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The in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we used single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induced transcriptional shifts by antigenic stimulation in vitro and took advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for reverse phenotyping. This allowed identification of SARS-CoV-2-reactive TCRs and revealed phenotypic effects introduced by antigen-specific stimulation. We characterized transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and showed correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.
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BackgroundDue to the ongoing COVID-19 pandemic, demand for diagnostic testing has increased drastically, resulting in shortages of necessary materials to conduct the tests and overwhelming the capacity of testing laboratories. The supply scarcity and capacity limits affect test administration: priority must be given to hospitalized patients and symptomatic individuals, which can prevent the identification of asymptomatic and presymptomatic individuals and hence effective tracking and tracing policies. We describe optimized group testing strategies applicable to SARS-CoV-2 tests in scenarios tailored to the current COVID-19 pandemic and assess significant gains compared to individual testing. MethodsWe account for biochemically realistic scenarios in the context of dilution effects on SARS-CoV-2 samples and consider evidence on specificity and sensitivity of PCR-based tests for the novel coronavirus. Because of the current uncertainty and the temporal and spatial changes in the prevalence regime, we provide analysis for a number of realistic scenarios and propose fast and reliable strategies for massive testing procedures. FindingsWe find significant efficiency gaps between different group testing strategies in realistic scenarios for SARS-CoV-2 testing, highlighting the need for an informed decision of the pooling protocol depending on estimated prevalence, target specificity, and high-vs. low-risk population. For example, using one of the presented methods, all 1{middle dot}47 million inhabitants of Munich, Germany, could be tested using only around 141 thousand tests if the infection rate is below 0{middle dot}4% is assumed. Using 1 million tests, the 6{middle dot}69 million inhabitants from the city of Rio de Janeiro, Brazil, could be tested as long as the infection rate does not exceed 1%. InterpretationAltogether this work may help provide a basis for efficient upscaling of current testing procedures, taking the population heterogeneity into account and fine grained towards the desired study populations, e.g. cross-sectional versus health-care workers and adapted mixtures thereof. FundingGerman Science Foundation (DFG), German Federal Ministry of Education and Research (BMBF), Chan Zuckerberg Initiative DAF and Austrian Science Fund (FWF). Research in contextO_ST_ABSEvidence before this studyC_ST_ABSThe concept of group testing goes back to mathematical ideas developed in the 1940s and has already been successfully implemented for various infectious diseases but also in non-medical settings such as testing for failures of electronic components. The issue of group testing for SARS-CoV-2 has been addressed in a number of very recent papers and preprints including feasibility studies of different laboratories and a few methodological overview papers. Nevertheless, to the best of our knowledge, no study provided a comprehensive comparison contrasting hierarchical testing, array testing, and informative testing strategies based on combined groups for stratified populations and relying on up-to-date data about the accuracy of PCR-based test - all of them feasible to be implemented in the current pandemic. Moreover, a discussion of massive informative testing strategies for pandemic scenarios, employing combined pools consisting of high-risk and low-risk individuals, was still missing in the public health literature. Added value of this studyWe analyse sensitivity, specificity, and throughput of group testing schemes in a series of scenarios tailored to realistic prevalence rates for SARS-CoV-2 in stratified populations and to the characteristics of the qRT-PCR tests used to diagnose COVID-19. Our analysis yields a comprehensive characterisation of a wide range of pooling schemes, broken down by population characteristics, that can serve as a guideline to be queried by testing entities to meet their settings. In particular, our findings demonstrate that a promising strategy to test asymptomatic or presymptomatic individuals in conjunction with high-risk individuals such as healthcare workers is to amalgamate them together in a suitable way, and we provide adequate pool configurations. Such strategies had not been explored for SARS-CoV-2 testing as of yet. We also provide insights on group testing under constraints, i.e. when the number of stages, maximum group size and false negative rate of the whole method are restricted to a certain range of realistic values. We introduce efficient paralleliz-able non-adaptive test procedures for simplified and fast large-scale test design in case of severe shortage of test components. We develop an intuitive web application that can be used by any researcher working on the front line of testing procedures to visualize all the different strategies and to design pooling schemes in an flexible manner according to their specific prevalence scenario and test configuration. Implications of all the available evidenceTesting for SARS-CoV-2 presents new challenges to authorities such as rapidly changing prevalence estimates and bottlenecks in testing capacity. In this context, applying an appropriately chosen group testing procedure can allow for up to 10-fold increase of the feasible throughput, such that the existing testing capacities can be used to test a significantly increased number of individuals. As a consequence, adequate tracing and quarantine strategies to reduce community transmission can be established and valuable epidemiological studies relying on accurate prevalence rates can be performed for the ongoing pandemic situation.
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The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, creates an urgent need for identifying molecular mechanisms that mediate viral entry, propagation, and tissue pathology. Cell membrane bound angiotensin-converting enzyme 2 (ACE2) and associated proteases, transmembrane protease serine 2 (TMPRSS2) and Cathepsin L (CTSL), were previously identified as mediators of SARS-CoV2 cellular entry. Here, we assess the cell type-specific RNA expression of ACE2, TMPRSS2, and CTSL through an integrated analysis of 107 single-cell and single-nucleus RNA-Seq studies, including 22 lung and airways datasets (16 unpublished), and 85 datasets from other diverse organs. Joint expression of ACE2 and the accessory proteases identifies specific subsets of respiratory epithelial cells as putative targets of viral infection in the nasal passages, airways, and alveoli. Cells that co-express ACE2 and proteases are also identified in cells from other organs, some of which have been associated with COVID-19 transmission or pathology, including gut enterocytes, corneal epithelial cells, cardiomyocytes, heart pericytes, olfactory sustentacular cells, and renal epithelial cells. Performing the first meta-analyses of scRNA-seq studies, we analyzed 1,176,683 cells from 282 nasal, airway, and lung parenchyma samples from 164 donors spanning fetal, childhood, adult, and elderly age groups, associate increased levels of ACE2, TMPRSS2, and CTSL in specific cell types with increasing age, male gender, and smoking, all of which are epidemiologically linked to COVID-19 susceptibility and outcomes. Notably, there was a particularly low expression of ACE2 in the few young pediatric samples in the analysis. Further analysis reveals a gene expression program shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues, including genes that may mediate viral entry, subtend key immune functions, and mediate epithelial-macrophage cross-talk. Amongst these are IL6, its receptor and co-receptor, IL1R, TNF response pathways, and complement genes. Cell type specificity in the lung and airways and smoking effects were conserved in mice. Our analyses suggest that differences in the cell type-specific expression of mediators of SARS-CoV-2 viral entry may be responsible for aspects of COVID-19 epidemiology and clinical course, and point to putative molecular pathways involved in disease susceptibility and pathogenesis.
