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\begin{document}

\title{Assessment of SARS-CoV-2 immunity in convalescent children and
adolescents}



\author[1]{Patrick Ip}%
\author[1]{Hing Wai Tsang}%
\author[1]{Gilbert T Chua}%
\author[2]{Kelvin KW}%
\author[3]{Joshua Wong SC}%
\author[1]{Tu WW}%
\author[4]{Janette Kwok SY}%
\author[1]{WHS Wong}%
\author[1]{Xiwei Wang}%
\author[1]{Yanmei Zhang}%
\author[1]{Jaime Rosa Duque}%
\author[1]{Godfrey Chan}%
\author[5]{Chu WK}%
\author[5]{Pang CP}%
\author[6]{Paul Tam KH}%
\author[1]{YL Lau}%
\author[7]{Ian Wong}%
\author[1]{Leung WH}%
\author[2]{Kwok-Yung Yuen}%
\author[3]{Mike Kwan YW}%
\affil[1]{The University of Hong Kong Department of Paediatrics and Adolescent Medicine}%
\affil[2]{State Key Laboratory for Emerging Infectious Diseases Carol Yu Centre for Infection Department of Microbiology Li Ka Shing Faculty of Medicine The University of Hong KongHong Kong SAR China}%
\affil[3]{Department of Paediatrics and Adolescent Medicine Prince Margaret Hospital Hospital Authority Hong Kong SAR China}%
\affil[4]{Department of Pathology Queen Mary Hospital Hospital Authority Hong Kong SAR China}%
\affil[5]{The Chinese University of Hong Kong Department of Ophthalmology and Visual Sciences}%
\affil[6]{Department of Surgery and Dr Li Dak Sam Research Centre Li Ka Shing Faculty of Medicine The University of Hong Kong Hong Kong SAR China}%
\affil[7]{The University of Hong Kong Department of Pharmacology and Pharmacy}%


\vspace{-1em}



  
  \date{May 21, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Background:} Persistence of protective immunity for SARS-CoV-2
is important against reinfection. Knowledge on SARS-CoV-2 immunity in
pediatric patients is currently lacking. We opted to assess the
SARS-CoV-2 adaptive immunity in recovered children and adolescents,
addressing the pediatrics specific immunity towards COVID-19.
\textbf{Method:} Two independent assays were performed to investigate
humoral and cellular immunological memory in pediatric convalescent
COVID-19 patients. Specifically, RBD IgG, CD4+, and CD8+ T cell
responses were identified and quantified in recovered children and
adolescents. \textbf{Results:} SARS-CoV-2-specific RBD IgG detected in
recovered patients had a half-life of 121.6 days and estimated duration
of 7.9 months compared with baseline levels in controls. The specific T
cell response was shown to be independent of recovery time. Both CD4+
and CD8+ T cells showed robust responses not only to spike (S) peptides
(a main target of vaccine platforms) but were also similarly activated
when stimulated by membrane (M) and nuclear (N) peptides. Importantly,
we found the differences in the adaptive responses were correlated with
the age of the recovered patients. The CD4+ T cell response to
SARS-CoV-2 S peptide in children aged \textless{}12 years correlated
with higher SARS-CoV-2 RBD IgG levels, whereas higher level of CD8+ T
cells in children aged [?]12 years, suggesting the importance of a T
cell-dependent humoral response in younger children under 12 years.
\textbf{Conclusion:} Both cellular and humoral immunity against
SARS-CoV-2 infections can be induced in pediatric patients. Our
important findings provide fundamental knowledge on the immune memory
responses to SARS-CoV-2 in recovered pediatric patients.%
\end{abstract}%



\sloppy


\textbf{Assessment of SARS-CoV-2 immunity in convalescent children and
adolescents}

Hing Wai Tsang, PhD\textsuperscript{*1}, Gilbert T Chua,
MBBS\textsuperscript{*1}, Kelvin KW To, MD\textsuperscript{*2}, Joshua
SC Wong, FHKAM\textsuperscript{3}, WW Tu, PhD\textsuperscript{1},
Janette SY Kwok, FHKAM\textsuperscript{4}, Wilfred HS Wong,
PhD\textsuperscript{1}, Xiwei Wang, PhD\textsuperscript{1}, Yanmei
Zhang, PhD\textsuperscript{1}, Jaime S Rosa Duque, MD,
PhD\textsuperscript{1}, Godfrey CF Chan, MD\textsuperscript{1}, WK Chu,
DPhil\textsuperscript{5}, CP Pang, DPhil\textsuperscript{5}, Paul KH
Tam, ChM\textsuperscript{6,7}, YL Lau, MD\textsuperscript{1}, Ian CK
Wong, PhD\textsuperscript{8}, WH Leung, PhD\textsuperscript{1},
Kwok-Yung Yuen, MD\textsuperscript{2}, Mike YW Kwan,
FHKAM\textsuperscript{\#3}, and Patrick Ip, MPH\textsuperscript{\#1}

\textsubscript{*}These 3 authors contributed equally to this work

\textsubscript{\#} Co-corresponding authors.

\textbf{Running Title:} COVID-19 immunity in convalescent children and
adolescents

\textbf{Affiliations:}

\textsubscript{1} Department of Paediatrics and Adolescent Medicine, Li
Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong
SAR, China

\textsubscript{2} State Key Laboratory for Emerging Infectious Diseases,
Carol Yu Centre for Infection, Department of Microbiology, Li Ka Shing
Faculty of Medicine, The University of Hong Kong,Hong Kong SAR, China

\textsubscript{3} Department of Paediatrics and Adolescent Medicine,
Prince Margaret Hospital, Hospital Authority, Hong Kong SAR, China

\textsubscript{4} Department of Pathology, Queen Mary Hospital, Hospital
Authority, Hong Kong SAR, China

\textsubscript{5} Department of Ophthalmology and Visual Sciences, The
Chinese University of Hong Kong SAR, Hong Kong, China

\textsubscript{6} Department of Surgery and Dr. Li Dak Sam Research
Centre, Li Ka Shing Faculty of Medicine, The University of Hong Kong,
Hong Kong SAR, China

\textsubscript{7} Faculty of Medicine, Macau University of Science and
Technology, Macau SAR, China

\textsubscript{8} Department of Pharmacology and Pharmacy, Li Ka Shing
Faculty of Medicine, The University of Hong Kong, HKSAR, China

\textbf{Corresponding address:}

Dr. Mike Yat-Wah Kwan, Consultant, Hospital Authority. Email address:
kwanyw1@ha.org.hk; Postal address: 3\textsuperscript{rd} Floor, Block S,
Hospital Authority Infectious Disease Centre, Lai King Hill Road, Kwai
Chung, Lai Chi Kok, Hong Kong SAR, China; Tel:(+852) 29903243

Dr. Patrick Ip, Clinical Associate Professor, The University of Hong
Kong (main correspondence). Email address: patricip@hku.hk; Postal
address: Room 115, 1/F, New Clinical Building, Queen Mary Hospital, 102
Pokfulam Road, Hong Kong SAR, China; Tel: (+852) 22554090, Fax: (+852)
28551523

\textbf{Funding:} This work was supported by the Hong Kong Collaborative
Research Fund (CRF) 2020/21 and the CRF Coronavirus and Novel Infectious
Diseases Research Exercises (Reference Number: C7149-20G), and the
Health and Medical Research Fund (Reference number: COVID190106). The
funding sources were not involved in the study design, data collection,
analysis, and interpretation; writing of the manuscripts; and the
decision to submit the manuscript for publication.

\textbf{Conflict of interest disclosures:} The authors declare that
there are no conflicts of interest relevant to this article

Total character count: 29312

Abstract Word Count: 249

Article Word Count: 2853

No. of table: 1

No. of figures: 4

\textbf{Abstract}

\textbf{Background:} Persistence of protective immunity for SARS-CoV-2
is important against reinfection. Knowledge on SARS-CoV-2 immunity in
pediatric patients is currently lacking. We opted to assess the
SARS-CoV-2 adaptive immunity in recovered children and adolescents,
addressing the pediatrics specific immunity towards COVID-19.

\textbf{Method:} Two independent assays were performed to investigate
humoral and cellular immunological memory in pediatric convalescent
COVID-19 patients. Specifically, RBD IgG, CD4+, and CD8+ T cell
responses were identified and quantified in recovered children and
adolescents.

\textbf{Results:} SARS-CoV-2-specific RBD IgG detected in recovered
patients had a half-life of 121.6 days and estimated duration of 7.9
months compared with baseline levels in controls. The specific T cell
response was shown to be independent of recovery time. Both CD4+ and
CD8+ T cells showed robust responses not only to spike (S) peptides (a
main target of vaccine platforms) but were also similarly activated when
stimulated by membrane (M) and nuclear (N) peptides. Importantly, we
found the differences in the adaptive responses were correlated with the
age of the recovered patients. The CD4+ T cell response to SARS-CoV-2 S
peptide in children aged \textless{}12 years correlated with higher
SARS-CoV-2 RBD IgG levels, whereas higher level of CD8+ T cells in
children aged [?]12 years, suggesting the importance of a T cell-dependent
humoral response in younger children under 12 years.

\textbf{Conclusion:} Both cellular and humoral immunity against
SARS-CoV-2 infections can be induced in pediatric patients. Our
important findings provide fundamental knowledge on the immune memory
responses to SARS-CoV-2 in recovered pediatric patients.

\textbf{Keywords:} COVID-19; SARS-CoV-2; Convalescence; Children;
Adolescents; T cell response; SARS-CoV-2 RBD IgG

\textbf{Abbreviations:} COVID-19: Coronavirus disease 2019; SARS-CoV-2:
Severe Acute Respiratory Syndrome Coronavirus 2; PBMCs: Peripheral Blood
Monocytic Cells; RBD: Receptor Binding Domain

\textbf{Declarations}

Ethics Approval: The study was approved by the Institutional Review
Board of the University of Hong Kong/ Hospital Authority Hong Kong West
Cluster {[}Reference: UW 20-292 and UW 21-157{]} and the Kowloon West
Cluster Research Ethics Committee {[}Reference: KW/FR-20-086(148-10){]}

\textbf{Availability of data and materials:} Raw data will be made
available upon reasonable request

\textbf{Author Contributions}

Patrick Ip is the Principal Investigator of the Collaborative Research
Fund, the funding source of this study. Patrick Ip, Hing Wai Tsang, and
Gilbert T Chua contributed to the study conception and research design.
Mike Kwan, Kelvin To, Wilfred Wong, and Wing Hang Leung contributed to
the data collection and analysis. Patrick Ip, Hing Wai Tsang, Gilbert T
Chua, Yu Lung Lau, WW Tu, and Janette Kwok contributed to the data
interpretation. Hing Wai Tsang, Xiwei Wang and Yanmei Zhang contributed
to the experimental sample preparation and processing. Hing Wai Tsang
and Gilbert T Chua drafted the manuscript and all co-authors commented
and contributed to the revisions and final manuscript.



\textbf{Introduction}

At the end of 2019, a pneumonia outbreak with unknown etiology was
reported in Wuhan, China.{[}1, 2{]} The World Health Organization (WHO)
officially named this disease Coronavirus Disease-2019 (COVID-19), which
was later identified to be caused by the severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2).{[}3{]} The worldwide pandemic has
significantly impacted public health and the global economy.{[}4{]}
Preventive measures were enforced to reduce social distancing, including
limited gatherings, school closures, and restricted travel to reduce
transmission.{[}3, 5{]}

The clinical spectrum of COVID-19 ranges from asymptomatic to fatal
disease. Unfavorable outcomes were associated with the age and
comorbidities of patients,{[}6, 7{]} particularly those older than 65
years and individuals with diabetes mellitus or renal disease.{[}8-10{]}
Children infected with SARS-CoV-2 generally have mild symptoms and a low
mortality rate,{[}11-13{]} with a lower likelihood of severe symptoms in
children than in adults.{[}14-16{]} The SARS-CoV-2 viral-host response
plays an important role in the pathogenesis of the disease, including
changes in the biological responses of peripheral immune cells and the
levels of proinflammatory cytokines. Lymphopenia is a common clinical
characteristic symptom observed in COVID-19 patients, especially in
critical cases,{[}2, 15-20{]} with up to 83.2\% of patients showing
lymphopenia during admission.{[}21{]} Moreover, symptomatic children
with COVID-19 were found to have higher viral load, lower total
lymphocyte count, lower lymphocyte subsets, and elevated interleukin 6
(IL-6), IL-10, tumor necrosis factor-alpha (TNF-\selectlanguage{greek}α\selectlanguage{english}), and interferon-gamma
(IFN-\selectlanguage{greek}γ\selectlanguage{english}) levels compared with asymptomatic patients.{[}22, 23{]} The data
collectively suggest that altered immune cell subsets could be a
prognostic factor for COVID-19{[}24{]}, especially in critical
cases.{[}25{]} There are knowledge gaps in degree of host immune
responses among patients in terms of age, which could help to identify
beneficial factors associated with lower disease severity due to
SARS-CoV-2 infections.

The long-term persistence of T cell memory is important in mediating
both cellular and humoral immunity against SARS-CoV-2
reinfections.{[}26, 27{]} Patients infected with SARS-CoV-2 virus show T
cell memory along with neutralizing antibodies and polyfunctional T cell
responses.{[}26, 28{]} This T cell memory is capable of being
reactivated in patients with mild symptoms up to 8 months after
recovery.{[}29, 30{]} Epitope identification studies of SARS-CoV-2 T
cells have demonstrated that both CD4+ and CD8+ T cells respond to a
broad spectrum of structural and non-structural proteins (NSP) of the
SARS-CoV-2 virus. T cells showed immunodominant responses to spike (S),
membrane (M), and nuclear (N) structural proteins, whereas B cells
showed sub-dominant responses to ORF-1 ab-encoded NSPs.{[}31, 32{]}
However, current knowledge of SARS-CoV-2 immune responses specific to
pediatric patients is still lacking, such as the immunodominance of
SARS-CoV-2 epitopes and durability of antibodies after an infection.

Given the fundamental differences in the immunity of adults and
children,{[}33{]} we assessed the adaptive SARS-CoV-2-specific immune
responses in children and adolescents recovered from COVID-19.

\textbf{Materials and Methods}

\textbf{Subject recruitment}

Children and adolescents under 18 years of age who had recovered from
COVID-19 were recruited to the study. These subjects were admitted and
managed in the Paediatric Infectious Disease Centre, Princess Margaret
Hospital, Hong Kong, China. Patients were confirmed to have COVID-19 by
a positive SARS-CoV-2 RT-PCR test of their nasopharyngeal swab(NPS).
Patients were confirmed to have recovered from COVID-19 by either two
consecutives negative NPS by SARS-CoV-2 RT-PCR or the seroconversion of
SARS-CoV-2 anti-NP antibody response. Details of the admission and
discharge criteria and the laboratory investigations have been
previously described.{[}23{]} Their demographics, clinical symptoms
during the infection, and time since recovery were retrieved.

Uninfected controls were recruited from pediatric patients admitted to
the Queen Mary Hospital for follow up of other medical conditions
unrelated to COVID-19 or from healthy individuals in the community
(Table S1). Subjects below 18 years of age with no history of COVID-19
and a negative SARS-CoV-2 RT-PCR on the day of recruitment were invited
to participate in the study. Exclusion criteria included participants
with other acute infections 2 weeks before recruitment, having received
any kinds of COVID-19 vaccines, known underlying primary or acquired
immunodeficiency, and autoimmune disease or other condition that
required immunosuppressants.

\textbf{Isolation of peripheral blood mononuclear cells}

Whole blood samples from recovered patients and controls were collected
in heparin-coated blood tubes. Peripheral blood mononuclear cells
(PBMCs) were isolated by Ficoll density gradient centrifugation as
previously described.{[}34{]} Isolated PBMCs were cryo-preserved in
storage medium containing 90\% heat-inactivated fetal bovine serum (FBS;
Gibco, Thermo Fisher Scientific, Inc., Waltham, MA) and 10\% cell
culture grade DMSO (Sigma Aldrich, Merck, Germany). Samples were stored
in liquid nitrogen until batch recovery for the assays.

\textbf{T cell stimulation assay and SARS-CoV-2 peptide pools}

In vitro T cell stimulation assays were carried out with spike (S),
membrane (M), and nuclear (N) structural proteins. Briefly, viable cell
numbers were determined in the thawed PBMCs by staining with crystal
violet and counting with a hemocytometer. For the assays,
10\textsuperscript{6} cells were resuspended in 100 \selectlanguage{greek}µ\selectlanguage{english}L RPMI 1640 medium
(Gibco) supplemented with 10\% heat-inactivated FBS and 1\%
penicillin/streptomycin. The SARS-CoV-2 peptide pools (Miltenyi Biotec,
Germany) were prepared according to the manufacturer's recommendations.
Next, 1 \selectlanguage{greek}µ\selectlanguage{english}g of peptide/mL (0.6 nmol) separately or in a mixture was
introduced to the T cells. Along with the peptide pools, 0.1 \selectlanguage{greek}µ\selectlanguage{english}g/mL
purified anti-human CD28 (Miltenyi Biotec, Clone: REA612) and 0.1 \selectlanguage{greek}µ\selectlanguage{english}g/mL
purified anti-human CD49d (Miltenyi Biotec, Clone: MZ18-24A9) as
coactivators of T cells were also added to the wells for the entire
stimulation period. The T cells and peptide mixtures were incubated at
37\textsuperscript{o}C in 5\% CO\textsubscript{2} for 16 hours.
Brefeldin A (Biolegend, San Diego, CA) at a concentration of 0.1 \selectlanguage{greek}µ\selectlanguage{english}g/mL
was added to the culture medium in the last 4 hours to enhance
intracellular cytokine staining signals. The negative control was 10\%
DMSO and the positive control was an activation cocktail (Biolegend))
containing 8.1 nM phorbol-12-myristate (PMA) and 1.3 mM ionomycin.

\textbf{Flow cytometry}

Stimulated PBMCs were recovered from the culture plates and resuspended
in 100 \selectlanguage{greek}µ\selectlanguage{english}L PBS. Cell viability was assessed by staining with
Viobility\textsuperscript{TM} Fixable Dyes (Miltenyi Biotec, Germany).
Cells were washed, fixed, permeabilized, and then stained with an
antibody cocktail containing Pacific Blue\textsuperscript{TM} anti-human
CD3 (Biolegend, clone: HIT3a), PE/Cyanine7 anti-human CD4 (Biolegend,
clone:A161A1) and PerCP/Cyanine5.5 anti-human CD8 (Biolegend, clone:
SK1) for T cell identification; APC anti-human CD69 (Biolegend, clone:
FN50) and PE anti-human IFN-\selectlanguage{greek}γ \selectlanguage{english}(Biolegend, clone:4S.B3) for the
activation analysis; and FITC anti-human CD14 (Biolegend, clone:HCD14)
and FITC anti-human CD20 (Biolegend, clone:2H7) for the exclusion of
non-specific signals and B cells. Fifty thousand events were analyzed by
a BD LSR-II flow cytometer. The gates applied for the quantification of
the stimulated T cells are illustrated in Fig. S1.

\textbf{SARS-CoV-2 RBD ELISA}

Serum was isolated from whole blood samples obtained from recovered
patients and controls. The RBD IgG antibody level was measured using an
Euroimmun anti-SARS-CoV-2 ELISA assay (Lubeck, Germany) according to
manufacturer's protocol. Data were expressed as semi-quantitative IgG
ratios.

\textbf{Quantification and statistical analysis}

Data analyses were performed using FlowJo (version 10.1, BD Bioscience,
Ashland, OR). Statistical analyses were performed using SPSS for Windows
(version 26.0, SPSS Inc., Chicago, IL) and Prism for Windows (version
8.0.1, GraphPad Software, San Diego, CA). Data are expressed as mean \selectlanguage{ngerman}±
standard deviation (SD), and statistical details are provided in the
respective figure legends. Comparison analysis was carried out by
two-tailed Student's t test with p\textless{}0.05 considered
statistically significant. The antigenicity effect size of the different
SARS-CoV-2 peptides on T cell activation was assessed by Cohen's
d.{[}35{]}

To examine SARS-CoV-2-specific T cell response in recovered patients, we
measured the upregulation status of the early activation marker CD69 and
expression of intracellular cytokine IFN-\selectlanguage{greek}γ, \selectlanguage{english}a functional T cell marker
for protective immunity and analyzed the double-positive status of
CD69/IFN-\selectlanguage{greek}γ \selectlanguage{english}in CD4+ and CD8+ T cells, normalized to DMSO
control.{[}36-38{]}. To estimate the half-life of SARS-CoV-2 RBD IgG, we
calculated t\textsubscript{1/2} = A\textsubscript{o}/2k, where
A\textsubscript{o} is the initial amount of the antibody obtained from
the y-intercept of the trendline and k is the slope of the trendline
obtained from the scattered plot of RBD IgG ratio against recovery time.
The recovery time is defined as the time between the date of the
patient's clinical diagnosis to the date of the blood sample
collections. To analyze the relationship between anti-RBD IgG level and
T cells response, we performed Spearman's correlations and expressed as
correlation coefficient (r).

\textbf{Ethics approval}

The study was approved by the Institutional Review Board of the
University of Hong Kong/Hospital Authority Hong Kong West Cluster
(Reference: UW 20-292 and UW 21-157) and the Kowloon West Cluster
Research Ethics Committee {[}Reference: KW/FR-20-086(148-10){]}. Written
consent was obtained from parents or legal guardians of the subjects.

\textbf{Results}

\textbf{Subject recruitment and clinical characteristics}

Between 1\textsuperscript{st} December 2020 to
31\textsuperscript{st}March 2021, 31 patients who had recovered from
COVID-19 were recruited from Princess Margret Hospital, Hong Kong SAR.
Fourteen (45.2\%) were boys and 17 (54.8\%) were girls with a median age
of 12 years (range 2.7-18 years). Twenty age-matched uninfected controls
were also recruited from Queen Mary Hospital, Hong Kong SAR, China and
from the community. Subject demographics and clinical characteristics
are shown in Table 1. The majority of subjects were Chinese (80.6\%).
Among the COVID-19 cases, 83.9\% were domestic cases, 32.3\% were
asymptomatic, and the remaining cases (67.7\%) had mild disease. Blood
samples were collected at 29 to 219 days after recovery.

\textbf{Quantification of SARS-CoV-2 RBD IgG level and identification of
SARS-CoV-2 reactive T cells in recovered children and adolescents}

We detected the presence of SARS-CoV-2 RBD IgG antibodies in 30/31
recovered COVID-19 patients compared with the 20 healthy unexposed cases
(p\textless{}0.001), with 1 patient showed negative in the RBD IgG
antibodies test (Fig. 1A). Stimulation of CD4+ and CD8+ T cells with the
mixed SARS-CoV-2 peptide pool (S + M + N peptides, representing the
reactive epitopes of the SARS-CoV-2 virus) showed significantly higher
numbers of CD69+, IFN-\selectlanguage{greek}γ\selectlanguage{english}+, and double-positive CD69+/IFN-\selectlanguage{greek}γ\selectlanguage{english}+ T cells in
recovered patients compared with controls. In agreement with this
observation, we also found significantly higher numbers of CD4+ and CD8+
T cells responding to stimulations by M, N and S peptide pools, with
exception of CD8+CD69+IFN-\selectlanguage{greek}γ\selectlanguage{english}+ subsets that showed statistically marginal
differences. These results reveal a strong type 1 T cell memory response
against the SARS-CoV-2 virus (Fig. 1C).

Next, the reactivity of the CD4+ and CD8+ T cells towards individual M,
N, and S peptide pools were analyzed in convalescent patients. (Fig.2)
Both CD4+ (Fig.2 left panel) and CD8+ (Fig.2 right panel) reactivity was
readily detectable in all patients towards each structural protein;
however, the CD4+ T cells responded more strongly to stimulation by S
peptide than to N (Cohen's d=0.53) or M peptides (Cohen's d=0.34). On
the other hand, CD8+ T cells responded less strongly to stimulation by M
peptides compared with N peptides (Cohen's d=-0.36) or S peptide
(Cohen's d=-0.23), where the difference in CD8+ T cell responses between
S and N peptides was small (Cohen's d=0.10).

\textbf{The dynamics of humoral and cellular immunity in recovered
children and adolescents}

SARS-CoV-2 specific humoral immunity was found to decay over time, but
not T cell immunity (Fig. 3). Linear regression analysis showed that the
level of SARS-CoV-2 RBD IgG was significantly associated with recovery
time (p=6.31\textsuperscript{e-07}, R\textsuperscript{2}=0.5808) (Fig.
3A), but not with the specific CD4+ (p=0.783) or CD8+ (p=0.915) T cell
responses (Fig. 3B). SARS-CoV-2 RBD IgG had a fast decay rate (-0.0377
anti-RBD IgG ratio/day) while CD4+ (-0.0022\%/day) and CD8+
(-0.0001\%/day) T cell responses persist over time, including the
patient with the longest follow-up time at 219 days who had undetectable
anti-RBD IgG but persistent SARS-CoV-2 specific CD4+ and CD8+ T-cell
response. The average SARS-CoV-2 RBD IgG half-life
(t\textsubscript{1/2}) decay was 121.6 days, and the presence of
antibodies was estimated to last for 237.7 days or 7.9 months. The same
estimation was not applicable to CD4+ and CD8+ T cell responses because
of the lack of association with time.

\textbf{Age is a factor associated with the measured RBD IgG level and T
cell activation magnitudes in recoveredchildren and adolescents}

Fifteen patients were younger than 12 years and 16 patients were 12
years or older. The results demonstrated differences in the immune
responses to SARS-CoV-2 between older and younger children. In
comparison to children older than 12 years, the younger patients had a
significantly higher level of SARS-CoV-2 RBD IgG ratio (p=0.041) (Fig
4A). While the frequency of CD4+ T cells reactive to mixed M, N and S
peptide pool was similar between the age groups (Cohen's d=0.071) (Fig
4B(i)), the frequency of S-peptide specific CD4+ T cells was higher in
younger children (Cohen's d=0.3058) (Fig 4B(ii)). Correlative analysis
showed that the four patients with highest level of anti-RBD IgG and
S-peptide specific CD4+ T cells were all from the younger age group (Fig
4D(i)). In contrast, no difference was observed between the two age
groups in SARS-CoV-2 S-reactive CD8+ T cells (Cohen's d=0.03164) (Fig 4C
and 4D(ii)).

\textbf{Discussion}

This is one of the first studies to characterize SARS-CoV-2-specific
humoral and cellular immunity in children recovered from COVID-19. There
was an acquired immunity established in children with either symptomatic
or asymptomatic infections. Both SARS-CoV-2-specific humoral and
cellular immunity were detectable at different time points during the
recovery period. Detection of SARS-CoV-2 RBD IgG and reactive CD4+ and
CD8+ T cells against the various peptide pools suggests both humoral and
cellular immunity are present that can counter re-infections.

Our study showed that there were both CD4+ and CD8+ T cell responses to
SARS-CoV-2 S, N, and M proteins. SARS-CoV-2-specific T cells were found
to respond to stimulation by all peptide pools. The detection of
intracellular levels of IFN-\selectlanguage{greek}γ \selectlanguage{english}suggests the protective cellular immunity
towards SARS-CoV-2 was similar between children and adults, in that both
groups developed T cell memory.{[}26, 30, 39{]} A larger-scale study
will be needed to confirm our observations.

The persistence of humoral and cellular responses against the SARS-CoV-2
virus is key to understanding the risk of re-infections. {[}40, 41{]} We
observed a decline in humoral immunity associated with recovery time.
The SARS-CoV-2 RBD IgG antibody level lasted on average 7.9 months with
a half-life of 121.6 days, which is similar to other studies across
different age groups.{[}42-47{]}. There have only been a few studies
demonstrating the longevity of SARS-CoV-2 T cell response in recovered
pediatrics patients. Dan et al., demonstrated that approximately 92\%
and 50\% of recovered patients had specific CD4+ and CD8+ responses,
respectively, up to 8 months after the primary infection.{[}30{]} Based
on our finding and the above study, the humoral immunity against
SARS-CoV-2 in recovered pediatrics patients can last up to 7-8 months
after the primary infection, which is likely much longer than for
cellular immunity. To prolong the humoral responses, we recommend
recovered pediatrics patients to receive a booster dose of a COVID-19
vaccine approximately 6 months after the primary infection, which is in
line with the current adult guidelines.{[}48{]}

Ding et al., demonstrated an age-specific variation in childhood CD4+
and CD8+ T cell subsets in healthy Chinese, suggesting differences in
immune composition across pediatric age groups.{[}49{]} Along with this
finding, our data demonstrated that the age of the pediatric patients is
an important factor influencing the level of SARS-CoV-2 RBD IgG and the
magnitude of the T cell response to SARS-CoV-2. Convalescent children
younger than 12 years had higher SARS-CoV-2 RBD IgG levels, whereas
convalescent adolescents aged 12 years and older had stronger SARS-CoV-2
CD8+ T cell response. Other viruses, such as the cytomegalovirus (CMV),
have contrasting immune responses, with the intact CMV inducing specific
CD8+ T cell response, but deficient CD4+ T cell response, in young
children and toddlers.{[}34, 50{]} There was also age-dependent CD4+ T
cell activity in the production of the RBD IgG antibody. Based on our
data, only younger children presented with stronger SARS-CoV-2 S CD4+ T
cells response and linked with higher level of anti-RBD IgG ratio. Our
novel findings on the immune responses in convalescent pediatrics
patients in younger age group outlined the importance of SARS-CoV-2 S
specific CD4+ dependent humoral response in relations to the level of
anti-RBD IgG against reinfections, which warrant further larger-scale
studies to confirm the observations.

The study findings need to be interpreted with the following caveats.
First, the number of patients and controls was relatively small.
However, all the controls demonstrated negative immune memory responses
with undetectable SARS-CoV-2 anti-RBD antibody titer, indicating immune
protection against SARS-CoV-2 in unvaccinated and uninfected children
was minimal. Second, the quantity of blood that can be obtained from
younger children is limited, hence, other subsets of T cell responses to
SARS-CoV-2 peptide pools were not evaluated in this study. Future
investigations should include other T cell subsets such as regulatory T
cells and T follicular helper cells (Tfh) to draw a more comprehensive
picture of the T cell response against SARS-CoV-2 in children.

\textbf{Conclusion}

SARS-CoV-2 infection induces immune memory in recovered pediatrics
patients. The T cell reactivity upon stimulation by M, N, S peptide
pools in recovered pediatric patients were similar. There were
differences in the level of SARS-CoV-2 RBD IgG and the magnitude of T
cell responses between younger and older children. Our findings pave the
way for large-scale studies, which could help explain the differences in
clinical findings between children and adults with COVID-19. Our
findings also have important implications for the development of
COVID-19 vaccines targeting younger children.



\textbf{Table 1 Demographics and clinical characteristics of recovered
pediatric COVID-19 patients and uninfected controls}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& Children recovered from COVID-19 (N = 31) & Uninfected Controls (N =
20)\tabularnewline
\midrule
\endhead
Median age in years & 12 & 14\tabularnewline
Age range & 2.7-18 & 8-15\tabularnewline
Sex (\%) & &\tabularnewline
Male & 45.2 (14/31) & 80.0 (16/20)\tabularnewline
Female & 54.8 (17/31) & 20.0 (4/20)\tabularnewline
Residence (\%) & &\tabularnewline
Hong Kong & 100 & 100\tabularnewline
Ethnicity (\%) & &\tabularnewline
Han Chinese & 80.6 (25/31) & 80.0 (16/20)\tabularnewline
Others & 19.4 (6/31) & 20.0 (4/20)\tabularnewline
Travel history (\%) & &\tabularnewline
Yes & 16.1 (5/31) & N/A\tabularnewline
No & 83.9 (26/31) & N/A\tabularnewline
Disease awareness (\%) & &\tabularnewline
Asymptomatic & 32.3 (10/31) & N/A\tabularnewline
Symptomatic & 67.7 (21/31) & N/A\tabularnewline
Signs/symptoms (\%) & &\tabularnewline
Fever & 61.9 (13/21) & N/A\tabularnewline
Cough & 47.6 (10/21) & N/A\tabularnewline
Runny nose & 28.6 (6/21) & N/A\tabularnewline
Ageusia & 19.0 (4/21) & N/A\tabularnewline
Vomit & 14.3 (3/21) & N/A\tabularnewline
Anosmia & 9.5 (2/21) & N/A\tabularnewline
Sputum & 4.9 (1/21) & N/A\tabularnewline
Headache & 4.9 (1/21) & N/A\tabularnewline
SARS-CoV-2 PCR positivity (\%) & &\tabularnewline
Positive & 100 (31/31) & N/A\tabularnewline
Negative & 0 (0/31) & 100(20/20)\tabularnewline
SARS-CoV-2 anti-NP IgG positivity (\%) & 100 & N/A\tabularnewline
Sample collection period & Dec2020 - March 2021 & Dec2020 - March
2021\tabularnewline
Recovery time (Days) & 29-219 (Median=46.5) & N/A\tabularnewline
\bottomrule
\end{longtable}

\textbf{Fig. 1. Comparison of SARS-CoV-2 RBD-specific antibodies and
SARS-CoV-2-specific T cell response in healthy controls and recovered
children and adolescents.} (A) Serological responses to recombinant RBD
protein in 31 recovered COVID-19 patients with median 46.5 recovery days
and ranging 29-219 days and 20 uninfected controls. Dash line indicated
the anti-RBD IgG ratio reference obtained from uninfected controls. (B)
Representative data of the T cell response towards SARS-CoV-2 peptide
pools in controls and recovered patients. (C) Immunophenotyping of PBMCs
for frequency of CD4+, CD8+, or CD69+ T cells, IFN-\selectlanguage{greek}γ\selectlanguage{english}+ cells, and
CD69+/IFN-\selectlanguage{greek}γ\selectlanguage{english}+ double-positive cells from uninfected individuals (n=20) or
convalescent children and adolescents (n=31). Data are presented as
mean\selectlanguage{ngerman}±SD and analyzed using two-sided Student's t-test between control
and patient groups. *p\textless{}0.05, **p\textless{}0.01,
***p\textless{}0.001

\textbf{Fig. 2.} \textbf{Measurement of SARS-CoV-2-specific T cell
response in recovered patients.} Total T cell responses towards
SARS-CoV-2 Membrane (M), Nuclear (N) and Spike (S) peptides and mixed
peptide pools in stacked columns representing the summation of different
measured immune subsets in CD4+ and CD8+ T cells after 16 hours of
incubation of PBMCs from recovered patient. Data are expressed as
mean±SD. Dash line in the stack columns indicated the corresponding
reference CD4+ and CD8+ T cells response stimulated by different
SARS-CoV-2 peptide in uninfected controls group.

\textbf{Fig. 3. SARS-CoV-2-specific RBD and T cell responses over
time.}(A) Regression analysis of the measured RBD IgG ratio in
convalescent serum was plotted against the post-infection time. The best
fitting trendline is shown. The calculated t\textsubscript{1/2} was
121.6 days and the estimated duration of antibodies was 7.9 months
compared with the average basal level obtained from uninfected
individuals. (B) Representative T cell subset frequencies in PBMC of
recovered patients were plotted against the post-infection period
showing a flat slope for (i) CD4+ and (ii) CD8+, indicating a sustained
T cell response to SARS-CoV-2 virus in recovered pediatric patients.

\textbf{Fig. 4. Age-dependent differences of SARS-CoV-2-specific S-RBD
IgG level and SARS-CoV-2-specific T cell response in recovered children
and adolescents} . The corresponding reference anti-RBD IgG ratio and T
cell response obtained from uninfected control was indicated as a dash
line in the figures. (A) Serological analysis in 15 patients who were
younger than 12 years and 16 patients who were 12 years or older. Data
was adjusted by recovery days and comparisons analyzed by two-sided
Student's t-test *p\textless{}0.05. (B) Comparison analysis of the total
measured CD4+ T cell responses to (i)mixed peptide pools and (ii) S
peptide between younger children and older children. (C) Comparison
analysis of the total measured CD8+ T cell responses to (i)mixed peptide
pools and (ii)S peptide between younger children and older children. (D)
Correlation analysis of anti-RBD IgG level against (i) CD4+ and (ii)
CD8+ T cells response in the recovery patients. Data was plotted as
age-subgroups with color-labelled dots in the scattered plots. A
trendline indicated the correlations direction of the analysis
parameters.\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
& Medical Conditions\tabularnewline
\midrule
\endhead
Control 1 & Obesity, Asthma, Allergic Rhinitis\tabularnewline
Control 2 & Diplopia (spontaneously recovered), no autoimmune causes
identified\tabularnewline
Control 3 & Syncope\tabularnewline
Control 4 & Healthy\tabularnewline
Control 5 & Healthy\tabularnewline
Control 6 & Healthy\tabularnewline
Control 7 & Healthy\tabularnewline
Control 8 & Healthy\tabularnewline
Control 9 & Healthy\tabularnewline
Control 10 & Healthy\tabularnewline
Control 11 & Healthy\tabularnewline
Control 12 & Healthy\tabularnewline
Control 13 & Healthy\tabularnewline
Control 14 & Healthy\tabularnewline
Control 15 & Healthy\tabularnewline
Control 16 & Healthy\tabularnewline
Control 17 & Healthy\tabularnewline
Control 18 & Healthy\tabularnewline
Control 19 & Healthy\tabularnewline
Control 20 & Healthy\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S1. Other medical conditions in the controls}

\textbf{Fig. S1. Representative gating analysis by flow cytometry}
.\textbf{a} . Forward scatter height (FSC-H) versus forward scatter area
(FSC-A) plot for single cell inclusion. \textbf{b} . Live cells were
gated based on live/dead discrimination dye staining. \textbf{c} . Side
scatter (SSC-A) versus Forward scatter area (FSC-A) plot for lymphocyte
identification. \textbf{d} . T cells were gated based on specific CD3
expression, excluding CD14 and CD20 expressing cells. \textbf{e,f} .
Subsets of T cells were gated based on the high expression of CD4 and
CD8, and were used in further marker analysis. Floating gates on CD69,
IFN-\selectlanguage{greek}γ, \selectlanguage{english}and double-positive CD69/IFN-\selectlanguage{greek}γ \selectlanguage{english}plots were based on the
corresponding expressions of the positive control cells.

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