A potent synthetic nanobody with broad-spectrum activity neutralizes SARS-Cov-2 virus and Omicron variant through a unique binding mode

  1. Dongping Zhao
  2. Liqin Liu
  3. Xinlin Liu
  4. Jinlei Zhang
  5. Yuqing Yin
  6. Linli Luan
  7. Dingwen Jiang
  8. Xiong Yang
  9. Lei Li
  10. Hualong Xiong
  11. Dongming Xing
  12. Qingbing Zheng
  13. Ningshao Xia
  14. Yuyong Tao
  15. Shaowei Li
  16. Haiming Huang

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Abstract

The major challenge to control COVID pandemic is the rapid mutation rate of the SARS-Cov-2 virus, leading to the escape of the protection of vaccines and most of the neutralizing antibodies to date. Thus, it is essential to develop neutralizing antibodies with broad-spectrum activity targeting multiple SARS-Cov-2 variants. Here, we reported a synthetic nanobody (named C5G2) obtianed by phage display and subsequent antibody engineering. C5G2 has a single digit nanomolar binding affinity to RBD domain and inhibits its binding to ACE2 with an IC 50 of 3.7 nM. Pseudovirus assay indicated that the monovalent C5G2 could protect the cells from the infection of SARS-Cov-2 wild type virus and most of the virus of concern, i.e. Alpha, Beta, Gamma and Omicron variants. Strikingly, C5G2 has the highest potency against Omicron among all the variants with the IC 50 of 4.9ng/mL. The Cryo-EM structure of C5G2 in complex with the Spike trimer showed that C5G2 bind to RBD mainly through its CDR3 at a conserved region that not overlapping with the ACE2 binding surface. Additionally, C5G2 bind simultaneously to the neighboring NTD domain of spike trimer through the same CDR3 loop, which may further increase its potency against the virus infection. Third, the steric hindrance caused by FR2 of C5G2 could inhibit the binding of ACE2 to RBD as well. Thus, this triple-function nanobody may be served as an effective drug for the prophylaxis and therapy against Omicron as well as future variants.

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  1. Version published to 10.1101/2022.04.11.487660v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2022.04.11.487660: (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

    Recombinant DNA
    SentencesResources
    C5 affinity maturation library construction: The sequence of C5 in phagemid pComb3XSS were used as template for library construction.
    pComb3XSS
    suggested: RRID:Addgene_63890)
    Nanobody expression and purification: The cDNA encoding the nanobodies in the pComb3XSS vector were PCR amplified and subcloned into vector pET22b to express 6×His tagged proteins.
    pET22b
    suggested: RRID:Addgene_84863)
    Software and Algorithms
    SentencesResources
    The coding cDNA of 1ZVH was chemically synthesized and subcloned into a modified phage display vector of pComb3XSS, in which the amber stop codon (TAG) were mutated to CAG to facilitate nanobody display in E. coli without gene supE, e.g. SS320(Genentech).
    Genentech
    suggested: (Genentech, RRID:SCR_003997)
    Standard variation values were calculated using a 3-parameter logistic regression fit using Prism Software (GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Data were acquired using the SerialEM software on an FEI Tecnai F30 transmission electron microscope (ThermoFisher Scientific) operated at 300 kV and equipped with a Gatan K3 direct detector.
    SerialEM
    suggested: (SerialEM, RRID:SCR_017293)
    Image processing and 3D reconstruction: Drift and beam-induced motion correction were performed with MotionCor2 [61] to produce a micrograph from each movie.
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    Contrast transfer function (CTF) fitting and phase-shift estimation were conducted with Gctf [62].
    Gctf
    suggested: (GCTF, RRID:SCR_016500)
    Local map resolution was estimated with ResMap [65].
    ResMap
    suggested: None
    We initially fitted the templates models into the corresponding final cryo-EM map using Chimera [67], and further corrected and adjusted them manually by real-space refinement in Coot [68].
    Chimera
    suggested: (Chimera, RRID:SCR_002959)
    Coot
    suggested: (Coot, RRID:SCR_014222)
    The resulting models were then refined with phenix.real_space_refine in PHENIX [69].
    PHENIX
    suggested: (Phenix, RRID:SCR_014224)
    The final atomic models were validated with Molprobity [70, 71].
    Molprobity
    suggested: (MolProbity, RRID:SCR_014226)
    All figures were generated with Chimera or ChimeraX [72, 73].
    ChimeraX
    suggested: (UCSF ChimeraX, RRID:SCR_015872)

    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.

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

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  3. Version published to 10.1101/2022.04.11.487660v1 on bioRxiv