Scientists identify a novel epitope at the SARS-CoV-2 spike furin cleavage site

By Dr. Sanchari Sinha Dutta, Ph.D.Reviewed by Benedette Cuffari, M.Sc.Dec 15 2022

Scientists have identified an immunogenic epitope at the furin cleavage site of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that induces robust antibody responses in coronavirus disease 2019 (COVID-19) patients. In their study published in the journal eBioMedicine, the researchers also identified a monoclonal antibody that binds to this epitope and protects mice against SARS-CoV-2 infection.

Study: Identification of an immunogenic epitope and protective antibody against the furin cleavage site of SARS-CoV-2. Image Credit: CI Photos / Shutterstock.com

Background

SARS-CoV-2 contains a unique four-amino acid insertion (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. The sequential cleavage of the spike protein at S1/S2 and S2’ cleavage sites by furin and TMPRSS2 is essential for viral entry and infectivity.

In the current study, scientists identify an immunogenic epitope at the furin cleavage site that specifically recognizes immunoglobulin G (IgG) and IgM antibodies in COVID-19 patients.

Identification and characterization of novel spike epitope

The scientists screened spike epitopes using the immune epitope database and analysis resource prediction, ultimately identifying seven peptides. The 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 the enzyme-linked immunosorbent assay (ELISA) to identify peptide-specific antibodies in COVID-19 patients and healthy individuals. The most potent antibody response was detected against the peptide at the 672-691 spike position, indicating that this peptide contains an immunogenic epitope.

A comparison of the peptide sequence with other human coronaviruses revealed that the peptide contains a PRRA between the S1 and S2 subunits absent in other coronaviruses.

The ELISA findings revealed that both IgG and IgM antibodies targeting the spike peptide 672-691 are present in COVID-19 patients and suspected patients at significantly higher concentrations than healthy individuals.

The scientists further tested the applicability of the spike peptide as an immunogenic antigen for the diagnosis of COVID-19. These experiments revealed that the serum titers of peptide-specific IgG antibodies could serve as potential biomarkers for diagnostic purposes.

Antiviral efficacy of the spike peptide

The scientists developed a synthetic version of the spike peptide 672-691 to determine its ability to prevent SARS-CoV-2 infection in cultured cells. To this end, 672-691 was found to block wild-type SARS-CoV-2 and the Omicron variant from infecting the cells.

Experimental mice were then treated with the peptide, followed by the collection of serum samples to determine its virus-neutralizing efficiency. To this end, serum samples obtained from 672-691-treated mice could prevent SARS-CoV-2 infection.

The researchers also introduced deletion mutations within the spike peptide to isolate a specific antibody-binding region. The identified immunogenic epitope of 672-691 was found to overlap with the PRRA, which is unique to SARS-CoV-2.

Spike peptide-specific monoclonal antibody response

Peripheral blood mononuclear cells derived from COVID-19 patients were screened for spike peptide-specific human monoclonal antibodies. Multiple phage clones were obtained, following which sequence analysis was performed to identify two independent clones.

A monoclonal antibody generated from one of these clones exhibited a strong binding affinity for the spike peptide derived from wild-type SARS-CoV-2 and the Omicron variant. However, further analysis with mutant viral strains revealed that the spike peptide lacking the PRRA insertion does not bind to this monoclonal antibody, thus indicating that the monoclonal antibody directly binds to the furin cleavage site of the spike protein.

Regarding antiviral efficacy, the monoclonal antibody significantly reduced viral ribonucleic acid (RNA) titers in the lungs of SARS-CoV-2-infected mice.

Study significance

The current study identifies an immunogenic epitope at the furin cleavage site of the SARS-CoV-2 spike protein. This epitope induces a robust antibody response in COVID-19 patients. The researchers also identified a human monoclonal antibody that targets this epitope and protects mice from developing SARS-CoV-2 infection.

Overall, the study findings highlight that, apart from creating a furin cleavage site between the S1 and S2 subunits, the PRRA in the spike protein generates a highly immunogenic epitope. Thus, antibodies binding to the spike protein outside of the well-established receptor-binding domain (RBD) may also have potent antiviral activity.