Two linear epitopes on the SARS-CoV-2 spike protein that elicit neutralising antibodies in COVID-19 patients

Abstract

Given the ongoing SARS-CoV-2 pandemic, identification of immunogenic targets against the coronavirus spike glycoprotein will provide crucial advances towards the development of sensitive diagnostic tools and potential vaccine candidate targets. In this study, using pools of overlapping linear B-cell peptides, we report two IgG immunodominant regions on SARS-CoV-2 spike glycoprotein that are recognised by sera from COVID-19 convalescent patients. Notably, one is specific to SARS-CoV-2, which is located in close proximity to the receptor binding domain. The other region, which is localised at the fusion peptide, could potentially function as a pan-SARS target. Functionally, antibody depletion assays demonstrate that antibodies targeting these immunodominant regions significantly alter virus neutralisation capacities. Taken together, identification and validation of these neutralising B-cell epitopes will provide insights towards the design of diagnostics and vaccine candidates against this high priority coronavirus.

Fig. 1. COVID-19 patient sera can neutralise pseudotyped lentiviruses expressing SARS-CoV-2 spike protein.

a Sera of COVID-19 patients (n = 25) at 1:1000 dilution were incubated with luciferase expressing lentiviruses pseudotyped with SARS-CoV-2 spike (S) glycoprotein protein for 1 hour prior to infection of CHO-ACE2 cells for 48 hours. Infection levels were determined by luciferase assay, and percentage of neutralisation is presented. Recalled SARS patients (n = 13) and healthy controls (HC) were also conducted in parallel. Dotted lines correspond to 50% neutralisation and baseline of HC. b Sera of recalled SARS patients (n = 13, 1:100 dilution) or a healthy control (Healthy) were mixed with pseudotyped lentivirus expressing SARS-CoV or SARS-CoV-2 S glycoprotein, prior to incubation with CHO-ACE2 cells for 48 hours. Infection levels were determined by luciferase assay, and percentage neutralisation was analysed. c Dose–response neutralisation of pseudotyped lentivirus titration curves of COVID-19 patients (n = 41, 1:50 to 1:12,800 dilutions). d Dose–response neutralisation of live SARS-CoV-2 virus titration curves of COVID-19 patients (n = 8, 1:16 to 1:65,536 dilutions). Comparison table of IC₅₀ values from two assays. Lines represent non-linear regression robust fit. Source data are provided as a Source Data File.

Fig. 2. COVID-19 patient sera recognise two linear epitopes in SARS-CoV-2 spike protein.

a Sera of COVID-19 (n = 6) patients at 1:1000 dilution were subjected to peptide-based IgG ELISA using peptide pools covering the entire S protein of SARS-CoV-2 in duplicates. Sera of pooled healthy donors (n = 13) were assessed in parallel. Data are presented as mean patient OD values subtracted of healthy control value are presented, negative values are plotted as zero. b Sera of COVID-19 patients (n = 6) were subjected to peptide-based ELISA for IgG detection using individual peptides of SARS-CoV-2 S peptide pools S14 and S21. The z score values of each patient were calculated using the formula [OD value of patient for peptide−average (OD values of patient)]/standard deviation (OD values of patient). Data shown are from two independent experiments and presented as mean. c Serum peptide binding response of COVID-19 patients on SARS-CoV-2 peptides S14P5 and S21P2, and the corresponding regions on SARS-CoV peptides S45P3 and S51P5, respectively, was determined by ELISA at 1:1000 dilution. Statistical analysis was carried out with paired parametric two-tailed t test (*p < 0.05). d Peptides S14P5 and S21P2 response in 41 COVID-19 patients and 29 healthy controls assessed by ELISA in 1% Triton X-treated plasma fraction at 1:1000 dilution. Data are presented as mean of baseline subtracted OD of two independent experiments and was analysed by Mann–Whitney U test (***p < 0.001). e Spearman correlation of ELISA response from 41 COVID-19 patients to individual peptides from d and sera IC₅₀ neutralisation against SARS-CoV-2 S pseudotyped lentiviruses (Supplementary Table 4) were shown. Line was drawn using non-linear regression with 1/Y² weighting. Source data are provided as a Source Data file.

Fig. 3. Antibodies against S14P5 and S21P2 linear B-cell epitopes neutralise SARS-CoV-2.

a–b Localisation and sequences of a SARS-CoV-2 specific S14P5 and b pan-CoV S21P2 epitopes on spike (S) protein (PDB: 6VSB) are shown. Each S monomer is denoted as either pink, blue or orange. c–e Pooled sera of COVID-19 patients (n = 6) were added to plates coated with the corresponding peptides to deplete specific antibodies. c–d Validation of depletion by peptide-based ELISA against c depleted or d non-depleted peptides. Data of depleted sera (white bar) were normalised to percentages of the non-depleted sera (grey bar). Data are from one experiment in duplicate. Dotted line represents healthy sera mean value. e Non-depleted and peptide-specific antibody-depleted pooled sera were mixed with SARS-CoV-2 pseudovirus for 1 hour before infection of CHO-ACE2 cells for 48 hours. Percentage of pseudovirus neutralisation relative to the non-depleted sera, are shown. Data are presented as mean ± SD in triplicates. Statistical analysis was carried out with one-sample t test for each experiment (*p < 0.05, **p < 0.01). Figure is representative of two independent experiments. Source data are provided as a Source Data File.