Identification of B-Cell Linear Epitopes in the Nucleocapsid (N) Protein B-Cell Linear Epitopes Conserved among the Main SARS-CoV-2 Variants

Abstract

The Nucleocapsid (N) protein is highlighted as the main target for COVID-19 diagnosis by antigen detection due to its abundance in circulation early during infection. However, the effects of the described mutations in the N protein epitopes and the efficacy of antigen testing across SARS-CoV-2 variants remain controversial and poorly understood. Here, we used immunoinformatics to identify five epitopes in the SARS-CoV-2 N protein (N(34–48), N(89–104), N(185–197), N(277–287), and N(378–390)) and validate their reactivity against samples from COVID-19 convalescent patients. All identified epitopes are fully conserved in the main SARS-CoV-2 variants and highly conserved with SARS-CoV. Moreover, the epitopes N(185–197) and N(277–287) are highly conserved with MERS-CoV, while the epitopes N(34–48), N(89–104), N(277–287), and N(378–390) are lowly conserved with common cold coronaviruses (229E, NL63, OC43, HKU1). These data are in accordance with the observed conservation of amino acids recognized by the antibodies 7R98, 7N0R, and 7CR5, which are conserved in the SARS-CoV-2 variants, SARS-CoV and MERS-CoV but lowly conserved in common cold coronaviruses. Therefore, we support the antigen tests as a scalable solution for the population-level diagnosis of SARS-CoV-2, but we highlight the need to verify the cross-reactivity of these tests against the common cold coronaviruses.

Role of SARS‑CoV‑2 nucleocapsid protein in affecting immune cells and insights on its molecular mechanisms

“…However, given the commonalities between model cells and primary cells, the current study could provide a theoretical basis for the immune regulatory activity and molecular mechanism of N protein at the cellular and molecular levels. B-cell-linear epitopes and T-cell epitopes of the N protein are conserved in the main SARS-CoV-2 variants including A (B.1.1.7), Ο (BA.1), Ο (XBB.1.5) ( 43-47 ), which means that the results of the current study may have similarities with in vitro immunomodulatory activity of the conserved N protein of the major variant strains of SARS-CoV-2. In addition, the findings that N protein has regulatory activity on RAW264.7, Jurkat and Raji, and can affect the expression of genes in innate immunity and acquired immunity supported the vaccination strategies designed to target the N protein that could generate immune responses which had cross-reactivity with SARS-CoV-2, as well as the potential ability to protect or modulate disease.…”

B-Cell Epitopes-Based Chimeric Protein from SARS-CoV-2 N and S Proteins Is Recognized by Specific Antibodies in Serum and Urine Samples from Patients

“…Bioinformatic tools to predict antigenic epitopes that are specific for the pathogens have been advancing the development of new diagnostics. Many epitopes have been identified by immunoinformatics prediction or immunoassays and the peptides validated by serological assays, such as ELISA [ 22 , 23 , 24 , 25 ]. Epitope-based chimeric proteins have been then proposed as recombinant proteins for the development of new COVID-19 vaccines or diagnostics, showing stronger immunogenicity with the possibility to express proteins in a simpler prokaryotic system [ 13 , 14 ].…”

“…Previous studies revealed that SARS-CoV-1 memory B cell responses tend to be short-lived after infection [ 31 ]. The main concern for antigen test cross-reactivity lies with the common cold coronaviruses (HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1), however, we found low similarity in the epitopes used here [ 24 ].…”