Linear B-cell epitopes in the spike and nucleocapsid proteins as markers of SARS-CoV-2 exposure and disease severity

Abstract

Background: Given the unceasing worldwide surge in COVID-19 cases, there is an imperative need to develop highly specific and sensitive serology assays to define exposure to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Methods: Pooled plasma samples from PCR positive COVID-19 patients were used to identify linear B-cell epitopes from a SARS-CoV-2 peptide library of spike (S), envelope (E), membrane (M), and nucleocapsid (N) structural proteins by peptide-based ELISA. Hit epitopes were further validated with 79 COVID-19 patients with different disease severity status, 13 seasonal human CoV, 20 recovered SARS patients and 22 healthy donors.

Findings: Four immunodominant epitopes, S14P5, S20P2, S21P2 and N4P5, were identified on the S and N viral proteins. IgG responses to all identified epitopes displayed a strong detection profile, with N4P5 achieving the highest level of specificity (100%) and sensitivity (>96%) against SARS-CoV-2. Furthermore, the magnitude of IgG responses to S14P5, S21P2 and N4P5 were strongly associated with disease severity.

Interpretation: IgG responses to the peptide epitopes can serve as useful indicators for the degree of immunopathology in COVID-19 patients, and function as higly specific and sensitive sero-immunosurveillance tools for recent or past SARS-CoV-2 infections. The flexibility of these epitopes to be used alone or in combination will allow for the development of improved point-of-care-tests (POCTs).

Funding: Biomedical Research Council (BMRC), the A*ccelerate GAP-funded project (ACCL/19-GAP064-R20H-H) from Agency of Science, Technology and Research (A*STAR), and National Medical Research Council (NMRC) COVID-19 Research fund (COVID19RF-001) and CCGSFPOR20002. ATR is supported by the Singapore International Graduate Award (SINGA), A*STAR.

Keywords: Biomarkers; COVID-19; Epitopes; Patients; SARS-CoV-2.

Fig. 1

Identification of IgG-specific linear B-cell epitopes on spike (S), envelope (E), membrane (M), and nucleocapsid (N) SARS-CoV-2 proteins with COVID-19 patient plasma. (a–c) Plasma samples of COVID-19 patients collected at timepoint of median 14 days post-illness onset (pio, n = 18), recovered SARS individuals (n = 20) and healthy donors (n = 22) were pooled, respectively. (a) Pooled plasma samples (1:1000 dilution) were tested in a peptide-based ELISA of peptide pools covering SARS-CoV-2 S, E, M, and N proteins for IgG. Each pool consists of 5–8 peptides, with an overlapping sequence of 10 amino acids. OD results are presented in a heatmap, with white and red colours denoting low and high OD values, respectively. The cut-off for positive pools was derived from mean + 3SD of pooled healthy donors. (b) Individual peptides of the positive peptide pools in (a) were screened with pooled plasma samples (1:1000 dilution) to determine the IgG hit peptides. OD results are presented in a heatmap, with white and red colours denoting low and high OD values, respectively. (c) Peptide-binding response of pooled plasma samples (1:1000 dilution) against the corresponding hit peptides on SARS-CoV, with bar graphs presented as mean ± SEM, and dotted line denoting mean + 3SD of pooled healthy donors. All data in (a–c) are of two independent experiments. (d,e) Diagrams showing the localisation of SARS-CoV-2 specific IgG peptides S6P2, S14P5, S20P2, S21P2, and N4P5 on (d) SARS-CoV-2 S (PDB: 6VSB), and (e) N (PDB: 6YVO) protein. Each monomer is denoted as either pink, blue, orange or green. All peptides are annotated on the blue monomer.

Fig. 2

Specificity and sensitivity of putative IgG epitopes to detect anti-SARS-CoV-2 antibodies from COVID-19 patients. (a) Plasma samples from COVID-19 patients collected at timepoints of median 10 days post-illness onset (pio, n = 59) and median 23 days pio (n = 66), sera from seasonal hCoV-infected patients (n = 13) either pre- or post-infection, and plasma samples from recovered SARS patients (n = 20) and healthy donors (n = 22) were screened at 1:1000 dilution against five IgG-specific putative epitopes: S6P2, S14P5, S20P2, S21P2, and N4P5. Data are shown as mean ± SD of two independent experiments, with dotted lines indicating mean + 3SD of healthy donors. (b) Percentage specificity and sensitivity of putative epitopes to detect SARS-CoV-2-specific antibodies in plasma samples from COVID-19 patients during the timepoints of median 10 days pio (left panel) and median 23 days pio (right panel). Specificity and sensitivity values were derived from a threshold determined by the maximisation of the Youden's J statistic with a constraint that the threshold value has to be positive. (c) Percentage recognition of epitopes during the timepoints of median 10 days pio (black bars) and median 23 days pio (white bars).

Fig. 3

Association of anti-SARS-CoV-2 antibody response of COVID-19 patients with clinical severity during SARS-CoV-2 infection. Antibody profiles of COVID-19 patients at timepoint of median 23 days post-illness onset (pio, n = 66) against significant IgG peptides under different clinical measures of (a) disease severity, (b) pneumonia, and (c) intensive care unit (ICU) admission and oxygen supply requirement. Samples in (a) were classified based on severity with green denoting mild (clinical severity 0; n = 28), blue denoting moderate (clinical severity 1; n = 22), and red denoting severe (clinical severity 2; n = 16). Data are presented as mean ± SD of two independent experiments. Negative and positive observations in (b-c) are denoted as - and +, respectively. Statistical analysis was carried out using Kruskal-Wallis tests, followed by post hoc Dunn's multiple comparison tests. (**P<0•01, ***P<0•001).