Characterization of the antibody response to SARS‐CoV‐2 in a mildly affected pediatric population

Abstract Background While children usually experience a mild course of COVID‐19, and a severe disease is more common in adults, the features, specificities, and functionality of the SARS‐CoV‐2‐specific antibody response in the pediatric population are of interest. Methods We performed a detailed analysis of IgG antibodies specific for SARS‐CoV‐2‐derived antigens S and RBD by ELISA in 26 SARS‐CoV‐2 seropositive schoolchildren with mild or asymptomatic disease course, and in an equally sized, age‐ and gender‐matched control group. Furthermore, a detailed mapping of IgG reactivity to a panel of microarrayed SARS‐CoV‐2 proteins and S‐derived peptides was performed by microarray technology. The capacity of the antibody response to block RBD‐ACE2 binding and virus neutralization were assessed. Results were compared with those obtained in an adult COVID‐19 convalescent population. Results After mild COVID‐19, anti‐S and RBD‐specific IgG antibodies were developed by 100% and 84.6% of pediatric subjects, respectively. No difference was observed in regards to symptoms and gender. Mounted antibodies recognized conformational epitopes of the spike protein and were capable to neutralize the virus up to a titer of ≥80 and to inhibit the ACE2‐RBD interaction by up to 65%. SARS‐CoV‐2‐specific IgG responses in children were comparable to mildly affected adult patients. Conclusion SARS‐CoV‐2 asymptomatic and mildly affected pediatric patients develop a SARS‐CoV‐2‐specific antibody response, which is comparable regarding antigen, epitope recognition, and the ability to inhibit the RBD‐ACE2 interaction to that observed in adult patients after mild COVID‐19.


| INTRODUC TI ON
The clinical manifestation of COVID-19 ranges from asymptomatic and mild course to severe respiratory impairments that may lead to death. Interestingly, an age-related difference in disease presentation was observed. [1][2][3] The older population was more prone to develop severe outcomes such as an acute respiratory distress syndrome (ARDS), [4][5][6] while the majority of children and young adults had mild to moderate clinical manifestations and rarely developed a severe course of disease in a form of the multisystem inflammatory syndrome (MIS-C) or thrombotic complications. 1,[7][8][9] This age-related discordance in the disease course is one of the most striking findings and may have important implications for the development of preventive and therapeutic strategies against SARS-CoV-2 infections.
Several hypotheses explaining this phenomenon were suggested.
One possibility may be different immune responses to SARS-CoV-2 in children and adults. It has been described that children had a reduced breadth of anti-SARS-CoV-2 antibodies, predominantly mounting anti-S but not anti-NCP specific IgG, and had reduced virus-neutralizing activity. 2 Furthermore, predominant activation of innate immune response represented in a pediatric population was associated with a diminished release of proinflammatory cytokines and a more favorable clinical course of the disease. 10,11 Another mutually nonexclusive possibility for the mild course of COVID-19 in children could be a lower expression of the angiotensin-converting enzyme 2 (ACE2) receptor, which is recognized by the receptorbinding domain (RBD) of SARS-CoV-2. [12][13][14][15] Furthermore, it has been suggested that ACE2 expression could be affected by co-morbidities, which may be associated with severe courses of COVID-19. 16 In majority of the SARS-CoV-2 convalescent adult patients, antibodies to S, RBD, and NCP were detected. 2,[17][18][19][20] Further investigation revealed that neutralizing antibodies recognize conformational, but neither linear epitopes nor S-derived peptides. 20 Interestingly, several groups reported additional non-RBD epitopes on the S1 that could be involved in virus neutralization. [21][22][23] It is still debated whether children develop antibodies to the same extent and recognizing the same epitopes as adults.
Here, we performed an in-depth analysis of the IgG antibody responses to S and RBD SARS-CoV-2-specific antigens and Sderived peptides by ELISA and microarray technology in representative samples from a previously undertaken study in a cohort of more than 2000 schoolchildren. 19 Additionally, we investigated the virus-neutralizing activity of antibodies and their ability to inhibit the interaction of RBD and ACE2 in a recently developed molecular Results: After mild COVID-19, anti-S and RBD-specific IgG antibodies were developed by 100% and 84.6% of pediatric subjects, respectively. No difference was observed in regards to symptoms and gender. Mounted antibodies recognized conformational epitopes of the spike protein and were capable to neutralize the virus up to a titer of ≥80 and to inhibit the ACE2-RBD interaction by up to 65%. SARS-CoV-2-specific IgG responses in children were comparable to mildly affected adult patients.
Conclusion: SARS-CoV-2 asymptomatic and mildly affected pediatric patients develop a SARS-CoV-2-specific antibody response, which is comparable regarding antigen, epitope recognition, and the ability to inhibit the RBD-ACE2 interaction to that observed in adult patients after mild COVID-19.

Key Message
Age-dependent diversity in course of COVID-19 was repeatedly observed, with children presenting rather mild or no symptoms and adults having more severe manifestations. Here, we performed an in-depth analysis of antibody responses in 26 previously reported seropositive children and an age-and gender-matched control group. After a mild course of COVID-19, children developed anti-S and anti-RBD-specific IgG antibodies directed towards conformational epitopes on spike protein that were neutralizing the virus and inhibiting ACE2-RBD interaction. The specificity and magnitude of the antibody response were comparable between children and adults after mild COVID-19, implying that the age-related clinical discrepancies might not be related to divergent properties of antibody response, as previously thought. interaction assay. 24 Moreover, we compared results obtained in children with data from a SARS-CoV-2-affected adult population collected at the same time period. 20,25 2 | ME THODS

| Study population
In a recent cross-sectional study, we performed a comprehensive seroprevalence analysis in a population of 2069 schoolchildren in Vienna, Austria. 19 The study was conducted from May to July 2020, while
Plates were subsequently blocked (PBS, 2% BSA, 0.05% Tween 20) and incubated overnight with 1:50 diluted serum samples. Plates were washed and incubated for 1 hour with 1:1000 diluted HRP-conjugated anti-human IgG (BD, San Jose, CA, USA) and developed with ABTS (Sigma-Aldrich, St. Louis, MO, USA). The optical density was measured at 405/492 nm with an Infinite F50 ELISA reader after 10 min (Tecan, Männedorf, Switzerland). Sera from historical controls were analyzed by ELISA and used to establish cut-off levels.

| Microarray containing SARS-CoV-2 proteins, control proteins, and synthetic S-derived peptides
Peptides with a length of 25-30 amino acids with an overlap of approximately 5 amino acids spanning the sequence of the complete spike protein (S1 and S2) were synthesized by solid-phase synthesis as described (Table S1). 24 The S protein-derived peptides as well as the following proteins (Insect cell-expressed folded S protein, Genscript, Piscataway, NJ; HEK cell-expressed, folded S1, Genscript; E. coli-expressed, unfolded S1 20 ; HEK cell-expressed, folded S2, Native Antigen Company, Oxford, UK; E. coli-expressed, unfolded S2 20 ; HEK cell-expressed, folded RBD, Genscript; E. coli-expressed, unfolded RBD 20 ) were spotted onto pre-activated glass slides as described. 20,27,28 The analysis of specific IgG levels was performed as described 20 on sera from six representative children exhibiting different VNT titers (P16, P17, P19, P21, P23, and P26). Additionally, sera from two control children (C5 and C12) were analyzed. Pre-pandemic sera was used for establishing cut-off values.

| SARS-CoV-2 virus neutralization test and inhibition of the RBD-ACE2 interaction
The SARS-CoV-2 neutralization test (VNT) was performed as described. 19,26 To determine the inhibition of RBD to bind the ACE2 receptor by antibodies present in patients' sera, a molecular interaction assay was used. 24 In this assay, ELISA-plate-bound ACE2 was exposed to recombinant His-tagged RBD which had been preincubated with patients' sera. Bound RBD was detected with a mouse anti-His antibody followed by the detection with a secondary HRP-

| Statistical analysis
All statistical analyses were performed using GraphPad Prism Version 5.00 (La Jolla, CA, USA).
Differences in immunoglobulin reactivity to proteins in different groups were determined using a two-tailed Mann-Whitney Utest. Kruskal-Wallis test with Dunn's post-test was used to compare data between children, mild and severe adults. Correlations of immunoglobulin reactivity, virus neutralization titers, and percentages of blocking RBD-ACE2 interaction were assessed by Spearman´s rank correlation coefficient. P values of <0.05 were considered as significant.

| Characterization of studied children
Serum samples from 26 seropositive children (median age 13 years, 50% males) and 26 age-and gender-matched controls were obtained from the cross-sectional study on schoolchildren from May to July 2020. 19 Fourteen (53.9%) seropositive children self-reported mild acute symptoms in questionnaire whereas twelve (46.1%) did not report any symptoms in the weeks prior to the study (Table 1). One third of the control group subjects experienced and self-reported some types of acute symptoms as well ( Table 1). The vast majority of seropositive subjects (ie, 13 out of 14) experienced symptoms 5-16 weeks prior to the collection of serum. For asymptomatic patients, we assume that the infection occurred earliest at the end of February when the first SARS-CoV-2 cases were registered in Austria, ie, 16-20 weeks prior to the collection of serum.

CoV-2 antigens are comparable regarding gender and symptoms
Anti-S and RBD-specific IgG antibodies in sera from SARS-CoV-2seropositive children and in an age-and gender-matched control group were determined ( Figure 1A, Table S2). S-specific IgG antibodies were detected in each of the seropositive children but not in the control group, except for one child ( Figure 1A, Table S2: C11).
Furthermore, 84.6% of the seropositive children showed RBDspecific IgG antibodies above the cut-off level ( Figure 1A, Table S2).
Conversely, none of the subjects in the control group had RBDspecific IgG antibodies. Furthermore, IgG antibody levels mounted to S protein were significantly higher than those of RBD in seropositive children (p < 0.0001; Figure 1A).
All four seropositive children without RBD-specific IgG antibodies (15.4%) were female (Table S2: P6, P8, P23, and P26). However, no significant differences in S-and RBD-specific IgG antibody levels were observed in respect of gender ( Figure 1B) or symptoms ( Figure 1C) in the seropositive group of children.

| IgG antibodies of COVID-19 convalescent children are mainly directed to folded S and RBD but not to S-derived sequential peptide epitopes
The IgG response to a panel of microarrayed, folded, and unfolded SARS-CoV-2 proteins and forty-six 25 to 30mer peptides spanning the S protein (Table S1) was assessed in a subset of six representative seropositive children exhibiting different VNT levels and two control children. Each of the tested subjects had antibodies directed to eukaryotic-expressed, folded S (100%), and the majority showed IgG reactivity to folded RBD (ie, 5 of 6, 83.3%). Folded subdomain S1 was recognized by all subjects, whereas folded S2 was recognized by only one (17%) of the COVID-19 convalescent children (Figure 2A).
IgG response to S-derived linear peptides were in general much lower than to folded S and RBD and occurred only in certain serum samples ( Figure 2B,C). Notably, the nine peptides spanning RBD were poorly recognized. Overall, an increased IgG response was only detected to peptides 32, 33, and 46 in S2 domain of the spike protein ( Figure 2C) and was the most prominent for the patient with the highest VNT titer.

| Sera of COVID-19 convalescent children show virus-neutralizing capacity and inhibit the RBD-ACE2 interaction
The functionality of the SARS-CoV-2-specific antibodies in the chil- antibodies ( Figure 3A, Table S2). Notably, the virus neutralization titers were irrespective of gender and symptom presentation (data not shown) and low. Two Blocking of RBD-binding to ACE2 was detected in all but three seropositive children (88.5%) ( Figure 3B, Table S2). Overall, sera from seropositive children inhibited RBD binding to ACE2 up to 65% with a median inhibition of 31.9% ( Figure 3B). Three children (ie, C9, C22, and C23) in the control group showed more than 10% blocking of RBD binding to ACE2 ( Figure 3B, Table S2).
Discordant results were found in 5 out of 26 seropositive children when comparing VNT test and assay of molecular inhibition of RBD-ACE2 binding ( Figure 3A,B). Three children of the control group who were capable to inhibit molecular interaction of RBD with ACE2 were VNT negative. Overall, the outcomes of VNT and RBD to ACE2-binding inhibition assay were comparable in 84.6% of cases.

| Correlation of functional assays with S-and RBD-specific IgG levels in children
A correlation of antibody levels to VNT and RBD to ACE2-binding inhibition assay was assessed in SARS-CoV-2 seropositive children.
VNT titers showed a significant positive correlation with S-specific IgG and to a lower extent with RBD-specific IgG ( Figure 4A,B). In contrast, the ability of antibodies to inhibit the RBD-ACE2 interaction correlated significantly with RBD-specific IgG but not with S-specific IgG ( Figure 4C,D). Furthermore, in the children, VNT titers and the extent of ACE2-RBD inhibition did not correlate (data not shown).

| Antibody responses to SARS-CoV-2 are comparable in pediatric and adult patients
Our population of children consisting of asymptomatic and mildly symptomatic subjects showed comparable S-specific IgG levels by ELISA as adult subjects after severe COVID-19, while they were significantly higher than those of adults after mild COVID-19 (Table 2).
This difference was not noted in a subset of children tested by chip technology ( Table 2, lower part). RBD-specific IgG antibody levels did not differ significantly between children and adults after mild symptoms as measured by ELISA and chip technology but were approximately twice lower than that of adults after severe COVID-19, and the difference was significant ( Table 2). The median inhibition of RBD binding to ACE2 did not differ between children and adults, whereas VNT titers were significantly lower in children. (Table 2).
When comparing the IgG response to microarrayed folded S and RBD antigens in a subset of our pediatric patients, levels were similar to those of adults with mild symptoms, but lower than those of adults after severe COVID-19 (Table 2).
In a recent adult study focusing on anti-S1-and anti-N-specific SARS-CoV-2 antibody responses, significant titer differences of neutralizing antibody responses were observed between asymptomatic and mild COVID-19-affected individuals/patients. 18 We could not observe such a divergence in neutralizing titers in our pediatric population, but the assays used in the studies differed. The difference between before mentioned and our study might be explained by a very mild course of the disease in our pediatric group with a generally low neutralizing titer expression or the fact that age directly correlates with more accurate antibody responses assignable to disease activity.
Using microarrayed folded and unfolded SARS-CoV-2 antigens, we found that IgG antibodies of COVID-19 convalescent children were directed against folded S, S1, and RBD but not to the unfolded proteins indicating that antibodies react with conformational  in particular, against RBD-derived peptide epitopes among children.
The same results were observed for an adult COVID-19 population from the same region whose serum samples were obtained at the same time of the pandemic. 20 We assessed the capability of the patients' antibodies to prevent RBD-ACE2 molecular interaction when RBD is pre-incubated with patients' sera by an in-house assay. In our seropositive study population, inhibition levels ranged from 17.7% up to 65.6% which was in concordance with previously reported results from adult patients. 24 Although the outcome directly correlated with anti-RBD titers in the pediatric population, adult patients after a severe disease course showed comparable inhibition capacity despite increased RBD IgG antibody levels. This may implicate that these patients due to longer virus exposure and higher virus load mount antibodies detectable by ELISA but not functional in terms of RBD-ACE2-binding inhibition.
Three out of 26 control patients' sera inhibited RBD-ACE2 molecular interaction and, therefore, were considered positive. This could be explained by cross-reactivity with seasonal human coronaviruses.
Another possible explanation is an undetected SARS-CoV-2 infection; however, we consider it highly unlikely as all other parameters (anti-S and -RBD IgG antibodies and VNT) were negative.
Although differences in antibody responses to SARS-CoV-2 antigens in children and adults with indications of reduced protective serological response in younger age were previously reported, 2 we did not observe consistent differences when comparing our results from children with an adult population assessed by same assays. 20 We observed that mildly affected children and adults expressed a similar magnitude of RBD-specific IgG antibody levels. Likewise, there were no big differences regarding S-specific antibody responses in children and adults. The ability to inhibit RBD binding to ACE2 did not differ significantly between children and adults. Only VNT titers in children were lower than in adults. Despite the comparable antibody titer levels in the pediatric and adult populations, virus neutralization was, in general, more pronounced in adults in this functional assay, suggesting that other factors may underlie this observation.
Even though our study is limited only to humoral aspect of immunity, it seems that SARS-CoV-2 pediatric patients exhibit SARS-