Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jan 5:13:1010105.
doi: 10.3389/fimmu.2022.1010105. eCollection 2022.

Immunodominant antibody responses directed to SARS-CoV-2 hotspot mutation sites and risk of immune escape

Jamille Ramos Oliveira  1   2   3 , Cesar Manuel Remuzgo Ruiz  2 , Rafael Rahal Guaragna Machado  4 , Jhosiene Yukari Magawa  1   2   3 , Isabela Pazotti Daher  1   2   3 , Alysson Henrique Urbanski  5 , Gabriela Justamante Händel Schmitz  1   2 , Helen Andrade Arcuri  6 , Marcelo Alves Ferreira  7 , Greyce Luri Sasahara  2 , Giuliana Xavier de Medeiros  1 , Roberto Carlos Vieira Silva Júnior  2 , Edison Luiz Durigon  4   8 , Silvia Beatriz Boscardin  9 , Daniela Santoro Rosa  2   10 , Deborah Schechtman  11 , Helder I Nakaya  8   12 , Edecio Cunha-Neto  1   2   3 , Gabriele Gadermaier  13 , Jorge Kalil  1   2   3 , Verônica Coelho  1   2   3 , Keity Souza Santos  1   2   3
Affiliations

Immunodominant antibody responses directed to SARS-CoV-2 hotspot mutation sites and risk of immune escape

Jamille Ramos Oliveira et al. Front Immunol. .

Abstract

Introduction: Considering the likely need for the development of novel effective vaccines adapted to emerging relevant CoV-2 variants, the increasing knowledge of epitope recognition profile among convalescents and afterwards vaccinated with identification of immunodominant regions may provide important information.

Methods: We used an RBD peptide microarray to identify IgG and IgA binding regions in serum of 71 COVID-19 convalescents and 18 vaccinated individuals.

Results: We found a set of immunodominant RBD antibody epitopes, each recognized by more than 30% of the tested cohort, that differ among the two different groups and are within conserved regions among betacoronavirus. Of those, only one peptide, P44 (S415-429), recognized by 68% of convalescents, presented IgG and IgA antibody reactivity that positively correlated with nAb titers, suggesting that this is a relevant RBD region and a potential target of IgG/IgA neutralizing activity.

Discussion: This peptide is localized within the area of contact with ACE-2 and harbors the mutation hotspot site K417 present in gamma (K417T), beta (K417N), and omicron (K417N) variants of concern. The epitope profile of vaccinated individuals differed from convalescents, with a more diverse repertoire of immunodominant peptides, recognized by more than 30% of the cohort. Noteworthy, immunodominant regions of recognition by vaccinated coincide with mutation sites at Omicron BA.1, an important variant emerging after massive vaccination. Together, our data show that immune pressure induced by dominant antibody responses may favor hotspot mutation sites and the selection of variants capable of evading humoral response.

Keywords: RBD; betacoronavirus; immune pressure; linear antibody epitopes; peptide array; sarbecovirus.

PubMed Disclaimer

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
(A) Heatmap representing the magnitude of recognition of SARS-CoV-2 RBD peptides tested for IgG and IgA reactivity using a peptide microarray (columns: ordered by the primary structure sequence) for all convalescents (rows: grouped by antibody isotype and level of neutralizing activity tested in the same samples, and clustered using median values within each group). Highlighted is peptide P44 (S415-429) and its respective amino acid sequence. High: individuals displaying serum with high neutralizing activity (≥1:160), Low: individuals displaying serum with low neutralizing activity (<1:160). MFI: mean fluorescence intensity. (B) Number of peptides recognized per individual considering high and low VNT for IgG and IgA (C, D) Selected immunodominant peptides recognized by IgG (P12, P14, P18, P30, P44, P50 and P66) and IgA (P6, P12, P14, P18, P30, P44 and P66), of at least 30% of the individuals in the cohort (n=71) are represented with their total percentage of recognition and neutralization titers ranges. P6: peptide 6 (S353-367), P12: peptide 12 (S365-379), P14: peptide 14 (S370-384) and P18: peptide 18 (S378-392), P30: peptide 30 (S397-401), P44: peptide 44 (S415-429), P50: peptide 50 (S427-441) and P66: peptide 66 (S459-473).
Figure 2
Figure 2
Structural and conservation evaluation of topmost recognized peptides (A) Conservation analysis of the top recognized peptides among RBD proteins from coronaviruses of the B lineage, SARS-CoV-2, SARS-CoV, SARS-SZ3, Rs3367-bat, CoV-pangolin, and RaTG13-bat. The height of the letters in the logo plot reflects the frequency of the amino acid in the multiple sequence alignment. (B) Entropy of aminoacid residues, top peptides highlighted, of the alignment made at (A, C). RBD binding to the therapeutic neutralizing antibody B38 (pdb: 7BZ5) and with ACE-2 (D) interacting with P44, with contact amino acid residues in red. SARS-CoV-2 RBD (light gray). Colored regions show the most frequently recognized peptides identified among convalescents, namely, orange: P44: peptide 44 (S415-429), silver P6: peptide 6 (S353-367), golden P12, P14 and P18, peptide 12, 14, and 18 (S365-379, S370-384, S378-392), blue P30: peptide 30 (S397-401), violet P50, peptide 50 (S427-441) and green P66, peptide 66 (S459-473).
Figure 3
Figure 3
Differential intensity of reactivity to selected RBD peptides, comparing convalescents with high or low neutralizing capacity. (A) IgG profile. (B) IgA profile. High N: individuals with high neutralization capacity (≥1:160 virus neutralization titer); Low N: individuals with low neutralizing capacity (<1:160 virus neutralization titer); MFI: mean fluorescence intensity; the median is shown as a solid line, the box indicates the 25th and 75th percentiles, whiskers range from the highest to the lowest value (all data points shown). Mann Whitney *p<0.05; **p<0.001; ***p<0.0001.
Figure 4
Figure 4
Profile of P44 recognition. (A) In silico epitope prediction using BepiPred. Highlighted is the region corresponding to P44, showing a high epitope predictive value. (B) Positive correlation of P44 MFI and RBD IgG recognition in ELISA. (C) Positive correlation of IgG and IgA MFI specific for P44 and virus neutralization titers (Spearman correlation for IgG r= 0.4846, p<0.0001, 95% confidence interval 0.2769 - 0.6491, and for IgA r=0.3103, p=0.0084, 95% confidence interval 0.07602 - 0.5121). (D) Mean fluorescence intensity (MFI) of P44 antibody recognition of hospitalized compared to non-hospitalized individuals, showing higher IgA reactivity in hospitalized individuals (p=0.0092, Mann Whitney).
Figure 5
Figure 5
Structural localization of the eight most recognized peptides and monoclonal binding faces of RBD. (A) Linear RBD sequence showing the RBM region, highlighting ACE-2 contact residues. Top eight IgG and/or IgA recognized peptides (P6, P12, P14, P18, P30, P44, P50 and P66) are shown according to RBD residues. The pink heatmap represents the frequency at which nAbs contact each amino acid in RBD, according to a previous review (18). Mutation sites are shown in green in upper part. (B) Frequency of nAbs in contact with each amino acid in the RBD depicted in (A, C). Surface structure of different RBD views showing regions where neutralizing antibodies bind to (in pink), and three non-neutralizing faces, in gray. (D) Top recognized peptides are shown in colors and non-neutralizing faces are highlighted in gray according to B.
Figure 6
Figure 6
RBD peptide recognition profile of vaccinated individuals. (A) Heatmap representing the magnitude of recognition of SARS-CoV-2 RBD peptides tested for IgG reactivity using a peptide microarray (columns: ordered by the primary structure sequence) for all individuals (rows: grouped by vaccine regimen and clustered using median values within each group). 2nd dose: individuals who received two doses of Coronavac. Booster: individuals who received two doses of Coronavac followed by one dose of MiRNA-273 (Comirnaty). (B) Selected immunodominant peptides recognized by IgG by at least 30% of the individuals in the vaccinated cohort (n=18), showing the total percentage of recognition and neutralization titer ranges. (C) List of top peptides among convalescents or vaccinated. (D) Conservation analysis of the top recognized peptides among RBD proteins from different VOCs (alfa, beta, gamma, delta and omicron BA.1) compared to Wuhan. The height of the letters in the logo plot reflects the frequency of the amino acid in the multiple sequence alignment. (E) Entropy of amino acid residues considering the alignment performed, as in D with top peptides highlighted.
Figure 7
Figure 7
Structural evaluation of topmost recognized peptides among vaccinated and analysis of occurrence at mutation sites. (A) RBD binding to the therapeutic neutralizing antibody COVA1-16 (pdb:7JMW) and with ACE-2 (B) interacting with P14/18, the most recognized region, with contact amino acid residues in black. SARS-CoV-2 RBD (light gray). Colored regions show the most frequently recognized peptides identified among vaccinated. (C) Timeline showing collection period of samples, vaccination and surge of VOCs in Brazil. (D) Linear RBD sequence showing mutation sites of Beta, Gamma, Delta and Omicron BA.1. Regions highlighted in yellow are the most recognized among vaccinated. Top thirteen from vaccinated (orange) and top seven (blue) IgG recognized peptides are shown according to RBD residues.
See this image and copyright information in PMC

References

    1. Tai W, He L, Zhang X, Pu J, Voronin D, Jiang S, et al. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol (2020) 17(6). doi: 10.1038/s41423-020-0400-4 - DOI - PMC - PubMed
    1. Huang Y, Yang C, Xu XF, Xu W, Liu SW. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. vol. 41. Acta Pharmacol Sin (2020). doi: 10.1038/s41401-020-0485-4 - DOI - PMC - PubMed
    1. Li Y, Ma ML, Lei Q, Wang F, Hong W, Lai DY, et al. Linear epitope landscape of the SARS-CoV-2 spike protein constructed from 1,051 COVID-19 patients. Cell Rep (2021) 34(13). doi: 10.1016/j.celrep.2021.108915 - DOI - PMC - PubMed
    1. Piccoli L, Park YJ, Tortorici MA, Czudnochowski N, Walls AC, Beltramello M, et al. Mapping neutralizing and immunodominant sites on the SARS-CoV-2 spike receptor-binding domain by structure-guided high-resolution serology. Cell (2020) 183(4). doi: 10.1016/j.cell.2020.09.037 - DOI - PMC - PubMed
    1. Gattinger P, Niespodziana K, Stiasny K, Sahanic S, Tulaeva I, Borochova K, et al. Neutralization of SARS-CoV-2 requires antibodies against conformational receptor-binding domain epitopes. Allergy: Eur J Allergy Clin Immunol (2022) 77(1). doi: 10.1111/all.15066 - DOI - PMC - PubMed

Publication types