Antibody conversion rates to SARS-CoV-2 in saliva from children attending summer schools in Barcelona, Spain

A cohort of 1907 children (age 0–14 years old) attending 22 summer schools and 2 pre-schools, and adult staff working at the same facilities, located in 27 different venues in the Barcelona metropolitan region, Spain, was followed up from June 29th to July 31st, 2020. Symptomatic children were defined as those with acute respiratory infection including fever, cough, headache, gastrointestinal symptoms, rhinorrhea or nasal congestion, anosmia or ageusia, dyspnea, and myalgia.

Saliva samples were collected weekly over a 5-week period with Oracol devices (Malvern, UK) for optimal harvesting of crevicular fluid enriched with serum antibodies [23, 24], centrifuged, heat inactivated (60 °C, 30 min), and frozen until antibody analysis.

Saliva SARS-CoV-2 RT-PCR detection was performed as described [25]. Levels of IgG, IgA, and IgM against SARS-CoV-2 nucleocapsid (N) full-length (FL) and C-terminus (CT) [26], spike (S), S2, and RBD proteins, were measured by Luminex assays [21] (details in Additional file 1: Detailed methods) in saliva samples diluted 1:10 in 384-well plates, with paired samples from the same individual run together. Pre-pandemic negative controls were not available. Samples were acquired on a Flexmap 3D xMAP® and median fluorescent intensities (MFI) were exported for each analyte using xPONENT.

Non-parametric Mann-Whitney U tests were used in boxplots to compare levels (log₁₀MFI) of each antibody/antigen pair between study groups. Radar plots were used to compare median MFIs of antibody responses between study groups by Mann-Whitney U test, adjusting p values for multiple comparison by Benjamini-Hochberg. Heatmaps with hierarchical clustering (by Euclidian or Canberra methods) were used to evaluate patterns of responses at the individual level. To quantify how many participants got infected during the study period, we considered at least a 3–4-fold increase (FC) in antibody levels between two consecutive visits [19]. To define the saliva antibody conversion rate, we applied the more stringent threshold of ≥ 4-FC in antibody levels from the first to the last week visit only in the subset of individuals in whom at least two samples were collected ≥ 6 days apart. Saliva antibody reversion was defined as a ≥4-FC decrease in antibody levels from the first to the last week visit in the same subset of individuals (see Additional file 1: Detailed methods).

Saliva antibody conversion rates were finally compared depending on the age, sex, and the presence or not of symptoms.

For the estimation of antibody conversion/reversion rates, only data from participants with minimum two visits and minimum 6 days in between visits were considered (1548 participants). Figure 1 and Additional file 3: Figure S1 show the subjects in whom saliva antibody levels to SARS-CoV-2 changed ≥ 3–4-fold from the first to the last visit. Computing those with a ≥ 4-fold increase in antibody levels to at least one Ig/antigen pair, the overall antibody conversion rate was 3.22% (49/1518) (Table 2). This represented a 6 times higher estimate of new SARS-CoV-2 infections than what RT-PCR detected in this subgroup (8/1518, 0.53%). Stratifying by age, IgG conversion rates were significantly higher in adults (2.94%, 11/374) than in children (1.31%, 15/1144) (p=0.035) (Additional file 4: Table S2). Antibody conversion was higher for IgA (2.37%, 36/1518) and IgG (1.71%, 26/1518) than for IgM (0.2%, 3/1518). The N FL and N CT proteins were the main targets of saliva antibodies, followed by the S2 protein. Excluding individuals who only increased SARS-CoV-2 N FL antibodies (0.86%, 13/1518), potentially cross-reactive with HCoV N FL [26], the final adjusted conversion rate was 2.36%. Antibodies were maintained at a wide range of levels in a large number of subjects, and in others, they decayed from the first to the last visit (Table 2, Fig. 1), but no one reverted for all isotypes/antigens (≥ 4-fold decrease). SARS-CoV-2 IgAs reverted more than IgGs. Additional file 5: Figure S2 shows the antibody levels in those diagnosed as RT-PCR positive and Additional file 6: Figure S3 shows all subjects, including those with only one sample collected.

Saliva IgA and IgG levels were significantly lower in children (n=2677) than in adults (n=800), while no differences were seen for IgM (Fig. 2A). IgG and IgA levels gradually increased statistically significantly with age (Additional file 7: Figure S4). Among RT-PCR positives (n=10), IgA and IgG levels tended to also be higher in adults compared to children (Additional file 8: Figure S5A). Children with COVID-19-compatible symptoms had statistically significantly lower IgA to S2 and IgM and IgG RBD than children not reporting symptoms (Fig. 2B). Antibody levels were higher in females than males (Additional file 9: Figure S6).

In a heatmap of FC antibodies from the first to the last visit, most individuals with high FC raised both IgG and IgA (very few IgM), and a smaller group showed a raised either IgG or IgA (Fig. 3A). Focusing on individuals with ≥ 4-FC, some increased IgG predominantly, some increased IgA predominantly, and others increased both isotypes (Fig. 3B). There was no clear pattern for age or symptoms.

Combining all antibodies and variables in all individuals, the strongest signal for the high responders mapped to IgG to N FL and S2 antigens, as seen in the antibody conversion analysis, which was accompanied by IgG to S and RBD responses, but lower IgA reactivity (Fig. 3C). Another group of antibody responders had a more predominant IgA than IgG reactivity, while others had a more predominant IgG than IgA reactivity. There were more adults among higher antibody responders and no clear pattern was seen for symptoms.