Comprehensive Evaluation of ACE2-Fc Combination with Neutralization Antibody on Broad Protection against SARS-CoV-2 and Its Variants

  1. Haoneng Tang
  2. Yong Ke
  3. Hang Ma
  4. Lei Han
  5. Lei Wang
  6. Huifang Zong
  7. Yunsheng Yuan
  8. Zhenyu Wang
  9. Yang He
  10. Yunsong Chang
  11. Shusheng Wang
  12. Junjun Liu
  13. Yali Yue
  14. Wenbo Xu
  15. Xiaoju Zhang
  16. Ziqi Wang
  17. Li Yang
  18. Hua Chen
  19. Yanlin Bian
  20. Baohong Zhang
  21. Yunji Liao
  22. Haiyang Yin
  23. Yi Chen
  24. En Zhang
  25. Xiaoxiao Zhang
  26. Hua Jiang
  27. Yueqing Xie
  28. John Gilly
  29. Mingyuan Wu
  30. Tao Sun
  31. Jianwei Zhu

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Abstract

Emerging SARS-CoV-2 variants are threatening the efficacy of antibody therapies. Combination treatments including ACE2-Fc have been developed to overcome the evasion of neutralizing antibodies (NAbs) in individual cases. Here we conducted a comprehensive evaluation of this strategy by combining ACE2-Fc with NAbs of diverse epitopes on the RBD. NAb+ACE2-Fc combinations efficiently neutralized HIV-based pseudovirus carrying the spike protein of the Delta or Omicron variants, achieving a balance between efficacy and breadth. In an antibody escape assay using replication-competent VSV-SARS-CoV-2-S, all the combinations had no escape after fifteen passages. By comparison, all the NAbs without combo with ACE2-Fc had escaped within six passages. Further, the VSV-S variants escaped from NAbs were neutralized by ACE2-Fc, revealing the mechanism of NAb+ACE2-Fc combinations survived after fifteen passages. We finally examined ACE2-Fc neutralization against pseudovirus variants that were resistant to the therapeutic antibodies currently in clinic. Our results suggest ACE2-Fc is a universal combination partner to combat SARS-CoV-2 variants including Delta and Omicron.

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  1. Version published to 10.1101/2022.01.17.475291v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2022.01.17.475291: (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
    ACE2-Fc and Antibodies: The sequence of ACE2-Fc the same as the sequence of mACE2-Ig [2] (catalytic activity reduced variant) was synthesized and cloned into pcDNA3.4 to express in ExpiCHO™ Expression System (ThermoFisher).
    ACE2-Fc
    suggested: None
    The next day, after washing and blocking, S1 protein (Sino Biological) biotin-labeled using EZ-Link™ Sulfo-NHS-LC-LC-Biotin (ThermoFisher) was mixed with 50 μg/mL competitor antibodies before adding into the coated plates.
    Sulfo-NHS-LC-LC-Biotin
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    In brief, lentivirus bearing ACE2 gene was transduced to 293T cells to attain ACE2-293T cell pool, which was subsequently sorted out the 1% highest ACE2 expression cells with FACS Aria III instrument (BD).
    293T
    suggested: None
    ACE2-293T
    suggested: None
    At 48 h post-transfection, culture supernatants were collected and filtered through 0.22 μ m filter into fresh BHK21 cells.
    BHK21
    suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)
    If typical cytopathic effect (CPE) was observed 2 - 3 days after VSV infection, BHK-21 cells were transfected with pCAGGS-VSV-G plasmid to assist in creating passage 1 (P1), supernatants were collected and viruses were plaque-purified in Vero E6 cells.
    BHK-21
    suggested: ATCC Cat# CRL-6281, RRID:CVCL_1914)
    Vero E6
    suggested: RRID:CVCL_XD71)
    Recombinant DNA
    SentencesResources
    The VL and VH sequences of antibodies were synthesized and cloned into pcDNA3.4 to express in ExpiCHO™ Expression System.
    pcDNA3.4
    suggested: RRID:Addgene_131198)
    This plasmid together with psPAX2 and pLVX-Luc2 were transfected into 293T cells using Lipofectamine 3000 Reagent (ThermoFisher) according to the manufacture’s protocol.
    psPAX2
    suggested: RRID:Addgene_12260)
    pLVX-Luc2
    suggested: None
    The package plasmids of escape mutants from literatures were constructed by site-directed mutagenesis based on pMD2.G encoding WT spike.
    pMD2.G
    suggested: RRID:Addgene_12259)
    Recovery of Replication-Competent VSV-SARS-CoV-2-S: Briefly, co-transfection of VSV constructs (pVSVΔMT-ΔG-SpikeΔ21) with helper plasmids, pBS-N, P, L and G, was performed into BHK21 cells infected with a recombinant vaccinia virus (vTF7-3) expressing T7 RNA polymerase.
    pVSVΔMT-ΔG-SpikeΔ21
    suggested: None
    pBS-N
    suggested: None
    If typical cytopathic effect (CPE) was observed 2 - 3 days after VSV infection, BHK-21 cells were transfected with pCAGGS-VSV-G plasmid to assist in creating passage 1 (P1), supernatants were collected and viruses were plaque-purified in Vero E6 cells.
    pCAGGS-VSV-G
    suggested: None
    Software and Algorithms
    SentencesResources
    IC50 were determined by four-parameter nonlinear regression using GraphPad Prism.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Escape Mutants Data Analysis: Escape mutants data were downloaded from COG-UK Mutation Explorer (COG-UK-ME).
    Mutation Explorer
    suggested: None

    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: We detected the following sentences addressing limitations in the study:
    However, there is a limitation that the neutralization ability of ACE2-Fc was not efficient enough. APN01, a recombinant human ACE2 drug, has failed to show significant protection in COVID-19 patients in a phase II clinical trial (NCT04335136), probably because of its lack of Fc fusion to enhance pharmacokinetics and low affinity with RBD [28,29]. So it is necessary to engineer the ACE2 decoy receptors to improve their affinity with RBD in the meantime maintaining their broad neutralization spectrum. In previous studies, scientists have mutated and screened out ACE2 decoy receptors with higher affinity with RBD, which were evaluated by co-incubation with authentic SARS-CoV-2 and observed no emergence of escape variants [9]. In other work, S19W, T27W and N330Y mutations were included in ACE2 to enhance its affinity with RBD and demonstrate neutralization against antibody-resistant viruses [11]. Scientists also used molecular dynamics simulation to predict and design ACE2 decoy with T27Y/H34A mutations and confirmed its enhanced affinity against both WT and mutated RBD [30]. Structure-based approaches were used to engineer ACE2, resulting in enhanced neutralization against authentic SARS-CoV-2 [7]. Other investigators demonstrated that an engineered decoy receptor broadly binds spike variants in spike mutant libraries with saturation mutagenesis of RBD [10]. So these engineered ACE2 decoy receptors are promising partners for SARS-CoV-2 neutralizing antibodies and whether their ...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04335136CompletedRecombinant Human Angiotensin-converting Enzyme 2 (rhACE2) a…

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

    About SciScore

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  3. Version published to 10.1101/2022.01.17.475291v1 on bioRxiv