Mapping the allosteric effects that define functional activity of SARS-CoV-2 specific antibodies

  1. Nikhil K. Tulsian
  2. Palur V. Raghuvamsi
  3. Xinlei Qian
  4. Gu Yue
  5. Bhuvaneshwari D/O Shunmuganathan
  6. Firdaus Samsudin
  7. Wong Yee Hwa
  8. Lin Jianqing
  9. Kiren Purushotorman
  10. Mary M. Kozma
  11. Bei Wang
  12. Julien Lescar
  13. Cheng-I Wang
  14. Ganesh S. Anand
  15. Peter J. Bond
  16. Paul A. MacAry

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Abstract

Previous studies on the structural relationship between human antibodies and SARS-CoV-2 have focused on generating static snapshots of antibody complexes with the Spike trimer. However, antibody-antigen interactions are dynamic, with significant binding-induced allosteric effects on conformations of antibody and its target antigen. In this study, we employ hydrogen-deuterium exchange mass spectrometry, in vitro assays, and molecular dynamics simulations to investigate the allosteric perturbations linked to binding events between a group of human antibodies with differential functional activities, and the Spike trimer from SARS-CoV-2. Our investigations have revealed key dynamic features that define weakly or moderately neutralizing antibodies versus those with strong neutralizing activity. These results provide mechanistic insights into the functional modes of human antibodies against COVID-19, and provide a rationale for effective antiviral strategies.

Teaser

Different neutralizing antibodies induce site-specific allosteric effects across SARS-CoV-2 Spike protein

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  1. ScreenIT

    SciScore for 10.1101/2021.12.27.474251: (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
    After 60 min incubation, plate wash step was repeated and the detection antibody, goat anti-human IgG-HRP (Thermo Fisher, SG) was added at 100 μL/well for an incubation of 60 min in dark.
    anti-human IgG-HRP
    suggested: None
    Similarly, HDX was performed for nine convalescent antibodies - LSI-CoVA-014, LSI-CoVA-015, LSI-CoVA-016, and LSI-CoVA-017 identified in this study; and CR3022, CoVA2-04, CoVA-39, 4A8, and 5A6 (previous studies).
    CR3022
    suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)
    CoVA-39
    suggested: None
    The peptides showing protection in the presence of antibody were provided as input under attractive amino acid residues at the protein-protein interaction interface in ClusPro, using a specific module for antigen-antibody docking.
    antigen-antibody docking.
    suggested: None
    Capture ELISA: Monoclonal human IgG1 antibodies LSI-CoVA-014, LSI-CoVA-015, LSI-CoVA-016, and LSI-CoVA-017 were conjugated individually to peroxidase as per manufacturer’s protocol (Abnova, TW).
    human IgG1
    suggested: None
    Monoclonal antibodies LSI-CoVA-014, LSI-CoVA-015, LSI-CoVA-016 and LSI-CoVA-017 were tested at several concentrations ranging from 0.01 ng/mL to 10 μg/mL.
    LSI-CoVA-015
    suggested: None
    LSI-CoVA-016
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2-Spike construct was expressed in Spodoptera frugiperda Sf9 cells following instructions from bac-to-bac baculovirus expression system (Thermo Fisher, SG).
    Sf9
    suggested: CLS Cat# 604328/p700_Sf9, RRID:CVCL_0549)
    Plasmid Sars-Cov-2 Spike HexaPro (addgene) was used to transfect HEK 293 cells with polyethylenimine (PEI).
    HEK 293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    At Day 1, 36 ×106 HEK293T cells were transfected with 27 μg pMDLg/pRRE (Addgene, US), 13.5 μg pRSV-Rev (Addgene, US), 27μg pTT5LnX-WHCoV-St19 (SARS-CoV2 Spike) and 54 μg pHIV-Luc-ZsGreen (Addgene, US) using Lipofectamine 3000 transfection reagent (Thermo Fisher, SG) and cultured in a 37 °C, 5% CO2 incubator.
    HEK293T
    suggested: None
    Pseudovirus neutralization assay (PVNT): On Day 0, CHO cell lines with stable expression of ACE2 were seeded at a density of 5 x104 cells in 100 μL of complete medium [DMEM/high glucose with sodium pyruvate (Thermo Fisher, SG), supplemented with 10% FBS (Thermo Fisher, SG), 10%
    CHO
    suggested: None
    The antibody:pseudovirus mixtures were then added to CHO-ACE2 cells.
    CHO-ACE2
    suggested: None
    Recombinant DNA
    SentencesResources
    Antibody and antigen expression and purification: Genes coding for variable regions of antibody heavy and light chain were synthesized and cloned into vector pTT5 expression vector (National Research Council Canada, NRCC) by Twist
    pTT5
    suggested: RRID:Addgene_52326)
    At Day 1, 36 ×106 HEK293T cells were transfected with 27 μg pMDLg/pRRE (Addgene, US), 13.5 μg pRSV-Rev (Addgene, US), 27μg pTT5LnX-WHCoV-St19 (SARS-CoV2 Spike) and 54 μg pHIV-Luc-ZsGreen (Addgene, US) using Lipofectamine 3000 transfection reagent (Thermo Fisher, SG) and cultured in a 37 °C, 5% CO2 incubator.
    pMDLg/pRRE
    suggested: RRID:Addgene_12251)
    pRSV-Rev
    suggested: RRID:Addgene_12253)
    pHIV-Luc-ZsGreen
    suggested: RRID:Addgene_39196)
    Software and Algorithms
    SentencesResources
    GraphPad Prism was used for plots and one-way ANOVA statistical analysis.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Homology modelling of single Fab arm: Homology models of Fab arms of LSI-CoVA-014, LSI-CoVA-015, LSI-CoVA-016, and LSI-CoVA-017 were modelled using Modeller version 9.21 (43).
    Modeller
    suggested: (MODELLER, RRID:SCR_008395)
    Position specific iterative - BLAST was used to identify the template structures with high sequence identity and structures available at PDB were chosen. 7K8R, 6DWZ, 6DF2, and 7JXE were used as template structures to model peptides spanning heavy chain and light chain of LSI-CoVA-014, LSI-CoVA-015, LSI-CoVA-016, and LSI-CoVA-017 respectively.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    Hetero dimers containing heavy and light chain for respective antibodies were complexed by aligning to the respective template structures in PyMol (Schrodiner Inc, USA)
    PyMol
    suggested: (PyMOL, RRID:SCR_000305)
    The epitope sites on RBD and NTD identified using HDXMS for each antibody were chosen to perform a biased docking using ClusPro 2.0 webserver (
    ClusPro
    suggested: (ClusPro, RRID:SCR_018248)
    A total of 20 Fab:RBD/NTD complexes from four different Fab arms were modelled using CHARMM-GUI and simulated in GROMACS package version 2018.4 (50).
    GROMACS
    suggested: (GROMACS, RRID:SCR_014565)

    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.

    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.

  2. Version published to 10.1101/2021.12.27.474251v1 on bioRxiv