Children develop strong and sustained cross-reactive immune responses against Spike protein following SARS-CoV-2 infection, with enhanced recognition of variants of concern

TL;DR: In this paper, the authors studied the profile of antibody and cellular immunity in children aged 3-11 years in comparison with adults and found that children were strong with high titres against spike protein and receptor binding domain (RBD).

Abstract: SARS-CoV-2 infection is generally mild or asymptomatic in children but the biological basis for this is unclear. We studied the profile of antibody and cellular immunity in children aged 3-11 years in comparison with adults. Antibody profiles in children were strong with high titres against spike protein and receptor binding domain (RBD). SARS-CoV-2 seroconversion in children strongly boosted antibody responses against seasonal beta-coronaviruses, partly through cross-recognition of the S2 domain, indicating a broad humoral response that was not seen in adults. T cell responses against spike were also >2-fold higher in children compared to adults and displayed a strong Th1 cytokine profile. SARS-CoV-2 spike-reactive cellular responses were present in more than half the seronegative children, indicating pre-existing cross-reactive responses or sensitization against SARS-CoV-2. Importantly, all children retained high antibody titres and cellular responses for more than 6 months after infection whilst relative antibody waning was seen in adults. Children thus distinctly generate robust, cross-reactive and sustained immune responses after SARS-CoV-2 infection with focussed specificity against spike protein. These observations demonstrate several novel features of SARS-CoV-2-specific immune responses in children and may provide insights into relative clinical protection in this group. Such information on the profile of natural infection will help to guide the introduction of vaccination regimens into the paediatric population.

Summary

Introduction

• The SARS-CoV-2 pandemic has resulted in over 4.2 million deaths to date and the most significant determinant of outcome is age at the time of primary infection 1 .
• The immunological basis for this condition remains unclear but is characterised by diffuse endothelial involvement and broad autoantibody production 10 .
• One potential determinant of differential immune responses to SARS-CoV-2 across the life course may be the timing of exposure to the four additional endemic human coronaviruses (HCoV).
• The authors demonstrate a markedly different profile of immune response after SARS-CoV-2 infection in children compared to adults.

Sample collection

• Public Health England (PHE) initiated prospective SARS-CoV-2 surveillance in primary schools across the UK after they reopened following the easing of national lockdown in June 2020.
• The protocol for the COVID-19 Surveillance in School KIDs is available online (https://www.gov.uk/guidance/covid-19-paediatric-surveillance).
• For each known SARS-CoV-2 seropositive individual, an age-matched (nearest age in years for students, nearest 10 years for teachers) and sex-matched participant also underwent blood sampling.
• In total 154 adults and 91 children had sufficient blood sample for serology and cellular responses (Table 1 ).
• Pre-pandemic plasma and PBMC were obtained from healthy children as part of an ethically approved study ; South of Birmingham Research Ethics Committee (REC: 17/WM/0453, IRAS: 233593).

MSD Serology assay

• Quantitative IgG antibody titres were measured against trimeric spike (S) protein, nucleocapsid preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 28, 2021.
• After incubation plates were washed and detection antibody added.
• Plates were washed and were immediately read using a MESO TM QuickPlex SQ 120 system.
• Data were generated by Methodological Mind software and analysed with MSD Discovery Workbench (v4.0) software.

Total IgG/A/M anti-spike SARS-CoV-2 ELISA

• A total GAM anti-SARS-CoV-2 spike ELISA kit 18 was purchased from Binding Site (Birmingham, UK).
• ELISA was performed following the manufacturer's instructions.
• Optical density (OD) was compared to a known calibrator and expressed as a ratio to the calibrator.
• Samples with a ratio above 1.0 were considered seropositive.

RBD-ACE-2 competitive binding assay.

• The concentration of antibodies which inhibited interaction between RBD and ACE-2 was determined using a SARS-CoV-2 neutralisation assay following the manufacturer's instructions.
• Briefly, plasma or positive control antibody were pre-incubated with biotinylated-Fc-chimera-S1-RBD protein prior to addition of bead bound ACE-2.
• Samples were run on a BD LSR-II flow cytometer and analysed using LEGENDplex v8.0 software .
• Results were related to a known RBD neutralising antibody standard and displayed as ng/ml.

Live virus and pseudotype-based neutralisation assays

• Clinical isolates used in the study were provided by Public Health England and Imperial College London.
• Serum was titrated starting at a 1:100 dilution.
• The specified virus was then incubated at an MOI 0.01 with the Serum for 1hr prior to infection.
• 72hrs later infection plates were fixed with 8% formaldehyde and stained with Coomassie blue for 30 mins.
• Plates were washed and dried overnight before quantification using a Celigo Imaging Cytometer to measure the staining intensity.

IFN-γ ELISpot

• Pepmixes pool containing 15-mer peptides overlapping by 10aa from either SARS-CoV-2 spike S1 or S2 domains and a combined pool of Nucleoprotein (N) and Membrane (M) and Envelope (E) were purchased from Alta Biosciences (University of Birmingham, UK).
• Briefly, fresh PBMC were rested overnight prior to assay and 0.25-0.3x10 6 PBMC were added in duplicate per well containing either pep-mix, anti-CD3 or DMSO.
• Preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 28, 2021.
• Supernatant was harvested and stored at -80°C.
• Cut off values were previously determined 21 .

Cross-reactive T cell assay

• Cells were then plated into a 48-well plate and cultured in RPMI+10%FBS+.
• Pen/Strep with the addition of 20U/ml IL-2 for 9 days, with frequent media changes.
• Cells were washed and divided across four wells of an ELISpot plate and then re-stimulated with either SARS-CoV-2 S2 pepmix or S2 pepmixes from either the Beta (OC43 and HKU-1) or Alpha (NL63 and 229E) HCoV.
• Results were read as for ELISpot and presented as expansion compared to the DMSO controls.

Cytokine measurement

• Supernatants from donors with a detectable response in overnight ELISpot cultures were assessed using a LEGENDplex Th-profile 12-plex kit following manufacturer's instructions.
• Data was analysed using LEGENDplex v8.0 Software .

Intracellular Cytokine Staining

• Cryopreserved PBMC were thawed and rested overnight.
• Cells were then stimulated with a combined spike S1 and S2 peptide pool, at a final concentration of 1μg/ml per peptide, or DMSO (Mock).
• After 1hr eBioscience Protein transport inhibitor cocktail (Thermofisher Scientific) was added and cells incubated for a further 5 hrs.
• Following stimulation, cells were washed (PBS+0.1%BSA) and surface stained at 4°C for 30min.
• Cells were then washed and fixed in 2% paraformaldehyde.

Data visualisation and statistics

• Statistical tests, including normality tests, were performed as indicated using GraphPad Prism v9 software.
• Only results found to be significant (p<0.05) are displayed.
• Preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 28, 2021.

Children develop robust and broad antibody responses after SARS-CoV-2 infection

• Blood samples were obtained from 91 children and 154 adults, including 35 children and 81 adults known to be seropositive in previous rounds of testing.
• All infections were asymptomatic or mild and no staff or students in the cohort required medical care or hospitalisation.
• To ensure the sensitivity of their assays the authors obtained convalescent plasma samples from 35 SARS-CoV-2 PCR-confirmed children.
• 34 of these were seropositive in the assay whilst one donor mounted no detectable antibody response to any antigen tested.

SARS-CoV-2 infection boosts antibodies responses against HCoV in children

• Pre-existing immune responses against seasonal coronaviruses might act to modulate clinical outcome following primary SARS-CoV-2 infection, and cross-reactive neutralising antibodies have been reported in SARS-CoV-2-seronegative children 15 .
• The authors compared antibody levels against the four HCoV in SARS-CoV-2 in seronegative and seropositive children and adults.
• Notably, the level of HCoV-specific antibodies in All rights reserved.
• Preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 28, 2021.

SARS-CoV-2-specific antibodies in children can cross-react with the S2 domain of Betacoronaviruses

• Given the increase in HCoV-specific antibody titres following SARS-CoV-2 infection in children the authors next assessed to what extent this was cross-reactive against SARS-CoV-2 or could represent a HCoV-specific response.
• As such, recombinant S1 or S2 domain protein from SARS-CoV-2 was used to pre-absorb plasma samples prior to assessment of antibody levels to both SARS-CoV-2 and the four HCoV subtypes.
• As expected, pre-absorption with both the S1 and S2 domains significantly reduced antibody titres against total spike (p<0.0001 and p=0.0024, respectively, Friedman test with Dunn's multiple comparisons test).
• Of note, the S1 domain did not significantly reduce antibody binding to any of the 4 HCoV subtypes indicating little evidence for cross-reactive antibodies against this domain.
• These data show that the S1 region is the immunodominant target of antibody responses in children.

CoV-2 infection

• Preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 28, 2021.
• The authors next assessed the magnitude and profile of the cellular immune response against SARS-CoV-2 in children and adults.
• Two children and one adult classed as seronegative had anti-nucleocapsid antibodies (Extended Data Table 2.) but this was considered insufficient to provide definitive serostatus.
• Matched plasma samples, available for three donors, showed that antibody responses against HKU-1 and 229E were 2 and 5-fold greater in a donor with high cellular responses compared to two children that lacked cellular responses.

Children maintain immune responses against SARS-CoV-2 for at least 12 months

• All children retained humoral immunity whilst 7% (6/81) of previously seropositive adults failed to show significant antibody responses.
• Samples were also obtained from 16 children who had seroconverted at least 12 months prior to analysis.
• The magnitude of the spike-specific response remained higher in All rights reserved.
• Matched samples at 6 and 12 months were available for 5 of these children and were stable.
• These data show that children broadly retain both antibody responses and cellular responses for extended periods following primary infection .

Antibodies from children show enhanced binding to spike protein from viral variants of concern but equivalent levels of viral neutralisation

• Given the development and maintenance of crossreactive high-level antibody responses in children, the authors were interested to assess their relative recognition of spike protein from VOC.
• As described above, children, compared to adults, maintain higher levels of antibody binding to Wuhan spike and this was also observed in binding to spike from the three VOC with 1.7, 1.8, and 2.1-fold higher geometric mean titres (GMT) against alpha, beta and gamma variants, respectively .
• Children also demonstrated higher binding to the RBD region of the three VOC, compared to adults, with 2.1, 1.8 and 2.9-fold higher GMT, respectively (p=0.029 and p=0.0114 against beta and gamma, respectively; Kruskal-Wallis test with Dunn's multiple comparison test).
• The authors next assessed the ability of sera from children and adults to neutralise infection by live virus.
• A similar profile was seen with a pseudotype-based neutralisation assay .

Discussion

• Age is the primary determinant of the clinical severity of SARS-CoV-2 infection and a life course assessment of virus-specific immunity is essential to understand disease pathogenesis and design vaccine strategies in children.
• It has also been reported that children do not mount effective antibody responses against nucleocapsid in the early post-infection period 3, 5 .
• SARS-CoV-2 infection in children also boosts HCoV-specific antibody responses that are not directly cross-reactive, as demonstrated by increased titres against Alpha-coronaviruses that could not be pre-absorbed.
• Preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 28, 2021.
• As such further long-term characterisation of the T cell response in children, and the potential mechanisms that may act to drive T cell activation, are required.

Figures

Figure 5. Higher spike-specific immune responses are maintained in children for at least six months after infection.

Figure 6. Plasma from children at 6+ months after infection shows superior antibody binding to spike and RBD from SARS-CoV-2 viral variants but comparable neutralisation of live virus

Figure 2. Antibody responses against the seasonal HCoV Beta-coronaviruses are back-boosted in children following SARS-CoV-2 infection.

Figure 3 Cross reactive antibodies against the spike S2 domain contribute to increased HCoV-specific antibody responses in SARS-CoV-2 seropositive children

Figure 4.

Table 1. Demographics and serostatus of study participants.

Figure 7. Model of antibody and cellular immunity to seasonal human coronaviruses (HCoV) and SARS-CoV-2 in children and adults.

Figure 1 Children and adults develop coordinated antibody responses to SARS-CoV-2

Full text

1
Children develop robust and sustained cross-reactive spike-specific
immune responses following SARS-CoV-2 infection
Alexander C. Dowell
1
, Megan S. Butler
1†
, Elizabeth Jinks
1†
, Gokhan Tut
1†
, Tara Lancaster
1
,
Panagiota Sylla
1
, Jusnara Begum
1
, Rachel Bruton
1
, Hayden Pearce
1
, Kriti Verma
1
, Nicola
Logan
2
, Grace Tyson
2
,
Eliska Spalkova
1
, Sandra Margielewska-Davies
1
, Graham S. Taylor
1
,
Eleni Syrimi
1
, Frances Baawuah
3
, Joanne Beckmann
4
, Ifeanyichukwu Okike
3,5
, Shazaad
Ahmad
6
, Joanna Garstang
7
, Andrew J Brent
8,9
, Bernadette Brent
8
, Georgina Ireland
3
, Felicity
Aiano
3
, Zahin Amin-Chowdhury
3
, Samuel Jones
3
, Ray Borrow
10
, Ezra Linley
10
, John Wright
11
,
Rafaq Azad
11
, Dagmar Waiblinger
11
, Chris Davis
2
, Emma Thomson
2
, Massimo Palmarini
2
,
Brian J. Willett
2
, Wendy S. Barclay
12
, John Poh
3
, Vanessa Saliba
3
, Gayatri Amirthalingam
3
,
Kevin E Brown
3
, Mary E Ramsay
3
, Jianmin Zuo
1
, Paul Moss
1
*, Shamez Ladhani
3,13
*
†
*These authors contributed equally
1. Institute of Immunology & Immunotherapy, College of Medical and Dental Sciences,
University of Birmingham, Birmingham, B15 2TT, UK
2. MRC-University of Glasgow Centre for Virus Research, 464 Bearsden Road,
Glasgow G61-1QH, UK
3. Public Health England, 61 Colindale Avenue, London NW9 5EQ, UK
4. East London NHS Foundation Trust, 9 Allie Street, London E1 8DE, UK
5. University Hospitals of Derby and Burton NHS Foundation Trust, Uttoxeter New
Road, Derby DE22 3NE, UK
6. Manchester University NHS Foundation Trust, Oxford Road, Manchester M13 9WL,
UK
7. Birmingham Community Healthcare NHS Trust, Holt Street, Aston B7 4BN, UK
8. Oxford University Hospitals NHS Foundation Trust, Old Road, Oxford OX3 7HE
9. University of Oxford, Wellington Square, Oxford OX1 2JD, UK
10. Public Health England, Manchester Royal Infirmary, Manchester, United Kingdom
11. Bradford Institute for Health Research, Bradford Teaching Hospitals NHS Foundation
Trust, Bradford, BD9 6RJ, UK.
12. Department of Infectious Disease, Imperial College, London, UK.
13. Paediatric Infectious Diseases Research Group, St. George’s University of London,
London, UK
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perpetuity.
preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in
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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
SARS-CoV-2 infection is generally mild or asymptomatic in children but the biological basis
for this is unclear. We studied the profile of antibody and cellular immunity in children aged
3-11 years in comparison with adults. Antibody responses against spike and receptor binding
domain (RBD) were high in children and seroconversion boosted antibody responses against
seasonal Beta-coronaviruses through cross-recognition of the S2 domain. Seroneutralisation
assays against alpha, beta and delta SARS-CoV-2 variants demonstrated comparable
neutralising activity between children and adults. T cell responses against spike were >2-fold
higher in children compared to adults and displayed a T
H
1 cytokine profile. SARS-CoV-2
spike-specific T cells were also detected in many seronegative children, revealing pre-existing
responses that were cross-reactive with seasonal Alpha and Beta-coronaviruses. Importantly,
all children retained high antibody titres and cellular responses at 6 months after infection
whilst relative antibody waning was seen in adults. Spike-specific responses in children also
remained broadly stable beyond 12 months. Children thus distinctly generate robust, cross-
reactive and sustained immune responses after SARS-CoV-2 infection with focussed
specificity against spike protein. These observations demonstrate novel features of SARS-
CoV-2-specific immune responses in children and may provide insight into their relative
clinical protection. Furthermore, this information will help to guide the introduction of
vaccination regimens in the paediatric population.
All rights reserved. No reuse allowed without permission.
perpetuity.
preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in
The copyright holder for thisthis version posted September 28, 2021. ; https://doi.org/10.1101/2021.04.12.21255275doi: medRxiv preprint

3
Introduction
The SARS-CoV-2 pandemic has resulted in over 4.2 million deaths to date and the most
significant determinant of outcome is age at the time of primary infection
1
. SARS-CoV-2
infection in children is generally asymptomatic or mild and contrasts with high rates of
hospitalisations and deaths among older adults
2
. As such, there is interest in understanding the
profile of the immune response to SARS-CoV-2 in children. Such studies are limited to date
but have reported reduced magnitude of both antibody and cellular responses in comparison to
adults, and an absence of nucleocapsid-specific antibody responses during or early post-
infection
3, 4, 5, 6
. One unique feature of SARS-CoV-2 infection in children is the development
of a rare complication known as pediatric inflammatory multisystem syndrome temporally
associated with SARS-CoV-2 (PIMS-TS), also known as paediatric multisystem inflammatory
syndrome (MIS-C), which shares features with Kawasaki disease and toxic shock syndrome
7,
8
. MIS-C develops approximately 2-4 weeks after infection in children with a median age of 9
years
9
. The immunological basis for this condition remains unclear but is characterised by
diffuse endothelial involvement and broad autoantibody production
10
.
One potential determinant of differential immune responses to SARS-CoV-2 across the life
course may be the timing of exposure to the four additional endemic human coronaviruses
(HCoV). These comprise the Beta-coronaviruses OC43 and HKU-1, which have 38% and 35%
amino acid homology with SARS-CoV-2, as well as the more distantly related Alpha-
coronaviruses NL63 and 229E, each with around 31% homology
11
. These coronaviruses cause
frequent mild childhood infections and antibody seroconversion occurs typically before the age
of 5 years. Infection with one of the Alpha or Beta-coronaviruses provides short-term immunity
against re-infection from coronaviruses and is believed to represent transient cross-reactive
immunity within the subtypes
12, 13, 14
. As such, recent HCoV infection might pre-sensitize
children against SARS-CoV-2 infection and may explain cross-reactive SARS-CoV-2-
neutralising antibodies in some seronegative children
15
. Immune responses against HCoV are
retained throughout life but do not provide sterilising immunity
13
. Consequently, recurrent
infections are common, generating concern that a similar pattern will be observed after SARS-
CoV-2 infection.
COVID-19 vaccines are now being administered widely to adult populations and are also being
delivered to children in some countries. It is therefore imperative to understand the profile of
SARS-CoV-2-specific immune responses in children after natural infection in order to inform
vaccination strategy. Here we provide a comprehensive characterisation of the convalescent
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4
humoral and cellular immune response in a cohort of 91 primary school-aged children
compared with 154 adults taking part in the SARS-CoV-2 Surveillance in School Kids (sKIDs)
study
16
. We demonstrate a markedly different profile of immune response after SARS-CoV-2
infection in children compared to adults. These findings have potential implications for
understanding protective or pathological immune responses to infection in children and may
help to guide and interpret COVID-19 vaccination regimens for children.
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perpetuity.
preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in
The copyright holder for thisthis version posted September 28, 2021. ; https://doi.org/10.1101/2021.04.12.21255275doi: medRxiv preprint

5
Methods
Sample collection
Public Health England (PHE) initiated prospective SARS-CoV-2 surveillance in primary
schools across the UK after they reopened following the easing of national lockdown in June
2020. The protocol for the COVID-19 Surveillance in School KIDs (sKIDs) is available online
(https://www.gov.uk/guidance/covid-19-paediatric-surveillance)
16
. Surveillance comprised
two arms, one involving weekly swabbing of primary school students and staff for SARS-CoV-
2 infection (from June to mid-July 2020) and the other comprising swabbing and blood
sampling taken in 3 rounds: beginning (1-19 June) and end (3-23 July) of the second half of
the summer term when primary schools were partially re-opened, and after full reopening of
all schools in September 2020, at the end of the autumn term (23 November-18 December).
Samples for extended humoral and cellular analysis were taken in round 3. Additional samples
from children found to be seropositive in round 1 were taken from 21 June-24 July 2021.
For each known SARS-CoV-2 seropositive individual, an age-matched (nearest age in years
for students, nearest 10 years for teachers) and sex-matched participant also underwent blood
sampling. In total 154 adults and 91 children had sufficient blood sample for serology and
cellular responses (Table 1). Convalescent plasma samples were also available from 35
children aged 10-13 years with PCR-confirmed SARS-CoV-2 infection, taken a median of 6
months (range 2-12 months) following PCR result, from the Born in Bradford study
17
.
Pre-pandemic plasma and PBMC were obtained from healthy children as part of an ethically
approved study (TrICICL); South of Birmingham Research Ethics Committee (REC:
17/WM/0453, IRAS: 233593).
PBMC and Plasma Preparation
Blood tubes were spun at 300g for 10mins prior to removal of plasma which was then spun
again at 800g for 10mins and stored at -80°C. Remaining blood was diluted 1:1 with RPMI
and PBMC isolated on a Sepmate (Stemcell) density centrifugation tube, washed with RPMI
and rested in RPMI+10% FBS overnight at 37°C.
MSD Serology assay
Quantitative IgG antibody titres were measured against trimeric spike (S) protein, nucleocapsid
(N) and other coronavirus using MSD V-PLEX COVID-19 Coronavirus Panel 2 (N05368-A1),
Coronavirus Panel 7 (N05428A-1) responses to other respiratory viruses were measured using
MSD V-PLEX COVID-19 Respiratory Panel 1 (N05358-A1). Multiplex MSD Assays were
performed according to manufacturer instructions. Briefly 96-well plates were blocked.
Following washing, samples diluted 1:5000 in diluent, as well as reference standard and
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preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in
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Frequently asked questions

Q1. What have the authors contributed in "Children develop robust and sustained cross-reactive spike-specific immune responses following sars-cov-2 infection" ?

Dowell et al. this paper provided a comprehensive characterisation of the immunization response of primary school children to SARS-CoV-2 infection. 

Q2. What are the future works mentioned in the paper "Children develop robust and sustained cross-reactive spike-specific immune responses following sars-cov-2 infection" ?

These antibodies could still have important effector potential through mechanisms such as antibody-directed cell cytotoxicity and further studies to examine the specificity and function of the SARS-CoV-2 B cell repertoire in children following natural infection or vaccination would be of value. 

Q3. When did the response to nucleocapsid show significant waning?

Spike-specific antibody responses were also largely maintained in children at 12 months, whereas responses against nucleocapsid showed significant waning. 

Q4. How many children were found to be seropositive?

Children develop robust and broad antibody responses after SARS-CoV-2 infection Blood samples were obtained from 91 children and 154 adults, including 35 children and 81 adults known to be seropositive in previous rounds of testing. 

Q5. What was the effect of the IL-2-specific T cells in children?

To examine the presence of cross-reactive T cells in seronegative children the authors hypothesised that expansion of T cells in response to SARS-CoV-2 would be associated with an associated increase in HCoV-reactive responses. 

Q6. What is the significant determinant of outcome?

The SARS-CoV-2 pandemic has resulted in over 4.2 million deaths to date and the most significant determinant of outcome is age at the time of primary infection1. 

Q7. How long did the blood tube be stored?

Blood tubes were spun at 300g for 10mins prior to removal of plasma which was then spun again at 800g for 10mins and stored at -80°C. 

Q8. What was used to transfect HEK293T cells?

HEK293T cells were transfected with the appropriate SARS-CoV-2 S gene expression vector in conjunction with lentiviral vectors p8.91 and pCSFLW using polyethylenimine (PEI, Polysciences, Warrington, USA). 

Q9. What are the likely coV-specific T cells to be present in children?

T cell responses were detectable in more than half the seronegative children, including samples taken pre-pandemic, and are likely to represent HCoV-specific T cell responses that cross react against SARS-CoV2 peptides 34, 35. 

Q10. What is the common complication of SARS-CoV-2 in children?

One unique feature of SARS-CoV-2 infection in children is the development of a rare complication known as pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS), also known as paediatric multisystem inflammatory syndrome (MIS-C), which shares features with Kawasaki disease and toxic shock syndrome 7, 8. 

Q11. What is the effect of the S2 domain on antibody binding to the two HCoV?

The S2 domain, however, selectively reduced antibody binding to the two HCoV Beta-coronaviruses, OC43 and HKU-1 (p <0.0001 and p=0.0014, respectively by repeated measure one-way ANOVA with Holm-Sidak’s multiple comparison test). 

Q12. What is the effect of the spike protein on the immune response in children?

The authors find that the virus-specific T cell response is higher in children compared to adults and this mirrored the humoral response in that responses against the spike protein were markedly increased compared to nucleocapsid and envelope proteins. 

Q13. What are the other endemic human coronaviruses?

These comprise the Beta-coronaviruses OC43 and HKU-1, which have 38% and 35% amino acid homology with SARS-CoV-2, as well as the more distantly related Alphacoronaviruses NL63 and 229E, each with around 31% homology 11. 

Q14. What is the S2 domain of SARS-CoV-2?

The S2 domain is more highly conserved between HCoV than S1 and this pattern is compatible with preferential targeting of structurally-conserved epitopes by HCoV-specific antibodies in children 30 with the potential for neutralising activity against SARS-CoV-2 15, 31. 

Q15. What is the effect of the sARS-CoV-2 antibody on children?

In conclusion, the authors show that children display a characteristic robust and sustained adaptive immune response against SARS-CoV-2 with substantial cross-reactivity against other HCoV. 

Q16. How many children have been reported to have higher responses to SARS-CoV-2?

Of note, young adults have been recently reported to have higher T cellresponses to HCoV than older people and these can cross-react with SARS-CoV-2 36. 

Q17. How did the authors determine the presence of cross-reactive T cells in children?

to definitively assess the presence of cross-reactive T cells in children the authors obtained pre-pandemic PBMC from children and observed that 50% of these had notable responses to spike by ELISpot but lacked cellular responses against N/M peptides. 

Q18. What was the reaction between ACE-2 and RBD?

plasma or positive control antibody were pre-incubated with biotinylated-Fc-chimera-S1-RBD protein prior to addition of bead bound ACE-2.