Human SARS-CoV-2 challenge resolves local and systemic response dynamics (preprint)

The COVID-19 pandemic is an ongoing global health threat, yet our understanding of the cellular disease dynamics remains limited. In our unique COVID-19 human challenge study we used single cell genomics of nasopharyngeal swabs and blood to temporally resolve abortive, transient and sustained infections in 16 seronegative individuals challenged with preAlpha-SARS-CoV-2. Our analyses revealed rapid changes in cell type proportions and dozens of highly dynamic cellular response states in epithelial and immune cells associated with specific timepoints or infection status. We observed that the interferon response in blood precedes the nasopharynx, and that nasopharyngeal immune infiltration occurred early in transient but later in sustained infection, and thus correlated with preventing sustained infection. Ciliated cells showed an acute response phase, upregulated MHC class II while infected, and were most permissive for viral replication, whilst nasal T cells and macrophages were infected non-productively. We resolve 54 T cell states, including acutely activated T cells that clonally expanded while carrying convergent SARS-CoV-2 motifs. Our novel computational pipeline (Cell2TCR) identifies activated antigen-responding clonotype groups and motifs in any dataset. Together, we show that our detailed time series data (covid19cellatlas.org) can serve as a 'Rosetta stone' for the epithelial and immune cell responses, and reveals early dynamic responses associated with protection from infection.

The emergence of goblet inflammatory or ITGB6hi nasal progenitor cells determines age-associated SARS-CoV-2 pathogenesis (preprint)

Children infected with SARS-CoV-2 rarely progress to respiratory failure, but the risk of mortality in infected people over 85 years of age remains high, despite vaccination and improving treatment options. Here, we take a comprehensive, multidisciplinary approach to investigate differences in the cellular landscape and function of paediatric (<11y), adult (30-50y) and elderly (>70y) nasal epithelial cells experimentally infected with SARS-CoV-2. Our data reveal that nasal epithelial cell subtypes show different tropism to SARS-CoV-2, correlating with age, ACE2 and TMPRSS2 expression. Ciliated cells are a viral replication centre across all age groups, but a distinct goblet inflammatory subtype emerges in infected paediatric cultures, identifiable by high expression of interferon stimulated genes and truncated viral genomes. In contrast, infected elderly cultures show a proportional increase in ITGB6hi progenitors, which facilitate viral spread and are associated with dysfunctional epithelial repair pathways. A video explaining this work can be found here - https://youtu.be/uExP4bx6D_A .

Microbial and human transcriptional profiling of coronavirus disease 2019 patients: Potential predictors of disease severity

The high morbidity of patients with coronavirus disease 2019 (COVID-19) brings on a panic around the world. COVID-19 is associated with sex bias, immune system, and preexisting chronic diseases. We analyzed the gene expression in patients with COVID-19 and in their microbiota in order to identify potential biomarkers to aid in disease management. A total of 129 RNA samples from nasopharyngeal, oropharyngeal, and anal swabs were collected and sequenced in a high-throughput manner. Several microbial strains differed in abundance between patients with mild or severe COVID-19. Microbial genera were more abundant in oropharyngeal swabs than in nasopharyngeal or anal swabs. Oropharyngeal swabs allowed more sensitive detection of the causative SARS-CoV-2. Microbial and human transcriptomes in swabs from patients with mild disease showed enrichment of genes involved in amino acid metabolism, or protein modification via small protein removal, and antibacterial defense responses, respectively, whereas swabs from patients with severe disease showed enrichment of genes involved in drug metabolism, or negative regulation of apoptosis execution, spermatogenesis, and immune system, respectively. Microbial abundance and diversity did not differ significantly between males and females. The expression of several host genes on the X chromosome correlated negatively with disease severity. In this way, our analyses identify host genes whose differential expression could aid in the diagnosis of COVID-19 and prediction of its severity via non-invasive assay.

A spatial multi-omics atlas of the human lung reveals a novel gland-associated immune niche (preprint)

To (re)define tissue architecture of the lung and airways at the cellular and molecular level, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and Visium Spatial Transcriptomics. Using computational data integration and analysis, we extend beyond the suspension cell paradigm of lung atlases to date, to define and discover macro and micro-anatomical tissue compartments. We describe novel cell types and states in vascular, stromal and nerve bundle microenvironments. From our spatial transcriptomics, we discover and validate a novel survival niche for IgA plasma cells in the airway submucosal glands (SMG). In this niche we define a supporting role for SMG epithelial cells in mucosal immunity through recruitment and maintenance of IgA plasma, B and CD4 T cells locally at the airway SMG. We identify an immune-supporting role for SMG duct and serous cells with distinct signalling circuits to recruit B cells and IgA plasma cells, promoting longevity and antibody secretion through expression of CCL28, APRIL and IL6. We find high expression of MHC-II in SMG duct and serous cells, which are localised closely with memory CD4 T cells, suggesting local modulation of antigen specific immune responses locally at the glands. This new tissue microenvironment, which we term the “gland-associated immune niche” (GAIN) has major implications for respiratory immunity and infection response. Our single cell and spatial data is available for download and query at lungcellatlas.org.

Mapping interindividual dynamics of innate immune response at single-cell resolution (preprint)

Common genetic variants modulate the cellular response to viruses and are implicated in a range of immune pathologies, including infectious and autoimmune diseases. The transcriptional antiviral response is known to vary between infected cells from a single individual, yet how genetic variants across individuals modulate the antiviral response (and its cell-to-cell variability) is not well understood. Here, we triggered the antiviral response in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-seq. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), the first statistical approach designed to identify dynamic eQTLs across a transcriptional trajectory of cell populations, without aggregating single-cell data into pseudo-bulk. This allows us to uncover the underlying architecture and variability of antiviral response across responding cells, and to identify more than two thousands eQTLs modulating the dynamic changes during this response. Many of these eQTLs colocalise with risk loci identified in GWAS of infectious and autoimmune diseases. As a case study, we focus on a COVID-19 susceptibility locus, colocalised with the antiviral OAS1 splicing QTL. We validated it in blood cells from a patient cohort and in the infected nasal cells of a patient with the risk allele, demonstrating the utility of GASPACHO to fine-map and functionally characterise a genetic locus. In summary, our novel analytical approach provides a new framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.

The local and systemic response to SARS-CoV-2 infection in children and adults (preprint)

While a substantial proportion of adults infected with SARS-CoV-2 progress to develop severe disease, children rarely manifest respiratory complications. Therefore, understanding differences in the local and systemic response to SARS-CoV-2 infection between children and adults may provide important clues about the pathogenesis of SARS-CoV-2 infection. To address this, we first generated a healthy reference multi-omics single cell data set from children (n=30) in whom we have profiled triple matched samples: nasal and tracheal brushings and PBMCs, where we track the developmental changes for 42 airway and 31 blood cell populations from infancy, through childhood to adolescence. This has revealed the presence of naive B and T lymphocytes in neonates and infants with a unique gene expression signature bearing hallmarks of innate immunity. We then contrast the healthy reference with equivalent data from severe paediatric and adult COVID-19 patients (total n=27), from the same three types of samples: upper and lower airways and blood. We found striking differences: children with COVID-19 as opposed to adults had a higher proportion of innate lymphoid and non-clonally expanded naive T cells in peripheral blood, and a limited interferon-response signature. In the airway epithelium, we found the highest viral load in goblet and ciliated cells and describe a novel inflammatory epithelial cell population. These cells represent a transitional regenerative state between secretory and ciliated cells; they were found in healthy children and were enriched in pediatric and adult COVID-19 patients. Epithelial cells display an antiviral and neutrophil-recruiting gene signature that is weaker in severe paediatric versus adult COVID-19. Our matched blood and airway samples allowed us to study the spatial dynamics of infection. Lastly, we provide a user-friendly interface for this data as a highly granular reference for the study of immune responses in airways and blood in children.

The cellular immune response to COVID-19 deciphered by single cell multi-omics across three UK centres (preprint)

The COVID-19 pandemic, caused by SARS coronavirus 2 (SARS-CoV-2), has resulted in excess morbidity and mortality as well as economic decline. To characterise the systemic host immune response to SARS-CoV-2, we performed single-cell RNA-sequencing coupled with analysis of cell surface proteins, providing molecular profiling of over 800,000 peripheral blood mononuclear cells from a cohort of 130 patients with COVID-19. Our cohort, from three UK centres, spans the spectrum of clinical presentations and disease severities ranging from asymptomatic to critical. Three control groups were included: healthy volunteers, patients suffering from a non-COVID-19 severe respiratory illness and healthy individuals administered with intravenous lipopolysaccharide to model an acute inflammatory response. Full single cell transcriptomes coupled with quantification of 188 cell surface proteins, and T and B lymphocyte antigen receptor repertoires have provided several insights into COVID-19: 1. a new non-classical monocyte state that sequesters platelets and replenishes the alveolar macrophage pool; 2. platelet activation accompanied by early priming towards megakaryopoiesis in immature haematopoietic stem/progenitor cells and expansion of megakaryocyte-primed progenitors; 3. increased clonally expanded CD8+ effector:effector memory T cells, and proliferating CD4+ and CD8+ T cells in patients with more severe disease; and 4. relative increase of IgA plasmablasts in asymptomatic stages that switches to expansion of IgG plasmablasts and plasma cells, accompanied with higher incidence of BCR sharing, as disease severity increases. All data and analysis results are available for interrogation and data mining through an intuitive web portal. Together, these data detail the cellular processes present in peripheral blood during an acute immune response to COVID-19, and serve as a template for multi-omic single cell data integration across multiple centers to rapidly build powerful resources to help combat diseases such as COVID-19.

Integrated Single-Cell Atlases Reveal an Oral SARS-CoV-2 Infection and Transmission Axis (preprint)

Despite signs of infection, the involvement of the oral cavity in COVID-19 is poorly understood. To address this, single-cell RNA sequencing datasets were integrated from human minor salivary glands and gingiva to identify 11 epithelial, 7 mesenchymal, and 15 immune cell clusters. Analysis of SARS-CoV-2 viral entry factor expression showed enrichment in epithelia including the ducts and acini of the salivary glands and the suprabasal cells of the mucosae. COVID-19 autopsy tissues confirmed in vivo SARS CoV-2 infection in the salivary glands and mucosa. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibiting ACE2 expression and SARS-CoV-2 RNA. Matched nasopharyngeal and saliva samples found distinct viral shedding dynamics and viral burden in saliva correlated with COVID-19 symptoms including taste loss. Upon recovery, this cohort exhibited salivary antibodies against SARS-CoV-2 proteins. Collectively, the oral cavity represents a robust site for COVID-19 infection andimplicates saliva in viral transmission.

SARS-CoV-2 Entry Genes Are Most Highly Expressed in Nasal Goblet and Ciliated Cells within Human Airways (preprint)

The SARS-CoV-2 coronavirus, the etiologic agent responsible for COVID-19 coronavirus disease, is a global threat. To better understand viral tropism, we assessed the RNA expression of the coronavirus receptor, ACE2, as well as the viral S protein priming protease TMPRSS2 thought to govern viral entry in single-cell RNA-sequencing (scRNA-seq) datasets from healthy individuals generated by the Human Cell Atlas consortium. We found that ACE2, as well as the protease TMPRSS2, are differentially expressed in respiratory and gut epithelial cells. In-depth analysis of epithelial cells in the respiratory tree reveals that nasal epithelial cells, specifically goblet/secretory cells and ciliated cells, display the highest ACE2 expression of all the epithelial cells analyzed. The skewed expression of viral receptors/entry-associated proteins towards the upper airway may be correlated with enhanced transmissivity. Finally, we showed that many of the top genes associated with ACE2 airway epithelial expression are innate immune-associated, antiviral genes, highly enriched in the nasal epithelial cells. This association with immune pathways might have clinical implications for the course of infection and viral pathology, and highlights the specific significance of nasal epithelia in viral infection. Our findings underscore the importance of the availability of the Human Cell Atlas as a reference dataset. In this instance, analysis of the compendium of data points to a particularly relevant role for nasal goblet and ciliated cells as early viral targets and potential reservoirs of SARS-CoV-2 infection. This, in turn, serves as a biological framework for dissecting viral transmission and developing clinical strategies for prevention and therapy.