Targeting conserved viral virulence determinants by single domain antibodies to block SARS-CoV2 infectivity

  1. Sudhakar Singh
  2. Surbhi Dahiya
  3. Yuviana J. Singh
  4. Komal Beeton
  5. Ayush Jain
  6. Roman Sarkar
  7. Abhishek Dubey
  8. Syed Azeez Tehseen
  9. Sharvan Sehrawat

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

We selected SARS-CoV2 specific single domain antibodies (sdAbs) from a previously constructed phage display library using synthetic immunogenic peptides of the virus spike (S) protein as bait. The sdAbs targeting the cleavage site (CS) and the receptor binding domain (RBD) in S protein efficiently neutralised the infectivity of a pseudovirus expressing SARS-CoV2 S protein. Anti-CS sdAb blocked the virus infectivity by inhibiting proteolytic processing of SARS-CoV2 S protein. Both the sdAbs retained characteristic structure within the pH range of 2 to 12 and remained stable upto 65°C. Furthermore, structural disruptions induced by a high temperature in both the sdAbs were largely reversed upon their gradual cooling and the resulting products neutralised the reporter virus. Our results therefore suggest that targeting CS in addition to the RBD of S protein by sdAbs could serve as a viable option to reduce SARS-CoV2 infectivity and that proteolytic processing of the viral S protein is critical for infection.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2021.01.13.426537: (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
    ) anti-mouse antibody was used to recognize VHH (4A12E6: Invitrogen Rockford, USA) followed by probing with anti-mouse IgG raised in goat (A3562: 1ml Sigma, USA) and conjugated with alkaline phosphatase for the development of the blot.
    anti-mouse
    suggested: (Sigma-Aldrich Cat# A3562, RRID:AB_258091)
    The blocked PVDF membrane was probed with the sdAb (having both 6x(HIS)-tag and biotin-tag) and then monoclonal anti-6x(HIS)-tag antibody was probed followed by detection with anti-mouse IgG conjugated with alkaline phosphatase (A3562: 1ml Sigma, USA) was used for developing the blots.
    anti-6x(HIS)-tag
    suggested: None
    Since the spike construct contains the Flag tag, immunoblotting was performed to detect the presence of spike protein in supernatant collected 24 hours post transduction as well as in cell lysate of 72 hours post transduced Vero E6 cells using anti-flag mouse antibody (F1804-50UG, USA).
    anti-flag
    suggested: None
    A secondary antibody anti-mouse IgG raised in goat and conjugated with alkaline phosphatase was used for the development of the blot.
    anti-mouse IgG
    suggested: None
    For detection of sdAb, anti-6x(HIS) antibody was incubated for 1 hr at RT followed by washing and incubation with anti-mouse antibody conjugated with alkaline phosphatase for 1.5 hrs.
    anti-6x(HIS
    suggested: None
    Biolayer interferometry (BLI): BLI was performed to determine the binding kinetics of anti-CS and anti-RBD antibody with the purified spike protein produced from transfected HEK293T cells (S construct with 3x FLAG-tag) using BLltz System.
    anti-CS
    suggested: None
    Different concentrations of S protein were incubated for 5 minutes to measure the binding affinity with the immobilised anti-CS and anti-RBD antibodies, and their dissociation kinetics was measured in PBS.
    anti-RBD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    TG1 cells were then infected with helper phage M13K07 under static conditions at 30°C for 40 mins.
    TG1
    suggested: None
    Above mixture was kept at RT for 15 minutes and co-transfected in HEK239T cells (Human Embryonic Kidney).
    HEK239T
    suggested: None
    Modified Vero cells (Vero E6) originated from kidney epithelial cells of African green monkey were used for measuring the internalization using concentrated pseudoviruses.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Vero E6 cells infected with bald particles or the LV (VSV-G) pseudovirus particles served as a control for such experiments.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Flow cytometry: After 72 hrs of transduction, Vero-E6 cells were treated with 1mM PBS-EDTA for 15 minutes at 37°C in CO2 incubator and the cells were removed from 96 well (flat bottom) plates by reverse pipetting.
    Vero-E6
    suggested: None
    Biolayer interferometry (BLI): BLI was performed to determine the binding kinetics of anti-CS and anti-RBD antibody with the purified spike protein produced from transfected HEK293T cells (S construct with 3x FLAG-tag) using BLltz System.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Software and Algorithms
    SentencesResources
    Prediction of B cell epitopes: Linear B cell epitopes were predicted by IEDB database using BepiPred 2.0 server.
    BepiPred
    suggested: (BepiPred-2.0, RRID:SCR_018499)
    The available data was analysed using flowJo X software (TreeStar)44.
    flowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Analysis and scaling of all the taken images were done using ImageJ software44
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Scanning electron microscopy: Field Emission Scanning Electron Microscope (FESEM) was used to measure the surface topography of LV(CoV2-S) and LV(VSV-G).
    Field Emission Scanning
    suggested: (Hitachi S4700 Field Emission Scanning Electron Microscope, RRID:SCR_020019)
    All statistical analysis were done using Graphpad prism 8.0.2(263).
    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.

    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 10.1101/2021.01.13.426537v1 on bioRxiv