understanding of the role of RNA silencing in insects during virus infection will allow the exploitation of this pathway for improvement of public health related problems such as arbovirus infection and disease.

Both antiviral immunity and susceptibility to viral infections are remarkably variable. Factors such as older age, increased body mass index, and male sex can increase the risk of developing severe viral disease, yet the impact of these features on antiviral immunity, particularly the type I interferon system (IFN-I), is not understood. Here we propose to use a systems immunology approach to identify features that influence IFN-I responses and understand their impact on infection susceptibility. We will use datasets and samples from the Milieu Interieur (MI) cohort of 1000 donors consisting 500 males and 500 females aged 20-69 years old. Using Simoa digital ELISAs, we have quantified IFN-I (IFNa and IFNb) in the supernatants of whole blood from the 1000 donors that was stimulated ex vivo with 7 agonists that activate the IFN-I system. Preliminary analysis of these results shows interesting age and sex differences in the IFN-I response. We will integrate the IFN-I cytokine data with existing demographic, microbiome, immune cell phenotyping and plasma protein data from the cohort and determine their associations with IFN-I levels. To investigate sex differences in IFN-I we will quantify sex hormones in the plasma of the 1000 donors. We will perform RNA-sequencing on IFNa-stimulated whole blood on a subset (n=200) of the donors to investigate the factors that shape the response to IFNa itself. We will perform genome wide association studies of the IFN-I response in the cohort to identify variants that influence IFN-I production using SNP genotyping data from MI. To determine the impact of these features on IFN-I and influenza infectivity in vitro, we will develop an organ on chip model encompassing features that we find to be associated with IFN-I levels in MI. Results from this project will identify novel regulators of IFN-I and shed light on new strategies to boost the antiviral response to better protect against viral infection.

Anopheles mosquitoes are the vectors of human malaria and also the alphavirus O’nyong nyong (ONNV), currently in emergence in Africa. Aedes transmit many arboviruses such as dengue Zika, and chikungunya (CHIKV), currently causing pandemics. We will compare the antiviral mechanisms of the poor arbovirus vector Anopheles coluzzii, and the efficient arbovirus vector, Aedes aegypti, against ONNV and CHIKV. The results will reveal host restriction factors, indicate the level of risk that an arbovirus could shift between hosts to exploit a new vector, and identify new tools to impede arbovirus transmission. Aim 1) Functionally screen a curated candidate gene panel to identify factors underlying differential antiviral function in Anopheles and Aedes; Aim 2) Dissect the mechanisms of action of selected candidates from Aim 1, with particular focus on a putative antiviral immune protein complex; and Aim 3) Detect the function and viral molecular interactions of candidate host restriction factor Rasputin in Anopheles and Aedes.

Malaria is a disease caused by Plasmodium protozoa and remains one of the major public health problems in the world. P. falciparum, one of the five species of Plasmodium which infects the man, kills up to 1 million people every year, the majority, children in Africa. The symptoms, as well as the complications of the disease are due to the multiplication of the parasites in the erythrocytes. The infection begin with a phase known as pre-erythrocytic, which is asymptomatic and extends since the injection of the parasites (called sporozoites) in the skin by the mosquito vector until the beginning of the erythrocytic infection. This pre-erythrocytic phase of the infection remains not very known, in particular, because of the technical difficulties, which its study comprises, like the dependence of a insectarium or the very low number and the great mobility of the sporozoites. Although it is clear that the sporozoites are injected into the skin and that they transform and multiply into forms that infect the erythrocytes in the hepatocytes, the exact destiny of the sporozoites in their mammalian host remains obscure. On the other hand, the malaria pre-erythrocytic phase received much attention in the immunological plan. It was indeed shown in the 60’s that the injection of live radiation-attenuated sporozoites protected the host from the injection of wild-type sporozoites, this vaccination is still today the most effective anti-malarial vaccine. Recently, the demonstration that genetically modified sporozoites could also protect from the natural infection, at least in the rodent, involved a new wave of enthusiasm for the use of live sporozoites as vaccines in the man. Until recently, the accepted vision of the infection by the sporozoites was that, once injected into the skin, it arrived in the liver through the blood flow, where they left the blood circulation to enter the parenchyma, finishing inside a hepatocyte. The way by which the sporozoites cross the endothelial barriers, to enter blood circulation in the skin and leaving circulation in the liver, remains unknown or disputed. These last years, my work, initially as a post-doctoral fellow, then as associate researcher at the Institut Pasteur, consisted to better understand the malaria pre-erythrocytic phase by especially using intravital imaging approaches in a rodent malaria model. One of our most surprising discovery was the observation that the sporozoites injected into the skin could invade cutaneous cells and develop into the forms capable of infecting erythrocytes. This observation has important immunological implications, since it shows that the whole of the pre-erythrocytic antigens, i.e. coming at the same time from the sporozoites and the intracellular forms, which was thought to exist only in the liver, are presented to the draining lymphnode. My project is divided in two parts. The first part focuses on the mechanism by which sporozoite crosses endothelial barriers in the skin and in the liver, at a cellular and molecular level, using a rodent model of malaria. The second part consists to test if the development of the skin stage, as seen in the rodent models, is also true for P. falciparum, the most lethal plasmodial species for the man