Identification of linear B cell epitopes in the SARS-CoV-2 proteome that are conserved in humans coronaviruses

By Sangeeta PaulMar 15 2022Reviewed by Danielle Ellis, B.Sc.

In a recent study published in the International Journal of Molecular Sciences, researchers identified and validated multiple linear B-cell epitopes in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteomes conserved in humans coronaviruses (HCoVs). The researchers used immunoinformatics and computational predictive tools and extrapolated the information obtained to examine cross-reactive humoral response in coronaviruses.

Background

The characterization of SARS-CoV-2 B- cell epitopes is fundamental to developing antibody-based therapeutics, rational vaccine design, rapid diagnostic tests, and other clinical approaches against SARS-CoV-2 infection. Various studies have identified several cross-reactive T lymphocytes epitopes from SARS-CoV-2 with other betacoronaviruses.

This study was designed to focus on universal B-cell candidate epitopes highly conserved in beta coronaviruses to depict their functional relevance in the evolutionary and epidemiological context in the coronavirus disease 2019 (COVID-19) pandemic.

Study design

The study included 21 COVID-19 affected health care workers at Hospital 12 de Octubre, Madrid, Spain, with SARS-CoV-2-positive reverse transcription-polymerase chain reaction (RT-PCR) test, and a further 40 pre-COVID-19 samples from 2016.

The authors predicted linear B-cell epitopes in the four structural sequences of SARS-CoV-2 by a consensus of eight bioinformatics methods. They retrieved a repository of experimentally detected multiple B- cell epitopes from SARS-CoV and SARS-CoV-2 from the immune epitope database (IEDB).

Reference sequences of structural coronavirus such as SARS-CoV, SARS-CoV-2, Middle Eastern respiratory syndrome (MERS), HCoV-HKU1, HCoV-229E, HCoV-NL63, and HCOV-OC43 were collected from the National Center for Biotechnology Information (NCBI) database. Total anti-SARS-CoV-2 immunoglobulin G (IgG) antibodies (Abs) and synthetic peptides from betacoronavirus were analyzed by enzyme-linked immunosorbent assay (ELISA).

Findings

The findings of this study demonstrated the presence of different regions enriched in epitopes for the four structural proteins of SARS-CoV-2 and other coronaviruses. The authors observed that B-cell epitope regions were abundant in the N (nucleocapsid) and S (spike) proteins of SARS-CoV-2 and were particularly longer in the N-protein. Most epitope zones were deemed conserved in SARS-CoV but declined sharply in MERS-CoV. While in betacoronavirus only a few zones (N: 7; M [membrane protein]: 2; S: 3) were conserved, none of the zones were conserved in Alphacoronavirus.

From the 12 conserved zones of Betacoronavirus, only four residues of N-proteins showed identical positions except for two in HCOV-HKU1. These residues in SARS-CoV-2 and HCOV-HKU1 were swapped to form two chimeric exchange (Exc-1) and Exc-2 peptides. Altogether the authors identified four conserved, surface-exposed, and hypothetical peptide regions - N-Ep1, N-Ep2, N-Ep3, and N-Ep4 exclusive for N-protein.

Serology analysis revealed that out of 21 SARS-CoV-2-positive healthcare workers, three and two healthcare workers showed few to nil anti-SARS-CoV-2 S protein IgG, respectively. One healthcare worker had an antibody titer of 400 while the remaining 14 healthcare workers had antibody titers of 1600 - 12,800. On the other hand, high titers of anti-SARS-CoV-2 N protein IgGs were observed in all healthcare workers except two.

The authors estimated the reactivity of four N-Ep peptides selected from the conserved regions between SARS-CoV-2 and HCoV-OC43 N proteins. They demonstrated that more than 50% of healthcare workers affected by COVID-19 showed reactivity with either of the four SARS-CoV-2 N peptides and four to five healthcare workers recognized all of the four N-derived peptides. Sera samples of three healthcare workers (sample C12+, C16+, and C21+) and one healthcare worker (sample C10+) showed reactivity with two and three SARS-CoV-2 peptides, respectively. This demonstrated that the four SARS-CoV-2 N protein regions were B cell epitopes from multiple subjects.

Moreover, HCoV-OC43 N peptides were recognized in the sera of three COVID-19-affected healthcare workers. Sera from samples C13+ and C21+ were positive with the N-Ep4 peptide and C10+ were reactive for N-Ep1, N-Ep3, and N-Ep4 peptides constituting 14.3% subjects.

Similarly, anti- SARS-CoV-2 IgG Abs were estimated in a cohort of 40 serum samples before the COVID-19 pandemic in 2016. Three serum samples showed very less anti-SARS-CoV-2 –S protein IgG and the other 37 samples showed no IgG response against SARS-CoV-2 S protein. Interestingly, sera from 14 pre-COVID-19 samples showed anti-SARS-CoV-2 N-specific IgG Ab titers (between 200 to 3200), while the other 26 samples showed very low to no response for SARS-CoV-2 N protein.

The researchers identified cross-reactivity against peptide candidates between N-proteins of SARS-CoV-2 and HCoV OC43 in 40 pre-COVID-19 samples. Four sera samples representing 10% of samples - C7-, C26-, C 43-, and C9 - recognized HCOV-V-OC43 N peptides. Samples C7- and C26- were positive for N-Ep1 peptide, sample C43- for N-Ep1, and N-Ep4 peptides and C9 sample detected N-Ep1, N-Ep3, and N-Ep4 peptides.

Conclusion

The findings of the study demonstrated the presence of four B-cell epitopes on SARS-CoV-2 N–protein (N-Ep1, N-Ep2, N-Ep3, and N-Ep4) that were recognized by the humoral response from multiple SARS-CoV-2-positive patients and were conserved in other human coronaviruses. Moreover, three surface-exposed sequences and peptide homologs were also identified from pre-COVID-19 serum samples highlighting cross-reactivity of antibodies against SARS-CoV-2 N protein.