Potent SARS-CoV-2 binding and neutralization through maturation of iconic SARS-CoV-1 antibodies

  1. Romain Rouet
  2. Ohan Mazigi
  3. Gregory J. Walker
  4. David B. Langley
  5. Meghna Sobti
  6. Peter Schofield
  7. Helen Lenthall
  8. Jennifer Jackson
  9. Stephanie Ubiparipovic
  10. Jake Y. Henry
  11. Arunasingam Abayasingam
  12. Deborah Burnett
  13. Anthony Kelleher
  14. Robert Brink
  15. Rowena A. Bull
  16. Stuart Turville
  17. Alastair G. Stewart
  18. Christopher C. Goodnow
  19. William D. Rawlinson
  20. Daniel Christ

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Abstract

Antibodies against coronavirus spike protein potently protect against infection and disease, however it remains unclear if such protection can be extended to variant coronaviruses. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here we explore antibody maturation strategies to change and broaden their specificity, enabling potent binding and neutralization of SARS-CoV-2. Using targeted mutagenesis as well as light chain shuffling on phage, we identified variants with considerably increased affinity and neutralization potential. The most potent antibody, derived from the NIH-developed mAb m396, neutralized live SARS-CoV-2 virus with a half-maximal inhibitory concentration (IC 50 ) of 160 ng/ml. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of antibodies can give rise to variants targeting diverse epitopes. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.14.422791: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Generation of site-directed mutagenesis antibody libraries: m396, CR3022, CR3014, and 80R scFv were gene synthesized (Genscript) and cloned into the pHEN1 phagemid vector.
    CR3022
    suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)
    CR3014
    suggested: None
    Plates were washed with PBST, incubated with HRP-conjugated anti-M13 antibody (GE Healthcare) for 1h and washed again.
    anti-M13
    suggested: None
    The plates were subsequently incubated with HRP-conjugated chicken anti c-myc antibody (ICL Lab) for 1h and washed again.
    anti c-myc
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The plasmid was transfected into Expi293 cells (Thermo Scientific) according to the manufacturer’s protocol and the protein expressed for 7 days at 37°C, 5% CO2.
    Expi293
    suggested: RRID:CVCL_D615)
    Retroviral SARS-CoV-1 and 2 pseudo-particles (SARS-2pp) were generated by co-transfecting expression plasmids containing SARS-CoV-1 or SARS-CoV-1 spike which were kindly provided by Prof Gary Whitaker and Dr Markus Hoffmannr, respectively, and the MLV gag/pol and luciferase vectors which were kindly provided by Prof. Francois-Loic Cosset, in CD81KO 293T cells, which were kindly provided by Dr Joe Grove30, using mammalian Calphos transfection kit (Takara Bio)
    293T
    suggested: None
    293T-ACE2 cells were seeded 24 hours earlier at 1.5 × 104 cells per well in 96-well white flat bottom plates (Sigma-Aldrich).
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Experimental Models: Organisms/Strains
    SentencesResources
    For the double-Fab 1:1:1 complex (RBD + CR3014-C8 + CR3022-B6), cell content analysis suggested that, should all components be present in the expected stoichiometric ratio, the solvent content would be 50%.
    RBD + CR3014-C8 + CR3022-B6
    suggested: None
    Software and Algorithms
    SentencesResources
    After 3 days, the plates were observed for cytopathic effect (CPE) and IC50 values were calculated from four parameter dose-response curves (GraphPad Prism).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Extracted particles were subjected to multiple rounds of 2D classification and ab initio reconstruction in cryoSPARC before their locations were exported to Relion 3.0 36.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Motion correction and CTF estimation was then implemented in Relion 3.0 and particles were reextracted and again subjected to 2D classification before 3D auto-refinement and Bayesian polishing.
    Relion
    suggested: (RELION, RRID:SCR_016274)

    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.
    • 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 10.1101/2020.12.14.422791 on bioRxiv