Seroprevalence and correlates of SARS-CoV-2 neutralizing antibodies from a population-based study in Bonn, Germany

To estimate the seroprevalence and temporal course of SARS-CoV-2 neutralizing antibodies, we embedded a multi-tiered seroprevalence survey within an ongoing community-based cohort study in Bonn, Germany. We first assessed anti-SARS-CoV-2 immunoglobulin G levels with an immunoassay, followed by confirmatory testing of borderline and positive test results with a recombinant spike-based immunofluorescence assay and a plaque reduction neutralization test (PRNT). Those with a borderline or positive immunoassay result were retested after 4 to 5 months. At baseline, 4771 persons participated (88% response rate). Between April 24th and June 30th, 2020, seroprevalence was 0.97% (95% CI: 0.72−1.30) by immunoassay and 0.36% (95% CI: 0.21−0.61) when considering only those with two additional positive confirmatory tests. Importantly, about 20% of PRNT+ individuals lost their neutralizing antibodies within five months. Here, we show that neutralizing antibodies are detectable in only one third of those with a positive immunoassay result, and wane relatively quickly.


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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Due to implementation of this serosurvey in an ongoing cohort study, the study group was already predefined and therefore no a priori sample size calculation was performed. This serosurvey was conducted in two groups. Group I consisted of all living participants who had been enrolled in the Rhineland Study until March 18th, 2020 (N=5427). Their participation in the study was therefore unrelated to attitude to or experience with SARS-CoV-2. Group II consisted of individuals who were eligible for but had not yet participated in the Rhineland Study. They actively approached us to indicate their willingness to participate in the serosurvey (N=597), which we allowed for those who agreed to become prospective participants in the Rhineland Study. Participation in this group was thus motivated by the prospect of being tested: we used this group to estimate the effect of selection bias in SARS-CoV-2 serosurveys.
This serosurvey was based on the Rhineland Study, an ongoing community-based cohort study in Bonn, Germany. All inhabitants aged 30 years and above of two geographically defined areas are invited to participate in the Rhineland Study. The sole exclusion criterion for participation in the Rhineland Study is insufficient command of the German language to provide informed consent. For the present study we included data on all individuals who participated in the serosuvey.
This serosurvey was an observational cross-sectional study concerning the seroprevalence and correlates of neutralizing antibodies against SARS-CoV-2 in Bonn, Germany, in the period between April 24th and June 30th, 2020. Therefore, there were no experimental findings that would be amenable to replication.
Given the observational design of the study, randomization procedures were not applicable.
Given the observational design of the study, blinding procedures were not applicable.
The levels of immunoglobulin G (IgG) antibodies against SARS-CoV-2 were measured using a commercially available ELISA (EUROIMMUN, Lübeck, Germany). According to the manufacture's product sheet, applying a cut-off of >1.1 for defining seropositivity results in an estimated sensitivity of 94.4% (at >10 days of infection) and specificity of 99.6%. Jackson, no. 109-545-088, Alexa Fluor488-conjugated AffiniPure Goat anti-human IgG (H+L) (Lot 147499) in a 1:200 dilution was used for the immunoassay validation experiments ( Supplementary Fig. 3 and Supplementary Figure 5).
Our independent in-house validation experiments of this assay using serum samples from 119 plasma donors after convalescence from mild to moderate COVID-19 (as documented by positive swab nucleic acid testing), as well as 110 healthy subjects (with either no history of COVID-19-typical symptoms and no risk contacts, or negative pharyngeal swab SARS-CoV-2 nucleic acid testing), were in line with the manufacturer's reported values yielding a sensitivity of 86.8% and a specificity of 100%. We performed two additional confirmatory tests in all those individuals whose ELISA assay results were either positive (i.e. >1.1) or borderline (i.e. between 0.8 and 1.1). Confirmatory tests consisted of an in-house recombinant immunofluorescence test and a plaque reduction neutralisation test (PRNT) to specifically check for the presence of neutralizing antibodies against SARS-CoV-2.