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Antibodies can have beneficial, neutral, or harmful effects so resolving an antibody repertoire to its target epitopes may explain heterogeneity in susceptibility to infectious disease. However, the three-dimensional nature of antibody-epitope interactions limits discovery of important targets. We describe and experimentally validated a computational method and synthetic biology pipeline for identifying structurally stable and functionally important epitopes from the SARS-CoV-2 proteome. We identify patterns of epitope-binding antibodies associated with immunopathology, including a non-isotype switching IgM response to a membrane protein epitope which is the strongest single immunological feature associated with severe COVID-19 to date (adjusted OR 72.14, 95% CI: 9.71 - 1300.15). We suggest the mechanism is T independent B cell activation and identify persistence (> 1 year) of this response in individuals with long COVID particularly affected by fatigue and depression. These findings highlight a previously unrecognized coronavirus host:pathogen interaction which is potentially an upstream event in severe immunopathology and this may have implications for the ongoing medical and public health response to the pandemic. The membrane protein epitope is a promising vaccine and monoclonal antibody target which may complement anti-spike vaccination or monoclonal antibody therapies broadening immunological protection. One-Sentence SummaryUsing a novel B cell epitope discovery method we have identified antibody signatures strongly associated with SARS-CoV-2 immunopathology and suggest the membrane protein is a pathological T independent antigen.
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Circulating microRNAs (miRNAs) are exceptional mechanism-based correlates of disease, yet their potential remains largely untapped in COVID-19. Here, we determined circulating miRNA and cytokine and chemokine (CC) profiles in 171 blood plasma samples from 58 hospitalised COVID-19 patients. Thirty-two miRNAs were differentially expressed in severe cases when compared to moderate and mild cases. These miRNAs and their predicted targets reflected key COVID-19 features including cell death and hypoxia. Compared to mild cases, moderate and severe cases were characterised by a global decrease in circulating miRNA levels. Partial least squares regression using miRNA and CC measurements allowed for discrimination of severe cases with greater accuracy (87%) than using miRNA or CC levels alone. Correlation analysis revealed severity group-specific associations between CC and miRNA levels. Importantly, the miRNAs that correlated with IL6 and CXCL10, two cardinal COVID-19-associated cytokines, were distinct between severity groups, providing a novel qualitative way to stratify patients with similar levels of proinflammatory cytokines but different disease severity. Integration of miRNA and CC levels with clinical parameters revealed severity-specific signatures associated with clinical hallmarks of COVID-19. Our study highlights the existence of severity-specific circulating CC/miRNA networks, providing insight into COVID-19 pathogenesis and a novel approach for monitoring COVID-19 progression.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulting in the clinical syndrome COVID-19 is associated with an exaggerated immune response and monocyte infiltrates in the lungs and other peripheral tissues. It is now increasingly recognised that chronic morbidity persists in some patients. We recently demonstrated profound alterations of monocytes in hospitalised COVID-19 patients. It is currently unclear whether these abnormalities resolve or progress following patient discharge. We show here that blood monocytes in convalescent patients at their 12 week follow up, have a greater propensity to produce pro-inflammatory cytokines TNF and IL-6, which was consistently higher in patients with resolution of lung injury as indicated by a normal chest X-ray and no shortness of breath (a key symptom of lung injury). Furthermore, monocytes from convalescent patients also displayed enhanced levels of molecules involved in leucocyte migration, including chemokine receptor CXCR6, adhesion molecule CD31/PECAM and integrins VLA-4 and LFA-1. Expression of migration molecules on monocytes was also consistently higher in convalescent patients with a normal chest X-ray. These data suggest persistent changes in innate immune function following recovery from COVID-19 and indicate that immune modulating therapies targeting monocytes and leucocyte migration may be useful in recovering COVID-19 patients with persistent symptoms.
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BackgroundEarly clinical reports have suggested that the prevalence of thrombotic complications in the pathogenesis of COVID-19 may be as high as 30% in intensive care unit (ICU)-admitted patients and could be a major factor contributing to mortality. However, mechanisms underlying COVID-19-associated thrombo-coagulopathy, and its impact on patient morbidity and mortality, are still poorly understood. MethodsWe performed a comprehensive analysis of coagulation and thromboinflammatory factors in plasma from COVID-19 patients with varying degrees of disease severity. Furthermore, we assessed the functional impact of these factors on clot formation and clot lysis. ResultsAcross all COVID-19 disease severities (mild, moderate and severe) we observed a significant increase (6-fold) in the concentration of ultra-large von Willebrand factor (UL-VWF) multimers compared to healthy controls. This is likely the result of an interleukin (IL)-6 driven imbalance of VWF and the regulatory protease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13). Upregulation of this key pro-coagulant pathway may also be influenced by the observed increase (~6-fold) in plasma -defensins, a consequence of increased numbers of neutrophils and neutrophil activation. Markers of endothelial, platelet and leukocyte activation were accompanied by increased plasma concentrations of Factor XIII (FXIII) and plasminogen activator inhibitor (PAI)-1. In patients with high FXIII we observed alteration of the fibrin network structure in in vitro assays of clot formation, which coupled with increased PAI-1, prolonged the time to clot lysis by the t-PA/plasmin fibrinolytic pathway by 52% across all COVID-19 patients (n=23). ConclusionsWe show that an imbalance in the VWF/ADAMTS13 axis causing increased VWF reactivity may contribute to the formation of platelet-rich thrombi in the pulmonary vasculature of COVID-19 patients. Through immune and inflammatory responses, COVID-19 also alters the balance of factors involved in fibrin generation and fibrinolysis which accounts for the persistent fibrin deposition previously observed in post-mortem lung tissue. What is new?O_LIIn all COVID-19 patients, even mild cases, UL-VWF is present in plasma due to the alteration of VWF and ADAMTS13 concentrations, likely driven by increased IL-6 and -defensins. C_LIO_LIIncreased plasma FXIII alters fibrin structure and enhances incorporation of VWF into fibrin clusters. C_LIO_LIDefective fibrin structure, coupled with increased plasma PAI-1 and 2-antiplasmin, inhibits fibrinolysis by t-PA/plasmin. C_LI What are the clinical implications?O_LIProphylactic anticoagulation and management of thrombotic complications in COVID-19 patients are ongoing challenges requiring a better understanding of the coagulopathic mechanisms involved. C_LIO_LIWe have identified FXIII and VWF as potential therapeutic targets for treating fibrin formation defects in COVID-19 patients. C_LIO_LIWe have identified a multifaceted fibrinolytic resistance in COVID-19 patient plasma with potential implications in the treatment of secondary thrombotic events such as acute ischaemic stroke or massive pulmonary embolism. C_LI
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BackgroundThe pathogenesis of COVID-19, caused by a novel strain of coronavirus (SARS-CoV-2), involves a complex host-virus interaction and is characterised by an exaggerated immune response, the specific components of which are poorly understood. Here we report the outcome of a longitudinal immune profiling study in hospitalised patients during the peak of the COVID-19 pandemic in the UK and show the relationship between immune responses and severity of the clinical presentation. MethodsThe Coronavirus Immune Response and Clinical Outcomes (CIRCO) study was conducted at four hospitals in Greater Manchester. Patients with SARS-CoV-2 infection, recruited as close to admission as possible, provided peripheral blood samples at enrolment and sequentially thereafter. Fresh samples were assessed for immune cells and proteins in whole blood and serum. Some samples were also stimulated for 3 hours with LPS and analysed for intracellular proteins. Results were stratified based on patient-level data including severity of symptoms and date of reported symptom onset. FindingsLongitudinal analysis showed a very high neutrophil to T cell ratio and abnormal activation of monocytes in the blood, which displayed high levels of the cell cycle marker, Ki67 and low COX-2. These properties all reverted in patient with good outcome. Unexpectedly, multiple aspects of inflammation were diminished as patients progressed in severity and time, even in ITU patients not recovering. InterpretationThis is the first detailed longitudinal analysis of COVID-19 patients of varying severity and outcome, revealing common features and aspects that track with severity. Patients destined for a severe outcome can be identified at admission when still displaying mild-moderate symptoms. We provide clues concerning pathogenesis that should influence clinical trials and therapeutics. Targeting pathways involved in neutrophil and monocyte release from the bone marrow should be tested in patients with COVID-19. FundingThe Kennedy Trust for Rheumatology Research, The Wellcome Trust, The Royal Society, The BBSRC, National Institute for Health Research (NIHR) Biomedical Research Centres (BRC). Research in contextO_ST_ABSEvidence before this studyC_ST_ABSAnalysis of the literature before the study via pubmed and bioRxiv searches using the terms COVID-19, SARS-CoV2, immune and inflammation (with the last search performed on 27th April 2020) showed evidence of an overactive immune response in a handful of studies in cross-sectional analyses all done at a single time point. Added value of this studyTo determine the role of the immune response in a disease process, it is necessary to correlate immune activity with clinical parameters dynamically. In this study patients presented to hospital at different stages of disease so we took samples at different time-points to provide an accurate picture of the relevant pathobiology. In order to avoid loss of large components of the immune system due to the processes of storage, longitudinal samples were interrogated in real time to reveal the full immune alterations in COVID-19. Implications of all the available evidenceRespiratory viruses continue to cause devastating global disease. The finding of altered myelopoiesis, with excess neutrophils and altered monocyte function, as dominant features in our study provides an incentive for clinical testing of therapeutics that specifically target this pathobiology. Given that inflammation is greatest prior to admission to intensive care, trials of specific immune-modulating therapies should be considered earlier in admission. Future studies of COVID-19 mechanisms should place more emphasis on longitudinal analyses since disease changes dramatically over time.
