Structural basis of a two-antibody cocktail exhibiting highly potent and broadly neutralizing activities against SARS-CoV-2 variants including diverse Omicron sublineages
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Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), especially the latest Omicron, have exhibited severe antibody evasion. Broadly neutralizing antibodies with high potency against Omicron are urgently needed for understanding the working mechanisms and developing therapeutic agents. In this study, we characterized the previously reported F61, which was isolated from convalescent patients infected with prototype SARS-CoV-2, as a broadly neutralizing antibody against all VOCs including Omicron BA.1, BA.1.1, BA.2, BA.3 and BA.4 sublineages by utilizing antigen binding and cell infection assays. We also identified and characterized another broadly neutralizing antibody D2 with epitope distinct from that of F61. More importantly, we showed that a combination of F61 with D2 exhibited synergy in neutralization and protecting mice from SARS-CoV-2 Delta and Omicron BA.1 variants. Cryo-Electron Microscopy (Cryo-EM) structures of the spike-F61 and spike-D2 binary complexes revealed the distinct epitopes of F61 and D2 at atomic level and the structural basis for neutralization. Cryo-EM structure of the Omicron-spike-F61-D2 ternary complex provides further structural insights into the synergy between F61 and D2. These results collectively indicated F61 and F61-D2 cocktail as promising therapeutic antibodies for combating SARS-CoV-2 variants including diverse Omicron sublineages.
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SciScore for 10.1101/2022.05.27.493682: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Euthanasia Agents: At the end point of the experiment, all remaining animals in the monoclonal antibody-administered group received an overdose of isoflurane and were humanely euthanized.
IACUC: Ethics statement: This study was approved by the Experimental Animal Welfare and Ethical Review Board of Wuhan Institute of Biological Products Co.Sex as a biological variable Male K18-hACE2 mice (6–8 weeks old, purchased from GemPharmatech Co., Ltd. Company.) were randomly distributed into groups (n = 3–6 mice per group). Randomization Male K18-hACE2 mice (6–8 weeks old, purchased from GemPharmatech Co., Ltd. Company.) were randomly distributed into groups (n = 3–6 mice per group). Blinding not detected. Po… SciScore for 10.1101/2022.05.27.493682: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Euthanasia Agents: At the end point of the experiment, all remaining animals in the monoclonal antibody-administered group received an overdose of isoflurane and were humanely euthanized.
IACUC: Ethics statement: This study was approved by the Experimental Animal Welfare and Ethical Review Board of Wuhan Institute of Biological Products Co.Sex as a biological variable Male K18-hACE2 mice (6–8 weeks old, purchased from GemPharmatech Co., Ltd. Company.) were randomly distributed into groups (n = 3–6 mice per group). Randomization Male K18-hACE2 mice (6–8 weeks old, purchased from GemPharmatech Co., Ltd. Company.) were randomly distributed into groups (n = 3–6 mice per group). Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Then cells were stained with anti-mouse IgG Taxes red conjugated antibody and anti-human IgG FITC-conjugated antibody (Sigma, USA) for another 30 min then analyzed by FACS Aria II (BD, USA). anti-mouse IgGsuggested: Noneanti-human IgGsuggested: NoneAntibody Binding Kinetics Measured by SPR: The binding kinetics of mAbs to SARS-CoV-2 Delta-RBD or Omicron-RBD monomer were analyzed using SPR (Biacore 8K; GE Healthcare). SARS-CoV-2suggested: NoneDelta-RBDsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cells, Viruses and Proteins: Cell lines (HEK293T and Vero E6 cells) were initially acquired from the American Type Culture Collection (ATCC; USA). Vero E6suggested: RRID:CVCL_XD71)HEK293T-hACE2-cells were generated via the overexpression of the human ACE2 receptor in HEK293T cells and were used in the neutralization assays of pseudoviruses. HEK293T-hACE2-cellssuggested: NoneThen mixtures were added to 2.5 × 105 HEK293T cells expressing ACE2 and incubated at 4 °C for another hour. HEK293Tsuggested: RRID:CVCL_HA71)HEK293-hACE2 cells (2.5 × 104 cells/100μL per well) were then added into the mixture and incubated at 37 °C in a humidified atmosphere with 5% CO2 for 23 h to 25 h. HEK293-hACE2suggested: NoneExperimental Models: Organisms/Strains Sentences Resources Male K18-hACE2 mice (6–8 weeks old, purchased from GemPharmatech Co., Ltd. Company.) were randomly distributed into groups (n = 3–6 mice per group). K18-hACE2suggested: RRID:IMSR_GPT:T037657)Recombinant DNA Sentences Resources The SARS-CoV-2 Spike ectodomain (1-1208) with a C-terminal Strep tag for purification and a foldon tag for trimerization was inserted into the pFastBac-Dual vector (Invitrogen) and was expressed using Bac-to-Bac baculovirus system (Invitrogen). pFastBac-Dualsuggested: RRID:Addgene_137166)The cDNA encoding SARS-CoV-2 Omicron Spike was synthesized (GenBank ID: ULC25168.1) and cloned into the pCAG vector. pCAGsuggested: RRID:Addgene_74288)Software and Algorithms Sentences Resources All of these data were analyzed using Flow Jo. Flow Josuggested: (FlowJo, RRID:SCR_008520)All statistical analysis was performed using GraphPad Prism 8. GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Coot v. Cootsuggested: (Coot, RRID:SCR_014222)Figures were generated using PyMOL 2.0.779 PyMOLsuggested: (PyMOL, RRID:SCR_000305)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: 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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