From Multiplex Serology to Serolomics-A Novel Approach to the Antibody Response against the SARS-CoV-2 Proteome

Abstract

The emerging SARS-CoV-2 pandemic entails an urgent need for specific and sensitive high-throughput serological assays to assess SARS-CoV-2 epidemiology. We, therefore, aimed at developing a fluorescent-bead based SARS-CoV-2 multiplex serology assay for detection of antibody responses to the SARS-CoV-2 proteome. Proteins of the SARS-CoV-2 proteome and protein N of SARS-CoV-1 and common cold Coronaviruses (ccCoVs) were recombinantly expressed in E. coli or HEK293 cells. Assay performance was assessed in a COVID-19 case cohort (n = 48 hospitalized patients from Heidelberg) as well as n = 85 age- and sex-matched pre-pandemic controls from the ESTHER study. Assay validation included comparison with home-made immunofluorescence and commercial enzyme-linked immunosorbent (ELISA) assays. A sensitivity of 100% (95% CI: 86-100%) was achieved in COVID-19 patients 14 days post symptom onset with dual sero-positivity to SARS-CoV-2 N and the receptor-binding domain of the spike protein. The specificity obtained with this algorithm was 100% (95% CI: 96-100%). Antibody responses to ccCoVs N were abundantly high and did not correlate with those to SARS-CoV-2 N. Inclusion of additional SARS-CoV-2 proteins as well as separate assessment of immunoglobulin (Ig) classes M, A, and G allowed for explorative analyses regarding disease progression and course of antibody response. This newly developed SARS-CoV-2 multiplex serology assay achieved high sensitivity and specificity to determine SARS-CoV-2 sero-positivity. Its high throughput ability allows epidemiologic SARS-CoV-2 research in large population-based studies. Inclusion of additional pathogens into the panel as well as separate assessment of Ig isotypes will furthermore allow addressing research questions beyond SARS-CoV-2 sero-prevalence.

Keywords: SARS-CoV-2; multiplex serology.

Figure 1

Schematic study diagram. COVID-19 cases were recruited at Heidelberg University Clinics between March and May 2020. Of the n = 192 serum samples, n = 18 samples were excluded due to technical errors, a negative SARS-CoV-2 PCR test, or missing information about onset of symptoms. Pre-pandemic controls were obtained from the ESTHER I study (“Epidemiologische Studie zu Chancen der Verhütung, Früherkennung und optimierten Therapie chronischer Erkrankungen in der älteren Bevölkerung”), a prospective cohort that enrolled subjects aged 50–75 years between 2000 and 2002 in Saarland, Germany. We randomly selected n = 88 participants to frequency match the age- and sex-distribution among the hospitalized n = 48 COVID-19 patients. Due to technical errors, n = 3 serum samples were excluded.

Figure 2

Antibody responses to SARS-CoV-2 proteins in n = 85 pre-pandemic controls and n = 48 hospitalized COVID-19 patients. (A) Antibody responses (median fluorescence intensity (MFI)) to nucleocapsid (N) and spike (S) proteins, as well as their respective fragments; (B) SARS-CoV-2 non-structural proteins (NSP); (C) other SARS-CoV-2 open reading frames (ORF). If multiple samples per individual were available only the sample with the latest time point after blood draw was considered for analysis. Boxes represent the 25th to 75th and whiskers the 10th to 90th percentiles, respectively. Wilcoxon rank sum test was applied to assess statistically significant differences in antibody responses (MFI) to SARS-CoV-2 proteins between pre-pandemic controls and hospitalized COVID-19 patients; * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.

Figure 3

Antibody responses to SARS-CoV-2 proteins N, S1, S2, and their respective sub-fragments (N-EP3, S1-receptor-binding domain (RBD), and S2′) in longitudinal samples of n = 32 hospitalized COVID-19 patients. The antibody response (MFI) is plotted against days post symptom onset. The dashed lines indicate the antigen-specific cut-offs defined as mean plus three times the standard deviation in n = 85 pre-pandemic controls.