key: cord-1046385-slka71eg
authors: Li, Lin; Zhao, Zhongpeng; Yang, Xiaolan; Li, Wendong; Chen, Shaolong; Sun, Ting; Wang, Lu; He, Yufei; Liu, Guang; Han, Xiaohan; Wen, Hao; Liu, Yong; Chen, Yifan; Wang, Haoyu; Li, Jing; Su, Zhongyi; Du, Chen; Wang, Yiting; Li, Xinyang; Yang, Zeqian; Wang, Jie; Li, Min; Wang, Tiecheng; Wang, Ying; Fan, Yubo; Wang, Hui; Zhang, Jing
title: Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies
date: 2020-12-13
journal: bioRxiv
DOI: 10.1101/2020.12.13.422550
sha: eccbedb1b7a57db233eed45f129bcebea519b1d9
doc_id: 1046385
cord_uid: slka71eg

SARS-CoV-2 unprecedentedly threatens the public health at worldwide level. There is an urgent need to develop an effective vaccine within a highly accelerated time. Here, we present the most comprehensive S-protein-based linear B-cell epitope candidate list by combining epitopes predicted by eight widely-used immune-informatics methods with the epitopes curated from literature published between Feb 6, 2020 and July 10, 2020. We find four top prioritized linear B-cell epitopes in the hotspot regions of S protein can specifically bind with serum antibodies from horse, mouse, and monkey inoculated with different SARS-CoV-2 vaccine candidates or a patient recovering from COVID-19. The four linear B-cell epitopes can induce neutralizing antibodies against both pseudo and live SARS-CoV-2 virus in immunized wild-type BALB/c mice. This study suggests that the four linear B-cell epitopes are potentially important candidates for serological assay or vaccine development.

The new coronavirus SARS-CoV-2 with documented person-to-person transmission has caused millions of confirmed cases and poses unprecedented threat to human lives [1] [2] [3] . The clinical features associated with SARS-CoV-2 infection include non-symptomatic infection, mild flu-like symptoms to pneumonia, severe acute respiratory distress syndrome or even deaths 3, 4 . It has been declared as a pandemic by World Health Organization (WHO) on 11 th March, 2020. To date, no effective treatment is available yet, even though great efforts are being put into the discovery of antiviral drugs. There is an urgent need to understand and develop vaccines against SARS-CoV-2.

At present, more than one hundred of vaccine development projects being carried out globally ranged from viral vector-based vaccines, mRNA and DNA vaccines, subunit vaccines, nano-particle-based vaccines, to inactivated-whole virus vaccines [5] [6] [7] [8] . Some vaccine candidates such as inactivated vaccine can inhibit virus replication and protect against upper respiratory tract disease, and other vaccine candidates such as Ad5-nCoV encoding the full spike of SARS-CoV-2 demonstrate good characteristics in both safety and immunogenicity [5] [6] [7] [8] [9] . However, some mild and self-limiting adverse reactions are still observed in some clinical trials 10 . It is necessary to carefully evaluate the effects and side-effects of these vaccine candidates, and meanwhile to have ongoing new vaccine development.

SARS-CoV-2 fuses and enters into the host cells through the spike (S) protein binding to the angiotensinconverting enzyme 2 (ACE2) receptor 11 . It would be efficient to prevent virus entry by blocking the binding of S protein to ACE2 [12] [13] [14] . The S protein consists of S1-subunit and the membrane fusion S2-subunit. The significant role of the S1 subunit receptor-binding domain (RBD) in the entry of virus to the host cell makes it wildly-investigated target for developing therapeutic antibodies and vaccines [15] [16] [17] . Other area of S protein may also elicit neutralizing antibodies 18, 19 .

Various immune-informatics methods have been used to predict large quantities of linear B-cell epitopes with high immunogenicity , but the selection of the in-silicon methods is mostly based on the researchers' experiences and preferences. Due to lack of validations from biological experiments, it is difficult to focus on a limited number of candidates for diagnostics and linear B-cell epitope-based vaccine development.

In this study, we presented a comprehensive S-protein-based linear B-cell epitope candidate list by combining epitopes predicted by eight widely-used immune-informatics methods with the epitopes curated from literature published between Feb 6, 2020 and July 10, 2020. The antigenicity, toxicity, stability and physiochemical properties were explored for all the identified linear B-cell epitopes. Based on integrative information from epitopes properties and 3D structure of S protein, the top prioritized linear B-cell epitopes were investigated for their binding affinity with serum antibodies from horse, mouse, and monkey inoculated with different SARS-CoV-2 vaccine candidates (S1-based vaccine for horse, RBD-based vaccines for mouse and monkey) and a patient recovering from COVID-19. The induction of neutralizing antibodies against both pseudo and live SARS-CoV-2 virus has been examined following immunizations in wild-type BALB/c mice. Four linear B-cell epitopes found able to elicit neutralizing antibodies are potentially important candidates for serological assay or vaccine development.

Spike protein is an important target for vaccine development due to its indispensable function in helping SARS-CoV-2 gain entry into host cells. B-cells can be guided through linear B-cell epitopes to recognize and activate defense responses against viral infection. To construct a comprehensive linear B-cell epitope candidate list, we first performed in-silicon prediction of B-cell epitopes from S protein through eight methods, obtaining a total of 4044 linear B-cell epitopes (256 for Bepipred and Bepipred2.0 with default parameter settings, Kolaskar and Tongaonkar antigenicity, Parker hydrophilicity, Chou and Fasman beta-turn, and Karplus and Schulz flexibility provided by IEDB (Immune-Epitope-Database And Analysis-Resource) 44 ; 128 for BcePred 45 using accessibility, antigenic propensity, exposed surface, flexibility, hydrophilicity, polarity, and turns; 3007 for the ANNpred-based server ABCpred 46 ; 44 for Ellipro 47 ; 176 for BCPREDS 48 ; 191 for AAP 49 ; 215 for FBCPRED 50 ; and 27 for COVIDep 51 ). We additionally extracted 279 linear B-cell epitopes from 24 articles or preprints published between Feb 6, 2020 and July 10, 2020. We finally established a full list of 3836 unique linear B-cell epitope candidates by combining predictions by the eight methods and those curated from literature (Supplementary Table 1) .

To obtain linear B-cell epitopes with high potential to initiate a defensive immune reaction, we adopted a series of high stringent criteria to filter out epitopes with low antigenicity. 614 linear B-cell epitopes were estimated by VaxiJen 2.0 52 to have high antigenicity scores (larger than 0.9 viewed adequate to initiate a defensive immune reaction) (Fig. 1A) with length varying from 6 amino acids to 30 amino acids (Fig. 1B) (Supplementary Table 1 ). The positions of the 614 linear B-cell epitopes on the S protein amino acid sequence were examined to exclude the epitopes locating on the non-outer surface based on the transmembrane topology of SARS-CoV-2 S protein predicted by TMHMM v2.0 (outside: were identified as hotspot regions containing highly antigenic linear B-cell epitopes identified by multiple methods (Fig. 1C) . Based on the conservation status of each residue of the S protein predicted by ConSurf using seven known coronaviruses incorporating SARS-CoV-2 (YP_009724390.1), SARS-CoV (NP_828851.1), MERS-CoV (YP_009047204.1), alpha coronavirus 229E (NP_073551.1), alpha coronavirus NL63 (AFV53148.1), beta coronavirus OC43 (YP_009555241.1) and beta coronavirus HKU1 (AAT98580.1), we discovered that RBD region (319-514) of the S protein was not conserved among the seven coronaviruses, consistent with the observation that more mutations were found in the RBD region by performing sequence alignment of S protein against 118,694 (20200927) sequences of SARS-CoV-2 in the NGDC database (Fig.   1C ). The linear B-cell epitopes from the 14 hotspot regions were mapped to the 3D structure of the SARS-CoV-2 S protein (PDB ID:6VSB), suggesting that hotspot regions locating on the exposed area of spike stem or spike head would harbor good B-cell epitope candidates ( Fig. 1D and E) .

From the hotspot regions of the exposed area of S protein, we selected 18 linear B-cell epitopes with few mutations reported in less than ten out of a total of 118,694 SARS-CoV-2 virus strains as the potentially optimal linear B-cell epitope candidates ( Fig. 2A) . As IgG and IgA are the most abundant isotypes in blood 53 , we examined the recently available target profiles (20-mer peptides tiling every 5 amino acids across the SARS-CoV-2 proteome) of IgG and IgA antibodies from 232 COVID-19 patient sera (101 for hospitalized, 131 for non-hospitalized) and 190 pre-COVID-19 era controls 53 . We discovered that the 20-mer peptides in the target profiles with more than 75% overlapping with the 18 linear B-cell epitopes exhibited non-negligible enrichment score in some hospitalized or non-hospitalized COVID-19 patient IgG, or IgA samples, suggesting 

Animal models are necessary to demonstrate efficacy and safety in the development of vaccines against SARS-CoV-2 infection 58, 59 . BALB/c mice is a good animal model for investigating SARS-CoV-2 infection in both upper and lower respiratory tracts 5 . Monkey, phylogenetically close to humans, has been used to test whether seroconversion provides protective immunity against SARS-CoV-2 58 . To assess the binding of the 18 linear Bcell epitopes with serum IgG antibodies against SARS-CoV-2, we immunized three model animals including horse, mouse, and monkey. Indirect enzyme-linked immunosorbent assay (ELISA) was performed between serum antibodies (including animals inoculated with different SARS-CoV-2 vaccine candidates and normal control animal models), and the 18 linear B-cell epitopes and arbitrary control peptides ('RRRRRRRRRRRRRRRR' and 'RRRRRRR'). We discovered that four linear B-cell epitopes reacted specifically and dose dependently with serum antibodies from the vaccinated horse ('YNSASFSTFKCYGVSPTKLNDLCFT', and three highly overlapped linear B-cell epitopes including 'GDEVRQIAPGQTGKIADYNYKLP', 'DEVRQIAPGQTGKIADYNYKLPDDFT' and 'APGQTGKIADYNYKLPDDFT' for horse (Fig. 3A) ; 'YQPYRVVVLSFELLH' and 'CVNFNFNGL' for mouse (Fig. 3B) ; 'CVNFNFNGL' and 'GDEVRQIAPGQTGKIADYNYKLP' for monkey (Fig. 3C) ), whereas the arbitrary control peptides had no effects. The normal sera from horse, mouse, monkey and human were not reactive. In addition, sera from a patient recovering from COVID-19 and the healthy persons were provided by Dr. Bin Su at Beijing Youan Hospital. Indirect ELISA results demonstrated that 'CVNFNFNGL' specifically bond to serum IgG antibodies in the patient recovering from COVID-19, but not in healthy human (Fig. 3D) .

These results suggest that the four linear B-cell epitopes ('YNSASFSTFKCYGVSPTKLNDLCFT', 'GDEVRQIAPGQTGKIADYNYKLP', 'YQPYRVVVLSFELLH', and 'CVNFNFNGL') may be able to induce antibodies against SARS-CoV-2.

To confirm that the four linear B-cell epitopes generated antibodies against SARS-CoV-2, we immunized six- In SARS-CoV and MERS-CoV 60 , pseudovirus neutralization assay is a sensitive and quantitative method. We therefore tested the concentration of neutralizing antibodies in immune sera from wild type mice 7 days after the fifth vaccination against SARS-CoV-2 pseudovirus using a pseudotyped virus-based neutralization assay developed recently for SARS-CoV-2 61 . 3F) , whereas no neutralizing antibodies were found in the two control group.

We also performed neutralization assay against SARS-CoV-2 live virus (BetaCoV/Beijing/IMEBJ01/2020) using immune sera from vaccinated mice 7 days after the fifth vaccination. We found the strongest neutralization activity with mean NT50 of 78.2 for 'YNSASFSTFKCYGVSPTKLNDLCFT';

'YQPYRVVVLSFELLH' also showed strong neutralization activity (mean NT50 = 47.4); we observed the neutralization activities for 'GDEVRQIAPGQTGKIADYNYKLP' (mean NT50 = 18.6) and 'CVNFNFNGL'

(mean NT50 = 15.2) (Fig. 3G) . Overall, these results demonstrated that the four linear B-cell epitopes can induce neutralizing antibodies against SARS-CoV-2. In this study, we predicted linear B-cell epitopes from S protein by eight widely-used immune-informatics methods including Bepipred and Bepipred2.0 with default parameter settings, Kolaskar and Tongaonkar antigenicity, Parker hydrophilicity, Chou and Fasman beta-turn, and Karplus and Schulz flexibility provided by IEDB (Immune-Epitope-Database And Analysis-Resource) 44 , BcePred 45 using accessibility, antigenic propensity, exposed surface, flexibility, hydrophilicity, polarity, and turns, ANNpred-based server ABCpred 46 published between Feb 6, 2020 and July 10, 2020. Interestingly, the linear B-cell epitopes predicted by different methods converged to some hotspot regions in the S protein, suggesting the pivotal role of these regions in diagnostics assay and vaccine development. Integrating antigenicity, toxicity, stability and physiochemical properties, 3D structure of S protein, the 3D conformation structure between RBD of S protein and ACE2, we selected 18 top prioritized linear B-cell epitopes for further investigation. Four out of 18 linear B-cell epitope-based synthetic peptides were found to specifically bind with serum antibodies from horse, mouse, and monkey inoculated with different SARS-CoV-2 vaccine candidates or a patient recovering from COVID-19. The serum antibodies we used here were generated by different regions of SARS-CoV-2 spike protein (S1-based vaccine for horse, RBD-based vaccines for mouse and monkey, unknown regions for a patient recovering from COVID-19), leading to the observation that the four peptides didn't consistently show specific binding with all antibodies produced across horse, mouse, monkey and a patient recovering from COVID-19. However, the four peptides were able to elicit neutralizing antibodies in immunized wild-type BALB/c mice against both pseudo and live SARS-CoV-2 virus.

To the best of our knowledge, the linear B-cell epitope candidate list we presented is one of the most comprehensive and valuable source for developing vaccines. Importantly, the four linear B-cell epitopes we identified able to elicit neutralizing antibodies against SARS-CoV-2 are promising and valuable candidates which have immediate usefulness for developing vaccines against SARS-CoV-2. Besides, more linear B-cell epitopes in the candidate list may also deserve being examined. The analysis workflow we adopted can be broadly applied to identify B-cell epitopes for a large repertoire of virus, not limited to coronavirus. In short, the results we presented here will be useful to guide the identification and prioritization of linear B-cell epitope-based diagnostics and vaccine designs during this unprecedented pandemic.

SARS-CoV-2 protein sequence (Accession number MN908947.3) 62 was extracted from the NCBI database.

Experimentally solved 3D structure of SARS-CoV-2 S protein (PDB ID: 6VSB) 11 And Analysis-Resource) 44 were applied upon SARS-CoV-2 S protein sequence to predict linear B-cell epitopes. Linear B-cell epitopes were also predicted by BcePred 45 using accessibility, antigenic propensity, were curated to obtain a total of 214 B-cell epitopes. All the linear B-cell epitopes including those predicted by the eight methods and those curated from literature were combined to the linear B-cell epitope candidate list.

According to the transmembrane topology of SARS-CoV-2 S protein predicted by TMHMM v2.0, the linear B-cell epitopes on the outer surface were retained for downstream analysis with intracellular epitopes eliminated. The linear B-cell epitopes consisting of less than six amino acids or more than 50 amino acids were further removed. The antigenicity of the remained linear B-cell epitopes was evaluated by VaxiJen 2.0 52 . A stringent criterion was employed to have linear B-cell epitopes with an antigenicity score larger than 0.9 viewed adequate to initiate a defensive immune reaction.

The 

The conservation status for each residue of SARS-CoV-2 were investigated by ConSurf 67 Blood samples were collected and used for ELISA and neutralization assays. 

A pseudotyped virus-based neutralization assay against SARS-CoV-2 in biosafety level 2 facilities was performed as previously described 61 

Microneutralization (MN) assay was performed to assess the neutralizing activity of sera from the mice. 50µl

(100 CCID 50 /0.05ml) of SARS-CoV-2 IME-BJ01 strain was incubated with serial dilution of heat-inactivated sera in 5% CO 2 environment at 37 o C for one hour. The complexes of antibody-virus (100TCID50/50µl) were added to pre-plated Vero cell monolayers in 96-well plates and incubated for 72 hours. The Reed-Muench method was applied to estimate the dilution of sera required for NT 50 . The initial dilution of sera (1:16) was set as the confidence limit of the assay. Seropositivity was defined as a titre ≥ 16. . e-f, The localizations of B cell discontinuous epitopes on SARS-CoV-2 S (PDB: 6VSB) protein (e) and ACE-RBD complex (f). The spike protein is grey, the RBD region is wheat color, the selected epitopes are green, the mutation sites are red, the human ACE domain is blue, and the discontinuous B-cell epitopes are purple. 

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We thank Dr. Weijin Huang at Institute for Biological Product Control, National Institutes for Food and Drug 

The authors declare no competing interests.

L.L., Z.P.Z, and X.L.Y. contribute equally to this work.Correspondence should be addressed to Y.W., Y.B.F., H.W., or J.Z.