Broadening a SARS-CoV-1–neutralizing antibody for potent SARS-CoV-2 neutralization through directed evolution

  1. Fangzhu Zhao
  2. Meng Yuan
  3. Celina Keating
  4. Namir Shaabani
  5. Oliver Limbo
  6. Collin Joyce
  7. Jordan Woehl
  8. Shawn Barman
  9. Alison Burns
  10. Quoc Tran
  11. Xueyong Zhu
  12. Michael Ricciardi
  13. Linghang Peng
  14. Jessica Smith
  15. Deli Huang
  16. Bryan Briney
  17. Devin Sok
  18. David Nemazee
  19. John R. Teijaro
  20. Ian A. Wilson
  21. Dennis R. Burton
  22. Joseph G. Jardine

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Abstract

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for strategies to rapidly develop neutralizing monoclonal antibodies that can function as prophylactic and therapeutic agents and to help guide vaccine design. Here, we demonstrate that engineering approaches can be used to refocus an existing antibody that neutralizes one virus but not a related virus. Through a rapid affinity maturation strategy, we engineered CR3022, a SARS-CoV-1–neutralizing antibody, to bind to the receptor binding domain of SARS-CoV-2 with >1000-fold increased affinity. The engineered CR3022 neutralized SARS-CoV-2 and provided prophylactic protection from viral challenge in a small animal model of SARS-CoV-2 infection. Deep sequencing throughout the engineering process paired with crystallographic analysis of engineered CR3022 elucidated the molecular mechanisms by which the antibody can accommodate sequence differences in the epitopes between SARS-CoV-1 and SARS-CoV-2. This workflow provides a blueprint for the rapid broadening of neutralization of an antibody from one virus to closely related but resistant viruses.

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  1. Version published to 10.1126/scisignal.abk3516
  2. ScreenIT

    SciScore for 10.1101/2021.05.29.443900: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Research protocol was approved and performed in accordance with Scripps Research IACUC Protocol #20-0003.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After washing, yeast cells were stained with FITC-conjugated chicken anti-C-Myc antibody (Immunology Consultants Laboratory, CMYC-45F), AF405-conjugated anti-V5 antibody (made in house), and streptavidin-APC (Invitrogen, SA1005) in 1:100 dilution for 20 min at 4 °C.
    anti-C-Myc
    suggested: None
    anti-V5
    suggested: None
    SA1005
    suggested: None
    In the neutralization assay, antibody samples were serially diluted with complete DMEM medium (Corning, 15-013-CV) containing 10% FBS (Omega Scientific, FB-02), 2 mM L-Glutamine (Corning, 25-005-Cl), and 100 U/mL of Penicillin/Streptomycin (Corning, 30-002-C).
    25-005-Cl
    suggested: None
    After washing, alkaline phosphatase-conjugated goat anti-human IgG Fcy secondary antibody (Jackson ImmunoResearch, 109-055-008) was added in 1:1000 dilution and incubated for 1h at RT.
    anti-human IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 109-055-008, RRID:AB_2337601)
    Animal study: Groups of twelve 6-8 week old Syrian hamsters were put into 6 treatment groups who each received an intraperitoneal (i.p.) infusion of either 10 mg, 2 mg, 0.5 mg, or 0.125 mg per animal of the eCR3022.7 monoclonal antibody or 10 mg per animal of the parental CR3022 monoclonal antibody or 10 mg per animal of an anti-dengue isotype matched control antibody (Den3).
    anti-dengue isotype matched control
    suggested: None
    Den3
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Yeast cells were firstly spun down and washed with PBS/1% BSA, then incubated with biotinylated SARS-CoV-2 RBD or S or HEK cell membrane protein at several non-depleting concentrations respectively for at least 30 min at 4°C.
    HEK
    suggested: None
    Then the mixture was transferred onto HEK 293T cells (ATCC, CRL-3216) in a 10 cm2 culture dish (Corning, 430293).
    HEK 293T
    suggested: ATCC Cat# CRL-3216, RRID:CVCL_0063)
    After that, 50 μL of Hela-hACE2 cells were added at 10,000 cells/well onto each well of the plates.
    Hela-hACE2
    suggested: None
    Authentic SARS-CoV-2 neutralization assay: Vero E6 cells were seeded in 96-well half-well plates at approximately 8000 cells/well in a total volume of 50 μL complete DMEM medium the day prior to the addition antibody and virus mixture.
    Vero E6
    suggested: None
    HEp2 epithelial cell polyreactive assay: Reactivity to human epithelial type 2 (HEp2) cells was determined by indirect immunofluorescence on HEp2 slides (Hemagen, 902360) according to manufacturer’s instructions.
    HEp2
    suggested: None
    Homogenized organs were titrated 1:10 over 6 steps and layered over Vero-E6 cells.
    Vero-E6
    suggested: None
    Recombinant DNA
    SentencesResources
    The libraries were displayed on the surface of yeast as molecular Fab using the pYDSI vector, a yeast display vector containing the bidirectional Gal1-10 promoter that was based on the design of a previously described vector (Wang et al., 2018), omitting the leucine-zipper dimerization domains.
    pYDSI
    suggested: None
    In brief, 12.5 μg of MLV gag/pol backbone (Addgene, 14887), 10 μg of MLV-CMV-Luciferase plasmid, and 2.5 μg of SARS-CoV-2-d18 spike plasmid were incubated with transfection reagent Lipofectamine 2000 (Thermo Fisher, 11668027) following manufacturer’s instructions for 20 min at RT.
    MLV-CMV-Luciferase
    suggested: None
    In brief, the heavy and light chains were cloned into phCMV3.
    phCMV3
    suggested: None
    Software and Algorithms
    SentencesResources
    Paired FASTQs were checked for sequence quality using the FastQC package (FastQC v0.11.9).
    FastQC
    suggested: (FastQC, RRID:SCR_014583)
    Merged reads with full sequence identity were clustered using VSEARCH (v2.15.1) (Rognes et al., 2016).
    VSEARCH
    suggested: None
    Pacbio sequencing: Long Amp Taq Polymerase (New England Biolabs) was used to PCR amplify Plasmid DNA after sort 4 according to manufacturer’s protocol with the following primers: First round PCR products were purified with SPRI beads (Beckman Coulter) and 10 uL of purified PCR product was used in a second round of index PCR with the following primers: DNA sample was again purified with SPRI beads, then submitted to GeneWiz, where a PacBio SMRTbell amplicon library was prepared per the manufacturer’s protocol and sequenced on the PacBio Sequel platform with v3.0 chemistry.
    GeneWiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    Sequence data that support the findings in this study are available at the NCBI Sequencing Read Archive (www.ncbi.nlm.nih.gov/sra) under BioProject number PRJNAXXXXXX.
    Sequencing Read Archive
    suggested: None
    BioProject
    suggested: (NCBI BioProject, RRID:SCR_004801)
    Python code will be available on github.
    Python
    suggested: (IPython, RRID:SCR_001658)
    Neutralization IC50 values were calculated using “One-Site Fit LogIC50” regression in GraphPad Prism 8.0.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    First, we plotted a standard curve of serially diluted virus (3000, 1000, 333, 111, 37, 12, 4, 1 PFU) versus RLU using four-parameter logistic regression (GraphPad Prism 8.0) below:

    (y: RLU, x: PFU, a,b,c and x0 are parameters fitted by standard curve) To convert sample RLU into PFU, use the equation below: (if y < a then x = 0)

    Percentage neutralization was calculated by the following equation:

    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Iterative model building and refinement were carried out in COOT (Emsley et al., 2010) and PHENIX (Adams et al., 2010), respectively.
    COOT
    suggested: (Coot, RRID:SCR_014222)
    PHENIX
    suggested: (Phenix, RRID:SCR_014224)

    Results from OddPub: Thank you for sharing your code.

    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 26. 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.

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

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.05.29.443900v1 on bioRxiv