In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19

Abstract

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N = 80 samples from persons with RT-PCR confirmed SARS-CoV-2 infection), and a specificity of 97.2% (N = 106 control samples).

Figure 1

Linear epitope BepiPred2 predictions: (A) summary count per protein with at least one predicted epitope; (B) length distribution.

Figure 2

Distribution of conservation levels (1 = 100% identity) of SARS-CoV-2 linear epitopes compared to our comprehensive database of protein sequences from HCoV endemic strains. Epitopes located on essential enzymes such as the RdRp and helicase are the most conserved.

Figure 3

Dominance score vs specificity of predicted linear B-cell epitopes (blue) and structural epitopes (orange). Specificity is calculated as 1—conservation. The dominance threshold (DiscoTope2 minimal score scaled to 0) is shown as a green line. Potentially dominant epitopes are labelled. Scores below − 3 have been omitted for clarity.

Figure 4

Amino acid variation in predicted epitopes, as average variability (SE100 = 100*Shannon entropy over epitope length) vs variant density. Epitopes with variants detected in over 80% of residues or variability above 5 are labelled. The high SE100 of orf3a_5 and nsp2_13 are due to single conserved variants.

Figure 5

ELISA results (mean optical density at 492 nm [OD₄₉₂], error bars are standard deviations of the mean) comparing plasma from 6 COVID-19 persons and 5 controls taken before November 2019 using anti-IgG conjugated to horseradish peroxidase (HRP). Performance of spike-receptor binding domain (S-RBD) and nucleoprotein in selective reaction with COVID-19 plasma IgG is far superior to nsp9, complexes of nsp7/8, complexes of nsp10/16, or nsp15. Note: the N-terminal portion of nucleoprotein (N-Nt) retains the superior signal of full-length nucleoprotein (Nuc) but improves the signal-to-noise ratio of the assay by nearly twofold, to 4.2 from 2.3, respectively. This implies that epitopes on the C-terminal portion of Nuc are non-specific for SARS-CoV-2, as predicted.