1
The local and systemic response to SARS-CoV-2 infection in children and adults
Masahiro Yoshida*
1,7
, Kaylee B. Worlock*
1
, Ni Huang*
2
, Rik G.H. Lindeboom*
2
, Colin R.
Butler
5
, Natsuhiko Kumasaka
2
, Cecilia Dominguez Conde
2
, Lira Mamanova
2
, Liam Bolt
2
,
Laura Richardson
2
, Krzysztof Polanski
2
, Elo Madissoon
2,10
, Josephine L. Barnes
1
, Jessica
Allen-Hyttinen
1
, Eliz Kilich
3
, Brendan C. Jones
5
, Angus de Wilton
3
, Anna Wilbrey-Clark
2
,
Waradon Sungnak
2
, J. Patrick Pett
2
, Elena Prigmore
2
, Henry Yung
1,3
, Puja Mehta
1,3
,
Aarash Saleh
4
, Anita Saigal
4
, Vivian Chu
4
, Jonathan M. Cohen
3
, Clare Cane
4
, Aikaterini
Iordanidou
4
, Soichi Shibuya
5
, Ann-Kathrin Reuschl
6
, A. Christine Argento
8
, Richard G.
Wunderink
8
, Sean B. Smith
8
, Taylor A. Poor
8
, Catherine A. Gao
8
, Jane E. Dematte
8
, NU
SCRIPT Study Investigators
8
, Gary Reynolds
13
, Muzlifah Haniffa
13
, Georgina S. Bowyer
11
,
Matthew Coates
11,12
, Menna R. Clatworthy
11
, Fernando J. Calero-Nieto
9
, Berthold
Göttgens
9
, Christopher O’Callaghan
5
, Neil J. Sebire
5
, Clare Jolly
6
, Paolo de Coppi
5
,
Claire M. Smith
5
, Alexander V. Misharin
8
, Sam M. Janes
1,3
, Sarah A. Teichmann
2,14
,
Marko Z. Nikolić
1,3 †
& Kerstin B. Meyer
2†
Correspondence to: m.nikolic@ucl.ac.uk, km16@sanger.ac.uk
Affiliations
1
UCL Respiratory, Division of Medicine, University College London, London, UK
2
Wellcome Sanger Institute, Cambridge, UK
3
University College London Hospitals NHS Foundation Trust, London, UK
4
Royal Free Hospital NHS Foundation Trust, London, UK
5
NIHR Great Ormond Street BRC and UCL Institute of Child Health, London, UK
6
UCL Division of Infection and Immunity, University College London, London, UK
7
Division of Respiratory Diseases, Department of Internal Medicine, Jikei University School
of Medicine, Tokyo, Japan
8
Division of Pulmonary and Critical Care Medicine, Northwestern University Feinberg
School of Medicine, Chicago, USA
9
Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
10
European Molecular Biology Laboratory - European Bioinformatics Institute, Cambridge,
UK
11
Department of Medicine, University of Cambridge, Cambridge Biomedical Campus, UK
12
Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK
13
Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK
14
Dept Physics/Cavendish Laboratory, University of Cambridge, JJ Thomson Ave,
Cambridge CB3 0HE, UK
*co-first authors
† co-senior authors
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NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
2
Abstract
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 data
1
as a highly
granular reference for the study of immune responses in airways and blood in children.
Introduction
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the
current coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV-2 infection is relatively
rare in children (1-2% of total cases reported <19 years)
2–4
, and generally presents with milder
severity compared to adults
2,3,5,6
. Hospitalisations, progression to symptomatic respiratory
failure requiring oxygen due to SARS-CoV-2 pneumonia, ventilatory support in intensive care
and death are all rare in children, with the most common clinical manifestation of the disease
reported as fevers, cough, rhinorrhea, myalgia and fatigue
7,8,9,10
. Differences in disease
progression between children and adults may hold clues for better treatment of severe SARS-
CoV-2 infection.
. CC-BY 4.0 International licenseIt is made available under a
perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint
The copyright holder for thisthis version posted March 15, 2021. ; https://doi.org/10.1101/2021.03.09.21253012doi: medRxiv preprint
3
SARS-CoV-2 employs a host cell surface protein, angiotensin-converting enzyme (ACE) 2, as
a receptor for cellular entry
11
. A number of recent studies have suggested that the expression
of ACE2 is both tissue and age-dependent
12,13
, and differences in ACE2 expression between
children and adults were proposed to contribute to less severe disease in children. At single cell
level, viral entry genes were shown to be most highly expressed in the nasal epithelium in
healthy adults
14
. In children, bulk RNA sequencing analysis revealed lower ACE2 gene
expression both in upper
15
and lower airways
13,16
compared to adults, although more recent
studies found no correlation with age or infection
17
.
Upon infection, 14% of symptomatic adults develop progressive respiratory failure displaying
a strong inflammatory immune response
18
. Recent single cell analysis of this response
demonstrated the involvement of various types of immune cells, including proinflammatory
monocytes/macrophages
19–21
, clonally expanded cytotoxic T cells
22–24
and neutrophils
22
.
Although these specific cell types are beginning to be resolved in adults
19–21,20,22,24,25
, they have
not been as comprehensively characterised in children. So far, only bulk RNA sequencing and
cytokine studies have compared immune responses between children and adults, suggesting a
more robust innate immune response, such as increased levels of interferon gamma (IFN-γ)
and interleukin-17 (IL-17A) in plasma
26
, and a reduced antibody response and neutralising
activity against SARS-CoV-2 in children
27
.
The immune landscape in early life has been shown to be distinct from that of adults
28,29,30
, in
keeping with the immune system maturing from an immune tolerant state in utero to a more
pro-inflammatory state with the increased exposure of new antigens and pathogens over the
years
31
. Changes in blood cell counts and immune cell composition throughout childhood are
well described
32
, and recently CyTOF (cytometry by time-of-flight) has facilitated a higher
multiplexed panel-dependent description of cell types, with studies focussing on either early
childhood
29
or reporting comparison to disease
33
. Unbiased transcriptomic analysis of healthy
paediatric blood immune cell types over the entire duration of childhood is lacking. Similarly,
the upper airway mucosa only reaches maturity after puberty, in keeping with the high
incidence of upper respiratory tract infections in children
34
, yet unbiased analysis of epithelial
maturation is still missing.
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is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint
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4
To address these gaps in our knowledge and to identify paediatric-specific responses in
COVID-19, we collected matched nasal, tracheal, bronchial and blood samples from patients
across infancy, childhood, adolescence and adulthood and analysed them with single cell
transcriptomics combined with protein profiling. We compare this with samples from
paediatric and adult COVID-19 patients and report markedly different responses. In paediatric
COVID-19, the biggest changes in blood are increased fractions of naive lymphocytes,
regulatory T cells (Tregs) and innate lymphoid cells (ILCs) and a loss of natural killer (NK)
cells and monocytes. In contrast, in the airways the proportion of activated macrophages,
neutrophils and CD8
+
T cells goes up whilst B lineage cells are underrepresented. In adult
blood, we see expanded T clones which encompass distinct T cell subtypes, whilst children
show less expanded T cell clones, particularly in COVID-19 patients. We also chart the
complex relationship between immune cell types present in the blood and the nose of the same
individuals.
Overall, the substantial differences in response to SARS-CoV-2 between children and adults
reflect the changes of the immune landscape over developmental time, which in children are
dominated by naive adaptive and innate cell responses, whilst those in adults are dominated by
memory cell recall adaptive responses, illustrated by expanded lymphocyte clones. Lastly, we
describe a novel epithelial cell type transitioning from secretory to ciliated cells that is present
in COVID-19 patients as well as healthy children which, among other epithelial cell types,
displays a striking antiviral and neutrophil-recruiting signature.
Results
Study cohort
We have assembled a cohort of 30 healthy children from the five World Health Organisation
(WHO) age ranges - neonates (0-30 days; n=6), infants (1-24 months; n=6), young children (2-
6 years; n=8), children (6-12 years; n=5), adolescents (12-18 years; n=5) - and profiled the
cellular landscape in the upper airways (nasal and tracheal brushings) and matching peripheral
blood mononuclear cells (PBMCs) from blood using single cell RNA sequencing (scRNA-seq)
(Figure 1a-b). For one individual a bronchial sample was included that matched both nasal
and blood samples. In addition, we sampled 4 paediatric COVID-19 patients spread across the
age categories and 18 adult COVID-19 patients with a range of disease severities, with lower
airways (bronchi) sampled in a subset (Figure 1b). Only patients who tested positive for
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5
SARS-CoV-2 by RT-qPCR were enrolled in the study, and symptom onset relative to RT-
qPCR testing and sampling is summarised in Extended Data Figure 1a. For some patients
blood was additionally sampled on the day of hospital discharge (labeled convalescent) and
our cohort included patients who were recalled 3 months after recovering from COVID-19
requiring respiratory support at the time of infection (non-invasive or invasive ventilation),
labeled as post-COVID (Extended Data Figure 1a-b). Patient characteristics and metadata
are in Extended Data Table 1a. This includes ethnicity data, but we also used our data set to
infer ethnicities and found that 48% of our cohort were of European, 16% of Asian, 10% of
African and 26% of mixed descent (Extended Data Figure 1c). Nasal, tracheal and bronchial
brushings were freshly processed by cold digestion and analysed by Chromium 10X 5’
sequencing. Where possible, isolated PBMCs from matching patients were frozen, thawed,
multiplexed and sequenced with 5’ 10X technology using a 192 antibody CITE-seq panel
(Figure 1b; Extended Data Table 2 for CITE-seq panel) generating matched transcriptional
and protein data.
For the upper airways, we generated 138,688 high quality single cell transcriptomes. Of these
115,726 were epithelial and 22,962 immune cells (Figure 1c, Extended Data Figure 2). For
the bronchi, a total of 11,672 cells were derived from 4 adult COVID-19 patients (Figure 1d).
For PBMCs we obtained 317,854 cells of which 181,346 were isolated from COVID-19
patients (Figure 1e). The full dataset is available at https://www.covid19cellatlas.org/ for easy
browsing and interactive data analysis.
Detection of SARS-CoV-2 reads
When aligning transcriptomes, we included the SARS-CoV-2 reference genome, and detected
viral reads (> 10 reads) in 4 individuals (3 nasal and 1 bronchial). In the upper airways, we
detected the highest levels of total viral expression in the two most abundant cell types: goblet
2 and ciliated 2 cell populations (Figure 1f). When examining the percentage of cells with viral
reads, fractions were similar across all nasal epithelial cell types (Figure 1g), which agrees
with the expression of ACE2 in our data set (Extended data Figure 3a). Viral reads were also
detected in lymphocytes and myeloid cells (mostly macrophages), either reflecting active
infection in macrophages
35
or merely the uptake of virions or infected dead cells.
These findings are consistent with previous studies that reported highest levels of expression
of ACE2 and TMPRSS2 in goblet and ciliated cells in healthy individuals
14
. Additional genes
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is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint
The copyright holder for thisthis version posted March 15, 2021. ; https://doi.org/10.1101/2021.03.09.21253012doi: medRxiv preprint