Therapeutic options in response to the SARS-Cov-2 outbreak are urgently needed and existing one are still limited. Facing pandemic development, actual strategy has been to repurpose some existing antiviral drugs used against respiratory diseases (SARS-Cov-1, MERS, Influenza) or chronic diseases (HIV-1, HCV, HBV, etc…). However, side effects are common and required short treatment mainly through intravenous injection, with a non optimal dose, and rarely through pulmonary route. A better formulation of such drug, adapted to this pulmonary disease, will allow an optimal efficacy of current drug or new candidates, such as protease inhibitors. Nanomedicine tools, such as used in HIV-1 treatment, could provide some solution by proposing innovative formulation, leading to the same efficacy with low dose of drugs and nasal/pulmonary delivery. Furthermore, nanoparticulate form favors drug stability, when present in aerosol via sprays or nebulizers. Thus, the CoviNanoMed project aims to formulate and test potential antiviral drugs or Host Targeted Agents using an existing robust nanoparticle platform in order to increase their potential efficacy, while diminishing their side effects. To this aim, we have identified six promising candidates, (non-exhaustive list which could be modulated according to ongoing clinical trials) and have gather four complementary research groups to: i) select the most promising for nanoformulation through in silico modeling and prepare reproducible and stable batch of nanodrugs ii) Assess their antiviral activities and toxicities using state of the art technique, adapted to their nanoparticulate form. It will allow us to classify them and to integrate in an iterative manner new ones according to collaborators iii) Compare nasal and pulmonary delivery in mice through devices used in clinical setting. We will use fluorescent particles and whole body imagine in mice and Non Human Primates to analyze the fate of particles in lung cells and tissue, and iv) Analyse drug release in bronchoalveolar fluids in mice and the most promising formulations in Non Human Primate. As this project concerns existing drugs, the analytical protocols and methods are already determined and will be quickly available. By this project, we expect to identify at least one promising candidate that could be quickly moved to clinical trial, as the biodegradable platform we propose will be considered as a new formulating excipient. Our system is solely based on poly–lactic acid (PLA), a biodegradable polymer approved by the FDA for medical devices such as sutures. It is therefore free of any controversial surfactant that frequently prevent the development of nanosystems to late phases. Additionally, the methodology we have set up could be adapted to new compounds that will emerge through ongoing clinical trials such as the multiple protease inhibitors that are undergoing studies.

NEO-COV-AM aims at developing a pediatric non-human primate (NHP) model of SARS-CoV-2 infection. We will use this model to study the mechanisms of infection, focusing on dynamics of viral replication and mucosal immunity in the respiratory tract and at sites, which also exhibit high expression of ACE2 receptor, like the gastrointestinal tract. Based on Partner 1 recent experience with SARS-CoV-2 infection in adult NHPs and compiled data collection, the consortium will characterize biomarkers of disease progression/resolution in infant vs adult. Key parameters of innate and adaptive immune cell responses will be studied longitudinally to understand SARS-CoV-2 immunopathology in pediatric populations and determine whether the immune tolerogenic environment is decisive in disease resolution. NEO-COV-AM will also study the changes in the gut and lung microbiota composition, which might modulate the differentiation of myeloid cells and distally impact innate immune defenses in the lung compartment. We will also work at establishing a model to investigate child to mother horizontal virus transfer while sharing a closed space for a long time. We surmise that such study is of primary importance to help understanding the mechanisms of SARS-CoV-2 infection and protection from severe disease in young infants. This pediatric preclinical model will help accelerating future treatments and vaccines developments, that need to demonstrate full safety and efficacy before their administration to young populations. It is therefore urgent to be prepared and respond quickly.

Multi-resistant bacteria regularly emerge throughout the world. They can be found in soldiers' wounds and in health care facilities where they cause nosocomial diseases. Some of these bacteria have become resistant to the best products available to treat multi-resistant bacteria. The consequences can be serious for the patients affected: prosthetic replacement, amputation or even death; but also for society with high induced costs. In general, there are no antibiotics currently available for clinicians for which no bacterial resistance has been reported. However, despite the introduction of 30 new antibiotics since 2000, the pipeline of new drugs for all pathogens is drying up. It is therefore essential to explore new approaches to the development of innovative antibacterial approaches. Innate immunity is the first-line barrier to prevent microbial invasion. The aim of this dual project is to develop multispecific proteins capable of stimulating cells of the innate immune system to inactivate two pathogenic bacteria of defence/civil interest. This innovative approach will help to reach bacteria regardless of their antibiotic resistance status and therefore those against which no effective antibiotic is available. It also offers the potential to be transposable to other pathogenic bacteria.

Despite extensive effort on vaccine development, protection against mucosally acquired viruses remains a difficult goal, most vaccines favoring systemic immune responses including T-cell immunity and IgG-based humoral responses. Indeed, to successfully protect against mucosal pathogens, vaccine strategies should combine the use of proper adjuvant and local immunization to trigger efficient and long lasting mucosal immune responses. We recently demonstrated that IL-7, secreted early and transiently after infection of mucosae, acts as a danger signal by inducing local expression of specific panels of chemokines that attracts immune cells at the infection sites. Similarly, locally administered IL-7 at the surface of vaginal mucosa or in the respiratory tract triggers local expression of chemokines and massive immune cell homing into these mucosae. Exploiting this property, VacMucIL7 project proposes that local delivery of IL-7, linked to poly(D, L-lactic acid) nanoparticles (PLA-NP) to favor mucosal barrier penetration, will prepare mucosae to further antigen delivery, improving immune responses to mucosal vaccines. We will establish the proof of concept of the IL-7/PLA-NP efficacy to adjuvant mucosal vaccines in mouse and non-human primate models of lung and vaginal infections (Influenza A and Herpes simplex viruses). In a first work package, we will study in details the immediate consequences of local administration of IL-7 in mice and define the best combinations of IL-7 and antigen, associated with PLA-NP to initiate a mucosal immune response using diphtheria toxoid and ovalbumin as model antigens in both the lungs and the vagina. The efficacy of these combinations will then be confirmed in non-human primates. Once established the best doses, time course and formulations for adequate mucosal vaccine delivery, we will test, both in mice and - non-human primate models, the efficacy of the best vaccine strategy to elicit protective mucosal immunity against respiratory tract infection with Influenza A virus or vaginal challenge with Herpes simplex virus type-2. The expected impacts of VacMuc7 project will be at different levels: - Develop an original approach to adjuvant mucosal vaccines by “preparing” the immunization site before vaccination using mucosal administration of a physiological dose of a natural molecule (IL-7) followed by local antigen delivery. This needle-free strategy will also avoid chemicals, presently disregarded by general population and participating to poor adhesion to vaccination campaigns. - Provide a comprehensive analysis of the IL-7 impact on mucosal immunity, both in lungs and the vagina, strengthening our knowledge concerning this particular arm of the immune system. Whether our approach of mucosal vaccination is successful to protect the animals not only against disease but, more importantly, against infection as a result of a quicker and better clearance at portals of entry of both airborne or sexually transmitted pathogens, it will be possible to adapt this strategy to the development of vaccines against other viral (HIV, Hepatitis…) and bacterial (Chlamydia…) infections that target mucosae and still represent major public health challenge. This project may also allow improving nanoparticles usage in vaccine development and permit elaborating new spray devices aimed at enhancing the efficiency of antigen availability/distribution at mucosal surfaces. VacMucIL7 project combines the complementary skills of 3 research teams, from mucosal immunity to nanomedicine and fully meets the requirements of the Axe 3.10 of the ANR program. Through a pre-clinical approach, we will develop an innovative adjuvant strategy to improve mucosal vaccines. Moreover, the molecular and cellular players implicated in the early phase of mucosal immune reactions will be studied in details not only in mice but also more importantly in non-human primates.

We have recently shown that glucose-dependent insulinotropic polypeptide (GIP)-dependent primary bilateral macronodular adrenal hyperplasia (PBMAH) with Cushing’s syndrome, is a genetic disease caused by germline inactivating mutations in lysine demethylase 1A (KDM1A or LSD1) with loss of heterozygosity of the second KDM1A locus in adrenal lesions, which occurs almost exclusively in women. KDM1A promotes a refractory chromatin state to gene expression by demethylating histone H3 on lysine 4 and can also activate gene transcription by demethylating histone H3 on lysine 9, or by interacting with transcription factors. We hypothesize that loss of KDM1A promotes the development of adrenal hyperplasia by activating the expression of sexually dimorphic genes involved in adrenocortical cell proliferation through chromatin remodeling associated with the loss of repressive histone marks. In mice, Kdm1a is required for pituitary organogenesis and pancreatic endocrine cell development. We therefore hypothesize that KDM1A is also required for the transition from fetal to adult adrenal cortex that occurs specifically in primates. Kdm1a is a master controller of gene transcription in spermatogonia and is essential for the maintenance and differentiation of spermatogonial stem cells in mice, but its involvement in (later stages of) spermatogenesis in men is unknown. We hypothesize, that the absence of functional KDM1A occurring during meiosis in 50% of spermatozoids in KDM1A-mutation carriers alters their H3K4 methylation marks and other sperm histones. Our objectives are to: 1) to decipher the molecular mechanism(s) by which loss of KDM1A promotes adrenal hyperplasia and investigate the underpinnings of the sex bias in this disease, with a special focus on the potential misexpression of the X-linked genes at single-cell resolution. 2) to study the expression and function of KDM1A and GIPR during adrenal cortex development in cortex of cynomolgus macaques, by performing repeated echo-guided adrenal gland biopsies in utero in 4 fetuses at 18 weeks gestational age, and further postnatally at 1-2 week and at 6 months of life. Prior to these biopsies, we will investigate the cortisol response to GIP injection in vivo. 3) to investigate adrenal function and morphology in apparently healthy carriers of KDM1A mutation and analyze histone H3 methylation in their sperm. We plan to genotype > 120 family members of patients with GIP-dependent PBMAH from our cohort, and to perform biochemical screening and adrenal CT scans in KDM1A-mutation carriers to describe the prevalence and the natural history of adrenal hyperplasia and multiple myeloma. If sperm from KDM1A-mutation carriers show altered H3K4 methylation, we will attempt to link the observed variations to specific genomic loci. This project is consistent with governmental plans and strategic research priorities for 2023, as it develops “Translational research in rare diseases”. The use of complementary experimental approaches including studies on adrenal samples derived from patients, cell lines, mouse models, and non-human primates, as well as clinical investigations in patients, illustrates the translational nature of the project. Our project will provide new insights into a key question in cell biology and medicine: how (adrenal) cells maintain a specific transcriptomic pattern that defines their identity during development and in pathological situations, focusing on KDM1A as an epigenetic regulator of cell fate determination. Genetic and biochemical screening in families of patients with KDM1A mutation is important, as it will allow for earlier detection of adrenal hyperplasia and prevent the insidious effects of progressive cortisol excess on health. The feasibility of the project is underlined by published and preliminary results and the complementarity of the consortium members, who are renowned researchers in their field.