SARS-CoV-2 antibody responses in children exhibit higher FcR engagement and avidity than in adults

Cohort description

Blood samples were obtained from opportunistic sampling of volunteers with RT-PCR confirmed SARS-CoV-2 primary infection, who were previously infection and vaccination-naïve. Participants were recruited between March 2020 and December 2020 and prior to the introduction of COVID-19 vaccines. Children (0.25–17 years) and adults (18-71 years) were recruited from hospitals based on RT-PCR positivity, lack of admission to ICU and by WHO severity score < 2 (mild)³⁴. Due to the zero-COVID policy in place in Hong Kong until late 2022, less than 1% of the population was infected in the first two years of the pandemic and even mild or asymptomatic individuals were hospitalised for isolation purposes. Adult samples were selected based on severity ( <2) and timepoints to match available samples from children, despite a higher proportion of mild cases (85.9% for adults than children 56.8%, p < 0.0001), than asymptomatic (Supplementary Fig. 1C). There was also more repeated sampling in children than adults (Table 1, p = 0.0018). No critical or fatal COVID-19 cases were included and those taking medications other than steroids or antivirals were excluded. Due to limited volume and sample availability, a subset of 160 children’s samples were included for non-Spike and antibody avidity assessment (with the same age range 0.25–17 years and average±SD 9.3 ± 4.7 years old). For multivariate analysis, 75 samples from 52 children and 111 samples from 80 adults who had data collected from most immune measures tested were included (Table 1).

Acute antibody responses are similar between children and adults

The early acute ((le)14 days) SARS-CoV-2 infection serological response was assessed in infected children, adults, and uninfected controls (Fig. 1). Infected children and adults had significantly higher acute S-IgG compared to uninfected controls, but there was no significant difference between infected age groups (mean 0.12 for children and 0.39 O.D. for adults, Fig. 1A). Similarly, there was no difference by age in infected groups in the magnitude of IgG responses against Nucleocapsid (N) (Fig. 1B) or replication dependent open reading frame 8 (ORF8) (Fig. 1C) with acute N-IgG at 0.063 for children and 0.14 O.D. for adults (Fig. 1B) and ORF8-IgG 0.32 for children and 0.58 O.D. for adults (Fig. 1C). At acute infection, most infected children and adults had S-IgG responses below the cut-off of detection, with only 37.3% of children and 47.5% of adults seropositive for S-IgG responses above the pre-pandemic responder cut-off (Fig. 1A), which was also similar for N (Fig. 1B) and ORF8 (Fig. 1C) IgG seropositivity.

Acute serum IgM responses, known as an early antibody immune response, to S, N and ORF8 proteins were equivalent in children and adults (Fig. 1D–F). Furthermore, uninfected children’s IgM levels were equivalent to those in the infected children group indicating either a high cross-reactive baseline, or an immature and non-specific response. The uninfected children’s IgM responses were significantly higher than uninfected adults (S-IgM 5.5-fold, p < 0.0001, N-IgM 4.5-fold, p < 0.0001, ORF8-IgM 3.4-fold, p = 0.0002, Fig. 1D–F), as previously seen by others for S-IgM³¹. SARS-CoV-2 infected participants response to CCCoV OC43 Spike showed no significant differences between infected and uninfected children (though from only a small number (n = 12) of uninfected children), but was significantly higher in infected than uninfected adults IgG (p = 0.0033) and IgM (p = 0.0325) (Fig. 1G, H).

Temporal patterns result in significantly lower SARS-CoV-2 antibodies in children than adults at convalescence

While neutralising antibodies at convalescent ( >14 days) timepoints were equivalent between children and adults (Fig. 2A), there was a significantly lower IgG magnitude response in children than adults for both SARS-CoV-2 Spike (p < 0.0001), Fig. 2B), and OC43 Spike (p = 0.0001, Fig. 2C). The difference in SARS-CoV-2 response was driven by significant differences (p < 0.0001) in S2 domain IgG (Fig. 2D), while Receptor Binding Domain (RBD) and S1-N-Terminal Domain (NTD) IgG were equivalent at acute and convalescent timepoints (Supplementary Fig 2A, B, D, E). ORF8-IgG convalescent responses were also lower in children (p = 0.0267), whilst there were no significant differences between children and adults for Nucleocapsid (Fig. 2E, F). IgG responses also increased with age as a continuous variable (S-IgG Spearman coefficient r = 0.336, p < 0.0001, Supplementary Fig 3). The higher response against SARS-CoV-2 Spike in adults was also observed in a longitudinal analysis, for which we fitted a Generalised Additive Mixed Model (GAMM) accounting for repeated samples from the same individuals. The S-IgG response was on average 66.9% decreased in children compared to adults (p < 0.0001), and 26.9% against Nucleocapsid (p = 0.046, Fig. 2G, H).

Children samples in red (PRNT50 n = 58 Spike (S) n = 111 OC43 S/ Nucleocapsid (N)/ ORF8 (n = 82), S2 (n = 81) and adults in black (PRNT50 n = 40, Spike n = 82, OC43 S/S2/N/ORF8 n = 80), neutralising antibody titres (A), IgG responses (B–F) at convalescent timepoints (from day 15 post symptom onset or hospital admission). Dotted lines represent a responder cut off based on the mean+2 SD response of 48 pre-pandemic controls, and % donors with responses above the cut off shown in grey boxes below. Individual data points are shown, with lines and error bars showing mean and standard deviations. Lower bars are not shown if at 0 or below. Differences between groups were assessed using a two-sided Mann-Whitney test with exact p values up to 4 significant figures shown. Antibody levels directed against SARS-CoV-2 S up to 180 days post symptom onset or hospital admission (G) or against SARS-CoV-2 N up to 138 days (H). For S-IgG,122 samples from 85 adults are depicted in black and 231 samples from 137 children are shown in red (G) while, for N-IgG, points represent 113 samples from 84 adults in black and 158 samples from 106 children in red (H). Ratio between IgG and IgM in infected children, adults, uninfected children and uninfected adults against S, N, ORF8 and OC43 S (I–L) with dotted likes at 1 for equal ratio. Differences between groups assessed with two-sided Kruskal-Wallis test with Dunn’s multiple comparisons correction and exact p values up to 4 significant figures shown. M Longitudinal plot of IgG-to-IgM ratios targeting S tracked until day 104. Data include 103 samples from 77 adults and 55 samples from 42 children. (N) Longitudinal plot of IgG-to-IgM ratios targeting nucleocapsid up to day 138. Points represent n = 104 samples from 79 adults in grey and n = 74 samples from 52 children in red. G, H, M, N Fitted values are derived from GAMM accounting for participants’ ID as a random effect and, as a fixed effect, age group, symptoms and sex (G), age group and symptoms (M) or only age group (H, N). 95% confidence intervals are depicted by the shaded areas. The p-value corresponds to the significance of age group as a parametric coefficient in the model and the average decrease of response in children compared to adults is indicated. Source data are provided as a Source Data file.

When defined in terms of the IgG/IgM ratio, indicative of a class switched and maturing antibody response, adults had significantly more S-specific SARS-CoV-2 and OC43 class-switched antibodies (IgG>IgM) than children at convalescent timepoints (p = 0.0074 for SARS-CoV-2, p < 0.0001 for OC43, Fig. 2I, J) and over time. Our GAM models confirmed lower IgG/IgM ratios in children, with ratios on average 58.1% and 74.6% lower than in adults for SARS-CoV-2 and OC43 Spikes respectively (Fig. 2M, N, Supplementary Fig 4A). While IgG/IgM ratios for SARS-CoV-2 N and ORF8 proteins were not significantly lower in children at convalescent timepoints (Fig. 2K, L), they were significantly lower over time compared to adults, as shown by our GAM models accounting for participants’ ID as random effects and age group as fixed effect. IgG/IgM ratios for children were indeed respectively 60.7% (N) and 61.9% (ORF8) of the adults’ ratios (Fig. 2L, Supplementary Fig 4B). SARS-CoV-2 S-IgM were equivalent between children and adults at convalescent timepoints and over time (Supplementary Fig 5A, G), thus higher ratios in adults are accounted for by increased IgG responses. OC43 S-IgM remained significantly higher in children (p = 0.0002, Supplementary Fig 5D) at convalescent timepoints and over time (on average +56.8% in children, p < 0.0001, Supplementary Fig. 5J). Children’s total serum IgM was not different compared to adults (Supplementary Fig 5E, K), and children also had higher RSV-F IgM at steady state, potentially suggesting the presence of non-specific IgM responses in children (Supplementary Fig 5F, L).

Therefore, while acute IgG and IgM responses are not different between children and adults, temporal patterns IgG magnitude and class switching resulted in significantly lower SARS-CoV-2 Spike IgG in children than adults by convalescence. This was not dependent on symptomatic illness, as there were no differences seen between symptomatic and asymptomatic children or adults, either longitudinally, or at convalescent timepoints (Supplementary Fig 6).

High SARS-CoV-2 and not OC43 IgG avidity suggests de novo antibody responses in children

The longitudinal avidity, representative of affinity maturation, while accounting for increased strength of binding by multivalent responses³⁵ of S-IgG demonstrated higher avidity which increased over time in children compared to adults (Fig. 3A, p = 0.0098), and S-IgG avidity at convalescence was significantly higher in children than adults (p < 0.0001,Fig. 3B). The proportion of the avidity response of high avidity ( >50%) was 56.0% in children compared to 22.8% adults. This was driven by higher RBD-IgG avidity in children than adults (p = 0.0440, Fig. 3C), while S1-NTD and S2 domains had equivalent or lower avidity in children than adults (Supplementary Fig 2F, G). Like S, the avidity of N-IgG is consistently higher in children than adults over time (23.8% higher avidity in children, p < 0.0001, Fig. 3D) and significantly higher at convalescence (children: 78.0% versus adults: 31.6%, p < 0.0001, Fig. 3E). The avidity of the OC43 S-IgG response showed the opposite pattern, with significantly higher avidity in adults (100%) than in children (79.6%, p < 0.0001) at convalescent timepoints and over time (on average 37.6% lower in children) (Figure. 3F, G). OC43-S IgG avidity was comparable between infected and uninfected adults (Fig. 3G), thus there is no further affinity maturation of the OC43-S B cell response during SARS-CoV-2 infection.

IgG avidity of response based on response following 8 M Urea treatment for removal of low avidity IgG against SARS-CoV-2 whole Spike, and RBD domains (A–C), and Nucleocapsid (D, E) and OC43 Spike (F, G). Data is representative of individual values in children (red) and adults (black). Data shown over time up to 138 days post infection with predictions from a generalised additive mixed model accounting for participants’ IDs as a random effect and age group as a fixed effect (A, D, F), and as convalescent avidities after day 14 post infection with mean and SD in error bars (BCEG). Lower bars are not shown if at 0 or below. For convalescent timepoints (BCEG), the sample sizes were n = 50 children’s samples and n = 57 adults’ samples (B), n = 73 children and 77 adults in (C), n = 51 children and n = 57 adults (E), or n = 51 infected children, 56 infected adults, 10 non-infected children, and 9 non-infected adults in (G). A, D, F Data for longitudinal graphs originated from 62 samples from 45 children and 91 samples from 68 adults in (A), 70 samples from 48 children and 93 samples from 69 adults in (D), and 69 samples from 47 children and 95 samples from 69 adults in (F). Dotted lines in graphs show 50% avidity with high avidity responses above the line. Percentage donors with high avidity responses are shown in grey boxes (B, C, E, G). Statistical differences in avidity were assessed with two-sided Mann-Whitney test (B, C, E) to compare 2 groups, and two-sided Kruskal-Wallis with Dunn’s multiple comparisons to include comparisons with uninfected children (-red open circles) and uninfected adults (black open circles) with significant p-values shown (G). P values in (A, D, F) correspond to the significance of age group as a parametric coefficient in the model and the average difference in percentage is indicated when significant. 95% confidence intervals are depicted by the shaded areas (ADF). Source data are provided as a Source Data file.

The IgA responses are not impacted by age for SARS-CoV-2 S, N and ORF8 IgA (Supplementary Fig 7A–C), but are significantly higher in children for OC43 S-IgA (p = 0.0245, Supplementary Fig 7D). The class switched response from IgM to IgA at convalescent timepoints was also significantly higher in adults than children against SARS-CoV-2 N (p = 0.0043, Supplementary Fig 7F).

Antibody FcR binding is higher in children compared to adults

The functional quality of antibodies to mediate effector functions was assessed by Fc gamma receptor (FcγR) binding. FcγRIIIa-binding correlates (r = 0.5114, p = 0.0005) with NK cell degranulation in adults (Supplementary Fig. 8) and is a proxy and correlate for NK cell mediated ADCC killing³⁶. This correlation is consistent with other pathogens like influenza virus and HIV in adults and children^37,38. Although used as in vitro assays, experimental ADCC is also a correlate of protection against severe and fatal SARS-CoV-2 infections in humans^17,18.

Adults and children had the same pattern of S-FcγRIIa longitudinal response for ADCP with an increase over time and maintained until at least 4 months, as shown by GAM model accounting for individual participants (Fig. 4A, p = 0.15). At convalescent timepoints, responses were comparable, with significantly lower responses in uninfected children and adults (p < 0.0001 for both, Fig. 4B). As a proportion of the total IgG response (measured as a ratio FcγRIIa-binding to total S-IgG), children had significantly higher proportion S-IgG with FcγRIIa-binding capacity (p = 0.0004, Fig. 4C), indicating a functional enrichment of ADCP responses.

Spike specific antibodies that bind to FcγRIIa (A–C) and FcγRIIIa (D–F) in infected children (red) and adults (black) (A, D) over time shown as individual values with predictions from a generalised additive mixed model and 95% CI and at (BCEF) convalescent ( > 14 days post infection) timepoints (n = 53 children and 71 adults in (B), 34 children and 71 adults in (C), and 111 (E) or 110 (F) children and 82 adults in (E, F)). 12 (B, E) or 10 (E, F) non-infected children and 10 non-infected adults were tested in (B, C, E, F). Responses shown with bars for mean ± SD and as (B, E) raw FcR binding responses and (C, F) ratio of FcR response to total IgG. Lower bars are not shown if at 0 or below. Groups were compared with two-sided Kruskal-Wallis with Dunn’s multiple comparisons. A, D Longitudinal responses are plotted up to 138 days and represent 75 samples from 52 children and 105 samples from 79 adults (A) or up to 180 days with 231 samples from 137 children and 122 samples from 85 adults (D). The GAMMs account for participants’ IDs as random effect and age group as fixed effects (A, D). The p-values correspond to the significance of age group as a parametric coefficient in the model and the average increase of response in children compared to adults is indicated. 95% confidence intervals are depicted by the shaded areas (A, D). Source data are provided as a Source Data file.

Meanwhile, S-FcγRIIIa-binding response for ADCC was significantly higher in children than adults over time (Fig. 4D), ( + 73.3% higher on average in children than adults, p < 0.0001, GAMM accounting for repeated measures). At convalescent timepoints, children thus had increased S-FcγRIIIa responses compared to adults (p < 0.0001). Similarly, the S-FcγRIIIa to IgG ratio was significantly higher in infected children than adults (p < 0.0001, Fig. 4F).

Multiparametric analysis confirms distinct coordinated immune responses in children and adults

Correlation analyses show distinct overall serological responses based on the parameters measured above. Adults have a larger cluster of positively correlated serological responses linking several IgG, FcγRIIa- and FcγRIIIa-binding responses against SARS-CoV-2 S, RBD, S1, N, OC43 and PRNT50 than children (Fig. 5A, B). Children’s SARS-CoV-2 S, N, ORF8 IgG and S FcR-binding responses are more strongly positively correlated together (r = 0.46 for S–N, 0.57 for FcγRIIa—S, and 0.54 for FcγRIIa—FcγRIIIa in adults against 0.72, 0.78 and 0.90 for children respectively). Children’s responses also show a strong positive correlation with increasing time post infection (0.60 N-IgG, 0.43 for FcγRIIIa and 0.50 for FcγRIIa, Fig. 5B). OC43-S-IgG is significantly positively correlated with SARS-CoV-2 proteins in adults (r = 0.36 for S-IgG, p < 0.05, 0.61 for N-IgG in adults, p < 0.0001, and 0.46 for ORF8-IgG, p < 0.0001, Fig. 5A) and with neutralisation (r = 0.53). On the contrary, no significant correlations for OC43 S-IgG were evident in children, except for age and avidity for OC43 (Fig. 5B). Univariate correlations similarly highlighted more positive correlations of antibody responses with increasing time for children, and negative correlations for adults (Supplementary Fig 9), whilst emphasising the strong positive correlations and link between IgG features (avidity and FcR) in adults.

A subset of samples with multiple measurements was selected for the multivariate analyses. It consists of 111 samples from 80 adults and 75 samples from 52 children. A, B Spearman correlations between all antibody measurements were calculated and depicted as a network for samples from adults (A) or children (B) participants. Each node corresponds to a variable, and edges between them represent positive correlations in red and negative correlations in blue. The thickness of the edge represents the correlation coefficient, with fine lines indicating a coefficient below 0.4. Only significant correlations after Bonferroni correction for multiple testing are shown. Nodes were grouped together when strongly correlating for children. Features are colour coded, with grey for donor information, blue for IgG, pink for IgM, green for IgA, orange for IgG avidity, purple for Fc-receptors and red for neutralisation (PRNT). C, D A principal component analysis with all numerical variables except age was performed and points for dimensions 3 and 4 are shown in (C) coloured by age group with children in red and adults in grey. 95% concentration ellipses are overlaid to indicate clustering patterns. A two-sided Analysis of Similarities test (ANOSIM) was used to assess if the two age groups were statistically different: p-value and R statistic are indicated. R ranges from -1 to 1, if around 0 the groups are similar, the closest to 1 the most the groups are separated. The contribution of each variable to each dimension of the PCA are shown in (D) with the size of the dots while the colour of the dots represents the loadings on each principal component. Only significant contributions are represented, where a contribution is considered significant if it exceeds 3.7%, corresponding to the expected value under a uniform distribution. E, F Supervised multiple factor analysis illustrating the distribution of samples colour-coded by sex, symptoms or age group (E). ANOSIM p-values and R statistic are indicated. The loadings of each variable are shown in (F) on the x-axis, with bars colour-coded to indicate their contribution to the principal component, non-significant contributions are in grey. Contributions are considered significant if they exceed 3%, corresponding to the expected value under a uniform distribution. Source data are provided as a Source Data file.

Unsupervised principal component analysis highlighted that, when taking into account all the antibody features measured in our study and the time post onset of disease, children’s and adults’ samples clustered separately on principal components 3 and 4 as confirmed by analysis of similarities (ANOSIM) for which p = 0.001 and R = 0.32 (the closest to 1 the most distinct are the groups, Fig. 5C and Supplementary Fig 10A). This clustering was not based on symptomatic or asymptomatic infection (Supplementary Fig. 10B), or on the sex of participants (Supplementary Fig 10C). The variables driving this separation on Dimension 3 were mostly avidity for S-OC43 IgG (16.2% contribution, -0.6 loading), S-FcγRIIIa (13.1% contribution, 0.59 loading), and RSV-IgM (11.9% contribution, 0.56 loading) (Fig. 5D). Positive loadings are associated with children while negative are associated with adults.

Finally, to consider both the quantitative and qualitative variables (age group, symptomology and sex), a multiple factor analysis (MFA) was performed (Fig. 5E). The MFA confirmed a distinct separation of samples along Dimension 1 based on age group (R = 0.56, p = 0.001). Specifically, samples from children clustered in the negative space of Dimension 1, while those from adults were positioned in the positive space. In contrast, separation based on symptoms or sex was less pronounced, despite a significant difference in symptoms between adults and children (Table 1); as both groups were mild/asymptomatic this did not drive major antibody differences. The main contributors to Dimension 1 after age groups, are IgG avidity for OC43 that contributes for 16.4% with +0.77 loading, avidity for N-IgG with 6.5% and -0.49 loading, and RSV-IgM with 5.3% contribution and -0.49 loading (Fig. 5F). Separation along Dimension 2 is mainly driven by Time (24.5%, −0.12 loading) and Fc-receptors responses, with S-FcγRIIa contributing for 16.1% and S-FcγRIIIa for 9.9%. Our multiparametric analyses thus confirms our previous univariate findings for distinct serological signatures between adults and children based on specificity, avidity and Fc effector functions.