Exploring beyond clinical routine SARS-CoV-2 serology using MultiCoV-Ab to evaluate endemic coronavirus cross-reactivity

Abstract

The humoral immune response to SARS-CoV-2 is a benchmark for immunity and detailed analysis is required to understand the manifestation and progression of COVID-19, monitor seroconversion within the general population, and support vaccine development. The majority of currently available commercial serological assays only quantify the SARS-CoV-2 antibody response against individual antigens, limiting our understanding of the immune response. To overcome this, we have developed a multiplex immunoassay (MultiCoV-Ab) including spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses. Compared to three broadly used commercial in vitro diagnostic tests, our MultiCoV-Ab achieves a higher sensitivity and specificity when analyzing a well-characterized sample set of SARS-CoV-2 infected and uninfected individuals. We find a high response against endemic coronaviruses in our sample set, but no consistent cross-reactive IgG response patterns against SARS-CoV-2. Here we show a robust, high-content-enabled, antigen-saving multiplex assay suited to both monitoring vaccination studies and facilitating epidemiologic screenings for humoral immunity towards pandemic and endemic coronaviruses.

Fig. 1. MultiCoV-Ab, a sensitive and specific tool to monitor SARS-CoV-2 antibody responses.

a Control sera (blue, n = 72) and sera from individuals with PCR-confirmed SARS-CoV-2 infection (red, n = 205) were screened in a multiplex bead-based assay using Luminex technology (MultiCoV-Ab) to quantify IgG or IgA responses to various antigens. Reactivity towards trimeric SARS-CoV-2 spike protein (Spike Trimer) or SARS-CoV-2 receptor binding domain of spike (RBD) was found to be the best predictor of SARS-CoV-2 infection. Data are presented as Box-Whisker plots of a sample’s median fluorescence intensity (MFI) on a logarithmic scale. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. Cut-off values for classification for single antigens are displayed as horizontal lines (Spike Trimer IgG: 3,000 MFI, IgA: 400 MFI; RBD IgG: 450 MFI, IgA: 250 MFI). b Sample set from a, was used to compare assay performance of the MultiCoV-Ab using Spike Trimer and RBD antigens with commercially available single analyte SARS-CoV-2 IVD assays which detect total Ig (Elecsys Anti-SARS-CoV-2 (Roche); ADVIA Centaur SARS-CoV-2 Total (COV2T) (Siemens Healthineers)) or IgG (Anti-SARS-CoV-2-ELISA - IgG (Euroimmun)) or IgA (Anti-SARS-CoV-2-ELISA - IgA (Euroimmun)). SARS-CoV-2 infection status of samples based on PCR diagnostic is indicated as SARS-CoV-2 positive or negative. Antibody test results were classified as negative (blue), positive (red), or borderline (gray) as per the manufacturer’s definition. Only samples with divergent antibody test results are shown. c Performance and specifications as stated in the manufacturer’s IVD assay manual. For the manufacturer sensitivity specification, information for samples >14 days post-infection are presented. Respective sensitivity and specificity values calculated in this study are given with 95% Clopper-Pearson confidence intervals. Positive and negative predictive values (PPV/NPV) were calculated based on a seropositivity of 3%. Source data are provided as a Source Data file.

Fig. 2. Combination of 2 spike protein variants and isotype profiling by multiplex assay increases accuracy to identify SARS-CoV-2 antibody-positive individuals.

a, b Scatterplot detailing MultiCoV-Ab cut-offs. Signal to cut-off (S/CO) values are displayed for Spike Trimer against RBD on a logarithmic scale. For IgG (a), cut-offs are visualized by straight lines and SARS-CoV-2-infected and uninfected samples are separated by color (black circles – SARS-CoV-2-uninfected; red circles – SARS-CoV-2-infected). For IgA (b) cut-offs are visualized as dashed lines and S/CO of 2 used for the combined cut-off is shown as straight lines. SARS-CoV-2-infected samples are split into IgG-positives and -negatives by color as indicated in the plot. c, d Scatterplots display IgG response to additional SARS-CoV-2 antigens contained in the MultiCoV-Ab panel: MFI for spike subdomains S1 vs S2 (c) or nucleocapsid antigens N vs N-NTD (d) are displayed on a logarithmic scale. SARS-CoV-2-uninfected samples are distinguished from SARS-CoV-2-infected and MultiCoV-Ab classification into positives or negatives as indicated by color. Source data are provided as a Source Data file.

Fig. 3. Multiplex-based seroprofiling allows in-depth characterization of SARS-CoV-2 antibody responses.

a Kinetic of SARS-CoV-2 antigen-specific IgA and IgG responses is shown for indicated days after symptom onset for six SARS-CoV-2-specific antigens for five different patients. Patients are indicated by color. b, c Samples of SARS-CoV-2-infected individuals were analyzed to identify antigen- and isotype-specific antibody responses based on hospitalization indicating disease severity (b) or age (c). Data is presented as Box-Whisker plots of sample MFI on a logarithmic scale. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. p-value (Mann–Whitney U test, two-sided) is displayed at the top of the boxes, indicating differences between signal distribution for respective groups. Cut-off values for MultiCoV-Ab classification are displayed as horizontal lines (Spike Trimer IgG: 3,000 MFI, IgA: 400 MFI; RBD IgG: 450 MFI, IgA: 250 MFI). Source data are provided as a Source Data file.

Fig. 4. Correlation of seasonal hCoV and SARS CoV-2 antibody responses.

a Correlation of IgG response for the entire sample set (n = 1176) is visualized as heatmap based on Spearman’s ρ coefficient; dendrogram on the right side displays antigens after hierarchical clustering was performed. b-c, Immune responses (IgG and IgA) towards hCoV S1 (b) and N (c) proteins are presented as Box-Whisker plots of sample MFI on a logarithmic scale for SARS-CoV-2-infected (red, n = 310) and uninfected (blue, n = 866) individuals. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. d-e, Relative levels of IgG-specific immune response towards hCoV S1 (d) and N (e) proteins are presented as Box-Whisker plots/strip chart overlays of log-transformed and per-antigen scaled and centered MFI for the sample subsets of Spike Trimer false positives (blue, n = 17) and combined IgG + IgA false negatives (red, n = 31). Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values, excluding outliers as determined by 1.5 times IQR. Source data are provided as a Source Data file.

Fig. 5. Analysis of seasonal hCoV high and low responders.

a From the entire study population, groups of α- or β-hCoV high and low responders were built as indicated. High responder were defined as samples with above average MFI values for S1 and N-specific IgGs of the respective hCoV clade. Low responders were defined with below MFI values, correspondingly. Responder groups (i) α-hCoV ↑, red, n = 233, (ii) β-hCoV ↑, green, n = 254, (iii) α-hCoV ↓, blue, n = 172 (iv) β-hCoV ↓, purple, n = 210 are shown as Box-Whisker plots of log-transformed and per-antigen scaled and centered MFI values across hCoV N and S1 antigens. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR are depicted as circles. b The over- or under-representation of SARS-CoV-2 responders (SARS-CoV-2 + , n = 279, as determined by positive MultiCoV-Ab classification) within the four sample groups is visualized in Venn diagrams, stochastic significance was calculated using Fisher’s exact test (two-sided). Source data are provided as a Source Data file.