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.

Hypoxia inducible factors regulate infectious SARS-CoV-2, epithelial damage and respiratory symptoms in a hamster COVID-19 model. (preprint)

Understanding the host pathways that define susceptibility to SARS-CoV-2 infection and disease are essential for the design of new therapies. Oxygen levels in the microenvironment define the transcriptional landscape, however the influence of hypoxia on virus replication and disease in animal models is not well understood. In this study, we identify a role for the hypoxic inducible factor (HIF) signalling axis to inhibit SARS-CoV-2 infection, epithelial damage and respiratory symptoms in Syrian hamsters. Pharmacological activation of HIF with the prolyl-hydroxylase inhibitor FG-4592 significantly reduced the levels of infectious virus in the upper and lower respiratory tract. Nasal and lung epithelia showed a reduction in SARS-CoV-2 RNA and nucleocapsid expression in treated animals. Transcriptomic and pathological analysis showed reduced epithelial damage and increased expression of ciliated cells. Our study provides new insights on the intrinsic antiviral properties of the HIF signalling pathway in SARS-CoV-2 replication that may be applicable to other respiratory pathogens and identifies new therapeutic opportunities.

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).

Poor Antibody Responses to SARS-CoV-2 Infection or Vaccination Are Associated With High Re-Infection Rates in Haemodialysis and Renal Transplant Patients (preprint)

Background: Patients with end-stage renal disease (ESRD) are vulnerable to SARS-CoV-2 infection and mount poor antibody responses to standard vaccines. We addressed whether ESRD patients could mount immune responses that protected against re-infection following natural SARS-CoV-2 infection or 2-dose vaccination.Methods: Haemodialysis (HD and renal transplant patients were recruited following SARS-CoV-2 infection (n=46) or before SARS-CoV-2 vaccination (n=94). SARS-CoV-2 IgG responses, surrogate neutralising antibody (NAb) titres to wildtype and VOCs, T cell responses and viral sequencing in the vaccine-naïve convalescent cohort were serially assessed following infection. Surrogate NAb titres were measured pre-vaccination and 33 days after 2nd vaccine. Incidence of breakthrough infection was assessed 180 days following 1st vaccination. Findings: 22% of vaccine-naive HD (n=9/36) and transplant patients (n=1/10) demonstrated PCR-positive re-infection (RI) at median 212 days (IQR 140-239) post 1st infection. Prior to RI episodes, RI patients demonstrated poor IgG Spike and RBD responses which were equivalent to levels in pre-pandemic sera (median RI titres: Spike 187 AU/ml, IQR 143-3432, p=0.96; RBD 145 AU/ml, IQR 85-938, p>0.99), unlike patients who developed a single infection only (SI) when compared to pre-pandemic sera (median SI titres: Spike 22826 AU/ml, IQR 1255-63811, p<0.0001; RBD 9588 AU/ml, IQR 270-21616, p=0.001). IgG Spike and RBD titres increased following RI compared to pre-pandemic sera (median RI titres: Spike 22611 AU/ml, IQR 4488-75509, p=0.0006; RBD 6354 AU/ml, IQR 1671-20962, p=0.01). T cell analysis revealed no differences between RI and SI cohorts. Following 2-dose vaccination, 5% of the HD cohort who received AZD1222 (n=3/61) developed breakthrough infection at 6 months following 1st vaccination, unlike those who received BNT162b2 (n=0/16). AZD1222-vaccinated, infection-naïve (I-N) HD patients (n=32) and immunosuppressed transplant recipients (n=17) made poor NAb responses to wildtype, alpha, beta and gamma when compared to infection-experienced (I-E) HD patients (n=29) (I-N vs I-E HD wildtype p<0.0001, alpha p=0.0007, beta p<0.0001, gamma p=0.002). NAb responses improved with BNT162b2 vaccination (n=16); RI patients mounted larger NAb responses to AZD1222 vaccination than SI patients (wildtype p=0.01, alpha p=0.02, beta p<0.02). Interpretation: ESRD patients are highly susceptible to SARS-CoV-2 re-infection, or breakthrough infection following vaccination, associated with poor protective antibody responses. SARS-CoV-2-specific IgG and surrogate NAb responses increase with repeated exposure (infection experience and/or vaccination) in patients who survive infections. Our findings support the case for specific booster regimens in such immune-incompetent patients. Funding Information: Oxford Transplant Foundation, Oxfordshire Health Services Research Committee, UK Department of Health and Social Care, Huo Family Foundation, NIHR (COV19-RECPLAS), UK Coronavirus Immunology Consortium, NIHR Oxford Biomedical Research Centre, WT109965MA.Declaration of Interests: We declare no competing interestsEthics Approval Statement: Haemodialysis (HD) and transplant cohorts: In this prospective, observational cohort study, HD and transplant patients within Oxford University Hospitals NHS Foundation Trust(OUH) were recruited under Oxford Radcliffe Biobank approved studies, “Biomarkers to stratify risk in Renal Transplant Recipients and Dialysis Patients with Covid-19” (ref: ORB 20/A056), and “Immunological responses to COVID-19 vaccines in transplant and haemodialysis patients” (ref: ORB 21/A014). The Oxford Radcliffe Biobank has a favorable ethics opinion from the South Central Oxford Committee C (REC: 19/SC/0173). Healthcare Worker cohort (HC, PITCH study): PITCH is a sub-study of the SIREN study which was approved by the Berkshire Research Ethics Committee, Health Research 250 Authority (IRAS ID 284460, REC reference 20/SC/0230), with PITCH recognised as a sub-study on 2 December 2020. SIREN is registered with ISRCTN (Trial ID:252 ISRCTN11041050)The study was conducted in compliance with all relevant ethical regulations for work with human participants, and according to the principles of the Declaration of Helsinki (2008) and the International Conference on Harmonization (ICH) Good Clinical Practice (GCP) guidelines. Written informed consent was obtained for all patients enrolled in the study.

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.