Versatile, Multivalent Nanobody Cocktails Efficiently Neutralize SARS-CoV-2

  1. Yufei Xiang
  2. Sham Nambulli
  3. Zhengyun Xiao
  4. Heng Liu
  5. Zhe Sang
  6. W. Paul Duprex
  7. Dina Schneidman-Duhovny
  8. Cheng Zhang
  9. Yi Shi

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Abstract

The outbreak of COVID-19 has severely impacted global health and the economy. Cost-effective, highly efficacious therapeutics are urgently needed. Here, we used camelid immunization and proteomics to identify a large repertoire of highly potent neutralizing nanobodies (Nbs) to the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD). We discovered multiple elite Nbs with picomolar to femtomolar affinities that inhibit viral infection at sub-ng/ml concentration, more potent than some of the best human neutralizing antibodies. We determined a crystal structure of such an elite neutralizing Nb in complex with RBD. Structural proteomics and integrative modeling revealed multiple distinct and non-overlapping epitopes and indicated an array of potential neutralization mechanisms. Structural characterization facilitated the bioengineering of novel multivalent Nb constructs into multi-epitope cocktails that achieved ultrahigh neutralization potency (IC50s as low as 0.058 ng/ml) and may prevent mutational escape. These thermostable Nbs can be rapidly produced in bulk from microbes and resist lyophilization, and aerosolization. These promising agents are readily translated into efficient, cost-effective, and convenient therapeutics to help end this once-in-a-century health crisis.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.24.264333: (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
    HRP-conjugated secondary antibodies against T7-tag (Thermo) were diluted 1:7500 and incubated with the well for 1 hr at room temperature.
    T7-tag
    suggested: None
    A validated SARS-CoV-2 antibody-negative human serum control, a validated NIBSC SARS-CoV-2 plasma control, was obtained from the National Institute for Biological Standards and Control, UK) and an uninfected cells control were also performed to ensure that virus neutralization by antibodies was specific.
    Control , UK
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The RBD (residues 319-541) of the SARS-Cov-2 S protein was expressed as a secreted protein in Spodoptera frugiperda Sf9 cells (Expression Systems) using the Bac-to-bac baculovirus method (Invitrogen).
    Sf9
    suggested: CLS Cat# 604328/p700_Sf9, RRID:CVCL_0549)
    Pseudotyped SARS-CoV-2 neutralization assay: The 293T-hsACE2 stable cell line (Cat# C-HA101) and the pseudotyped SARS-CoV-2 (Wuhan-Hu-1 strain) particles with GFP (Cat# RVP-701G, Lot#CG-113A) or firefly luciferase (Cat# RVP-701L, Lot# CL109A, and CL-114A) reporters were purchased from the Integral Molecular.
    293T-hsACE2
    suggested: None
    The serum–virus mixes (220 μl total) were incubated at 37 °C for 1 h, after which they were added dropwise onto confluent Vero E6 cell monolayers in the six-well plates.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    The raw data was processed by Prism 7 (GraphPad) to fit into a 4PL curve and to calculate logIC50.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    After the MS analysis, the data was searched by pLink for the identification of cross-linked peptides.
    pLink
    suggested: (PLINK, RRID:SCR_001757)
    Each Nb model was then docked to the RBD structure (PDB 6LZG) by an antibody-antigen docking protocol of PatchDock software that focuses the search to the CDRs and optimizes CXMS-based distance restraints satisfaction (Schneidman-Duhovny, 2012 #52; Schneidman-Duhovny, 2020 #53).
    PatchDock
    suggested: (PatchDock, RRID:SCR_017589)
    The antigen interface residues (distance <6Å from Nb atoms) among the top 10 scoring models, according to the SOAP score, were used to determine the epitopes.
    SOAP
    suggested: (SOAP, RRID:SCR_000689)
    The initial model was refined in Phenix (Adams, 2010 #61)and adjusted in COOT (Emsley, 2004 #62).
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    COOT
    suggested: (Coot, RRID:SCR_014222)
    The model quality was checked by MolProbity (Williams, 2018 #63).
    MolProbity
    suggested: (MolProbity, RRID:SCR_014226)
    Nb21 comparative modeling was done using the Nb20 structure as a template in MODELLER.
    MODELLER
    suggested: (MODELLER, RRID:SCR_008395)

    Results from OddPub: Thank you for sharing your data.

    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 10.1101/2020.08.24.264333 on bioRxiv