Peptide derived nanobody inhibits entry of SARS-CoV-2 variants

  1. Nivya Mendon
  2. Rayees Ganie
  3. Shubham Kesarwani
  4. Drisya Dileep
  5. Sarika Sasi
  6. Prakash Lama
  7. Anchal Chandra
  8. Minhajuddin Sirajuddin

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Abstract

Emergence of the new escape mutants of the SARS-CoV-2 virus has escalated its penetration among the human population and has reinstated its status as a global pandemic. Therefore, developing effective antiviral therapy against emerging SARS variants and other viruses in a short period of time becomes essential. Blocking the SARS-CoV-2 entry into human host cells by disrupting the spike glycoprotein-ACE2 interaction has been already exploited for vaccine development and monoclonal antibody therapy. Unlike the previous reports, our study used a 9 amino acid peptide from the receptor-binding motif (RBM) of Spike (S) protein as an epitope. We report the identification of an efficacious nanobody N1.2 that blocks the entry of pseudovirus containing SARS-CoV-2 spike as the surface glycoprotein. Moreover, we observe a more potent neutralizing effect against both the hCoV19 (Wuhan/WIV04/2019) and the Omicron (BA.1) pseudotyped spike virus with a bivalent version of the nanobody. In summary, our study presents a faster and efficient methodology to use peptide sequences from a protein-receptor interaction interface as epitopes for screening nanobodies against potential pathogenic targets. This approach can also be widely extended to target other viruses and pathogens in the future.

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    SciScore for 10.1101/2022.04.21.489021: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The cells were incubated with 100μM of the respective peptide and 1:200 dilution of rabbit anti-HA tag antibody (Sigma; catalogue no. Cat # H6908) at 4°C for 1hr.
    anti-HA
    suggested: None
    Cells were incubated with 1:200 dilution of goat anti-rabbit Alexa fluor-647 antibody (Invitrogen) and 1:100 dilution of neutravidin fluorescein conjugate (Invitrogen; FITC, catalogue no. A2662, used for the first FACS) at 4°C for 30min.
    anti-rabbit
    suggested: None
    antibody (Invitrogen)
    suggested: (Rockland Cat# 00-8844-25, RRID:AB_2610705)
    30min
    suggested: None
    Cells were incubated with 1:500 dilution of mouse anti-FLAG monoclonal antibody (Merck, catalogue no. F3165) overnight at 4°C.
    anti-FLAG
    suggested: None
    The blots were probed with 1:5000 anti Flag antibody (Sigma, F3165) overnight at 4□C followed by 1:10000 anti-mouse secondary at room temperature for 1hr.
    anti Flag
    suggested: None
    anti-mouse
    suggested: None
    The surface of the well was washed twice with blocking buffer and incubated with HRP-conjugated rabbit anti-6xHis tag antibody (Abcam; catalogue no. AB1187), 1:10,000 dilution at 4°C overnight.
    anti-6xHis tag
    suggested: (Abcam Cat# ab1187, RRID:AB_298652)
    eGFP-ACE2/HEK293T immunostained with FLAG antibody was imaged at 60x oil objective of FV3000 confocal microscope (Figure 2B) and 100x oil objective of H-TIRF microscope (Supplementary Figure 2A) using 405nm, 488nm and 647nm laser lines for DAPI, eGFP, and Alexa fluor-647 fluorophores.
    eGFP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture experiments: Wild type mammalian HEK293T cells and LentiX-293T cells (Takara Bio, catalogue no. 632180) were used in this study for pseudotyped spike virus production and viral transduction assay.
    HEK293T
    suggested: None
    LentiX-293T
    suggested: None
    Caco2 cells were lysed for total RNA purification using the Trizol method.
    Caco2
    suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)
    Pseudoviral transduction assay: The GFP/HEK293T cells or eGFP-ACE2/HEK293T cells were grown up to 60-70% confluency in complete media before viral transduction.
    GFP/HEK293T
    suggested: None
    eGFP-ACE2/HEK293T
    suggested: None
    In this assay, the viral titre was used in a concentration such that to obtain more than 70% transduction efficiency in the eGFP-ACE2/HEK293T or eGFP/HEK293T cell line.
    eGFP/HEK293T
    suggested: None
    Recombinant DNA
    SentencesResources
    Cloning and protein purification: The nanobody gene was amplified from isolated yeast colonies and cloned between HindIII and XhoI sites in a pET-22b(+) plasmid containing a C-terminal 6x histidine tag.
    pET-22b(+)
    suggested: RRID:Addgene_12651)
    For viral particle production, 5 µg pHR lentiviral vector cloned with mCherry fluorescent protein, 3.75 µg packaging plasmid psPAX2 (Addgene; #12260), and 2.5 µg envelope plasmid for the expression of Spike glycoprotein (obtained as a kind gift from Prof. Nevan Krogan, UCSF, USA) of SARS-CoV-2 were mixed in 500 µl OptiMEM media and 20 µl PLUS reagent (Invitrogen; LTX transfection reagent, catalogue no. L15338100) and kept for incubation at room temperature for 5min.
    pHR
    suggested: RRID:Addgene_16514)
    We also generated control lentiviral particles by replacing Spike plasmid with VSV-G envelope protein, pmDG2 (Addgene; #12259) plasmid.
    pmDG2
    suggested: None
    Omicron pseudotyped virus production: Omicron pseudotyped viruses were produced similarly as described above for spike pseudoviruses but instead used omicron envelope plasmid along with packaging plasmid (psPAX2) and lentiviral plasmid (pHR mCherry) in the following ratio: psPAX2 (1.3pmol), pHR mCherry: 1.64pmol, SARS-CoV-2 Omicron Strain S gene (Genscript, Cat # MC_0101274): 0.72pmol.
    psPAX2
    suggested: RRID:Addgene_12260)
    This construct contains amino-terminus EGFP followed by self-cleaving 2A peptide sequence followed by ACE2 and carboxy-termini SNAP-tag and FLAG tags (eGFP-ACE2/HEK293T) Generation of stable HEK293T cell line for over-expression of ACE2: The lentiviral pTRIP vector cloned with eGFP-ACE2/HEK293T under CMV enhancer and chicken β-actin promoter (CAG promoter) flanked with 5′and 3′ long terminal repeat (LTR) sequences (39), were used to produce lentiviral particles as per the method described before (35).
    pTRIP
    suggested: RRID:Addgene_127663)
    Software and Algorithms
    SentencesResources
    The following peptide sequences from the receptor-binding domain (RBD) of the spike were synthesized from LifeTein with a biotin tag: Peptide-1: [FNCYFPLQS]S-K-Biotin Peptide-2: Biotin-[GFQPTNGVGY] Sequence Alignment for Covid Variants The hCoV19 spike (Wuhan/WIV04/2019), GISAID (EPI_ISL_402124) construct is a kind gift from Prof. Nevan Krogan, UCSF, USA (38).
    LifeTein
    suggested: (LifeTein, RRID:SCR_012626)
    For viral particle production, 5 µg pHR lentiviral vector cloned with mCherry fluorescent protein, 3.75 µg packaging plasmid psPAX2 (Addgene; #12260), and 2.5 µg envelope plasmid for the expression of Spike glycoprotein (obtained as a kind gift from Prof. Nevan Krogan, UCSF, USA) of SARS-CoV-2 were mixed in 500 µl OptiMEM media and 20 µl PLUS reagent (Invitrogen; LTX transfection reagent, catalogue no. L15338100) and kept for incubation at room temperature for 5min.
    Addgene
    suggested: (Addgene, RRID:SCR_002037)
    Images were analysed on Fiji software to calculate mean fluorescence intensity (MFI) for eGFP (ACE2 expression) and mCherry (viral transduction) channel from the z-projected stacks.
    Fiji
    suggested: (Fiji, RRID:SCR_002285)

    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.

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  2. Version published to 10.1101/2022.04.21.489021 on bioRxiv