SARS-CoV-2 variants of concern: spike protein mutational analysis and epitope for broad neutralization

  1. Dhiraj Mannar
  2. James W. Saville
  3. Zehua Sun
  4. Xing Zhu
  5. Michelle M. Marti
  6. Shanti S. Srivastava
  7. Alison M. Berezuk
  8. Steven Zhou
  9. Katharine S. Tuttle
  10. Michele D. Sobolewski
  11. Andrew Kim
  12. Benjamin R. Treat
  13. Priscila Mayrelle Da Silva Castanha
  14. Jana L. Jacobs
  15. Simon M. Barratt-Boyes
  16. John W. Mellors
  17. Dimiter S. Dimitrov
  18. Wei Li
  19. Sriram Subramaniam

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Abstract

Mutations in the spike glycoproteins of SARS-CoV-2 variants of concern have independently been shown to enhance aspects of spike protein fitness. Here, we describe an antibody fragment (V H ab6) that neutralizes all major variants including the recently emerged BA.1 and BA.2 Omicron subvariants, with a unique mode of binding revealed by cryo-EM studies. Further, we provide a comparative analysis of the mutational effects within previously emerged variant spikes and identify the structural role of mutations within the NTD and RBD in evading antibody neutralization. Our analysis shows that the highly mutated Gamma N-terminal domain exhibits considerable structural rearrangements, partially explaining its decreased neutralization by convalescent sera. Our results provide mechanistic insights into the structural, functional, and antigenic consequences of SARS-CoV-2 spike mutations and highlight a spike protein vulnerability that may be exploited to achieve broad protection against circulating variants.

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  1. Version published to 10.1038/s41467-022-32262-8
  2. ScreenIT

    SciScore for 10.1101/2021.12.17.473178: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Electron Microscopy Sample Preparation and Data Collection: For cryo-EM, S protein samples were prepared at 2.25 mg/mL, with and without the addition of ACE2 or antibody (1:1.25 S protein trimer:ACE2 molar ratio, 1:3 S protein trimer:antibody molar ratio).
    ACE2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Pseudovirus Neutralization Assay: Variant and D614G spike pseudotyped retroviral particles were produced in HEK293T cells as described previously34.
    HEK293T
    suggested: None
    The gating strategy employed along with results for un-transfected (negative control) Expi293 cells are available in figure S10B.
    Expi293
    suggested: RRID:CVCL_D615)
    The antibody-virus mixture was then incubated at 37°C in a 5% CO2 incubator for 1 hour and added to the Vero E6 cell seeded monolayers, in duplicate.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Recombinant DNA
    SentencesResources
    The HexaPro expression plasmid was a gift from Jason McLellan (Addgene plasmid #154754; http://n2t.net/addgene:154754; RRID:Addgene_154754) which we used to construct the D614G mutant via site directed mutagenesis (Q5 Site-Directed Mutagenesis Kit, New England Biolabs).
    detected: RRID:Addgene_154754)
    NTD constructs were cloned into pcDNA 3.1 using Nhel and Mssl restriction enzyme cloning, while the RBD constructs were introduced in frame to the mu phosphatase signal sequence and incorporated within pcDNA3.1 via Gibson assembly (NEBuilder HiFi DNA Assembly Cloning Kit, New England Biolabs)
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    Software and Algorithms
    SentencesResources
    Patch mode motion correction (EER upsampling factor 1, EER number of fractions 40), patch mode CTF estimation, reference free particle picking, and particle extraction were carried out on-the-fly in cryoSPARC live.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    0.9.339, followed by iterative rounds of refinement in COOT and Phenix v.1.1940.
    COOT
    suggested: (Coot, RRID:SCR_014222)
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    Figures were prepared using UCSF Chimera, UCSF ChimeraX v.1.1.142, and PyMOL (v.2.2 Schrodinger, LLC)
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    Next, the cells were incubated in 100 microliters of a 1:300 dilution of secondary antibody (Thermo Fisher Cat# A-21235) in incubation buffer for 10 minutes.
    Thermo Fisher Cat#
    suggested: None
    Data was analyzed using FlowJo.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    To estimate the neutralizing capability of each antibody, IC50s were calculated by non-linear regression using the sigmoidal dose response equation in GraphPad Prism 9.
    GraphPad Prism
    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: We did not find any issues relating to colormaps.

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

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2021.12.17.473178v1 on bioRxiv