Scientists have identified an immunogenic epitope at the furin cleavage site of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that induces a robust antibody response in patients with coronavirus disease 2019 (COVID-19). In his study published in the journal eBiomedicine, researchers also identified a monoclonal antibody that binds to this epitope and protects mice from SARS-CoV-2 infection.
Study: Identification of an immunogenic epitope and a protective antibody against the furin cleavage site of SARS-CoV-2. Image credit: CI Photos / Shutterstock.com
SARS-CoV-2 contains a unique insertion of four amino acids (PRRA) between the S1 and S2 subunits of the spike protein. This unique insertion creates a transmembrane protease serine 2 (TMPRSS2)/furin cleavage site in the spike protein, which increases the infectivity of SARS-CoV-2. Sequential cleavage of the spike protein at the S1/S2 and S2′ cleavage sites by furin and TMPRSS2 is essential for virus entry and infectivity.
In the current study, scientists identify an immunogenic epitope at the furin cleavage site that is specifically recognized by immunoglobulin G (IgG) and IgM antibodies in patients with COVID-19.
Identification and characterization of a new spiked epitope
The scientists screened for peak epitopes using the Immune Epitope Database and Predictive Analysis Resources, ultimately identifying seven peptides. Structural models of these proteins were then constructed using a computer-guided homology modeling approach.
Furthermore, the researchers chemically synthesized these peptides and subjected them to an enzyme-linked immunosorbent assay (ELISA) to identify peptide-specific antibodies in COVID-19 patients and healthy individuals. The strongest antibody response was detected against the peptide at spike position 672-691, indicating that this peptide contains an immunogenic epitope.
Comparison of the peptide sequence with other human coronaviruses revealed that the peptide contains a PRRA between the S1 and S2 subunits that is not present in other coronaviruses.
ELISA findings revealed that both IgG and IgM antibodies targeting spike peptide 672-691 are present in patients with and suspected of having COVID-19 at significantly higher concentrations than healthy individuals.
The scientists further tested the applicability of the spiked peptide as an immunogenic antigen for the diagnosis of COVID-19. These experiments revealed that serum titers of peptide-specific IgG antibodies can serve as potential biomarkers for diagnostic purposes.
Antiviral efficacy of spiked peptide
The scientists developed a synthetic version of the 672-691 spike peptide to determine its ability to prevent SARS-CoV-2 infection in cultured cells. To this end, 672-691 was found to prevent wild-type SARS-CoV-2 and the Omicron variant from infecting cells.
Mice were then treated with the peptide, followed by collection of serum samples to determine its virus-neutralizing efficacy. To this end, serum samples obtained from mice treated with 672-691 could prevent SARS-CoV-2 infection.
The researchers also introduced deletion mutations within the spike peptide to isolate the specific antibody binding region. The identified immunogenic epitope 672-691 was found to overlap with PRRA, which is unique to SARS-CoV-2.
Spike peptide-specific monoclonal antibody response
Peripheral blood mononuclear cells obtained from patients with COVID-19 were screened for spike peptide-specific human monoclonal antibodies. Multiple phage clones were obtained, followed by sequence analysis to identify two independent clones.
A monoclonal antibody generated from one of these clones showed strong binding affinity to the spike peptide derived from wild-type SARS-CoV-2 and the Omicron variant. However, further analysis with mutant virus strains revealed that the spike peptide without the PRRA insertion did not bind to this monoclonal antibody, indicating that the monoclonal antibody binds directly to the furin cleavage site of the spike protein.
In terms of antiviral efficacy, the monoclonal antibody significantly reduced viral ribonucleic acid (RNA) titers in the lungs of mice infected with SARS-CoV-2.
Significance of the study
The current study identifies an immunogenic epitope at the furin cleavage site of the SARS-CoV-2 spike protein. This epitope elicits a strong antibody response in patients with COVID-19. The researchers also identified a human monoclonal antibody that targets this epitope and protects mice from developing SARS-CoV-2 infection.
Overall, the findings of the study highlight that, in addition to creating a furin cleavage site between the S1 and S2 subunits, PRRA in the spike protein creates a highly immunogenic epitope. Therefore, antibodies that bind to the spike protein outside the well-established receptor binding domain (RBD) may also have potent antiviral activity.
- Li, L., Gao, M., Li, J., et al. (2022). Identification of an immunogenic epitope and a protective antibody against the furin cleavage site of SARS-CoV-2. eBiomedicine. doi:10.1016/j.ebiom.2022.104401.