The SARS-CoV-2 antibody landscape is lower in magnitude for structural proteins, diversified for accessory proteins and stable long-term in children

Abstract

Background: Children are less clinically affected by SARS-CoV-2 infection than adults with the majority of cases being mild or asymptomatic and the differences in infection outcomes are poorly understood. The kinetics, magnitude and landscape of the antibody response may impact the clinical severity and serological diagnosis of COVID-19. Thus, a comprehensive investigation of the antibody landscape in children and adults is needed.

Methods: We tested 254 plasma from 122 children with symptomatic and asymptomatic SARS-CoV-2 infections in Hong Kong up to 206 days post symptom onset, including 146 longitudinal samples from 58 children. Adult COVID-19 patients and pre-pandemic controls were included for comparison. We assessed antibodies to a 14-wide panel of SARS-CoV-2 structural and accessory proteins by Luciferase Immunoprecipitation System (LIPS).

Findings: Children have lower levels of Spike and Nucleocapsid antibodies than adults, and their cumulative humoral response is more expanded to accessory proteins (NSP1 and Open Reading Frames (ORFs)). Sensitive serology using the three N, ORF3b, ORF8 antibodies can discriminate COVID-19 in children. Principal component analysis revealed distinct serological signatures in children and the highest contribution to variance were responses to non-structural proteins ORF3b, NSP1, ORF7a and ORF8. Longitudinal sampling revealed maintenance or increase of antibodies for at least 6 months, except for ORF7b antibodies which showed decline. It was interesting to note that children have higher antibody responses towards known IFN antagonists: ORF3b, ORF6 and ORF7a. The diversified SARS-CoV-2 antibody response in children may be an important factor in driving control of SARS-CoV-2 infection.

Keywords: Antibody; COVID-19; IFN; accessory; asymptomatic; longitudinal; pediatric; structural.

Figure 1.. Comparison of antibody responses to SARS-CoV-2 structural proteins in children and in adults with COVID-19.

Antibodies against the SARS-CoV-2 structural proteins Spike S1 subunit (S1) (a), Spike S2 subunit (b), Spike S2’ subunit (c), Nucleocapsid (N) (d), Membrane (M) (e), and Envelope (E) (f) were measured by LIPS from samples from pediatrics COVID-19 (n=254) or adult patients (n=36), and negative controls (n=33). Background no plasma values were subtracted. Experiments were repeated twice. All data represents individual responses, and the mean +/− stdev. Two-sided P values were calculated using the Mann-Whitney U test. * shows statistical significance between COVID-19 patients versus negative controls. **p<0.01, ***p<0,001, **** p<0,0001.

Figure 2.. Antibody responses to SARS-CoV-2 non-structural proteins and ORFs are lower in magnitude in children than in adults with COVID-19 but represent globally a higher proportion of the SARS-CoV-2 humoral response.

Antibodies against NSP1 (a) (in ORF1ab), and other ORFs (ORF3a (b), ORF3b (c), ORF6 (d), ORF7a (e), ORF7b (f), ORF8 (g) and ORF10 (h)) were measured in pediatric (n=254) and adult (n=36) COVID-19 cases and negative controls (n=33) by LIPS to cover all the ORFs of the virus. (i) A heatmap comparing the mean titres (LU) for structural (N, S, S1, S2’, S2, M, E) and accessory proteins (NSP1, ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF10) responses in the COVID-19 pediatric and adult populations and Negatives. (j) Percentages of single antibody levels to SARS-CoV-2 antigens of the cumulative SARS-COV-2 antibody response in COVID-19 children and adults for the 14 antigens. Experiments were repeated twice. Two-sided P values were calculated using the Mann-Whitney U test. * shows statistical significance between COVID-19 patients versus negative controls. *p<0.05, **p<0.01, ***p<0.005, **** p<0,0001. Data in (a-h) represents the individual responses and mean +/− stdev, data in (i) represents mean values (LU), data in (j) represents percentages.

Figure 3.. Representation of the pediatric COVID-19 population as a cluster of points for relevant antibody combinations and Principal Component Analysis (PCA).

(a-b). Cluster representation of S1, S2’, S2 antibodies combination. (a) shows the pediatric COVID-19 population versus the adult COVID-19 population, (b) shows the pediatric COVID-19 population versus the negative population. (c) Cluster representation of N, ORF3b, ORF8 antibodies combination, for the pediatric COVID-19 population versus the adult COVID-19 population and the negative population. Patients are presented according to their values of SARS-CoV-2 individual LIPS antibodies as (x, y, z) in the space. Pediatric COVID-19 patients (n=144) are represented as red dots. COVID-19 adult patients (n=36 in (a-b) and n=24 in (c)) are represented in blue. The negative population (n=28) is represented in gray. (d-f) PCA of 14 antibodies analyzed in COVID-19 pediatric patients. Dim1 explains 21% of the variation, while Dim2 explains 15% of the variation. (d) Correlation circle and contributions. The scale of contributions is indicated (right). (e) Contribution of variables on dimensions 1 and 2. The red dashed line on the graph above indicates the expected average contribution. (f) Factorial plot of PCA on dimension 1 and 2. The plot is colored by sample types, the largest point in shape in each group is the group mean point (circle is for Adult positives, triangle for Negatives and squares for Pediatric positives).

Figure 4.. Asymptomatic and mildly symptomatic children do not display different antibody landscapes.

Pediatric and adult samples were stratified according to the symptom score of the patients (asymptomatic « asympto » (pediatric COVID-19 n=98, adults COVID-19 n=9) versus symptomatic « sympto » (pediatric COVID-19 n=156, adults COVID n=27)), data from Figure 1 and 2 were analyzed according to “asympto” and “sympto”. (a) Antibodies against the SARS-CoV-2 structural proteins S1, S2, S2’, N, E, and M by LIPS. (b) Antibodies against SARS-COV-2 NSP1 (in ORF1ab), and all other ORFs (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8 and ORF10). Two-sided P values were calculated using the Mann-Whitney U test. * shows statistical significance between COVID-19 patients versus negative controls. *p<0.05, **p<0.01, ***p<0.005, **** p<0,0001. All data represent individual responses and the mean +/− stdev.

Figure 5.. A unique antibody landscape is specific of early time-point samples (< day 14).

Pediatric samples were stratified according to the time-point of collection, and data from Figure 1 and 2 were analyzed according to acute (

Figure 6.. Longitudinal stability of antibody responses for structural and non-structural SARS-CoV-2 proteins in COVID-19 children.

(a) Number of longitudinal patients with either 2, 3 or 4 blood draws from 58 pediatric COVID-19 cases. (b) Sample collection time-line (days post infection). (c) A linear trend on log₁₀ LIPS values was fitted for longitudinal samples for S1, S2’, N, M E, NSP1, ORF3a, ORF3b, ORF7a, ORF7b, ORF8 (n=58 pediatric COVID-19 patients).

Figure 7.. IFN-α producing pediatric patients display a different landscape of antibody response to SARS-CoV-2 accessory proteins.

(a) Cluster representation of antibodies to the accessory proteins ORF3b, ORF7a, ORF6 (x, y, z) for the COVID-19 pediatric samples (red, n= 144) versus the COVID-19 adult samples (blue, n= 27). (b) Plasma IFN-α concentrations (pg/ml) in pediatric (n=48) and adult COVID-19 cases (n=18) early timepoint samples (< day 7). Data represents individual responses and the mean +/− stdev. (c) Pie charts of the cumulative antibody responses to the relevant SARS-CoV-2 structural and non-structural protein antigens (excluding N) in COVID-19 pediatric cohort stratified (positive/negative) by their IFN-α responses. (d) Individual data for IFN-α⁺ pediatric cases viral loads by RT-PCR, ORF3, ORF6, and ORF7b LIPS LU. P values were calculated using Chi-squared test between the mean of IFNα- pediatric COVID-19 patients (N=45) and the IFNα⁺ (N=3). ns, p=0.0591 *p<0.05, **p<0.01, ***p<0.001, **** p<0.0001.