Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies

  1. Lin Li
  2. Zhongpeng Zhao
  3. Xiaolan Yang
  4. Wendong Li
  5. Shaolong Chen
  6. Ting Sun
  7. Lu Wang
  8. Yufei He
  9. Guang Liu
  10. Xiaohan Han
  11. Hao Wen
  12. Yong Liu
  13. Yifan Chen
  14. Haoyu Wang
  15. Jing Li
  16. Zhongyi Su
  17. Chen Du
  18. Yiting Wang
  19. Xinyang Li
  20. Zeqian Yang
  21. Jie Wang
  22. Min Li
  23. Tiecheng Wang
  24. Ying Wang
  25. Yubo Fan
  26. Hui Wang
  27. Jing Zhang

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Abstract

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.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.13.422550: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Characterization of the 18 selected linear B-cell epitopes: The target profiles of IgG or IgA antibodies from 232 COVID-19 patient sera (101 for hospitalized, 131 for non-hospitalized) and 190 pre-COVID-19 era controls were generated in duplicates by a recent research53, using coronavirus 20-mer libraries which included 20-mer peptides tiling every 5 amino acids across the SARS-CoV-2 proteome.
    IgA
    suggested: None
    The 4-6 year old, healthy brown horses (300-350kg in weight) that had no detectable antibodies against SARS-CoV-2, were provided by Chifeng Boen Pharmacy Co., LTD (InnerMongolia, China) The Balb/C mice and monkeys were intramuscularly (i.m.) immunized with the RBD-IgG1 Fc subunit vaccine candidate (10μg per mouse, 40μg per monkey), or Al(OH)3 adjuvant as a control.
    SARS-CoV-2
    suggested: None
    The complexes of antibody-virus (100TCID50/50μl) were added to pre-plated Vero cell monolayers in 96-well plates and incubated for 72 hours.
    antibody-virus (100TCID50/50μl)
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and viruses: Vero cells (ATCC,CCL-81) and 293T cells (ATCC,CRL-11268) were maintained in Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10% fetal bovine serum (Hyclone, USA), penicillin (Hyclone, USA, 100 units/mL) and streptomycin (Hyclone, USA, 100 μg/mL) (complete medium) in 5% CO2 environment at 37 °C and passaged every 2-3 days.
    293T
    suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
    The complexes of antibody-virus (100TCID50/50μl) were added to pre-plated Vero cell monolayers in 96-well plates and incubated for 72 hours.
    Vero
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Specific pathogen-free female BALB/c mice aged 6–8 weeks were obtained from Beijing Vital River Laboratory Animal Technologies Co., Ltd (Beijing, China) and were housed and bred in the temperature-, humidity and light cycle-controlled animal facility (20 ± 2 °C; 50 ± 10%; light, 7:00–19:00; dark,19:00–7:00).
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    Software and Algorithms
    SentencesResources
    Data retrieval and structural analysis: SARS-CoV-2 protein sequence (Accession number MN908947.3)62 was extracted from the NCBI database.
    NCBI
    suggested: (NCBI, RRID:SCR_006472)
    The transmembrane topology of S protein was examined by TMHMM.v2.0 (http://www.cbs.dtu.dk/services/TMHMM/).
    http://www.cbs.dtu.dk/services/TMHMM/
    suggested: (TMHMM Server, RRID:SCR_014935)
    VaxiJen v2.052 was used to estimate the antigenicity of full-length SARS-CoV-2 S protein.
    VaxiJen
    suggested: (VaxiJen, RRID:SCR_018514)
    The locations of the selected linear B-cell epitopes on the 3D structure of SARS-CoV-2 S protein or the interacting conformation of S protein RBD and human ACE254–56 were examined through open-source Pymol.
    Pymol
    suggested: (PyMOL, RRID:SCR_000305)
    Conservation analysis of selected B-cell epitopes: The conservation status for each residue of SARS-CoV-2 were investigated by ConSurf67 using the amino acid sequences of S protein from seven known coronaviruses including 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).
    SARS-CoV-2
    suggested: (Active Motif Cat# 91351, RRID:AB_2847848)
    The half maximal effective concentration (EC50) was calculated for the tested samples using the Reed-Muench method in GraphPad Prism 8 (GraphPad software, Inc., San Diego, CA, USA).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 21. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1101/2020.12.13.422550 on bioRxiv