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. 2022 May 26:13:879946.
doi: 10.3389/fimmu.2022.879946. eCollection 2022.

A SARS-CoV-2 Spike Receptor Binding Motif Peptide Induces Anti-Spike Antibodies in Mice andIs Recognized by COVID-19 Patients

Federico Pratesi  1 , Fosca Errante  2 , Lorenzo Pacini  3 , Irina Charlot Peña-Moreno  4 , Sebastian Quiceno  4 , Alfonso Carotenuto  5 , Saidou Balam  6   7 , Drissa Konaté  6 , Mahamadou M Diakité  6 , Myriam Arévalo-Herrera  8 , Andrey V Kajava  9 , Paolo Rovero  2 , Giampietro Corradin  10 , Paola Migliorini  1 , Anna M Papini  3 , Sócrates Herrera  4
Affiliations

A SARS-CoV-2 Spike Receptor Binding Motif Peptide Induces Anti-Spike Antibodies in Mice andIs Recognized by COVID-19 Patients

Federico Pratesi et al. Front Immunol. .

Expression of concern in

Abstract

The currently devastating pandemic of severe acute respiratory syndrome known as coronavirus disease 2019 or COVID-19 is caused by the coronavirus SARS-CoV-2. Both the virus and the disease have been extensively studied worldwide. A trimeric spike (S) protein expressed on the virus outer bilayer leaflet has been identified as a ligand that allows the virus to penetrate human host cells and cause infection. Its receptor-binding domain (RBD) interacts with the angiotensin-converting enzyme 2 (ACE2), the host-cell viral receptor, and is, therefore, the subject of intense research for the development of virus control means, particularly vaccines. In this work, we search for smaller fragments of the S protein able to elicit virus-neutralizing antibodies, suitable for production by peptide synthesis technology. Based on the analysis of available data, we selected a 72 aa long receptor binding motif (RBM436-507) of RBD. We used ELISA to study the antibody response to each of the three antigens (S protein, its RBD domain and the RBM436-507 synthetic peptide) in humans exposed to the infection and in immunized mice. The seroreactivity analysis showed that anti-RBM antibodies are produced in COVID-19 patients and immunized mice and may exert neutralizing function, although with a frequency lower than anti-S and -RBD. These results provide a basis for further studies towards the development of vaccines or treatments focused on specific regions of the S virus protein, which can benefit from the absence of folding problems, conformational constraints and other advantages of the peptide synthesis production.

Keywords: COVID-19; SARS-CoV-2; immunized animals; neutralizing Abs; receptor binding motif; spike (S) protein.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
3D structure of the RBD-ACE2 complex and CD of RBM436-507. (A) Atomic 3D structure of the S trimer in the prefusion conformation (27). RBD is shown in ribbon representation (dark green). Region 476-507 is in orange. (B) Complex between RBD (green-orange) and ACE2 receptor (light cyan) (12). (C) Conformation of the RBM peptide (436-507) within the RBD. Cysteine residues are shown as spheres. (D) Circular Dichroism of RBM436-507 synthetic peptide used in this work.
Figure 2
Figure 2
Immunogenicity of S, RBD and RBM436-507 in mice. Analysis of the antibody response in mice immunized at days 0, 20, 40. (A) Anti-S, (B) Anti-RBD and (C) Anti-RBM436-507. SD is < 20% of the mean.
Figure 3
Figure 3
Homologous and cross reactivity of the S, RBD and RBM436-507 antigens with antibodies elicited upon mice immunization. (A–C) show reactivity of anti-S, anti-RBD and anti-RBM436-507 produced in mice with their homologous antigens. (D, G) show cross reactivity of anti-S with RBM and RBD antigens. (E, H) of anti-RBD with S and RBM antigens. (F, I), of anti RBM436-507 with S and RBD, respectively. SD is < 20% of the mean.
Figure 4
Figure 4
Cross-reactivity of S and RBD with ELISA captured RBM436-507. ELISA captured mice anti-RBM436-507 antibodies were eluted with Gly pH 2.5 and used to determine the reactivity with RBM (homologous), and with S and RBD (heterologous) antigens ELISA reaction was developed using rabbit anti-mouse alkaline phosphatase conjugate.
Figure 5
Figure 5
Anti-Spike and anti-RBD antibodies in COVID-19 patients. Distribution of anti-S IgG (A) and anti RBD IgG (B) in COVID-19 patients as compared to normal controls (NHS). Correlation of anti-S IgG and anti-RBD IgG in COVID-19 patients (C). p < 0.05 was considered significant.
Figure 6
Figure 6
Anti-RBM436-507 Ig isotypes in COVID-19 patients. Distribution of anti RBM IgG (A), IgM (B) and IgA (C) in COVID-19 patients is shown compared to normal controls (NHS). Correlation of anti-RBM IgG with anti-Spike (D) or anti-RBD (E) IgG in COVID-19 patients (D). Distribution of anti-RBM antibody isotypes (F). p < 0.05 was considered significant.
Figure 7
Figure 7
Neutralizing ability of antibodies in mice. Neutralizing ability of anti-S (A), anti-RBD (B) and anti-RBM (C) antibodies from immunized mice. Results are shown as the percentage of inhibition of specific antibodies at different days (0, 40, and 115) post-immunization.
Figure 8
Figure 8
Fine specificity of anti-RBM436-507 antibodies in COVID-19 patients. Reactivity of anti-RBM positive COVID-19 sera with 20-mers overlapping peptides (P11-P16) covering the entire RBM436-507 sequence. Results are shown as percentage of anti-RBM positive sera reacting with the specific peptide.
Figure 9
Figure 9
Neutralizing ability of antibodies in Covid-19 patients. Neutralizing ability of antigen eluted anti-S, anti-RBD and anti-RBM antibodies in COVID-19 patients. Results are shown as the percentage of inhibition of ACE-HRP binding to RBD.
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