Atypical B cells and impaired SARS-CoV-2 neutralisation following booster vaccination in the elderly.

Age is a major risk factor for hospitalization and death after SARS-CoV-2 infection, even in vaccinees. Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here we show that individuals 70 or older who received a primary two dose schedule with AZD1222 and booster third dose with mRNA vaccine achieved significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared to those younger than 70. One month after the booster neither the concentration of serum binding anti spike IgG antibody, nor the frequency of spike-specific B cells showed differences by age grouping. However, the impaired neutralization potency and breadth post-third dose in the elderly was associated with enrichment of circulating "atypical" spike-specific B cells expressing CD11c and FCRL5. Single cell RNA sequencing confirmed an expansion of TBX21-, ITGAX-expressing B cells in the elderly that enriched for B cell activation/receptor signalling pathway genes. Importantly we also observed impaired T cell responses to SARS-CoV-2 spike peptides in the elderly post-booster, both in terms of IFNgamma and IL2 secretion, as well as a decrease in T cell receptor signalling pathway genes. This expansion of atypical B cells and impaired T cell responses may contribute to the generation of less affinity-matured antibodies, with lower neutralizing capacity post-third dose in the elderly. Altogether, our data reveal the extent and potential mechanistic underpinning of the impaired vaccine responses present in the elderly after a booster dose, contributing to their increased susceptibility to COVID-19 infection.

Obesity associated with attenuated tissue immune cell responses in COVID-19

Obesity is common and associated with more severe COVID-19, proposed to be in part related to an adipokine-driven pro-inflammatory state. Here we analysed single cell transcriptomes from bronchiolar lavage in three adult cohorts, comparing obese (Ob, body mass index (BMI) >30m2) and non-obese (N-Ob, BMI <30m2). Surprisingly, we found that Ob subjects had attenuated lung immune/inflammatory responses in SARS-CoV-2 infection, with decreased expression of interferon (IFN), IFN{gamma} and tumour necrosis factor (TNF) alpha response gene signatures in almost all lung epithelial and immune cell subsets, and lower expression of IFNG and TNF in specific lung immune cells. Analysis of peripheral blood immune cells in an independent adult cohort showed a similar, but less marked, reduction in type I IFN and IFN{gamma} response genes, as well as decreased serum IFN, in Ob patients with SARS-CoV-2. Nasal immune cells from Ob children with COVID-19 also showed reduced enrichment of IFN and IFN{gamma} response genes. Altogether, these findings show blunted tissue immune responses in Ob COVID-19 patients, with clinical implications.

SARS-CoV-2 Omicron spike mediated immune escape, infectivity and cell-cell fusion

The SARS-CoV-2 Omicron BA.1 variant emerged in late 2021 and is characterised by multiple spike mutations across all spike domains. Here we show that Omicron BA.1 has higher affinity for ACE2 compared to Delta, and confers very significant evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralising antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralisation. Importantly, antiviral drugs remdesevir and molnupiravir retain efficacy against Omicron BA.1. We found that in human nasal epithelial 3D cultures replication was similar for both Omicron and Delta. However, in lower airway organoids, Calu-3 lung cells and gut adenocarcinoma cell lines live Omicron virus demonstrated significantly lower replication in comparison to Delta. We noted that despite presence of mutations predicted to favour spike S1/S2 cleavage, the spike protein is less efficiently cleaved in live Omicron virions compared to Delta virions. We mapped the replication differences between the variants to entry efficiency using spike pseudotyped virus (PV) entry assays. The defect for Omicron PV in specific cell types correlated with higher cellular RNA expression of TMPRSS2, and accordingly knock down of TMPRSS2 impacted Delta entry to a greater extent as compared to Omicron. Furthermore, drug inhibitors targeting specific entry pathways demonstrated that the Omicron spike inefficiently utilises the cellular protease TMPRSS2 that mediates cell entry via plasma membrane fusion. Instead, we demonstrate that Omicron spike has greater dependency on cell entry via the endocytic pathway requiring the activity of endosomal cathepsins to cleave spike. Consistent with suboptimal S1/S2 cleavage and inability to utilise TMPRSS2, syncytium formation by the Omicron spike was dramatically impaired compared to the Delta spike. Overall, Omicron appears to have gained significant evasion from neutralising antibodies whilst maintaining sensitivity to antiviral drugs targeting the polymerase. Omicron has shifted cellular tropism away from TMPRSS2 expressing cells that are enriched in cells found in the lower respiratory and GI tracts, with implications for altered pathogenesis.

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

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

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 paediatric 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 data1 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

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

Despite signs of infection, the involvement of the oral cavity in COVID-19 is poorly understood. To address this, single-cell RNA sequencing data-sets 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 and implicates saliva in viral transmission.

INSIGHT: a scalable isothermal NASBA-based platform for COVID-19 diagnosis

We present INSIGHT (Isothermal NASBA-Sequencing-based hIGH-througput Test): a two-stage COVID-19 testing strategy, using a barcoded isothermal NASBA reaction that combines point-of-care diagnosis with next generation sequencing, aiming to achieve population-scale COVID-19 testing. INSIGHT combines the advantages of near-patient with centralised testing. Stage 1 allows a quick decentralised readout for early isolation of pre-symptomatic or asymptomatic patients. The same reaction products can then be used in a highly multiplexed sequencing-based assay in Stage 2, confirming the near-patient testing results and facilitating centralised data collection. Based on experiments using commercially acquired human saliva with spiked-in viral RNA as input, the INSIGHT platform gives Stage 1 results within one to two hours, using either fluorescence detection or a lateral flow (dipstick) readout, whilst simultaneously incorporating sample-specific barcodes into the amplification product. INSIGHT Stage 2 can be performed by directly pooling and sequencing all post-amplification barcoded Stage 1 products from hundreds of thousands of samples with minimal sample preparation steps. The 95% limit of detection (LoD-95) for INSIGHT is estimated to be below 50 copies of viral RNA per 20 l of reaction. Our two-stage testing strategy is suitable for further development into a rapid home-based and point-of-care assay, and is potentially scalable to the population level.