The novel coronavirus SARS-CoV-2 has recently emerged as a human pathogen in China’s Hubei province, causing severe respiratory illness and pneumonia. According to the World Health Organization (WHO) on March 17th, 2020, there have been over 180,000 cases globally, leading to at least 7,500 deaths. The development of a vaccine will take at least one/two year(s) and the production of a sufficient number of vaccinal doses to protect the world population will represent a big industrial challenge. Thus, there is an urgent need to develop antivirals targeting SARS-CoV-2 and relatives. The objective of the project is to generate nanobinders (nanobodies/VHH and highly stable artificial proteins called aREP) targeting the spike and able to neutralize SARS-CoV-2 in vitro and protect from an infection in an animal infection model. For this goal, we will specifically target its receptor binding domain (RBD) that is known to induce high-titer neutralizing antibodies in others coronaviruses and to fold independently from other spike domains. Neutralizing nanobodies/VHH will be generated after alpaca immunization with the SARS-CoV-2 RBD and neutralizing aREP will be isolated from several independent libraries. Next, biological properties of the VHH and aREP will be characterized (neutralization activity, binding affinity to the (highly glycosylated) spike, thermo-stability, resistance to trypsin and to other proteases present in the lung) and sequence-optimized (VHH sequence humanization, construction of VHH-Fc and aREP-Fc to functionalize them for the immune system,…). Several highly potent candidates will be selected to analyze their prophylactic and therapeutic efficacies (through the intranasal route) in a transgenic mice infection model. Thus, we expect that this general approach targeting the spike RBS will allow the obtention of antivirals or hits for antivirals able to inhibit or block SARS-CoV-2 multiplication in the lung.

With increasing global population, feeding the world in a sustainable manner is a major challenge. The fast-growing fish farming industry is playing a vital role by providing healthy nutrients while stimulating local economy and preserving wild fish stocks. Fish infectious diseases are one of the major bottlenecks for aquaculture development and new strategies for maintaining health while avoiding antibiotics are highly needed. The bacterial pathogen Flavobacterium psychrophilum is the etiological agent of bacterial cold-water disease (BCWD). In rainbow trout, a septicaemic form of the disease severely affects fry, leading to high mortality that often rises up to 70%. The bacterium is able to survive in stream water for months while preserving its virulence, a property that likely favours the recurrent outbreaks observed in fish farms. Nowadays F. psychrophilum is one of the main pathogenic bacteria that French fisheries have to face. Due to the difficulty to develop efficient vaccines, treatments are exclusively based on the oral administration of antibiotics and there is a need for alternative preventive strategies. Bacterial genomics, molecular epidemiology and host transcriptomics studies allowed important progress regarding both the pathogen and host sides of BCWD, however much work remains to be done to understand F. psychrophilum biology and studies reporting the characterization of virulence factors are still scarce. In this context, FlavoPatho aims to investigate the underlying bacterial and host factors favouring pathogenesis of the fish pathogen F. psychrophilum. How bacteria adapt to long-term starvation in water, invade and colonize fish are the main issues that will be addressed by a multidisciplinary approach combining in vivo imaging, transcriptomics and comparative genomics of in vitro evolved bacterial strains. The infectious process will be monitored using bioluminescent imaging to identify the portals of entry and to track colonization of bath-infected rainbow trout. The expression pattern of F. psychrophilum will be analysed under starvation in water to characterize molecular determinants of this adaptation process, expected to play a key role in the recurrent fish farms disease outbreaks. We will also monitor simultaneously expression profiles of the pathogen and of its infected host by dual RNA sequencing using a rainbow trout infection model. This approach will allow establishing a list of genes putatively linked to virulence, and in vivo-induced surface-exposed proteins may represent promising targets for future vaccines. In parallel, we will run adaptive evolution experiments by in vitro sub-cultivations of F. psychrophilum isolates. By combining phenotypic and genotypic data, we will explore functional links between genes and specific physiological traits of F. psychrophilum, with a particular focus on pathogenicity. The in vitro evolved lineages that lost their virulence will be characterised by comparative genomics to identify relevant genetic markers. Several bacterial isolates representative of the two clonal-complexes reported to be dominant in French fish farms will be studied in the project. With the help of advanced research, FlavoPatho will generate fundamental results and significant insight into the molecular traits underlying pathogenesis for the etiological agent of BCWD, an important infectious disease affecting aquaculture worldwide. The project will contribute to describe the infectious process and to decipher mechanisms and determinisms of pathogenesis. By providing basic knowledge on the main factors favouring pathogenesis and outbreaks, FlavoPatho will bring perspectives for the development of promising preventive alternatives for a more sustainable fish industry. By implementing cutting-edge methodologies, the project will also bring knowledge beyond BCWD that will certainly benefit the whole field of bacterial infectious diseases.

By the end of 2021, there likely will be hundreds of millions of people infected with COVID-19 worldwide. The loss of smell (anosmia) will affect more than half of them. While most of them will rapidly recover their sense of smell, about 10% will not 6 months after the onset of the disease. Thus millions of people will suffer of long lasting anosmia linked to COVID-19. As this sense is among others very important for food intake, its loss will have a very important impact on life quality. While the cellular events leading to anosmia begin to be unravelled, its origin remains to be explored. The ability to detect odours starts in the olfactory neuroepithelium localized in the dorsal part of the nasal cavity. This neuroepithelium is composed mainly of olfactory neurons detecting odours, supportive cells and basal cells enabling a continuous regeneration of the epithelium. Among these cells, only the supportive cells express the receptors required for SARS-CoV-2 infection. Using the golden Syrian hamster as a model, it was shown that only 2 days after nasal instillation of SARS-CoV-2, supporting cells are massively infected by the virus. Furthermore, the olfactory epithelium structure is profoundly altered by the virus presence with most of the epithelium being desquamated. Along the disorganization of the olfactory epithelium, there is an important invasion of immune cells. The role of the immune cells in this process is not explored yet. Are they actively participating in the elimination of the infected cells? Are they present following cellular death induced by the virus to ensure its clearance? Following the disorganization of the olfactory epithelium induced by SARS-CoV-2 infection, it regenerates rapidly thanks to basal cells. After 14 days, approximately 50% of the epithelium is recovered and it is almost completely repaired in 21 days. This regeneration kinetic of the olfactory epithelium is consistent with the fast recovery of most COVID-19 related anosmia occurring for 40% of the patients in less than two weeks. However it cannot explain why 10% of these patients are still suffering from olfactory disturbance at 6 months following the onset of the disease. Clinicians treat the COVID-19 anosmic patients empirically by corticoids and/or by olfactory training but without any evidence of the effectiveness of these treatments on olfactory epithelium recovery. As corticoid treatments are known to reduce inflammation, their use could limit the desquamation of the olfactory epithelium if immune cells are active in this process. However, immune cells are also known to help the regeneration of the olfactory epithelium and the use of corticoid may thus disturb the recovery of olfaction. Using golden Syrian hamster as a model, the present project aims at: 1/Understanding the role of the immune system in the desquamation of the olfactory epithelium. 2/ Explore precisely the kinetic of the olfactory epithelium recovery from basal cells. 3/ Study the efficiency of corticoid treatment and/or olfactory training in order to improve the recovery from anosmia. Our study will improve our knowledge of the importance of immune cells in the COVID-19 related anosmia and its recovery as well as the efficiency of currently used treatment at a cellular level.

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.

Human Respiratory Syncytial Virus (RSV) is the main viral agent causing bronchiolitis in young children and a major pathogen of elderly and immunocompromised people, responsible for acute respiratory infections. Despite this burden, no vaccine or effective treatment against RSV is currently available. The RSV genome replication and transcription take place in cytoplasmic viral factories (VFs) where viral and cellular proteins involved in these activities concentrate. The genome, encapsidated by the nucleoprotein N into a helical nucleocapsid (NC), serves as template for the viral RNA synthesis by the polymerase L, its phosphoprotein cofactor P and the elongation factor M2-1. Together, the helical NC, P, L and M2-1 form the ribonucleoprotein (RNP) responsible for viral RNA synthesis. Although the structures of these proteins have been solved and the protein-protein interactions involved in the polymerase functioning characterised, the molecular architecture of the RNPs within VFs and the structural states of the NCs supporting the distinct activities of the polymerase remain unknown. Here, we propose to unravel the nanoscale organisation of the RSV VFs and the different functional states of the NCs by a parallel investigation of NCs in VFs and pseudo-VFs inside the cell, purified or reconstituted in vitro. This goal is challenging due to the inherent compositional heterogeneity, dynamics and environment of the VFs, and conformational flexibility of the NCs. We aim to achieve it by using an original multi-scale approach that relies on complementary state-of-the-art techniques such as advanced cellular imaging, cryo-CLEM, cryo-FIB/SEM, cryo-ET, cryo-EM, FAPS, and biochemical and biophysical characterisation. The project outcome will improve our understanding of the regulation of the RSV RNAs synthesis and contribute to innovative rational design of new antivirals.