Résumé: SARS-CoV-2 is a betacoronavirus that has recently emerged as a human pathogen in the city of Wuhan in China’s Hubei province. The disease caused by this newly identified virus has been named COVID-19 and symptoms include fever, severe respiratory illness and pneumonia. As of March 2020, the World Health Organization (WHO) has declared that SARS-CoV-2 is pandemic, and the number of confirmed cases is exponentially increasing. SARS-CoV-2 virus is closely related to SARS-CoV, which was responsible for the Severe Acute Respiratory Syndrome (SARS) in 2002. Similarly, to SARS-CoV, SARS-CoV-2 is of zoonotic origin and was demonstrated to cause life-threatening diseases in humans. So far, there is no vaccine or treatment for COVID-19. It is therefore critical to generate vaccines and drugs that will either prevent or treat COVID-19. At present, there are 8 candidate vaccines in clinical evaluation and more than 100 candidate vaccines in preclinical evaluation. Vaccine in clinical trials are represented by inactivated SARS-CoV-2 virus, non-replicating viral vectors, DNA and RNA vaccines (https://www.who.int). Surprisingly, no protein subunit vaccine are reported to be tested currently in clinical trial, as proposed in this project, which are generally safer and easier to produce. Moreover, all Human coronaviruses enter their host cells using the trimeric transmembrane spike (S) glycoprotein. The coronavirus’ S protein represents a major target for the human humoral immune response following SARS-CoV-2 infection. However, a large set of data from previous SARS-CoV-1 or CoV-2 infected individuals, or generated in preclinical models, pointed out the potential protective effect of cellular immunity. In this study, we propose to test the immunogenicity and the preventative effect of a combination of two vaccine platforms already in phase 1 to 3 clinical development; i.e the DNA-derived DREP platform and the anti-Dendritic cell (DC) targeting epitope-based vaccine. A series of DREP and DC-targeting constructs against SARS-CoV-2 are already available. A large set of data showed that these vaccines, either administered alone, or in a prime boost combination, elicited strong and durable T and B-cell immune responses against infectious agents. We develop here an original strategy aimed to induce a polyepitopic T and B cell responses. These vaccines are ready to be tested in two preclinical models, in humanized mice (mice reconstituted with a human immune system), allowing to study in depth human immune responses of different vaccine combinations, and in transgenic knock-in mice expressing the human CD40 and human ACE2, the receptor of SARS-CoV-2 to demonstrate the protective effect of these vaccines. The overreaching goal of this study is to identify within the 12-months time line of this project, vaccine (s) that will be moved forward to the clinical development.

In order to understand what causes severe cases of Covid19, and therefore to improve their medical management, it is essential to better understand the natural history of this disease caused by the SARS-CoV2 coronavirus. Specifically, we propose to solve the puzzle of the much debated role of type I interferons (IFN-I) and of their major cellular source during many viral infections, namely plasmacytoid dendritic cells (pDCs). We will take advantage of mutant mouse models that can be infected with Covid-Cov2 SARS, develop a disease very similar to Covid19, and have been modified for tracing or inactivating pDCs in vivo and for following IFN-I-producing cells with a high degree of resolution. This approach will allow us testing the hypothesis that IFN-I and pDCs play either beneficial or deleterious roles, depending on their activation dynamics in the first days following infection; this activation dynamics being itself dependent on the infectious dose of the virus received or on the initial efficacy of viral control.

Young mice show no pronounced pathology upon SARS-CoV-1 infection whereas ubiquitous expression of the human angiotensin-converting enzyme 2 (hACE2), the SARS-CoV-1 receptor, via transgenesis enhances clinical signs of disease in young mice. The interest for mouse models for SARS-CoV-1 infection faded away due to SARS-CoV-1 eradication. The few laboratories that still host them are overwhelmed with requests and cannot meet the need generated by the SARS-CoV-2 pandemic. Moreover, the level and tissue distribution of hACE2 are poorly controlled in the existing transgenic hACE2 models. The Centre d’Immunophénomique (CIPHE; INSERM US12, CNRS/AMU UMS3367) has a long history of expertise in the development of gene-edited preclinical mouse models and in the high-content analysis of the function of their immune cells. Four teams of CIPHE coordinated by B. Malissen propose to develop in parallel and in a fast-track mode (11 months) a resource of five humanized mouse models expressing hACE2 at different levels and in a tissue-specific or ubiquitous manner for testing vaccines and antiviral therapies for SARS-CoV-2, and to further our knowledge of the physiopathology of COVID-19 disease. CIPHE will immediately made available this resource to the scientific community by expanding them under a specific pathogen-free format and depositing them to the EMMA European repository. CIPHE operates a Biosafety Level 3 laboratory capable of hosting up to 500 cages of mice infected with respiratory viruses. Through a collaboration with Pr. B. La Scola (IHU Mediterranée Infection, Marseille), the hAC2-expressing mouse models will be infected with SARS-CoV-2 virus isolates to evaluate whether they are permissive to robust SARS-CoV-2 infection and support severe respiratory and generalized illness. Morbidity, mortality and distribution of SARS-CoV-2 studies will be conducted. The blood, primary and secondary lymphoid organs, and barrier organs will be subjected to high dimensional immunophenotyping and up to 23 inflammatory cytokines and chemokines measured using Bio-Plex cytometric bead array. The fast-track gene editing approach to be specifically used to construct the five hACE2-expressing mouse model has been developed with the laboratory of Y. Liang at Xinxiang Medical University (Henan Province, China), and our ongoing collaboration will help speeding up the development of the proposed resource. Due to the demand of the scientific community, the first model to be developed corresponds to mice expressing hACE2 in a ubiquitous manner. Mice ubiquitously overexpressing a catalytically inactive form of hACE2 will be also developed as a backup. In absence of enzymatic activity, the overexpressed hACE2 molecules will not affect the action of the mouse ACE and ACE2. Little is known about the human cell types co-expressing the SARS-CoV-2 receptor ACE2 and the TMPRSS2 serine protease that cooperate to viral entry. In lungs of mice intranasally infected with a mouse-adapted SARS-CoV-1 virus, viral antigens were heavily distributed in type I and II pneumocytes. A recent series of scRNAseq studies involving human subjects strongly suggests that airway goblet secretory cells and club cells also co-express ACE2 and the proteases TMPRSS2 are thus potentially highly vulnerable for SARS-CoV-2 infection. These findings constitute the rationale basis for the development of 3 additional mouse models specifically expressing hACE2 in type I and II pneumocytes and in lung club cells. The 5 hACE2 mouse models have been prioritized to meet the expected need of the scientific community. However, to take in account that several human proteins cooperate with hACE2 for virus entry, the integrated CIPHE pipeline should permit to coincidently develop mice expressing hACE2 together with candidate human co-receptors as well as mice expressing hACE2 in goblet cells and validate their response to SARS-CoV-2.

Type I interferons (IFN-I) are key cytokines in vertebrate antiviral defense. However, if excessive or chronic, their production can contribute to the pulmonary immunopathology caused by respiratory infections with influenza (Flu) or coronaviruses. Treatment with glucocorticoids induces a broad immunosuppression at the risk of increased viral replication or susceptibility to other infections. More specific treatments may be achieved by selective manipulation of IFN-I production or responses. This implies determining the deleterious versus beneficial cellular sources and functions of IFN-I. IFN-I can be produced from infected cells and plasmacytoid dendritic cells (pDC). pDC produce high levels of all IFN-I, without being infected therefore escaping inhibition by viral immune evasion genes. Therefore, pDC are considered to be crucial for antiviral defense. Yet, in human and mouse infections with Flu or SARS-CoV2, whether IFN-I and their production by pDC are beneficial or deleterious is debated. This controversy is in part due to lack of tools for specific and penetrant targeting of pDC. We have overcome this bottleneck by generating pDC-less mice, constitutively and specifically lacking pDC, and harnessing them for conditional gene inactivation in pDC. These innovative approaches enabled us demonstrating a deleterious role of pDC in Flu and Covid19. To understand the underlying mechanisms, we will: 1) determine how pDC contribute to severe immunopathology during Flu infection, 2) test whether viral restriction by Mx1 mitigates IFN-I/pDC deleterious effects in Flu infection, and 3) dissect how IFN-I protects against SARS-CoV2 despite pDC redundant/deleterious functions. Our project will contribute characterizing cellular and molecular actors contributing to the hyperactivation/misfiring of IFN-I responses causing severe lung immunopathology during respiratory viral infections, hence providing novel levers to manipulate immune responses to promote health.

SARS-CoV-2 entry into host cells depends on binding of the viral spike (S) protein with the surface receptor ACE2 and subsequent priming by TMPRRS2 allowing membrane fusion. Soluble recombinant ACE2 was proven to bind to the S protein, and reduce SARS-CoV-2 entry in Vero-E6 cells and engineered human organoids. ACE2, however, is synthesized as a transmembrane protein. In a proof-of -concept study, we have demonstrated that ACE2 on the surface of extracellular vesicles (EVs) results in better efficacy as decoy to capture SARS-CoV-2-Spike containing lentivirus as compared with recombinant ACE2 ectodomain. Following up on these results, the proposed project aims at developing an innovative approach to obtain second generation EVs bearing the human receptor for the SARS-CoV-2 virus (hACE2). Engineered EVs will be used in vitro and in vivo to prevent initial infection or further internal dissemination of the virus and its variants, and thus improve the outcome of the infection