key: cord-1013859-lfp9o017
authors: Qi, Huan; Ma, Ming-liang; Jiang, He-wei; Ling, Jian-ya; Chen, Ling-yun; Zhang, Hai-nan; Lai, Dan-yun; Li, Yang; Guo, Zi-wen; Hu, Chuan-sheng; Guo, Shu-juan; Meng, Qing-feng; Ren, Yan; Yang, Xiao; Wang, Wei; Zhou, Jie; Zhao, Xiao-dong; Li, Hua; Tao, Sheng-ce
title: Systematic profiling of SARS-CoV-2-specific IgG epitopes at amino acid resolution
date: 2021-03-04
journal: Cell Mol Immunol
DOI: 10.1038/s41423-021-00654-3
sha: 2b4724d5032fba707348dac4c49df1a29116562b
doc_id: 1013859
cord_uid: lfp9o017

nan

S protein. These results are highly consistent with a peptide microarray-based study, in which~4000 samples were analyzed against a set of 197 peptides that covers the entire S protein. 6, 7 In addition, we took the S protein as an example to demonstrate the necessity of enriching SARS-CoV-2-specific antibodies from sera ( Fig. S3 ). We defined epitopes with a frequency >=3 as significant epitopes and obtained 28 epitopes (Fig. S4A) ; we mapped them to the 3D structure of the S protein 8 monomer (Fig. 1B) and trimer (Fig. S4B ). It is clear that most of these epitopes are located on the surface of the S protein monomer and trimer (Fig. 1C , D and Table S4 ). High homology was observed among SARS-CoV-2, SARS-CoV and BtCoV-RaTG13, especially between SARS-CoV-2 and SARS-CoV ( Fig. 1E and Table S4) .

Biotinylated RBD was also applied to enrich specific antibodies from COVID-19 convalescent sera (Fig. S1 ). One relatively significant epitope was identified (Fig. S5A) . To visualize the conformational location of this epitope, we mapped the critical epitope residues (yellow) to the 3D structure of the RBD (Fig. S5B ). It is clear that the critical epitope residues are not on the exact binding interface of RBD and ACE2 but adjacent to it (Fig. S5C) , which was similar to the results reported for several well-studied antibodies ( Fig. S5D-J) . Antibodies targeting this epitope may cause conformational changes or spatial hindrance, thus interfering with the binding of ACE2 and these neutralizing antibodies to the RBD.

To explore the roles of the critical epitope residues, we matched the mutation sites with the epitope map of the S protein ( Fig. 1A ) and found that six mutation sites are also critical epitope residues ( Fig. S6A and Table S5 ), of which D936Y and P1263L could cause 60-70% and 98-99% infectivity loss, respectively. 9 These mutation sites were also of high frequency (Fig. S6B ). Mutations with high frequency, i.e., D614G, 10 were not identified as critical epitope residues. Furthermore, most of the identified critical residues have a low frequency of mutation. Overall, it is reasonable to argue that these critical residues, especially D936 and P1263, may be useful for vaccine development.

To present the distribution of the epitopes on the N protein, we also matched the epitopes to the N protein sequence. The epitopes were distributed across but not evenly on the N protein.

One area rich in significant epitopes was readily identified (Fig. S7A) , and 6 significant epitopes were identified when the Fig. 1 Significant epitopes on the S protein identified by AbMap and validated by peptide microarray. A The distribution of the sequencematched epitopes on the S protein. B The distribution of the significant epitopes (frequency ≥ 3) on the 3D structure of the S protein monomer. Red amino acids represent key residues of epitopes. C A representative significant epitope. D The epitope was matched to the S protein, and critical residues were labeled yellow. E Homology analysis of the epitope among coronaviruses. F Validation of epitope identification procedures by a peptide microarray: comparison of sera and protein-enriched antibodies. Peptide 1, which corresponds to Epitope-S1-8 (KLFPFQQF), was selected as an example. The ratio of signal intensity (S1 enriched)/signal intensity (serum) for each residue was plotted (right) Systematic profiling of SARS-CoV-2-specific IgG epitopes at amino acid. . . H Qi et al. cutoff was set as frequency >=3 (Fig. S7B ). High homology for the epitopes and the critical epitope residues was observed among SARS-CoV-2, SARS-CoV and BtCoV-RaTG13 for all 6 significant epitopes (Fig. S7C) .

To validate the key residues of the significant epitopes, we selected four epitopes, i.e., Epitope-S1-8 and Epitope-S1-12, which are of the highest frequency, Epitope-RBD-1 from the RBD, and an epitope of low frequency, Epitope-S2-15, as a control. We then mutated the amino acids of these epitopes to alanine one by one, synthesized the mutated peptides, conjugated them to BSA, and fabricated a peptide microarray (Fig. S8) . To validate the epitopes on the peptide microarray, we selected 5 sera, from which at least one of the significant epitopes among Epitope-S1-8, Epitope-S1-12 and Epitope-RBD-1 was identified by AbMap (Fig. S9A) . For all three epitopes, when the corresponding samples were tested, significant binding signal loss was observed when any of the critical residues was mutated to alanine (Figs. 1F and S9B ). In addition, the results in Figs. 1F and S10 further confirmed the necessity for the enrichment of antibodies from sera by specific proteins for epitope mapping.

The aim of this study was to promote our understanding of the SARS-CoV-2-specific IgG response in a more precise way. Currently, no traditional platforms can provide epitope information at the amino acid level. Taking advantage of AbMap, we constructed the first map of SARS-CoV-2-specific IgG binding epitopes at amino acid resolution. We identified several critical epitope residues on the S protein that are highly correlated with the infectivity of SARS-CoV-2 and the binding of ACE2/neutralizing antibodies to the S protein.

There are a variety of possible applications for the significant epitopes/critical residues identified in this study. For example, researchers can select one or a few epitopes based on neutralization activity to generate site-specific antibodies or vaccines on demand for S protein as well as other proteins. Since the COVID-19 pandemic is still unfolding and the sequenced genomes of SARS-CoV-2 are accumulating, undoubtedly, more functionally important amino acid sites will be identified, and we can match those sites to the identified critical epitope residues, thus linking mutation or SARS-CoV-2 evolution with immune responses.

Taken together, these results facilitate the in-depth understanding of SARS-CoV-2-specific IgG responses and provide a basis for the precise development of diagnostic reagents, therapeutic antibodies and even vaccines.

A new coronavirus associated with human respiratory disease in China

SARS-CoV-2 proteome microarray for global profiling of COVID-19 specific IgG and IgM responses

SARS-CoV-2 proteome microarray for mapping COVID-19 antibody interactions at amino acid resolution

ReScan, a multiplex diagnostic pipeline, pans human sera for SARS-CoV-2 antigens

Antibody binding epitope Mapping (AbMap) of hundred antibodies in a single run

Linear epitopes of SARS-CoV-2 spike protein elicit neutralizing antibodies in COVID-19 patients

Systematic evaluation of IgG responses to SARS-CoV-2 spike proteinderived peptides for monitoring COVID-19 patients

Characterization of the SARS-CoV-2 S protein: biophysical, biochemical, structural, and antigenic analysis

The impact of mutations in SARS-CoV-2 spike on viral infectivity and antigenicity

SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41423-021-00654-3.Competing interests: The authors declare no conflicts of interest.