A Public Health Antibody Screening Indicates a 6-Fold Higher SARS-CoV-2 Exposure Rate than Reported Cases in Children

Abstract

Background: Antibody responses to virus reflect exposure and potential protection.

Methods: We developed a highly specific and sensitive approach to measuring antibodies against SARS-CoV-2 for population-scale immune surveillance. Antibody positivity was defined as a dual-positive response against both the receptor-binding domain and nucleocapsid proteins of SARS-CoV-2. Antibodies were measured by immunoprecipitation assays in capillary blood from 15,771 children aged 1 to 18 years living in Bavaria, Germany, and participating in a public health type 1 diabetes screening program (ClinicalTrials.gov: NCT04039945), in 1,916 dried blood spots from neonates in a Bavarian screening study (ClinicalTrials.gov: NCT03316261), and in 75 SARS-CoV-2-positive individuals. Virus positive incidence was obtained from the Bavarian health authority data.

Findings: Dual-antibody positivity was detected in none of the 3,887 children in 2019 (100% specificity) and 73 of 75 SARS-CoV-2-positive individuals (97.3% sensitivity). Antibody surveillance in children during 2020 resulted in frequencies of 0.08% in January to March, 0.61% in April, 0.74% in May, 1.13% in June, and 0.91% in July. Antibody prevalence from April 2020 was 6-fold higher than the incidence of authority-reported cases (156 per 100,000 children), showed marked variation between the seven Bavarian regions (p < 0.0001), and was not associated with age or sex. Transmission in children with virus-positive family members was 35%. 47% of positive children were asymptomatic. No association with type 1 diabetes autoimmunity was observed. Antibody frequency in newborns was 0.47%.

Conclusions: We demonstrate the value of population-based screening programs for pandemic monitoring.

Funding: The work was supported by funding from the BMBF (FKZ01KX1818).

Keywords: RBD; SARS-CoV-2 antibody; nucleocapsid antigen; public health screening; receptor binding domain; seroprevalence; type 1 diabetes.

Graphical abstract

Figure 1

Number of Individuals Tested for SARS-CoV-2 Antibodies The total numbers from each group, the numbers positive at the screening, and confirmation stages are shown. AU, arbitrary unit; RBD, receptor binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. See also Table S1.

Figure 2

SARS-CoV-2 Antibody Sensitivity and Specificity (A) SARS-CoV-2 RBD antibody titers in 3,321 children sampled between August and December 2019, in 75 people with a confirmed SARS-CoV-2 antibody-positive virus test, and 666 children sampled in April or May 2019. The red broken line shows the logarithmic mean plus 5 SD at >0.9 AU. Samples with values >0.9 AU are shown as large circles. (B) Antibodies to SARS-CoV-2 nucleocapsid protein (y axis) and RBD antibodies (x axis) in children sampled in 2019 with RBD antibodies >0.9 AU (open circles) and in SARS-CoV-2 virus-positive cases (filled circles). The broken lines indicate the threshold for positivity assigned to each antibody. (C) Inhibition of binding to Nluc-SARS-CoV-2 RBD protein by competition with purified RBD protein (y axis) in children (open circles) and virus-positive cases (filled circles) with RBD antibodies >0.9 AU. The samples were separated into nucleocapsid protein antibody-negative (left) and -positive (right) samples. The broken line indicates 50% inhibition. (D) Antibodies to the RBD of the beta coronavirus OC43 in children (y axis) with SARS-CoV-2 RBD antibodies <0.9 AU (open circles) and >0.9 AU (gray filled circles), and in virus-positive cases (dark gray filled circles). AU, arbitrary unit; PCR, polymerase chain reaction; RBD, receptor binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. See also Figures S1 and S2.

Figure 3

Immune Surveillance for SARS-CoV-2 Antibodies in the Fr1da Study for Children with Samples Obtained in 2020 (A) SARS-CoV-2 RBD antibody values in 11,884 children sampled between January and July 2020. Samples with antibody titers >0.9 AU are shown as large circles. Samples fulfilling the definition of antibody-positive status (RBD antibody titers >0.9 AU and positive for anti-nucleocapsid antibodies) are indicated as filled circles. The red broken lines indicate the logarithmic mean plus 5 SD at >0.9 AU. (B) Frequency of SARS-CoV-2 antibody-positive children per month in 2020 (gray bars and left y axis) and the cumulative incidence of health authority reported virus-positive children by the end of each month from March through to July (blue dots and lines, right y axis). Error bars show the upper 95% confidence interval for the antibody prevalence. AU, arbitrary unit; IgG, immunoglobulin G; RBD, receptor binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. See also Tables S2, S3 and S4 and Figures S3 and S4.

Figure 4

Regional Variation in SARS-CoV-2 Antibody Prevalence and Health Authority-Reported Incidence of Virus-Positive Cases (A) The frequency of antibody-positive children for the period of April to July 2020 in the seven administrative regions of Bavaria is indicated as a heatmap. The number of antibody-positive children out of the total number tested in the regions is also indicated. (B) The incidence of health authority-reported virus-positive children per 100,000 children inhabitants by July 31, 2020 in the seven administrative regions indicated as a heatmap. See also Tables S5, S6, and S7.

Figure 5

Serum Inhibition of RBD Binding to Its Receptor ACE2 (A) The ability of SARS-CoV-2 antibody-negative sera (open circles, n = 22 children) and -positive sera (filled green circles, n = 74 children) to inhibit the binding of nanoluciferase-tagged RBD to biotinylated recombinant ACE2 coated streptavidin Sepharose beads. Maximum RBD binding to ACE2-Sepharose beads corresponded to approximately 90,000 light units and background binding of RBD to uncoated beads corresponded to approximately 300 light units. (B) Inhibition of binding (y axis) is shown against the SARS-CoV-2 RBD antibody titer (x axis) for the antibody-positive children (n = 74).

Figure 6

SARS-CoV-2 Antibodies in Relation to Family Communicated Virus Positivity and Symptoms The questionnaire data from 4,859 children are shown as a Venn diagram depicting children who were indicated as virus positive (pale red, n = 12), having COVID-19-like or flu-like symptoms (blue, n = 514), having a virus-positive family member (n = 88), and those without virus, symptoms, or a virus-positive family member (n = 4,296). The number of children in each of the Venn diagram sectors is indicated and the number of the children in each sector who was SARS-CoV-2 antibody-positive is given in parentheses. See also Table S8 and Figure S5.

Figure 7

SARS-CoV-2 Antibodies in Newborns (A) Relationship between SARS-CoV-2 RBD antibody titers in serum (x axis) and the blood spot eluate (x axis) from the same blood draw (n = 45) obtained by the luciferase immunoprecipitation assay used to measure SARS-CoV-2 antibodies, which was adapted to measure eluates from dried blood spots. The dried blood spots were stored at −80°C for a median of 51 days (range, 14 to 66 days) prior to elution and testing. (B) SARS-CoV-2 RBD antibody titers in the blood spot eluates in 1,916 neonates born between April and June 2020. Samples with RBD antibody titers >0.9 are shown as large circles. Samples fulfilling the definition of positive (anti-RBD >0.9 AU and positive for nucleocapsid protein antibodies) are indicated as filled circles. The dried blood spots from neonates were stored at −80°C for a median of 34 days (range, 7 to 80 days) prior to elution and testing. The red broken line indicates the logarithmic mean plus 5 SD at >0.9 AU. AU, arbitrary unit; RBD, receptor binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. See also Table S9.