Combating the SARS-CoV-2 Omicron variant with non-Omicron neutralizing antibodies

  1. Yingdan Wang
  2. Xiang Zhang
  3. Jiangyan Liu
  4. Yanqun Wang
  5. Wuqiang Zhan
  6. Mei Liu
  7. Meng Zhang
  8. Qimin Wang
  9. Qianying Liu
  10. Tongyu Zhu
  11. Yumei Wen
  12. Zhenguo Chen
  13. Jincun Zhao
  14. Fan Wu
  15. Lei Sun
  16. Jinghe Huang

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Abstract

The highly mutated and transmissible Omicron variant has provoked serious concerns over its decreased sensitivity to the current coronavirus disease 2019 (COVID-19) vaccines and evasion from most anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies (NAbs). In this study, we explored the possibility of combatting the Omicron variant by constructing bispecific antibodies based on non-Omicron NAbs. We engineered ten IgG-like bispecific antibodies with non-Omicron NAbs named GW01, 16L9, 4L12, and REGN10987 by fusing the single-chain variable fragments (scFvs) of two antibodies through a linker and then connecting them to the Fc region of IgG1. Surprisingly, eight out of ten bispecific antibodies showed high binding affinity to the Omicron receptor-binding domain (RBD) and exhibited extreme breadth and potency against pseudotyped SARS-CoV-2 variants of concern (VOCs) including Omicron, as well as authentic Omicron(+R346K) variants. Six bispecific antibodies containing the cross-NAb GW01 neutralized Omicron variant and retained their abilities to neutralize other sarbecoviruses. Bispecific antibodies inhibited Omicron infection by binding to the ACE2 binding site. A cryo-electron microscopy (cryo-EM) structure study of the representative bispecific antibody FD01 in complex with the Omicron spike (S) revealed 5 distinct trimers and one unique bi-trimer conformation. The structure and mapping analyses of 34 Omicron S variant single mutants elucidated that two scFvs of the bispecific antibody synergistically induced the RBD-down conformation into 3-RBD-up conformation, enlarged the interface area, accommodated the S371L mutation, improved the affinity between a single IgG and the Omicron RBD, and hindered ACE2 binding by forming bi-trimer conformation. Our study offers an important foundation for anti-Omicron NAb design. Engineering bispecific antibodies based on non-Omicron NAbs may provide an efficient solution to combat the Omicron variant.

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    SciScore for 10.1101/2022.01.30.478305: (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
    Memory B-cell staining, sorting and antibody cloning: CD19+IgA–IgD–IgM-primary B cells were sorted out from peripheral blood mononuclear cells (PBMC) of recovered patients of COVID-19 and expanded in vitro in MEM medium with 10% FBS in the presence of irradiated 3T3-msCD40L feeder cells, IL-2 and IL-21 as previously described 18.
    IL-21
    suggested: None
    After 3 washing steps with PBS 0.05% Tween 20 (PBS-T), 1:2500 diluted HRP-conjugated goat anti-human IgG antibody (Jackson Immuno Research Laboratories, USA) was added for 1 hour at room temperature.
    anti-human IgG
    suggested: None
    After permeabilized with 0.2% Triton X-100 for 20 min at room temperature, the plates were sequentially stained with cross-reactive rabbit anti-SARS-CoV-2 N IgG (Cat. No.: 40143-T62, Sino Biological Inc) as the primary antibody and HRP-conjugated goat anti-rabbit IgG(H+L) (No.: 109-035-088, Jackson ImmunoResearch) as the secondary antibody in 37°C for 1 hour respectively.
    anti-SARS-CoV-2 N IgG
    suggested: None
    anti-rabbit IgG(H+L
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines, proteins, viruses and plasmids: The human primary embryonic kidney cell lines (HEK293T) and 293T-hACE2 cells were cultured in DMEM medium with 10% fetal bovine serum (FBS).
    HEK293T
    suggested: None
    African green monkey kidney-derived Vero E6 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS).
    Vero E6
    suggested: None
    Pseudoviruses were generated by co-transfection of 293T cells with an env-deficient HIV backbone pNL4-3.
    293T
    suggested: None
    104 293T-hACE2 cells were then added to the mixture and cultured for 48 h at 37 °C.
    293T-hACE2
    suggested: None
    From the wells with SARS-CoV-2 neutralization activities, the variable regions of the antibody (VH and VL) genes were amplified by RT–PCR. mAbs were expressed as human IgG1 by HEK293F cells and purified using a protein G column (Smart-Lifesciences).
    HEK293F
    suggested: RRID:CVCL_6642)
    3. 293F cells were transiently transfected with bispecific Abs plasmid.
    293F
    suggested: RRID:CVCL_D615)
    Recombinant DNA
    SentencesResources
    Alpha (containing 69–70 and 144 deletions and N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H substitutions), Beta (containing D80A, D215G, 241-243 deletions and K417N, E484K, N501Y, D614G and A701V substitutions), Gamma (containing L18F,T20N,P26S,D138Y, R190S,K417T, E484K,N501Y, D614G.H655Y,T1027I, and V1176F substitutions), Delta (containing T19R, 157-158 deletions and L452R, T478K, D614G, P681R and D950N substitutions), and Omicron (containing A67V, 69-70del, T95I, G142D, 143-145del, N211I, 212del, ins215EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F substitutions), SARS-CoV, bat SARSr-CoVs (WIV1 and Rs3367) were synthesized by BGI and constructed in pcDNA3.1 vector.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    Pseudoviruses were generated by co-transfection of 293T cells with an env-deficient HIV backbone pNL4-3.
    pNL4-3
    suggested: None
    Software and Algorithms
    SentencesResources
    The IC50s of NAbs were calculated using the GraphPad Prism 7.04 software (La Jolla, CA, USA)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Automated data acquisition was carried out with SerialEM software21 through beam-image shift method22.
    SerialEM
    suggested: (SerialEM, RRID:SCR_017293)
    A total of 4,363 movie stacks was binned 2 × 2, dose weighted, and motion corrected using MotionCor2 25 within RELION.
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    RELION
    suggested: (RELION, RRID:SCR_016274)
    Remaining 3,817 good images were imported into cryoSPARC for further patched CTF-estimating, blob-picking and 2D classification.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Model building and refinement: For model building of SARS-CoV-2 Omicron S FD01 complex, the SARS-CoV-2 Omicron S trimer model and the antibody model generated by swiss-model 31 were fitted into the map using UCSF Chimera and then manually adjusted with COOT 32.
    COOT
    suggested: (Coot, RRID:SCR_014222)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 20. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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 10.1101/2022.01.30.478305 on bioRxiv