Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the current coronavirus disease 2019 (COVID-19) pandemic. This virus emerged in China at the end of 2019 and has, since then, dramatically spread across the world. As of today (18th of June 2020), SARS-CoV-2 has caused more than 445,000 deaths worldwide, general lockdowns in many countries across the globe and an unprecedented global economic crisis. SARS-CoV-2 is the seventh coronavirus identified as capable of infecting humans and is closely related to the highly pathogenic SARS-CoV (or SARS-CoV-1), isolated in 2002. Similarly to SARS-CoV disease, COVID-19 is characterized by fever, breathing difficulty, acute respiratory distress syndrome and death in the most severe cases. There is currently no treatment or vaccine available. Strategies for vaccine development are being developed with several ongoing clinical trials in phase II and III, but even if some of these strategies prove to be efficient, a large-scale vaccine will take months to produce. There is therefore a dire need to better understand the requirements for SARS-CoV-2 replication at the cellular level in order to identify potential drug targets and rapidly help developing treatments. Genetic screens with CRISPR (clustered regularly interspaced short palindromic repeats) technology are powerful approaches to identify genes and pathways involved in various biological processes, such as viral infections. With the CRISPR-TARGET-CoV project, we propose to rapidly perform genome-scale CRISPR screens to identify both cellular dependency factors and inhibitors of SARS-CoV-2 in close collaboration with Dr. John Doench, a world leader in the CRISPR field. This work will establish a map of new possible drug targets for COVID-19. We will then screen selected drugs of interest, which will be relevant with respect to the identified cellular factors. We will focus on approved molecules, that could be used in a repositioning strategy in order to rapidly identify new potential therapeutic avenues for COVID-19. The effect of the drugs will be validated ex vivo, in primary human airway epithelial cells, one of the best models for analysis of SARS-CoV-2 replication.

Along evolution, intracellular bacterial pathogens have developed elaborated means to counter the immune response of their host in order to establish optimal replicative niches in which they replicate in high numbers. This is achieved by the secretion of bacterial effector proteins that divert the function of host cell proteins to create a pathogen-specific compartment and to dampen the cell response resulting from their invasion. Understanding the host/pathogen interactions that prevent detection and clearance of bacterial pathogens is thus of prime importance to counter infections. Coxiella burnetii is a highly infectious class 3 pathogen causing the zoonosis Q fever. The bacterium develops in the placental cells of contaminated wild animals or cattle without causing any symptoms of disease. Following delivery or stillbirths, Coxiella is shed in the environment and humans usually are infected by inhalation of Coxiella-contaminated particles. Acute Q Fever first develops as a flu-like disease but can evolve to a chronic form that can be lethal, as the bacterium is capable of colonising aortic and endocardial tissues. In humans, Coxiella can invade and develop in alveolar macrophages, hepatocytes and trophoblasts. Key to Coxiella infections is the re-routing of multiple trafficking pathways of the infected cell for the formation of an intracellular replicative niche named Coxiella-Containing Vacuole (CCV) as well as the inhibition of apoptosis. These processes are driven by effector proteins secreted by the bacterium into the host cell cytoplasm via a Dot/Icm Type 4b Secretion System (T4SS). Our laboratory and others have shown that Coxiella can dampen the inflammatory response of infected cells allowing its persistence in the host, as a stealth pathogen. Indeed, Coxiella modulates innate immune signalling in a T4SS-dependent manner, with effectors NopA and IcaA playing a role in the inhibition of nuclear transport of NF-kB and non-canonical inflammasome activity, respectively. Additionally, analysis of our Coxiella mutant library led to the identification of bacterial genes required for cytoprotection of the infected cells. Among the identified mutants, I focused on Coxiella icaB::Tn mutant and discovered that IcaB is the first Coxiella effector protein capable of interacting with a set of cytosolic sensors of the NOD-like receptors family (NLRs). These NLR proteins play a crucial role in the recognition of intracellular pathogens and the inflammatory response of immune and placental cells. It is thus of prime importance to study the interactions of Coxiella with host NLRs and how this microbe manipulates the inflammasome pathway to evade cytosolic microbial sensors. With the present project, I aim at: 1) characterising the NLR-interacting effector protein IcaB, 2) determining which NLRs are manipulated by Coxiella during infection, 3) identifying additional bacterial effectors targeting NLRs. This project will help to further understand how innate immune sensing is manipulated during Coxiella infections and unveil the mechanisms that render Coxiella stealth in vivo. It will open the path for the development of antimicrobial molecules to treat Coxiella infections and for the repurposing of bacterial products as new anti-inflammatory molecules.

Infections caused by Non-Tuberculous Mycobacteria (NTM) are on the rise worldwide, not only in cystic fibrosis and immuno-compromised patients, but also in immune-competent individuals. These infections represent a major healthcare challenge with treatment using current antibiotics remaining less than optimal and associated with high therapeutic failure. The primary NTMs involved in human pulmonary diseases are Mycobacterium avium Complex (MAC) and Mycobacterium abscessus (MABS) complex, notorious for their inherent resistance to most antibiotic classes. With the added complexity of increasing acquired antibiotic resistance, it is evident that novel drugs are urgently needed. Proteins of the electron transport chain (ETC) have in recent years emerged as attractive new targets for the development of novel efficacious antibiotics against mycobacteria, though genetic differences between mycobacterial species mean that not all ETC targeting inhibitors are active on MAC and MABS. Recently, members of the current consortium have discovered and worked extensively on the development of a novel class of anti-tuberculosis molecules, named the Tricyclic-Spirolactams (TriSLa), that are potent inhibitors of type II-NADH dehydrogenases (Ndh-2) of the ETC of mycobacteria. During this extensive early drug development research, medicinal chemistry efforts have generated more than 250 TriSLa analogues, and in vitro profiling found that these novel inhibitors maintain strong activity on both MAC and MABS, making them an attractive alternative tool for targeting the NTM ETC. This offers the potential to develop a novel candidate to be included in a poly-therapy treatment for these extremely difficult-to-manage mycobacteria. Based on the extensive knowledge that the consortium has already accumulated regarding the favourable and largely optimised physicochemical and pharmacokinetic properties of the more potent TriSLas, TriSLa-4-NTM proposes to accelerate the development of an efficacious TriSLa-based preclinical candidate for the treatment of pulmonary infections by M. abscessus and M. avium. To achieve the goal of the TriSLa-4-NTM, the consortium of 4 partners sharing complementary expertise in drug discovery & development, medicinal chemistry, pharmacology, mycobacteriology, NTM genetics and animal models, has structured its R&D pipeline around 3 inter-connected work packages. Firstly, the mechanism of action and in vitro profile of TriSLa on MAC and MABS will be evaluated; this includes the understanding of the importance of media composition on TriSLa activity, and how TriSLa acts in combination with other antibiotics. Secondly, the in vitro and in vivo essentiality and vulnerability of Ndh-2 in MAC and MABS will be assessed using functional genomics. Of interest is that MAC have two Ndh-2 homologues while MABS only have one. This section will also investigate if any genes in MABS impact the efficacy of TriSLa. Thirdly, a large focus of the program will be on the synthesis and pharmacokinetic/ADME profiling of lead TriSLa inhibitors for selection of optimised candidates for in vivo efficacy studies, their efficacy evaluation on MAC/MABS infected zebrafish, and critically, the evaluation of TriSLa efficacy on murine models of pulmonary MABS/MAC infection, determination of their minimal effective dose, and finally, their efficacy in combination with other antibiotics. The goal of TriSLa-4-NTM research program will be to deliver a well-characterised and optimised set of TriSLa inhibitors efficacious against murine models of NTM infection, ready to complete pre-clinical studies, with the ultimate goal of developing a clinical candidate for the treatment of pulmonary NTM infections. In addition, this research will strengthen the expertise in France in animal models for NTM infections, an expertise that is essential for antibiotic drug development for NTMs.

The UnSETTLE-NET project aims to bring together 4 international experts to submit a highly competitive proposal at the forthcoming ERC Synergy Grant 2019 (call will close late autumn 2018). The objective of the ERC Synergy Grants is to enable two to four Principal Investigators to work in synergy in order to jointly address ambitious research questions that could not be addressed by the individual Principal Investigators working alone. The UnSETTLE project will bring together partners with unique and complementary expertise to address a key problem in the field of chronic infections: how to train and boost innate immune cells to detect and eliminate stealth pathogens (viruses that establish latency, and intracellular bacteria that establish chronic infection)? Some pathogens have evolved multiple evasion strategies through co-existing evolution with their host to evade both innate and adaptive immunities, and then persist in their host by suppressing immune responses. These so-called stealth pathogens take refuge in a subcellular compartment and maintain undetectable replication levels to continue propagating in their host, all the while suppressing or thwarting host immunity. The aims of our synergy project are 1) to quantify and boost distress signals emitted by latently and chronically infected cells and 2) to understand the dysregulation of the immune system associated with latent and chronic infections. The UnSETTLE project will open up an entirely new and unconventional direction in the field of chronic infections, that will make a break with conventional approaches that seek to boost pathogen replication to trigger its detection, or to train acquired immunity to locate specific antigens. The key to the project is to boost sentinel innate immune cells to distinguish latently or chronically infected cells from self. The project already brings together 3 internationally-recognized Principal Investigators with unique and complementary expertise to address the ambitious project objectives: virology (HIV molecular virology, AIDS pathogenesis, endogenous retroelements, evolutionary virology), microbiology (intracellular bacteria, Coxiella, Brucella), innate immunity (innate signalling, intrinsic immunity, and innate immune cells), high-throughput screening of nuclear translocation events, endogenous peptides with antiviral activity. Our main goal is to include a fourth Principal Investigator expert in immunology and to prepare a solid and competitive version of the UnSETTLE proposal. The aim of the UnSETTLE-NET project will be (i) to identify the 4th PI, (ii) visit two key technical platforms for mass cytometry and animal facility, (iii) organise a workshop with all participants, and (iv) outsource the scientific editing of our proposal.

The recent SARS-CoV-2 pandemic has highlighted the need to develop new antivirals against RNA viruses that are responsible for more than a third of new emerging or re-emerging infections. As many RNA viruses use cellular helicases during their infectious cycle, these proteins have become promising therapeutic targets. VIR2RHA is a collaborative research project aimed at developing RHA inhibitors to effectively fight against a broad spectrum of RNA viruses. Thus, using drug design approaches, we have developed new original molecules which bind specifically to RHA, which are non-toxic and which demonstrate broad-spectrum antiviral properties in vitro against Chikungunya virus (CHIKV), the virus Dengue fever (DENV), influenza virus (IAV), human immunodeficiency virus type -1 (HIV-1), T-cell leukemia virus type 1 (HTLV-1) and also the SARS-CoV-2 responsible for the COVID-19 pandemic. In the VIR2RHA project, our objectives are to study the mechanisms of action of our inhibitors in order to continue their preclinical development. This program is also designed to elucidate the proviral functions of RHA in the infectious cycle of these RNA viruses. Our program is part of the pandemic preparness action and adapts to the axes "Life, Health and Well-being" and "Medical innovation, Nanotechnologies, Regenerative medicine, Innovative therapies and vaccines" of the ANR 2022 call