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

The study addresses the need to identify immunogenic targets on the SARS-CoV-2 spike (S) glycoprotein that elicit neutralising antibodies, to inform diagnostics and vaccine design amid the COVID-19 pandemic. In December 2019, a cluster of pneumonia cases of unknown aetiology in Wuhan was attributed to a novel coronavirus, SARS-CoV-2. SARS-CoV-2 uses the S glycoprotein to bind ACE2 with higher affinity than SARS-CoV, and blocking ACE2 binding or S cleavage/fusion is a plausible antiviral strategy. Several studies had assessed immunogenicity of S domains, and emerging data indicated potent humoral responses in infection. This work investigates whether COVID-19 convalescent sera recognise linear B-cell epitopes on S and whether antibodies to such epitopes contribute to virus neutralisation, using pseudotyped lentivirus and validation with live virus.

Prior work has shown SARS-CoV-2 infection elicits strong immune responses and several studies assessed immunogenicity of recombinant S domains. Blocking ACE2 binding or proteolytic activation of S is an established strategy to prevent coronavirus entry. The fusion peptide region is highly conserved among coronaviruses and has been targeted by pan-coronavirus inhibitors in SARS-CoV and MERS-CoV. Computational predictions had suggested segments overlapping S14P5 could be immunogenic, and a recent preprint reported an epitope overlapping S21P2.

Study design and samples: Convalescent COVID-19 patient sera were evaluated alongside recalled SARS patient sera and healthy controls. For neutralisation, 41 COVID-19 patient sera were titrated (pseudovirus assay), with eight also tested against live SARS-CoV-2 under BSL3 for validation. Additional cohorts included 25 COVID-19 sera assessed at fixed dilution, 13 recalled SARS patients (controls), and 28–29 healthy controls for serology. Neutralisation assays: A BSL-2 pseudotyped lentivirus expressing SARS-CoV-2 S and a luciferase reporter was used to measure neutralisation in CHO-ACE2 cells after 48 h infection. Initial screening used fixed dilutions (e.g., 1:1000 or 1:100), followed by dose–response titrations to determine IC50. A subset of eight sera underwent parallel live virus neutralisation under BSL3, demonstrating comparable IC50 values to the pseudovirus assay. Linear epitope mapping: Overlapping 15-mer peptide libraries covering the full S glycoproteins of SARS-CoV-2 and SARS-CoV were organised into pools of five peptides. Sera from six selected COVID-19 patients (with sufficient titres) and five recalled SARS patients were screened by peptide-based IgG ELISA to identify reactive pools. Healthy donor sera were run in parallel. Reactive pools were deconvoluted to individual peptides to pinpoint epitopes. ELISA and data analysis: Peptide-based ELISAs quantified IgG binding. Responses were background-corrected using healthy control means, Z-scores were computed, and statistical analyses included paired t-tests and Mann–Whitney U tests. Spearman correlations assessed relationships between peptide-specific ELISA signals and neutralisation IC50 values. Antibody depletion: To assess functional contribution, pooled COVID-19 sera (n = 6) were affinity-depleted using peptide-coated plates targeting specific epitopes (S14P5, S21P2). Depletion efficacy was confirmed by ELISA. Depleted and non-depleted sera were then tested in the pseudovirus neutralisation assay to evaluate changes in neutralisation capacity. Cell culture and virus production: 293T, Vero E6, and CHO-ACE2 cells were maintained under standard conditions. Pseudotyped lentiviruses bearing SARS-CoV-2 or SARS-CoV S proteins were produced by plasmid co-transfection and concentrated. ACE2 expression on CHO-ACE2 cells was verified by flow cytometry/immunostaining. Ethics: Written informed consent was obtained; studies were approved by relevant IRBs (DSRB numbers 2012/0197, 2019/0017, 2020/00243).

- COVID-19 convalescent sera broadly neutralised SARS-CoV-2 S pseudotyped lentivirus at screening dilutions, whereas recalled SARS patient sera did not neutralise SARS-CoV-2 but retained low-level activity against SARS-CoV. - Two immunodominant linear B-cell epitope regions on SARS-CoV-2 S were identified: pools S14 and S21; deconvolution pinpointed peptides S14P5 (near the receptor-binding domain) and S21P2 (within/overlapping the fusion peptide) as major targets of IgG from COVID-19 patients. - Binding to S14P5 and S21P2 was stronger for SARS-CoV-2 peptides than corresponding SARS-CoV peptides; S21P2 aligns with a conserved fusion peptide region, suggesting pan-SARS potential. - Across 41 COVID-19 patients, peptide-specific ELISA signals for S14P5 and S21P2 positively correlated with neutralisation IC50 against SARS-CoV-2 S pseudotyped lentivirus (Spearman correlation reported), indicating these antibodies constitute a significant component of the neutralising response. - Affinity depletion of antibodies against S14P5 or S21P2 from pooled COVID-19 sera led to significant reductions in pseudovirus neutralisation (greater than 20% decrease relative to non-depleted sera), demonstrating functional contribution of these epitope-specific antibodies. - Dual depletion of both epitope-specific antibodies did not show synergistic loss compared to single depletions, suggesting independent, non-synergistic contributions. - Pseudovirus and live virus neutralisation IC50 values were comparable for tested samples, validating the pseudovirus assay as a surrogate for live virus neutralisation.

The research question—whether linear B-cell epitopes on the SARS-CoV-2 S protein elicit neutralising antibodies—was addressed by mapping sera reactivity and linking it to functional neutralisation. Identification of S14P5 (proximal to RBD) suggests a mechanism of neutralisation via steric hindrance of ACE2 binding or allosteric effects on the RBD. S21P2, overlapping the conserved fusion peptide, indicates interference with the membrane fusion process and offers potential as a pan-coronavirus target. The positive correlation between epitope-specific IgG levels and neutralisation supports development of quantitative serologies targeting these epitopes as proxies for exposure and protective immunity. Depletion experiments confirmed that antibodies to these linear regions contribute materially to neutralisation, although their effects were not synergistic. These findings are relevant for vaccine antigen selection prioritising conserved, functionally critical regions and for enhancing serological assay sensitivity and specificity, with a lower susceptibility to false negatives due to mutations reported to have low-to-moderate impact in these regions.

This study maps two immunodominant linear B-cell epitopes on the SARS-CoV-2 spike—S14P5 (near RBD) and S21P2 (fusion peptide)—that are recognized by COVID-19 convalescent sera and contribute significantly to neutralisation. The pseudotyped lentivirus assay, validated against live virus, facilitates safe and rapid assessment of neutralising activity. The results inform design of epitope-focused diagnostics and vaccines, including potential pan-coronavirus strategies leveraging the conserved fusion peptide region. Future work should isolate and characterise monoclonal antibodies to these epitopes, quantify their neutralisation potency relative to RBD and conformational epitopes, assess persistence of epitope-specific responses, and validate epitope-based serology across diverse populations and pathogens.

- The analysis focuses on linear epitopes; conformational epitopes on S may account for additional neutralising activity not captured by linear peptide ELISAs. - Although depletion showed >20% reduction in neutralisation, epitope-specific antibodies likely represent a subset of the total anti-S response. - Combined depletion of S14P5 and S21P2 did not exhibit synergistic effects, indicating other neutralising determinants may be involved. - Validation was performed on moderate sample sizes and primarily with pseudovirus assays (albeit with live virus confirmation in a subset). - Generalisability to broader and more diverse cohorts and cross-pathogen specificity of epitope-based serology require further validation.