Recently, the World Health Organization (WHO) has declared the novel coronavirus (2019-nCoV) outbreak a Public Health Emergency of International Concern (PHEIC),1 which is now formally named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).2 As of 27 February 2020, a total of 82,178 cases of SARS-CoV-2 infection have been confirmed across the world, with 78,630 cases in China (https://ncov.dxy.cn/ncovh5/view/pneumonia?source=). The SARS-CoV-2 has been determined as the seventh member of the coronaviruses infected humans.3 Moreover, similar to severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), the SARS-CoV-2 could also cause severe and fatal illness.3 Since the SARS-CoV-2 outbreak, there have been approximately 14,792 clinically severe cases and 2800 dead cases.
Due to the fast spread of SARS-CoV-2 and shortage of specific therapy, many efforts have focused on neutralizing antibody and vaccine development.4 Vaccines prevent disease largely depending on inducing neutralizing antibodies against vulnerable epitopes on antigen. Among the structural proteins of coronavirus, the spike glycoprotein contains receptor-binding domain (RBD) to mediate coronavirus entering host cells, which makes spike protein the primary antigenic target of neutralizing antibody and vaccine.5
Recently, it has been reported that the genome of SARS-CoV-2 have 79.5% nucleotide sequence identify to that of SARS-CoV.6 The genome relatedness indicates the possibilities that pre-clinical drugs against SARS-CoV might be effective to SARS-CoV-2. Also, a recent study was focused on cross-protective epitope between the spike proteins of SARS-CoV-2 and SARS-CoV, and successfully found the cross-protective epitopes in the RBDs of the spike proteins.7 Moreover, another study found that the spike RBD of SARS-CoV-2 bound potently to angiotensin-converting enzyme 2 (ACE2), the host cell receptor of SARS-CoV.5 However, in spite of the same binding target to ACE2, three of four monoclonal antibodies capable of binding potently to the SRAS-CoV RBD failed to show evident binding to the SARS-CoV-2 RBD.5 The limited antibody cross-reactivity suggests the importance to investigate the difference of antibody epitopes between the spike proteins of SARS-CoV and SARS-CoV-2.
In our study, we found the SARS-CoV-2 spike protein had approximately 24.5% amino acid (a.a.) sequence non-conserved to that of SARS-CoV (Supplementary Fig. 1). Because of the divergence of spike proteins, the non-conserved regions of spike proteins might have the main responsibility for the antigenic difference. Thus, to solve the problem, we conducted antibody epitope analysis that focused on the comparison of the conserved and non-conserved regions of spike glycoproteins between MERS-CoV, SRAS-CoV, and SARS-CoV-2.
The spike proteins of SARS-CoV-2 from Wuhan, Zhejiang, and Guandong in China and other countries of the United States, France, Australia, and Germany were nearly 100% conserved (Supplementary Fig. 2). Next, alignment and phylogenetic analysis of the amino acid sequences of spike proteins in SARS-CoV-2, MERS-CoV, and SARS-CoVs showed the difference of sequence conservancy (Fig. 1a and Supplementary Fig. 1). As spike proteins of those five SARS-CoVs had approximately 99.5% homologous a.a. sequence (Supplementary Fig. 2), we used SARS-NS1 as a representative SARS-CoV for further analysis.
Currently, bioinformatic approaches of epitope analysis are well-developed and successfully proved to identify both weak and strong epitopes that might be experimentally ignored.8 In our study, using antibody epitope bioinformatic tools (Supplementary Materials and Methods), we computed sequence-based antibody epitope scores in spike proteins of MERS-CoV, SARS-CoV, and SARS-CoV-2 (Fig. 1b). The SARS-CoV-2 had significantly lower antibody epitope score compared with MERS-CoV (p < 0.0001; Fig. 1c) and significantly higher antibody epitope score compared with SARS-CoV (p < 0.01; Fig. 1c), indicating the spike proteins have significantly variable antigenicity. Next, we conducted sequence alignment to acquire the conserved and non-conserved regions of spike proteins (Fig. 1d and Supplementary Fig. 1). Compared with the conserved regions, the non-conserved regions had significantly higher antibody epitope score (Fig. 1e, f), indicating the non-conserved regions of spike proteins are more antigenic.
As the surface accessibility of epitope is also important for the interaction of antibody and antigen, we evaluated the surface epitope accessibility of spike proteins (Fig. 1g), no significant difference was observed in the total protein level (Fig. 1h). However, non-conserved regions showed significantly higher surface epitope accessibility score (Fig. 1i, j), indicating the non-conserved regions of spike proteins are more available for antibody recognition.
Furthermore, we identified the antibody epitopes considering both the antibody epitope and surface epitope accessibility scores (Fig. 1k and Supplementary Materials and Methods). The antibody epitopes of spike proteins were compared between MERS-CoV, SARS-CoV, and SARS-CoV-2 (Fig. 1l), and the unique, shared, and public epitopes were identified (Fig. 1m). No public epitope could be found. Although five epitopes were shared between SARS-CoV and SARS-CoV-2, there were apparent dominances of unique epitopes in SRAS-CoV (83.9%) and SRAS-CoV-2 (85.3%) (Fig. 1n). Moreover, among these unique epitopes, 92.7% of them were derived from the non-conserved regions and the combinations of the conserved and non-conserved regions (Fig. 1o), indicating the divergence of spike proteins could lead to major changes in the antibody epitopes.
Next, according to the cryo-electron microscopy structure of the SARS-CoV spike protein complexed with human ACE2 protein (PDB accession: 6ACJ),9 we used Swiss-model bioinformatic tool10 to model the three-dimensional complex structure of the SARS-CoV-2 spike protein binding to its host cell receptor ACE2 (Fig. 1p). We discovered that the SARS-CoV-2 spike RBD was in the interaction interface with ACE2 (Fig. 1p). In the RBD of SARS-CoV-2 spike protein, we found seven epitopes and only one of them was from the conserved region homologous to SARS-CoV, yet the rest are novel epitopes using the combinations of conserved and non-conserved regions (Fig. 1q). Furthermore, we identified the high-score epitopes with both high epitope and surface accessibility score (Fig. 1r and Supplementary Fig. 3). Finally, we found 11 high-score epitopes for SARS-CoV-2 and only 1 of them was from the conserved region, but located outside RBD; nevertheless, we identified two novel high-score epitopes located in RBD (Fig. 1s), which might be used to block the spike-ACE2 interaction to inhibit the SARS-CoV-2 infection.
In summary, our study showed that, although SARS-CoV-2 spike protein displayed high (75.5%) homology toward that of SARS-CoV, the novel epitopes contributed to 85.3% of all the antibody epitopes, 85.7% of the RBD antibody epitopes, and 90.9% of the high-score antibody epitopes in SARS-CoV-2, implying remarkable alterations in the antigenicity. Notably, these results might explain why the most of the antibodies against SRAS-CoV spike protein were invalidated for SARS-CoV-2 in the previous study5 and indicate the necessity to develop new antibodies and vaccines specific for SARS-CoV-2. Importantly, we discovered novel and high-score antibody epitopes for SARS-CoV-2 spike protein and analyzed their RBD locations, which should be potent and specific targets for developing antibody drugs and vaccines of SARS-CoV-2 in the future. Taken together, our study found that the antigenicity of SARS-CoV-2 spike protein is remarkably dominated and altered by novel antibody epitopes, which provides promising leads for the research and development of vaccine for SARS-CoV-2.
Supplementary information
Acknowledgements
This project is supported by the National Natural Science Foundation of China (numbers 31570758, 91743115, and 31270797) to L.S.
Author contributions
M.Z. conceived the project, developed the method, conducted data analysis, and wrote the manuscript. L.S. supervised the study and wrote the manuscript with M.Z.
Competing interests
The authors declare no competing interests.
Contributor Information
Ming Zheng, Email: mmzheng@fmmu.edu.cn.
Lun Song, Email: lunsong0752@163.com.
Supplementary information
The online version of this article (10.1038/s41423-020-0385-z) contains supplementary material.
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