High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2

  1. Bernard Vanhove
  2. Odile Duvaux
  3. Juliette Rousse
  4. Pierre-Joseph Royer
  5. Gwénaëlle Evanno
  6. Carine Ciron
  7. Elsa Lheriteau
  8. Laurent Vacher
  9. Nadine Gervois
  10. Romain Oger
  11. Yannick Jacques
  12. Sophie Conchon
  13. Apolline Salama
  14. Roberto Duchi
  15. Irina Lagutina
  16. Andrea Perota
  17. Philippe Delahaut
  18. Matthieu Ledure
  19. Melody Paulus
  20. Ray T. So
  21. Chris Ka-Pun Mok
  22. Roberto Bruzzone
  23. Marc Bouillet
  24. Sophie Brouard
  25. Emanuele Cozzi
  26. Cesare Galli
  27. Dominique Blanchard
  28. Jean-Marie Bach
  29. Jean-Paul Soulillou

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Abstract

Perfusion of convalescent plasma (CP) has demonstrated a potential to improve the pneumonia induced by SARS-CoV-2, but procurement and standardization of CP are barriers to its wide usage. Many monoclonal antibodies (mAbs) have been developed but appear insufficient to neutralize SARS-CoV-2 unless two or three of them are being combined. Therefore, heterologous polyclonal antibodies of animal origin, that have been used for decades to fight against infectious agents might represent a highly efficient alternative to the use of CP or mAbs in COVID-19 by targeting multiple antigen epitopes. However, conventional heterologous polyclonal antibodies trigger human natural xenogeneic antibody responses particularly directed against animal-type carbohydrate epitopes, mainly the N-glycolyl form of the neuraminic acid (Neu5Gc) and the Gal α1,3-galactose (αGal), ultimately forming immune complexes and potentially leading to serum sickness or allergy. To circumvent these drawbacks, we engineered animals lacking the genes coding for the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) and α1,3-galactosyl-transferase (GGTA1) enzymes to produce glyco-humanized polyclonal antibodies (GH-pAb) lacking Neu5Gc and α-Gal epitopes. We found that pig IgG Fc domains fail to interact with human Fc receptors and thereby should confer the safety advantage to avoiding macrophage dependent exacerbated inflammatory responses, a drawback possibly associated with antibody responses against SARS-CoV-2 or to avoiding a possible antibody-dependent enhancement (ADE). Therefore, we immunized CMAH/GGTA1 double knockout (DKO) pigs with the SARS-CoV-2 spike receptor-binding domain (RBD) to elicit neutralizing antibodies. Animals rapidly developed a hyperimmune response with anti-SARS-CoV-2 end-titers binding dilutions over one to a million and end-titers neutralizing dilutions of 1:10,000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV-19, neutralized Spike/angiotensin converting enzyme-2 (ACE-2) interaction at a concentration < 1μg/mL and inhibited infection of human cells by SARS-CoV-2 in cytopathic assays. These data and the accumulating safety advantages of using glyco-humanized swine antibodies in humans warranted clinical assessment of XAV-19 to fight against COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.25.217158: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line AuthenticationAuthentication: After this step, the pellets were placed on ice and resuspended with propidium iodide, which is a viability marker of cells and the cytotoxic activity of antibodies was determined by flow cytometry, after gating on lymphocytes, based on their morphology.

    Table 2: Resources

    Antibodies
    SentencesResources
    After washing, bound FcγR molecules were revealed with peroxidase conjugated anti-tag antibody (Miltenyi, 120-003-811; 1/5000 in 2% bovine serum albumin) and tetramethylbenzidine (TMB) reagent.
    anti-tag
    suggested: None
    Bound pig IgG were revealed with a secondary anti-pig-HRP-conjugated antibody (Bethyl Laboratories, USA) diluted in washing buffer, at 1:1000, incubated 1h at RT and washed 3 times.
    anti-pig-HRP-conjugated
    suggested: None
    The human Fc tag was then revealed with a specific HRP-conjugated anti-human IgG secondary antibody (diluted in in PBS-Tween-0.05%-1% skimmed milk powder at 1:1000, incubated 1h at RT and washed 3 times).
    anti-human IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Recombinant proteins from SARS-CoV-2 have been manufactured from Hek-293 cells and were purchased from Interchim, Monluçon, France.
    Hek-293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Swine serum dilutions were mixed with equal volumes of SARS-CoV-2 (BetaCoV/Hong Kong/VM20001061/2020 [KH1], corresponding to the D614 variant) or SARS-CoV (strain HK39849, SCoV) at a dose of 200 plaque-forming units determined by Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    Binding to human FcγR: The binding of IgG from DKO swine to the FcγRI, FcγRIIa and FcγRIIb human receptors was tested using an ELISA assay and BIAcore surface plasmon resonance (SPR).
    BIAcore
    suggested: (Biacore T100 System, RRID:SCR_019679)
    His-Tagged FcγRI, FcγRIIa and FcγRIIb receptors (CD64/32a/32b, R&D Systems; 100μL, 2.4 μg/mL in 2% bovine serum albumin) were then added and incubated for 2h at room temperature (RT).
    R&D Systems
    suggested: None
    All statistical analyses were performed on GraphPad Software (GraphPad Software, San Diego, CA).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04431219RecruitingFirst in Human Study: LIS1, an Induction Treatment in Kidney…
    NCT04453384RecruitingStudy to Evaluate the Safety and Efficacy of XAV-19 in Patie…

    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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.07.25.217158 on bioRxiv