ICARE

The recent outbreak of pneumonia-inducing and potentially lethal COVID-19 has become a major world-wide public health issue with dramatic societal and economical consequences. The SARS-CoV-2-inducing COVID-19 belongs to the Betacoronavirus family, and is close to the previously emerged SARS-CoV and MERS-CoV. The knowledge of initial cell targets in a viral infection as well as the early cell response are key elements in the understanding of the physio-pathogenesis of the induced disease. SARS-CoV and MERS-CoV interact with different types of receptors and co-receptors and present incompletely characterized tropisms for lung epithelial cells, as well as for monocyte/macrophages and dendritic cells, which are major cells bridging innate to adaptive and defensive immunity. The tropism of these viruses has been inferred from autopsy late in infection when multi-organ failure is reached, from in vitro cell cultures and at best, from organ fragment cultures, which do not recapitulate the complexity of the host-viral interaction in the whole lung structure. In ICARE, we propose to infect with SARS-CoV-2 whole human lung in the ex vivo lung perfusion (EVLP) system, which has been established to recondition marginal lungs for transplantation; this model offers a unique opportunity to identify the initial cell type targets in the most physiologically possible way in humans. Lungs refused for clinical transplantation will be used and infected by SARS-CoV-2 in our BSL3+ facility. In order to identify the initial targeted cell types and their early response, biopsies will be collected at 0H and 10H post infection and subjected to cell dissociation for single cell RNA-seq (scRNA-seq) using the total cell fraction and fractions enriched for monocyte/macrophages and dendritic cells. The different cell types will be identified from gene expression signatures which are characteristic of known subsets of epithelial cells (bronchial or alveolar cells), monocyte/macrophages and dendritic cells and viral RNA types will be analyzed in the different subsets. The viral tropism analysis will be completed by viral protein detection in immuno-histo-fluorescence of cleared tissue-biopsies and biphotonic microscopy. Finally the functional genomic analysis of the scRNA-seq will reveal the early innate response in the different lung subsets. Altogether ICARE will provide the proof of concept that EVLP can be used to provide highly relevant information on SARS-CoV-2 cell tropism and response, with possible extension to other SARS strains and respiratory viruses of different pathogenicity. The knowledge of the different cell targets and their relative importance may help to fight the disease by guiding prophylactic and therapeutic strategies aiming at blocking viral entry in key cell types. Additionally the early response to the infection, at the cell subset level, may reveal mechanisms implicated in viral escape to host control and mechanisms leading to disease severity which might be targetable by adapted treatments.