Broadly neutralizing antibodies to SARS-related viruses can be readily induced in rhesus macaques

  1. Wan-ting He
  2. Meng Yuan
  3. Sean Callaghan
  4. Rami Musharrafieh
  5. Ge Song
  6. Murillo Silva
  7. Nathan Beutler
  8. Wen-Hsin Lee
  9. Peter Yong
  10. Jonathan L. Torres
  11. Mariane Melo
  12. Panpan Zhou
  13. Fangzhu Zhao
  14. Xueyong Zhu
  15. Linghang Peng
  16. Deli Huang
  17. Fabio Anzanello
  18. James Ricketts
  19. Mara Parren
  20. Elijah Garcia
  21. Melissa Ferguson
  22. William Rinaldi
  23. Stephen A. Rawlings
  24. David Nemazee
  25. Davey M. Smith
  26. Bryan Briney
  27. Yana Safonova
  28. Thomas F. Rogers
  29. Jennifer M. Dan
  30. Zeli Zhang
  31. Daniela Weiskopf
  32. Alessandro Sette
  33. Shane Crotty
  34. Darrell J. Irvine
  35. Andrew B. Ward
  36. Ian A. Wilson
  37. Dennis R. Burton
  38. Raiees Andrabi

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Abstract

To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting broadly neutralizing antibody responses to CoVs. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein with a two-shot protocol generated potent serum receptor binding domain cross-neutralizing antibody responses to both SARS-CoV-2 and SARS-CoV-1. Furthermore, responses were equally effective against most SARS-CoV-2 variants of concern (VOCs) and some were highly effective against Omicron. This result contrasts with human infection or many two-shot vaccination protocols where responses were typically more SARS-CoV-2 specific and where VOCs were less well neutralized. Structural studies showed that cloned macaque neutralizing antibodies, particularly using a given heavy chain germline gene, recognized a relatively conserved region proximal to the angiotensin converting enzyme 2 receptor binding site (RBS), whereas many frequently elicited human neutralizing antibodies targeted more variable epitopes overlapping the RBS. B cell repertoire differences between humans and macaques appeared to influence the vaccine response. The macaque neutralizing antibodies identified a pan-SARS–related virus epitope region less well targeted by human antibodies that could be exploited in rational vaccine design.

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  1. Version published to 10.1126/scitranslmed.abl9605
  2. ScreenIT

    SciScore for 10.1101/2021.07.05.451222: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: All human donors were assessed for medical decision-making capacity using a standardized, approved assessment, and voluntarily gave informed consent prior to being enrolled in the study.
    Field Sample Permit: Ethics Statement: The mice and rhesus macaque animal studies were approved and carried out in accordance with protocols provided to the Institutional Animal Care and Use Committee (IACUC) respectively at The Scripps Research Institute (TSRI; La Jolla, CA) under approval number 19-0020 and at Alphagenesis Institutional Animal Care and Use Committee (IACUC) under approval number AUP 19-10.
    IACUC: Ethics Statement: The mice and rhesus macaque animal studies were approved and carried out in accordance with protocols provided to the Institutional Animal Care and Use Committee (IACUC) respectively at The Scripps Research Institute (TSRI; La Jolla, CA) under approval number 19-0020 and at Alphagenesis Institutional Animal Care and Use Committee (IACUC) under approval number AUP 19-10.
    Sex as a biological variableOne group of 5 mice (C57BL/6 mice (Jackson Laboratory): 3 females and 2 males), aged ∼8 weeks, were immunized with 20µg SARS-CoV-2 S protein immunogen along with 5µg of SMNP adjuvant per animal per immunization.
    RandomizationFor conjugation, 500 µL (500 µg/mL) of randomly biotinylated SARS-CoV-2-RBD solution was mixed with 5 mg of cleaned T1 beads.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ELISA: 96-well half-area plates (Corning cat. #3690, Thermo Fisher Scientific) were coated overnight at 4°C with 2 μg/mL of mouse anti-His-tag antibody (Invitrogen cat. #MA1-21315-1MG, Thermo Fisher Scientific) in PBS.
    anti-His-tag
    suggested: None
    After washes, a secondary antibody conjugated with alkaline phosphatase (AffiniPure goat anti-human IgG Fc fragment specific, Jackson ImmunoResearch Laboratories cat. #109-055-008) diluted 1:1000 in 1% BSA/PBST, was added to each well.
    anti-human IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 109-055-008, RRID:AB_2337601)
    Percentage of neutralization was calculated according to the equation:

    The 50% pseudovirus neutralizing (IC50) or binding (ID50) antibody titer was calculated by non- linear fitting the plots of luciferase signals against antibody concentrations or sera dilution ratio in Graph Pad Prism.

    ID50
    suggested: (bNAber Cat# bNAberID_50, RRID:AB_2491067)
    For neutralization of anti-RBD antibody depleted macaque sera, the immune sera were depleted by two rounds of sequential incubation with SARS-CoV-2 RBD-conjugated with magnetic beads.
    anti-RBD
    suggested: None
    Phylogenetic analysis: Heavy chain sequences of neutralizing and non-neutralizing antibodies collected from animals K398 and K288 were processed using DiversityAnalyzer tool (92).
    K288
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Transient transfection: To express antibodies, the corresponding HC and LC plasmids were transiently transfected into the Expi293 cell (Life Technologies) at 3 x 106 cells/mL with FectoPRO PolyPlus transfection reagent (Polyplus Cat # 116-040).
    Expi293
    suggested: RRID:CVCL_D615)
    To express the soluble S ectodomain proteins from SARS-CoV-1, SARS-CoV-2 and their truncations, plasmids were transfected into HEK293F cells (Life Technologies) at 1 million cells/mL.
    HEK293F
    suggested: RRID:CVCL_6642)
    Cell lines: To generate HeLa-hACE2 cells, the human ACE2 lentivirus was transduced into HeLa cells.
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    Pseudovirus production: To generate the pseudoviruses, the MLV-gag/pol and MLV-CMV-Luciferase plasmids were co- transfected with full-length or variant SARS-CoV-1 or SARS-CoV-2 plasmid into HEK293T cells by using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019).
    HEK293T
    suggested: None
    Pseudovirus entry and serum neutralization assays: To test the inhibition of pseudovirus infection by serum or mAbs, we used the stable cell line HeLa-hACE2 generated by lentivirus transduction with consistent ACE2 expression level to carry out the assay.
    HeLa-hACE2
    suggested: None
    The truncated heavy chains were co-transfected with the corresponding light chains in 293Expi cells to produce the Fab.
    293Expi
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    One group of 5 mice (C57BL/6 mice (Jackson Laboratory): 3 females and 2 males), aged ∼8 weeks, were immunized with 20µg SARS-CoV-2 S protein immunogen along with 5µg of SMNP adjuvant per animal per immunization.
    C57BL/6
    suggested: None
    Recombinant DNA
    SentencesResources
    The ectodomains of SARS-CoV-1 and SARS-CoV-2 were constructed by PCR amplification and Gibson assembly (NEB, E2621L) cloning into the vector phCMV3 (Genlantis, USA).
    phCMV3
    suggested: None
    To generate gene fragments encoding SARS-CoV-1 RBD (residue 307-513), SARS-CoV-2 NTD (residue 1- 290), RBD (residue 320-527), RBD-SD1 (residue 320-591), and RBD-SD1-2 (residue 320-681) subdomains, PCR-amplifications were carried out from the SARS-CoV-1 and SARS-CoV-2 plasmids.
    SARS-CoV-2
    suggested: RRID:Addgene_164583)
    To produce the ACE2 lentivirus, the pBOB-hACE2 plasmid was co-transfected into HEK293T cells with lentiviral packaging plasmids pMDL, pREV, and pVSV-G (Addgene #12251, #12253, #8454) by Lipofectamine 2000 (Thermo Fisher Scientific, 11668019).
    pBOB-hACE2
    suggested: None
    pMDL
    suggested: None
    pREV
    suggested: None
    pVSV-G
    suggested: RRID:Addgene_138479)
    Crystal structure determination of Fab-RBD complexes: The coding sequence for receptor binding domain (RBD; residues 333-529) of the SARS-CoV-2 spike (S) protein was synthesized and cloned into a customized pFastBac vector (84), which was designed to fuse an N-terminal gp67 signal peptide and C-terminal His6-tag to the target protein.
    pFastBac
    suggested: RRID:Addgene_1925)
    Software and Algorithms
    SentencesResources
    Protocol was approved by the UCSD Human Research Protection Program.
    UCSD Human Research Protection Program
    suggested: None
    After incubation, the Protein A Sepharose was loaded into Econo-Pac columns (BioRad #7321010).
    BioRad
    suggested: None
    The biotinylated proteins were evaluated by BioLayer Interferometry using the SA biosensor.
    BioLayer
    suggested: (Harvard Medical School Center for Macromolecular Interactions Core Facility, RRID:SCR_018270)
    Micrographs were collected on a ThermoFisher Tecnai Spirit microscope operating at 120kV with a FEI Eagle CCD (4k) camera at 52,000 magnification using Leginon automated image collection software (81).
    Leginon
    suggested: (Leginon, RRID:SCR_016731)
    Particles were picked using DogPicker (82) and 3D classification was done using Relion 3.0 (83).
    Relion
    suggested: (RELION, RRID:SCR_016274)
    Iterative model building and refinement were carried out in COOT (90, 91), respectively.
    COOT
    suggested: (Coot, RRID:SCR_014222)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 23. 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.

    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.

  3. Version published to 10.1101/2021.07.05.451222 on bioRxiv