The severity of the global COVID-19 pandemic poses an urgent need for the development of efficient therapeutic strategies. To complete the available therapeutic arsenal, targeting the SARS-CoV-2 genome by antisense RNA therapy should be deeply investigated. We designed in silico antisense oligonucleotides (ASO) targeting viral genome to block the viral replication and transcription. The objective of the project is to validate the best ASO firstly by in vitro experiments on infected Vero E6 cultures, and secondly to test the best oligonucleotides antisense in vivo on infected animal model to perform a preclinical trial.

The pneumovirus family contains several viruses that infect the respiratory tract and induce severe or fatal pneumonia and bronchiolitis in humans and animals. Pneumoviruses include human respiratory syncytial virus (RSV), bovine RSV, human metapneumovirus, avian metapneumovirus, and the recently discovered swine orthopneumovirus (SOV). There are no effective vaccines against these viruses and antivirals are restricted to Ribavirin, a toxic and nonspecific nucleoside analogue. Developing new strategies against these viruses requires improving our knowledge of the molecular mechanisms of viral replication. The genome of Pneumoviruses is composed of an non segmented, single-stranded RNA with a negative polarity of around 15 kb and which encodes 9-11 genes. This RNA genome is always enwrapped by the viral nucleoprotein which prevents any detection of RNA by the cellular innate immunity sensors and protects it against cellular attacks. Pneumoviruses are enveloped viruses that replicate in the cytoplasm of the cells. Once the viral and cellular membranes are fused together, the virus is released into the cytoplasm in the form of a holo-nucleocapsid including the viral polymerase L, capable on the one hand of synthesizing viral mRNAs that will be translated by the cellular machinery, and on the other hand to replicate its genome to create new viral particles that will bud at the plasma membrane thanks to the neo-synthesized glycoproteins. In this project we want to elucidate the mechanisms of viral RNAs synthesis as well as the mechanisms of encapsidation of genomes by nucleoproteins. The viral machinery synthesizing the viral RNAs is autonomous and specific and formed by a multi-protein complex (no cellular equivalent). This complex consists of 3 central proteins: the large polymerase L (250 kDa), the phosphoprotein P, the nucleoprotein N which surrounds the genomic RNA. Pneumoviruses also have transcription (M2-1) or replication (M2-2) co-factors. To synthesize the mRNAs or replicate the genome, the polymerase must temporarily access the viral RNA by "opening" the nucleocapsid. Mirroring, after synthesis of the new genomes, they will be encapsidated by neosynthesized N proteins. In this project, we want to elucidate the molecular mechanisms and regulation of this complex process by associating 4 teams with complementary skills and expertise: (1) an INRA team with the molecular tools to synthesize and purify N, P, M2-1 and L proteins in recombinant form as well as genomic RNAs. (2) an IBS team able to solve structures by electron microscopy and cryo-microscopy. (3) an IBMM team (CNRS, Montpellier University, ENSCM) specialized in the synthesis of modified and varied RNAs at the level of their sequence (methylation at different nucleoside sites) or at the 5 'end by adding various caps. (4) a CNRS-U Aix-Marseille team with the tools and know-how to measure enzymatic activities carried by L proteins. Our main specific objectives will be: To determine the mechanisms controlling the specificity (sequence, chemical nature) of encapsidation of the RNAs by the N proteins To characterize the atomic structure of the N-RNA protein complexes To characterize the epitranscriptomic modifications of the RNAs mediated by the L protein (capping and methylations). Ultimately, we want to develop an autonomous system of in vitro synthesis of RNAs that will permit studying the functioning of this complex and testing antiviral compounds targeting the replication / transcription steps in vitro.

Lung transplantation is the last therapeutic option of chronic respiratory diseases. Its management has improved due to the normothermic ex vivo lung perfusion (EVLP) which extends the number of suitable donor lungs. However the procedure should be improved for increasing the conversion rate from EVLP to transplantation to further expand the number of suitable grafts. Indeed standard EVLP with positive pressure ventilation drive to 1) pulmonary edema, 2) heterogeneity in the distribution of pulmonary ventilation and 3) strong modification of gene expression of inflammatory and stress pathways leading to failure of the procedure. Several therapies have been tested for mitigating the side effects and the inflammatory response during EVLP however the negative pressure ventilation (NPV) and the mobilization of the grafts (MG) are key points that we will focus in REVOLUTION. We propose to develop a new device bridging the gap on the standard EVLP strategy combining NPV and MG to assess the potential beneficial effects of the procedure. The prototype will be tested on a pig model for evaluating physiological parameters (ventilatory and hemodynamic) and inflammatory responses in different EVLP protocols. The screening methods will include Luminex/multiplex cytokine assays, LDH/lactate and ROS detection, immune-histo-fluorescence analyses, and bulk RNA-seq. The most promising protocol regarding physiological parameters and anti-inflammatory parameters will be tested on a preclinical human lung model. The cellular response will be analyzed by single cell RNA-seq in order to identify the functions and signaling pathways modified by standard EVLP in comparison to NPV + MG, at the cell subset level in human lung. The implementation of the REVOLUTION project will be conducted in optimal conditions by the rare combination of complementary expertise with biomedical engineers, thoracic surgeons, immunologists, bio-informaticians and statisticians. The new device developed during the REVOLUTION project is promising impact at the medical level by increasing the number and quality of grafts, at the socio-economic level by reducing the costs due to EVLP failures and by creation of a spin-off, and at the scientific level by an improved understanding of the biological response to ex vivo organ maintenance. The EVLP with the REVOLUTION device may be in the future the new gold standard for optimizing non-optimal lung and improving the results of transplantation.

The development of a safe and efficient RSV vaccine for infants in the first six months of life is a public health challenge for reducing the severe burden of respiratory diseases and hospitalizations. Globally, it is estimated that RSV causes > 30 million lower respiratory tract infections each year resulting in >3 million hospitalizations, making it the most common cause of hospitalizations in children under 5 years old. Since the failure of formalin-inactivated virus vaccines, a large array of alternative vaccination strategies (vaccine candidates and routes of administration) has been explored against RSV without providing satisfactory solutions. There are major challenges, unique to RSV, related to the young age of infection, the failure of natural infection to induce immunity that prevent reinfection and the risk of immune-mediated disease exacerbation. However, there are currently nor adequate treatment nor vaccine commercially available. The RSV-NanoViaSkin project aims to develop a pre-clinical proof of concept for an innovative efficient and safe pediatric RSV vaccine able to overcome all the barriers of current RSV vaccination strategies. Several innovations are gathered in the project to develop the epicutaneous RSV pediatric vaccine: 1) an original patented carrier derived from the viral nucleoprotein, produced and purified by the team of VIM-INRA which forms ring-shaped nanostructures (Nring) and is decorated with epitopes from the viral fusion protein (eF), and 2) an original patented epicutaneous delivery system (Viaskin®) loaded according to an innovative process, the electrospray, developed by DBV Technologies. In fact, the expected breakthroughs concern several parts of the project: o Novel immunogenic antigens (N-eF proteins), targeting CTL and neutralizing antibody mediated immunity to RSV, using well characterized immunogenic nanostructures (Nring) based on patented/published pre-clinical results, carried out by VIM-INRA o Innovative way to administer the vaccine: the epicutaneous route of administration using an original adapted technology (Viaskin®) able to overcome the hurdles of interference of maternal antibodies and the immaturity of the immune system. The new technology has the advantage that it does not require any preparation of the skin and adjuvant to facilitate the passage of the antigen through the skin. o An innovative loading process: the Electrospray allows loading of very small amounts of antigen protected from biological/physical degradation. This robust industrial process is already used in the production of cutaneous device. o Validation by preclinical tests of efficiency and safety in animal models adapted to the issues of epicutanous neonatal RSV vaccine (neonate mice, piglet skin, cotton rat, rabbit) This preclinical research will enable to define the clinical development strategy for the first non-invasive and adjuvant-free epicutaneous RSV pediatric vaccine that is able to protect the respiratory tract of newborns and infants against viral infection. The new approach will offer great benefits in terms of ease to use and painlessness compared to other pediatric vaccines in development. The project will associate industrial (DBV Technologies) and academic (VIM-INRA) partners to bring together all the complementary competences needed for the success of the project. The project will be conducted for 30 months. This project is the first step before entering in a clinical development process. The results will enable us to enter in the first-in-man clinical phase.

Chronic Wasting Disease (CWD) is a prion disease that affects wild and farmed cervids. CWD is a highly contagious: over the last 15 years the disease has spread across the whole United States of America and Canada. The CWD epidemics reached a stage where it now threatens the long term survival of cervid populations. Beside the ecological disaster it represents, major concerns exist with regard to the risk that CWD prions might represent for human (zoonosis) and other animal species (propagation in farmed ruminants) During three decades Europe was considered to be free of CWD. However, CWD cases have now been identified in three European countries. The goals of this project are: • to provide the necessary elements for an in depth assessment of the public health risks that are associated with the emergence of CWD prions in Europe. • to identify allele that would be associated with genetic resistance/ susceptibility to the disease in the cervid populations.