One year health outcomes associated with systemic corticosteroids for COVID-19: a longitudinal cohort study (preprint)

BackgroundIn patients with COVID-19 requiring supplemental oxygen, dexamethasone reduces acute severity and improves survival, but longer-term effects are unknown. We hypothesised that systemic corticosteroid administration during acute COVID-19 would be associated with improved health-related quality of life (HRQoL) one year after discharge. MethodsAdults admitted to hospital between February 2020 and March 2021 for COVID-19 and meeting current guideline recommendations for dexamethasone treatment were included using two prospective UK cohort studies. HRQoL, assessed by EQ-5D-5L utility index, pre-hospital and one year after discharge were compared between those receiving corticosteroids or not after propensity weighting for treatment. Secondary outcomes included patient reported recovery, physical and mental health status, and measures of organ impairment. Sensitivity analyses were undertaken to account for survival and selection bias. FindingsIn 1,888 participants included in the primary analysis, 1,149 received corticosteroids. There was no between-group difference in EQ-5D-5L utility index at one year (mean difference 0.004, 95% CI: -0.026 to 0.034, p = 0.77). A similar reduction in EQ-5D-5L was seen at one year between corticosteroid exposed and non-exposed groups (mean (SD) change -0.12 (0.22) vs -0.11 (0.22), p = 0.32). Overall, there were no differences in secondary outcome measures. After sensitivity analyses modelled using a larger cohort of 109,318 patients admitted to hospital with COVID-19, EQ-5D-5L utility index at one year remained similar between the two groups. InterpretationSystemic corticosteroids for acute COVID-19 have no impact on the large reduction in HRQoL one year after hospital discharge. Treatments to address this are urgently needed. Take home messageSystemic corticosteroids given for acute COVID-19 do not affect health-related quality of life or other patient reported outcomes, physical and mental health outcomes, and organ function one year after hospital discharge

Cohort Profile: Post-hospitalisation COVID-19 study (PHOSP-COVID) (preprint)

Abstract [bullet] PHOSP-COVID is a national UK multi-centre cohort study of patients who were hospitalised for COVID-19 and subsequently discharged. [bullet] PHOSP-COVID was established to investigate the medium- and long-term sequelae of severe COVID-19 requiring hospitalisation, understand the underlying mechanisms of these sequelae, evaluate the medium- and long-term effects of COVID-19 treatments, and to serve as a platform to enable future studies, including clinical trials. [bullet] Data collected covered a wide range of physical measures, biological samples, and Patient Reported Outcome Measures (PROMs). [bullet] Participants could join the cohort either in Tier 1 only with remote data collection using hospital records, a PROMs app and postal saliva sample for DNA, or in Tier 2 where they were invited to attend two specific research visits for further data collection and biological research sampling. These research visits occurred at five (range 2-7) months and 12 (range 10-14) months post-discharge. Participants could also participate in specific nested studies (Tier 3) at selected sites. [bullet] All participants were asked to consent to further follow-up for 25 years via linkage to their electronic healthcare records and to be re-contacted for further research. [bullet] In total, 7935 participants were recruited from 83 UK sites: 5238 to Tier 1 and 2697 to Tier 2, between August 2020 and March 2022. [bullet] Cohort data are held in a Trusted Research Environment and samples stored in a central biobank. Data and samples can be accessed upon request and subject to approvals.

Unbiased single cell spatial analysis localises inflammatory clusters of immature neutrophils-CD8 T cells to alveolar progenitor cells in fatal COVID-19 lungs (preprint)

Single cell spatial interrogation of the immune-structural interactions in COVID -19 lungs is challenging, mainly because of the marked cellular infiltrate and architecturally distorted microstructure. To address this, we developed a suite of mathematical tools to search for statistically significant co-locations amongst immune and structural cells identified using 37-plex imaging mass cytometry. This unbiased method revealed a cellular map interleaved with an inflammatory network of immature neutrophils, cytotoxic CD8 T cells, megakaryocytes and monocytes co-located with regenerating alveolar progenitors and endothelium. Of note, a highly active cluster of immature neutrophils and cytotoxic CD8 T cells, was found spatially linked with alveolar progenitor cells, and temporally with the diffuse alveolar damage stage. These findings provide new insights into how immune cells interact in the lungs of severe COVID-19 disease. We provide our pipeline [Spatial Omics Oxford Pipeline (SpOOx)] and visual-analytical tool, Multi-Dimensional Viewer (MDV) software, as a resource for spatial analysis.

The effects of sleep disturbance on dyspnoea and impaired lung function following COVID-19 hospitalisation: a prospective multi-centre cohort study (preprint)

Background Sleep disturbance is common following hospitalisation both for COVID-19 and other causes. The clinical associations are poorly understood, despite it altering pathophysiology in other scenarios. We, therefore, investigated whether sleep disturbance is associated with dyspnoea along with relevant mediation pathways. Methods Sleep parameters were assessed in a prospective cohort of patients (n=2,468) hospitalised for COVID-19 in the United Kingdom in 39 centres using both subjective and device-based measures. Results were compared to a matched UK biobank cohort and associations were evaluated using multivariable linear regression. Findings 64% (456/714) of participants reported poor sleep quality; 56% felt their sleep quality had deteriorated for at least 1-year following hospitalisation. Compared to the matched cohort, both sleep regularity (44.5 vs 59.2, p<0.001) and sleep efficiency (85.4% vs 88.5%, p<0.001) were lower whilst sleep period duration was longer (8.25h vs 7.32h, p<0.001). Overall sleep quality (effect estimate 4.2 (3.0-5.5)), deterioration in sleep quality following hospitalisation (effect estimate 3.2 (2.0-4.5)), and sleep regularity (effect estimate 5.9 (3.7-8.1)) were associated with both dyspnoea and impaired lung function (FEV1 and FVC). Depending on the sleep metric, anxiety mediated 13-42% of the effect of sleep disturbance on dyspnoea and muscle weakness mediated 29-43% of this effect. Interpretation Sleep disturbance is associated with dyspnoea, anxiety and muscle weakness following COVID-19 hospitalisation. It could have similar effects for other causes of hospitalisation where sleep disturbance is prevalent.

Nasal IgA wanes 9 months after hospitalisation with COVID-19 and is not induced by subsequent vaccination. (preprint)

Background Most studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. Methods Plasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. Findings Strong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months. Nasal and plasma anti-S IgG remained elevated for at least 12 months with high plasma neutralising titres against all variants. Of 180 with complete data, 160 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal. Samples 12 months after admission showed no association between nasal IgA and plasma IgG responses, indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. Interpretation The decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity.

Device-assessed sleep and physical activity in individuals recovering from a hospital admission for COVID-19: a prospective, multicentre study (preprint)

ObjectivesTo describe physical behaviours following hospital admission for COVID-19 including associations with acute illness severity and ongoing symptoms. Methods1077 patients with COVID-19 discharged from hospital between March and November 2020 were recruited. Using a 14-day wear protocol, wrist-worn accelerometers were sent to participants after a five-month follow-up assessment. Acute illness severity was assessed by the WHO clinical progression scale, and the severity of ongoing symptoms was assessed using four previously reported data-driven clinical recovery clusters. Two existing control populations of office workers and type 2 diabetes were comparators. ResultsValid accelerometer data from 253 women and 462 men were included. Women engaged in a mean{+/-}SD of 14.9{+/-}14.7 minutes/day of moderate-to-vigorous physical activity (MVPA), with 725.6{+/-}104.9 minutes/day spent inactive and 7.22{+/-}1.08 hours/day asleep. The values for men were 21.0{+/-}22.3 and 755.5{+/-}102.8 minutes/day and 6.94{+/-}1.14 hours/day, respectively. Over 60% of women and men did not have any days containing a 30-minute bout of MVPA. Variability in sleep timing was approximately 2 hours in men and women. More severe acute illness was associated with lower total activity and MVPA in recovery. The very severe recovery cluster was associated with fewer days/week containing continuous bouts of MVPA, longer sleep duration, and higher variability in sleep timing. Patients post-hospitalisation with COVID-19 had lower levels of physical activity, greater sleep variability, and lower sleep efficiency than a similarly aged cohort of office workers or those with type 2 diabetes. ConclusionsPhysical activity and regulating sleep patterns are potential treatable traits for COVID-19 recovery programmes.

Clinical characteristics with inflammation profiling of Long-COVID and association with one-year recovery following hospitalisation in the UK: a prospective observational study (preprint)

Background There are currently no effective pharmacological or non-pharmacological interventions for Long-COVID. To identify potential therapeutic targets, we focussed on previously described four recovery clusters five months after hospital discharge, their underlying inflammatory profiles and relationship with clinical outcomes at one year. Methods PHOSP-COVID is a prospective longitudinal cohort study, recruiting adults hospitalised with COVID-19 across the UK. Recovery was assessed using patient reported outcomes measures (PROMs), physical performance, and organ function at five-months and one-year after hospital discharge. Hierarchical logistic regression modelling was performed for patient-perceived recovery at one-year. Cluster analysis was performed using clustering large applications (CLARA) k-medoids approach using clinical outcomes at five-months. Inflammatory protein profiling from plasma at the five-month visit was performed. Findings 2320 participants have been assessed at five months after discharge and 807 participants have completed both five-month and one-year visits. Of these, 35.6% were female, mean age 58.7 (SD 12.5) years, and 27.8% received invasive mechanical ventilation (IMV). The proportion of patients reporting full recovery was unchanged between five months 501/165 (25.6%) and one year 232/804 (28.9%). Factors associated with being less likely to report full recovery at one year were: female sex OR 0.68 (95% CI 0.46-0.99), obesity OR 0.50 (95%CI 0.34-0.74) and IMV OR 0.42 (95%CI 0.23-0.76). Cluster analysis (n=1636) corroborated the previously reported four clusters: very severe, severe, moderate/cognitive, mild relating to the severity of physical, mental health and cognitive impairments at five months in a larger sample. There was elevation of inflammatory mediators of tissue damage and repair in both the very severe and the moderate/cognitive clusters compared to the mild cluster including interleukin-6 which was elevated in both comparisons. Overall, there was a substantial deficit in median (IQR) EQ5D-5L utility index from pre-COVID (retrospective assessment) 0.88 (0.74-1.00), five months 0.74 (0.60-0.88) to one year: 0.74 (0.59-0.88), with minimal improvements across all outcome measures at one-year after discharge in the whole cohort and within each of the four clusters. Interpretation The sequelae of a hospital admission with COVID-19 remain substantial one year after discharge across a range of health domains with the minority in our cohort feeling fully recovered. Patient perceived health-related quality of life remains reduced at one year compared to pre-hospital admission. Systematic inflammation and obesity are potential treatable traits that warrant further investigation in clinical trials.

Neutrophil Extracellular Traps, Local IL-8 Expression, and Cytotoxic T-Lymphocyte Absence in the Lungs of Fatal COVID-19: A Retrospective Histopathology Cohort Study (preprint)

Background: Excessive inflammation is pathogenic in pneumonitis associated to severe COVID-19. Neutrophils are among the most abundantly present leukocytes in the inflammatory infiltrates and may form neutrophil extracellular traps (NETs) under the local influence of cytokines. NETs constitute a defence mechanism against bacteria but have also been shown to mediate tissue damage in a number of diseases. Methods: In this retrospective cohort study, sixteen immediate post-mortem lung biopsies were methodologically analysed as exploratory and validation cohorts. NETs were quantitatively analysed by multiplexed immunofluorescence and correlated with local levels of IL-8 mRNA expression and the density of CD8+ T-cell infiltration. SARS-CoV-2 presence in tissue was quantified by RT-PCR and immunohistochemistry.Findings: NETs were found in the lung interstitium and surrounding the bronchiolar epithelium with interindividual and spatial heterogeneity. NET density did not correlate with SARS-CoV-2 tissue viral load. NETs were associated with local IL-8 mRNA levels. NETs were also detected in pulmonary thrombi and in only one out of eight liver tissues in spatial fashion. NET focal presence negatively correlated with CD8+ T-cell infiltration in the lungs. Interpretation: Abundant neutrophils undergoing NETosis are found in the lungs of patients with fatal COVID-19, showing no correlation with viral loads. The strong association between NETs and IL-8 focal expression points to this chemokine as the potential causative factor. The function of cytotoxic T-lymphocytes in the immune responses against SARS-CoV-2 may be interfered by the presence of NETs.Funding Information: This study was supported by Banco Bilbao Vizcaya (BBVA) Foundation, “Ayudas a Equipos de Investigación Científica SARS-CoV-2 y COVID-19”. Declaration of Interests: I.M. reports receiving commercial research grants from BMS, Bioncotech, Alligator, Pfizer, Leadartis and Roche; has received speakers bureau honoraria from MSD; and is a consultant or advisory board member for BMS, Roche, Genmab, F-Star, Bioncotech, Bayer, Numab, Pieris, Alligator, and Merck Serono. C.E.A reports research grants from AstraZeneca. All other authors declare no competing interests.Ethics Approval Statement: This study was approved by the ethics committee of the University of Navarra, Spain (Approval 2020.192). Tissue collections were obtained with consent from a first-degree relative, following a protocol approved by the ethics committee of the University of Navarra (Protocol 2020.192p).

Symptom Persistence Despite Improvement in Cardiopulmonary Health - Insights from longitudinal CMR, CPET and lung function testing post-COVID-19 (preprint)

BackgroundThe longitudinal trajectories of cardiopulmonary abnormalities and symptoms following infection with coronavirus disease (COVID-19) are unclear. We sought to describe their natural history in previously hospitalised patients, compare this with controls, and assess the relationship between symptoms and cardiopulmonary impairment at 6 months post-COVID-19. MethodsFifty-eight patients and thirty matched controls underwent symptom-questionnaires, cardiac and lung magnetic resonance imaging (CMR), cardiopulmonary exercise test (CPET), and spirometry at 3 months following COVID-19. Of them, forty-six patients returned for follow-up assessments at 6 months. FindingsAt 2-3 months, 83% of patients had at least one cardiopulmonary symptom versus 33% of controls. Patients and controls had comparable biventricular volumes and function. Native cardiac T1 (marker of inflammation) and late gadolinium enhancement (LGE, marker of focal fibrosis) were increased in patients. Sixty percent of patients had lung parenchymal abnormalities on CMR and 55% had reduced peak oxygen consumption (pVO2) on CPET. By 6 months, 53% of patients remained symptomatic. On CMR, indexed right ventricular (RV) end-diastolic volume (-4{middle dot}3 mls/m2, P=0{middle dot}005) decreased and RV ejection fraction (+3{middle dot}2%, P=0{middle dot}0003) increased. Native T1 and LGE improved and was comparable to controls. Lung parenchymal abnormalities and peak VO2, although better, were abnormal in patients versus controls. 31% had reduced pVO2 secondary to fatigue and submaximal tests. Cardiopulmonary symptoms in patients did not associate with CMR, lung function, or CPET measures. InterpretationIn patients, cardiopulmonary abnormalities improve over time, though some measures remain abnormal relative to controls. Persistent symptoms at 6 months post-COVID-19 did not associate with objective measures of cardiopulmonary health. FundingNIHR Oxford and Oxford Health BRC, Oxford BHF CRE, UKRI and Wellcome Trust.

Immunodominant NP105-113-B*0702 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease (preprint)

NP 105-113 -B*07:02 specific CD8 + T-cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP 105-113 -B*07:02 specific T-cell clones and single cell sequencing were performed concurrently, with functional avidity and anti-viral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with TCR usage, transcriptome signature, and disease severity (acute N=77, convalescent N=52). We demonstrated a beneficial association of NP 105-113 -B*07:02 specific T-cells in COVID-19 disease progression, linked with expansion of T-cell precursors, high functional avidity and anti-viral effector function. Broad immune memory pools were narrowed post-infection but NP 105-113 -B*07:02 specific T-cells were maintained 6 months after infection with preserved anti-viral efficacy to the SARS-CoV-2 Victoria strain, as well as new Alpha, Beta and Gamma variants. Our data shows that NP 105-113 -B*07:02 specific T-cell responses associate with mild disease and high anti-viral efficacy, pointing to inclusion for future vaccine design.

Spatial transcriptomic characterization of COVID-19 pneumonitis identifies immune pathways related to tissue injury (preprint)

Severe lung damage in COVID-19 is known to involve complex interactions between diverse populations of immune and stromal cells. In this study, we applied a spatial transcriptomics approach to better delineate the cells, pathways and genes responsible for promoting and perpetuating severe tissue pathology in COVID-19 pneumonitis. Guided by tissue histology and immunohistochemistry we performed a targeted sampling of dozens of regions representing a spectrum of diffuse alveolar damage from the post-mortem lung of three COVID-19 patients. Application of a combination of differential gene expression, weighted gene correlation network, pathway and spatial deconvolution analysis stratified the sampled regions into five distinct groups according to degree of alveolar damage, levels of cytotoxic inflammation and innate activation, epithelial reorganization, and fibrosis. Integrative network analysis of the identified groups revealed the presence of proliferating CD8 T and NK cells in severely damaged areas along with signatures of cytotoxicity, interferon signalling and high expression of immune cell chemoattractants (including CXCL9/10/11 and CCL2). Areas of milder damage were marked by innate immune signalling (including TLR response, IL-1, IL-6) together with signatures of antigen presentation, and fibrosis. Based on these data we present a cellular model of tissue damage in terminal COVID-19 that confirms previous observations and highlights novel opportunities for therapeutic intervention.

Namilumab or infliximab compared to standard of care in hospitalised patients with COVID-19 (CATALYST): a phase 2 randomised adaptive trial (preprint)

Summary Background Dysregulated inflammation is associated with poor outcomes in Coronavirus disease 2019 (COVID-19). We assessed the efficacy of namilumab, a granulocyte-macrophage colony-stimulating factor inhibitor and infliximab, a tumour necrosis factor inhibitor in hospitalised patients with COVID-19 in order to prioritise agents for phase 3 trials. Methods In this randomised, multi-arm, parallel group, open label, adaptive phase 2 proof-of-concept trial (CATALYST) we recruited hospitalised patients ≥ 16 years with COVID-19 pneumonia and C-reactive protein (CRP) ≥ 40mg/L in nine UK hospitals. Participants were randomly allocated with equal probability to usual care, or usual care plus a single 150mg intravenous dose of namilumab (150mg) or infliximab (5mg/kg). Randomisation was stratified for ward versus ICU. The primary endpoint was improvement in inflammation in intervention arms compared to control as measured by CRP over time, analysed using Bayesian multi-level models. ISRCTN registry number 40580903. Findings Between 15 th June 2020 and 18 th February 2021 we randomised 146 participants: 54 to usual care, 57 to namilumab and 35 to infliximab. The probabilities that namilumab and infliximab were superior to usual care in reducing CRP over time were 97% and 15% respectively. Consistent effects were seen in ward and ICU patients and aligned with clinical outcomes, such that the probability of discharge (WHO levels 1-3) at day 28 was 47% and 64% for ICU and ward patients on usual care, versus 66% and 77% for patients treated with namilumab. 134 adverse events occurred in 30/55 (54.5%) namilumab patients compared to 145 in 29/54 (53.7%) usual care patients. 102 events occurred in 20/29 (69.0%) infliximab patients versus 112 events in 17/34 (50.0%) usual care patients. Interpretation Namilumab, but not infliximab, demonstrated proof-of-concept evidence for reduction in inflammation in hospitalised patients with COVID-19 pneumonia which was consistent with secondary clinical outcomes. Namilumab should be prioritised for further investigation in COVID-19. Funding Medical Research Council.

A blood atlas of COVID-19 defines hallmarks of disease severity and specificity (preprint)

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete understanding of potentially druggable immune mediators of disease. To advance this, we present a comprehensive multi-omic blood atlas in patients with varying COVID-19 severity and compare with influenza, sepsis and healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity revealed cells, their inflammatory mediators and networks as potential therapeutic targets, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Tensor and matrix decomposition of the overall dataset revealed feature groupings linked with disease severity and specificity. Our systems-based integrative approach and blood atlas will inform future drug development, clinical trial design and personalised medicine approaches for COVID-19.

Physical, cognitive and mental health impacts of COVID-19 following hospitalisation: a multi-centre prospective cohort study (preprint)

Background The impact of COVID-19 on physical and mental health, and employment following hospitalisation is poorly understood. Methods PHOSP-COVID is a multi-centre, UK, observational study of adults discharged from hospital with a clinical diagnosis of COVID-19 involving an assessment between two- and seven-months later including detailed symptom, physiological and biochemical testing. Multivariable logistic regression was performed for patient-perceived recovery with age, sex, ethnicity, body mass index (BMI), co-morbidities, and severity of acute illness as co-variates. Cluster analysis was performed using outcomes for breathlessness, fatigue, mental health, cognition and physical function. Findings We report findings of 1077 patients discharged in 2020, from the assessment undertaken a median 5 [IQR4 to 6] months later: 36% female, mean age 58 [SD 13] years, 69% white ethnicity, 27% mechanical ventilation, and 50% had at least two co-morbidities. At follow-up only 29% felt fully recovered, 20% had a new disability, and 19% experienced a health-related change in occupation. Factors associated with failure to recover were female, middle-age, white ethnicity, two or more co-morbidities, and more severe acute illness. The magnitude of the persistent health burden was substantial and weakly related to acute severity. Four clusters were identified with different severities of mental and physical health impairment: 1) Very severe (17%), 2) Severe (21%), 3) Moderate with cognitive impairment (17%), 4) Mild (46%), with 3%, 7%, 36% and 43% feeling fully recovered, respectively. Persistent systemic inflammation determined by C-reactive protein was related to cluster severity, but not acute illness severity. Interpretation We identified factors related to recovery from a hospital admission with COVID-19 and four different phenotypes relating to the severity of physical, mental, and cognitive health five months later. The implications for clinical care include the potential to stratify care and the need for a pro-active approach with wide-access to COVID-19 holistic clinical services. Funding: UKRI and NIHR

Medium-term effects of SARS-CoV-2 infection on multiple vital organs, exercise capacity, cognition, quality of life and mental health, post-hospital discharge (preprint)

BackgroundThe medium-term effects of Coronavirus disease (COVID-19) on multiple organ health, exercise capacity, cognition, quality of life and mental health are poorly understood. MethodsFifty-eight COVID-19 patients post-hospital discharge and 30 comorbidity-matched controls were prospectively enrolled for multiorgan (brain, lungs, heart, liver and kidneys) magnetic resonance imaging (MRI), spirometry, six-minute walk test, cardiopulmonary exercise test (CPET), quality of life, cognitive and mental health assessments. FindingsAt 2-3 months from disease-onset, 64% of patients experienced persistent breathlessness and 55% complained of significant fatigue. On MRI, tissue signal abnormalities were seen in the lungs (60%), heart (26%), liver (10%) and kidneys (29%) of patients. COVID-19 patients also exhibited tissue changes in the thalamus, posterior thalamic radiations and sagittal stratum on brain MRI and demonstrated impaired cognitive performance, specifically in the executive and visuospatial domain relative to controls. Exercise tolerance (maximal oxygen consumption and ventilatory efficiency on CPET) and six-minute walk distance (405{+/-}118m vs 517{+/-}106m in controls, p<0.0001) were significantly reduced in patients. The extent of extra-pulmonary MRI abnormalities and exercise tolerance correlated with serum markers of ongoing inflammation and severity of acute illness. Patients were more likely to report symptoms of moderate to severe anxiety (35% versus 10%, p=0.012) and depression (39% versus 17%, p=0.036) and a significant impairment in all domains of quality of life compared to controls. InterpretationA significant proportion of COVID-19 patients discharged from hospital experience ongoing symptoms of breathlessness, fatigue, anxiety, depression and exercise limitation at 2-3 months from disease-onset. Persistent lung and extra-pulmonary organ MRI findings are common. In COVID-19 survivors, chronic inflammation may underlie multiorgan abnormalities and contribute to impaired quality of life. FundingNIHR Oxford and Oxford Health Biomedical Research Centres, British Heart Foundation Centre for Research Excellence, UKRI, Wellcome Trust, British Heart Foundation.

Adult Stem Cell-derived Complete Lung Organoid Models Emulate Lung Disease in COVID-19 (preprint)

SARS-CoV-2, the virus responsible for COVID-19, causes widespread damage in the lungs in the setting of an overzealous immune response whose origin remains unclear. We present a scalable, propagable, personalized, cost-effective adult stem cell-derived human lung organoid model that is complete with both proximal and distal airway epithelia. Monolayers derived from adult lung organoids (ALOs), primary airway cells, or hiPSC-derived alveolar type-II (AT2) pneumocytes were infected with SARS-CoV-2 to create in vitro lung models of COVID-19. Infected ALO-monolayers best recapitulated the transcriptomic signatures in diverse cohorts of COVID-19 patient-derived respiratory samples. The airway (proximal) cells were critical for sustained viral infection whereas distal alveolar differentiation (AT2[->]AT1) was critical for mounting the overzealous host immune response in fatal disease; ALO monolayers with well-mixed proximodistal airway components recapitulated both. Findings validate a human lung model of COVID-19 which can be immediately utilized to investigate COVID-19 pathogenesis, and vet new therapies and vaccines.

3D reconstruction of SARS-CoV-2 infection in ferrets emphasizes focal infection pattern in the upper respiratory tract (preprint)

The visualization of viral pathogens in infected tissues is an invaluable tool to understand spatial virus distribution, localization, and cell tropism in vivo. Commonly, virus-infected tissues are analyzed using conventional immunohistochemistry in paraffin-embedded thin sections. Here, we demonstrate the utility of volumetric three-dimensional (3D) immunofluorescence imaging using tissue optical clearing and light sheet microscopy to investigate host-pathogen interactions of pandemic SARS-CoV-2 in ferrets at a mesoscopic scale. The superior spatial context of large, intact samples (> 150 mm3) allowed detailed quantification of interrelated parameters like focus-to-focus distance or SARS-CoV-2-infected area, facilitating an in-depth description of SARS-CoV-2 infection foci. Accordingly, we could confirm a preferential infection of the ferret upper respiratory tract by SARS-CoV-2 and emphasize a distinct focal infection pattern in nasal turbinates. Conclusively, we present a proof-of-concept study for investigating critically important respiratory pathogens in their spatial tissue morphology and demonstrate the first specific 3D visualization of SARS-CoV-2 infection.

Molecular mechanism of SARS-CoV-2 inactivation by temperature (preprint)

Recent studies have shown that SARS-CoV-2 virus can be inactivated by effect of heat, even though, little is known about the molecular changes induced by the temperature. Here, we unravel the basics of such inactivation mechanism over the SARS-CoV-2 spike glycoprotein by executing atomistic molecular dynamics simulations. Both the closed down and open up states, which determine the accessibility to the receptor binding domain, were considered. Results suggest that the spike undergoes drastic changes in the topology of the hydrogen bond network while salt bridges are mainly preserved. Reorganization in the hydrogen bonds structure produces conformational variations in the receptor binding subunit and explain the thermal inactivation of the virus. Conversely, the macrostructure of the spike is preserved at high temperature because of the retained salt bridges. The proposed mechanism has important implications for engineering new approaches to inactivate the SARS-CoV-2 virus.

Recovery of monocyte exhaustion is associated with resolution of lung injury in COVID-19 convalescence. (preprint)

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.

Additional analyses exploring the hypothesized transdifferentiation of plasmablasts to developing neutrophils in severe COVID-19 (preprint)

We thank Alquicira-Hernandez et al. for their reanalysis of our single-cell transcriptomic dataset profiling peripheral immune responses to severe COVID-19. We agree that careful analysis of single-cell sequencing data is important for generating cogent hypotheses but find several aspects of their criticism of our analysis to be problematic. Here we respond briefly to misunderstandings and inaccuracies in their commentary that may have led to misinformed interpretation of our results.