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.

Keywords: COVID-19; SARS-CoV-2 variants; antigen test; immunoinformatic.

Figure 1

Location of predicted epitopes in SARS-CoV-2 N protein 3D structure. The protein chain is indicated by a gray cartoon and transparent surface. The locations of epitopes N_(34–48), N_(89–104), N_(185–197), N_(277–287), and N_(378–390) were indicated by colors blue, teal, purple, red, and orange, respectively. In the cartoon, round helices, flat sheets, and smooth loops are applied to allow better visualization of the predicted structure. Different rotations of the protein are shown in (a) and (b).

Figure 2

Heatmap of IgM and IgG reactivity indexes against synthetic epitopes. Values higher than 1 represent responder individuals and were indicated in the color scale, and non-responders were indicated by a light blue color.

Figure 3

Evaluation of natural immunogenicity of predicted epitopes. (a) Frequencies of IgM, IgG, and overall responders to N_(34–48) (blue bar), N_(89–104) (green bar), N_(185–197) (gray bar), N_(277–287) (red bar), and N_(378–390) (light blue bar). The frequencies of responders to epitopes were compared by Fisher’s exact test, and statistical differences were indicated by asterisks: (*) = p < 0.05; (**) = p < 0.01; (***) = p < 0.001. (b) IgM (white dots) and IgG (black dots) reactivity indexes against predicted epitopes. Responders to peptides were indicated above the traced line.

Figure 4

B-cell epitopes and key mutations in SARS-CoV-2 N protein—3D structure. The N protein was presented as cartoon (gray), in which the identified epitopes were represented by colored sticks: N_(34–48) (Blue), N_(89–104) (teal), N_(185–197) (magenta), N_(277–287) (red), and N_(378–390) (orange). The positions of key mutations (D3, Q9, P13, Del31/33, D63, P80, E136, R203, G204, T205, G215, L230, S235, D377, and S413) were represented by green spheres. In the cartoon, round helices, flat sheets, and smooth loops are applied to allow better visualization of the 3D structure.