MILD SARS-COV-2 INFECTIONS AND NEUTRALIZING ANTIBODY TITERS
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Mild SARS-CoV-2 Infections and
Neutralizing Antibody Titers
Francesco Bonfante, DVM,a,* Paola Costenaro, MD, DTM&H,b,* Anna Cantarutti, PhD,c Costanza Di Chiara, MDb
Alessio Bortolami, DVM, PhD,a Maria Raffaella Petrara, PhD,d Francesco Carmona, BSc,e Matteo Pagliari, PhD,a
Chiara Cosma, MD,f Sandra Cozzani, MD,b Eva Mazzetto, DVM,a Giovanni Di Salvo, MD,g Liviana Da Dalt, MD,g
Paolo Palma, MD,h,i Luisa Barzon, MD,j Giovanni Corrao,k,c Calogero Terregino, MD,a Andrea Padoan, MD,l
Mario Plebani, MD,l,f Anita De Rossi, PhD,d,e Daniele Dona, MD, PhD,b Carlo Giaquinto, MDb
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BACKGROUND: Recent evidence suggests that neutralizing antibodies (nAbs) to severe acute abstract
respiratory syndrome coronavirus 2 may persist over time; however, knowledge regarding
pediatric subjects is limited.
METHODS:A single-center, prospective observational study was conducted on 57 family clusters
of coronavirus disease 2019, including children of neonatal and pediatric age attending the
University Hospital of Padua (Italy). For each patient, blood samples were collected for both
the quantification of nAbs through a plaque reduction neutralizing test and the detection of
antinucleocapsid-spike protein immunoglobulin G and/or immunoglobulin M.
RESULTS:We analyzed 283 blood samples collected from 152 confirmed coronavirus disease
2019 cases (82 parents and 70 children or older siblings of median age of 8 years,
interquartile range: 4–13), presenting asymptomatic or with mildly symptomatic disease.
Despite the decrease of immunoglobulin G over time, nAbs were found to persist up to 7 to
8 months in children, whereas adults recorded a modest declining trend. Interestingly,
children aged
European countries have been facing naturally acquired nAbs among a Data Collection and Definitions
a third wave of the novel cohort of family clusters of COVID- Information collected during the
coronavirus disease 2019 (COVID- 19, including adults and children clinic was entered into a Web-based
19) pandemic and the spread of who recovered from asymptomatic database by using the Research
several severe acute respiratory or mild symptomatic infections. Electronic Data Capture platform
syndrome coronavirus 2 (SARS-CoV- (Vanderbilt University, Tennessee)
2) variants. With the advent of METHODS hosted in the server of the
vaccines,1 longitudinal studies of University of Padova. For this
both convalescent and vaccinated Study Design and Population
study, data were collected
patients are of fundamental A single-center, prospective study retrospectively from the existing
importance to understand the
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was conducted on Italian family clinical files and analyzed
kinetics of humoral response and clusters of COVID-19 attending the anonymously. Subjects were
infer correlates of protection for COVID-19 Family Cluster Follow-up considered patients with confirmed
both infection and disease. In this Clinic, at the Department of COVID-19 if they had a record of
respect, the titration of neutralizing virological positivity for SARS-CoV-
Women’s and Children’s Health of
antibodies (nAbs) is key to
the University Hospital of Padua 2 by real-time polymerase chain
determine the concentration of
(Veneto Region, Italy). From March reaction (RT-PCR) according to
antibodies preventing cells to be
1, 2020 to September 4, 2020, 57 routine diagnostic molecular
infected by SARS-CoV-2.2
families were enrolled meeting the protocols16 and/or resulted
following inclusion criteria: (1) positive by either of the 2
Studies including convalescent
having children of pediatric age serological tests adopted in this
adults reported that humoral
(aged 18 years, including older
adolescents are key in the confirmed SARS-CoV-2 family
siblings and parents, and legally outbreak (Supplemental Fig 6).
transmission of infection.15 Little is
authorized representatives of Subjects who were asymptomatic
known about the kinetics of SARS-
subjects aged
(>15 years). These age classes samples, stratified by age classes validation of immunoassays for
were deemed instrumental for a and by time between serological SARS-CoV-2, we calculated measures
translation of results into the sampling and baseline, categorizing of diagnostic accuracy of the CLIA
context of school-targeted subjects into 3 intervals, namely 1 assay.
vaccination and sero-surveillance to 2, 3 to 6 and 7 to 8 months. The
campaigns. 1-way analysis of variance and the Analyses were performed by using
independent samples t test were the Statistical Analysis System
Serological Assays performed, when appropriate. software (version 9.4; SAS Institute,
Plasma was stored at 80 C before Associations between antibody Inc, Cary, NC). Statistical
testing for the quantification of titers, baseline intervals and age, significance was set at the .05 level.
nAbs through a high-throughput were assessed with linear All P values were 2-sided. Graphs
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method for plaque reduction regression models. Strength of were made by using GraphPad
neutralizing test (PRNT).18 Another associations between variables was Prism version 9 (GraphPad
aliquot was analyzed with the assessed by Pearson correlation Software, Inc, La Jolla, CA).
chemiluminescence immunoassay coefficient by using the logarithm
(CLIA) MAGLUMI 2019-nCoV (base 10) of the antibody titers, RESULTS
Immunoglobulin M (IgM) and given data skew. From March 1, 2020, to December
Immunoglobulin G (IgG).18 Further 3, 2020, we prospectively evaluated
Use of the robust variance
details on the 2 assays are 57 family clusters of COVID-19
estimator to account for
reported in the Supplemental (Supplemental Fig 5). A serological
correlations within patients with
Information. assessment was performed at least
multiple blood samplings did not
once on 209 recruited subjects.
SARS-CoV-2 Viral Load Measurement change the CIs considerably in the
Subjects who had previously tested
unadjusted analyses, so correlation
A selection of NPSs of enrolled positive for SARS-CoV-2 by real-
structures were omitted from all
subjects that had been originally time RT-PCR (111 of 209) were
analyses. Among a subcohort of
screened at the Padova University considered to have confirmed
subjects that agreed to be sampled
Hospital were made available for COVID-19, together with individuals
again after enrollment, a
quantification of the viral load. who had no record of virological
dependent t test for subject-paired
Copies of SARS-CoV-2 were positivity but showed evidence of
samples was used to compare the
quantified by a homemade multiplex seropositivity by either of the 2
GMT and 95% CI.
quantitative assay on the basis of a serological tests adopted in this
1-step digital droplet polymerase To test the robustness of our data study (44 of 209). Descriptive
chain reaction (ddPCR).19 Results sets against selection bias, we analysis and additional information
were expressed as SARS-CoV-2 conducted a x2 test and verified on baseline identification are
copies per 5 ml. Further details are the homogeneity within each age provided as Supplemental
reported in the Supplemental class and time window of (1) the Information (Supplemental Table 2,
Information. temporal distribution of serological Supplemental Fig 6). Three out of
samplings (P 5 .4363) and (2) the 73 children were excluded from the
Statistical Analyses analyses (see Supplemental Fig 5).
proportion of cases identified by
Descriptive statistics were used virological or serological methods In total, 152 confirmed COVID-19
for comparing the distribution of (P 5 .6568). Moreover, we cases were studied: 70 children or
sex, age, disease-related conducted a x2 test to verify older siblings and 82 parents with
symptoms, and pediatric among subjects who contributed median ages of 8 (interquartile
comorbidities between patients with either 1, 2, or 3 samples the range [IQR], 4–13) and 42 years
infected with COVID-19 and homogeneity of sex (P 5 .6082), (IQR, 34–46), respectively. Of 152
uninfected patients. age (P 5 .0973), family position cases, 38, 97, and 17 were sampled
(P 5 .3971) and severity of once, twice and 3 times,
The humoral response was symptoms (P 5 .6947). respectively.
assessed by comparing the
geometric mean titer (GMT) and The diagnostic sensitivity of the Analyzing all 283 blood samples
the 95% confidence interval (CI) of CLIA and PRNT assays were collected from confirmed COVID-19
IgM, IgG, and PRNT50 values in the assessed on subjects with a positive cases, we observed that nAbs
overall data set, including both NPS result. Considering the PRNT persisted in the population,
independent and subject-paired assay as reference method for the (Fig 1A) recording a modest
PEDIATRICS Volume 148, number 3, September 2021 3
Downloaded from http://publications.aap.org/pediatrics/article-pdf/148/3/e2021052173/1227777/peds_2021052173.pdf by guest on 11 January 2022 FIGURE 1 Stability of SARS-CoV-2 nAb titers over time. A, PRNT50 titers from 283 serum samples collected at a median time of 132 days (IQR, 79–187) from infection onset, overall and stratified by 3 age classes, including children aged
Downloaded from http://publications.aap.org/pediatrics/article-pdf/148/3/e2021052173/1227777/peds_2021052173.pdf by guest on 11 January 2022 FIGURE 3 Performance of SARS-CoV-2 CLIA IgG and PRNT titers over time. A, Decreasing levels of SARS-CoV-2 CLIA IgG levels observed for all classes of age (aged
Downloaded from http://publications.aap.org/pediatrics/article-pdf/148/3/e2021052173/1227777/peds_2021052173.pdf by guest on 11 January 2022 FIGURE 4 nAb titers according to COVID-19 disease severity. A, Clinical presentation of COVID-19 in children aged
TABLE 1 Subject-Paired Serological Data of 76 Subjects Who Were Sampled Twice at Periods of 72 Days (SD ±22) and 169 Days (SD ±26) From
Baseline and Data From 50 Subjects, for Whom Paired Samples Were Available at 99 Days (SD ±35) and 234 Days (SD ±10) From Baseline
A B
a
First Sample Second Sample (5–6 mo) P First Sample Latest Sample (7–9 mo) Pa
Agedclusters of COVID-19. In our immunodominant proteins, blunting decrease between 3 and 6 months cohort, antibodies neutralizing the neutralizing potential.26 from infection for mild cases. In SARS-CoV-2 virus persisted over a Recently, this mechanism has been addition, Chia et al9 identified 5 period of 2 to 8 months from explored for influenza, proving that profiles of antibody responses and infection, recording only a modest children aged
19,32
analyses confirmed our shedding and transmission. It is
conclusions against the exclusion of the utmost importance to identify
ABBREVIATIONS
of few cases. age- and time-matched correlates of CI: confidence interval
protection to finally translate CLIA: chemiluminescence
In the absence of correlates of serological data into useful elements immunoassay
protection for nAbs acquired after for the design of vaccines and COVID-19: coronavirus disease
infection, it is not advisable to immunization campaigns for SARS- 2019
translate our data into predictions CoV-2. ddPCR: digital droplet
of a superior immunity of children polymerase chain
to reinfection. According to clinical ACKNOWLEDGMENTS reaction
studies and experimental animal We are grateful to Dr Franco Pisetta GMT: geometric mean titer
Downloaded from http://publications.aap.org/pediatrics/article-pdf/148/3/e2021052173/1227777/peds_2021052173.pdf by guest on 11 January 2022
work, superior nAbs for SARS-CoV-2 and to all the family pediatricians of IgG: immunoglobulin G
might translate into protection from Veneto Region for collaborating in IgM: immunoglobulin M
COVID-19 disease and higher viral this study. Moreover, we are grate- IQR: interquartile range
clearance in the upper respiratory ful to all families that participated nAb: neutralizing antibody
tract, leading to a reduction in to the study. NPS: nasal-pharyngeal swab
PRNT: plaque reduction
neutralizing test
RT-PCR: real-time polymerase
chain reaction
SARS-CoV-2: severe acute
respiratory
syndrome
coronavirus 2
PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275).
contributed to the writing and supervised the project; Drs Palma, Da Dalt, Di Salvo, and Terregino supervised the project; and all authors approved the final
manuscript as submitted.
DOI: https://doi.org/10.1542/peds.2021-052173
Accepted for publication Jun 14, 2021
Address correspondence to Francesco Bonfante, DVM, Istituto Zooprofilattico Sperimentale delle Venezie, viale dell’Universita 10, 35020 Legnaro, Padua, Italy. E-mail:
FBonfante@izsvenezie.it
PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275).
Copyright © 2021 by the American Academy of Pediatrics
FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to the article to disclose.
FUNDING: Partially supported by the ORCHESTRA project, Horizon 2020 research and innovation program under grant agreement 101016167 to Dr Giaquinto.
In addition, this work has received funding from the EU Horizon 2020 (RECOVER) under grant agreement 101003589 to Dr Giaquinto, and Fondazione Cassa
di Risparmio di Padova e Rovigo, Progetti di Ricerca COVID-19 (Dr De Rossi participant).
POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.
COMPANION PAPER: A companion to this article can be found online at www.pediatrics.org/cgi/doi/10.1542/peds.2021-052173.
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