The ongoing SARS-CoV-2 pandemic necessitates the identification of immunogenic targets on the coronavirus spike glycoprotein for diagnostic tools and vaccine development. SARS-CoV-2 utilizes its spike (S) glycoprotein to bind to the angiotensin-converting enzyme 2 (ACE2) receptor, facilitating entry into host cells. Blocking this binding or the host protease cleavage of the S glycoprotein to release the fusion peptide is a key strategy for preventing viral entry. Previous studies have investigated the immunogenicity of SARS-CoV-2 spike protein domains, showing that infection elicits potent immune responses. This study aims to identify and characterize immunodominant linear B-cell epitopes within the SARS-CoV-2 spike glycoprotein that elicit neutralizing antibodies, potentially contributing to the development of improved diagnostic tools and vaccines.

Several studies have assessed the immunogenicity of structural domains of recombinant SARS-CoV-2’s spike glycoprotein. Findings indicate that SARS-CoV-2 infection elicits potent immune responses, including the identification of immunodominant linear B-cell epitopes in the S glycoprotein. These epitopes could potentially be used for designing more sensitive serological assays for epidemiological or vaccine efficacy assessments.

The study employed a pseudotyped lentivirus assay expressing SARS-CoV-2 S glycoprotein tagged with a luciferase reporter to investigate the ability of sera from convalescent COVID-19 patients to elicit neutralizing antibodies. Sera from COVID-19 convalescent patients and SARS patients were tested. A linear B-cell peptide library spanning the entire S glycoprotein of SARS-CoV-2 and SARS-CoV was used to map linear antigenic targets. Enzyme-linked immunosorbent assays (ELISAs) were performed to detect IgG antibodies against specific peptides. Antibody depletion assays were conducted to assess the functional capacity of antibodies targeting identified epitopes. Neutralization titrations were performed using both pseudotyped lentiviruses and live SARS-CoV-2 virus under BSL3 conditions to validate the results. Statistical analysis included non-linear regression, paired parametric t-tests, Mann-Whitney U tests, and Spearman correlation.

Two distinct peptide pools (S14 and S21) from the SARS-CoV-2 library were strongly recognized by sera from COVID-19 patients but not by SARS patients or healthy controls. These pools were further narrowed down to specific peptides: S14P5 and S21P2. Peptide S14P5 is located near the receptor-binding domain (RBD), while S21P2 partially overlaps with the fusion peptide sequence, suggesting it could be a pan-SARS epitope. Antibody depletion assays showed that removing antibodies targeting S14P5 or S21P2 significantly reduced virus neutralization capacity. A positive correlation was observed between IgG levels against these epitopes and neutralizing antibody titers. The study also compared the neutralization capacity of the pseudotyped lentivirus assay with live virus neutralization assays, finding comparable IC50 values. This validates the safer pseudotyped lentivirus system for high-throughput screening.

The identification of two immunodominant linear B-cell epitopes, S14P5 and S21P2, on the SARS-CoV-2 spike protein, which elicit neutralizing antibodies, provides valuable insights for developing diagnostic tools and vaccines. The proximity of S14P5 to the RBD suggests that antibodies binding to this region may sterically hinder ACE2 receptor binding. S21P2's location within the fusion peptide region implies that antibodies targeting this region could interfere with viral fusion. The positive correlation between IgG levels against these epitopes and neutralizing antibody titers suggests that serological assays targeting these epitopes could serve as a proxy for virus exposure and protection levels. The relatively low mutation rate of these epitopes suggests a reduced risk of false negatives in serological assays.

This study successfully identified two immunodominant linear B-cell epitopes on the SARS-CoV-2 spike protein that elicit neutralizing antibodies. These epitopes represent promising targets for developing sensitive serological assays and vaccines. Future research should focus on isolating monoclonal antibodies targeting these epitopes to quantify their contribution to the overall neutralizing antibody response and investigate the long-term persistence of these antibodies.

The study used a pseudotyped lentivirus system, which may not perfectly mimic live virus infection. The sample size, while substantial, might be considered a limitation for certain analyses. Further investigation is needed to fully understand the role and neutralization capacity of antibodies targeting these regions in the context of diverse viral variants and populations.