Vaccination with SARS-CoV-2 Spike Protein and AS03 Adjuvant Induces Rapid Anamnestic Antibodies in the Lung and Protects Against Virus Challenge in Nonhuman Primates

  1. Joseph R. Francica
  2. Barbara J. Flynn
  3. Kathryn E. Foulds
  4. Amy T. Noe
  5. Anne P. Werner
  6. Ian N. Moore
  7. Matthew Gagne
  8. Timothy S. Johnston
  9. Courtney Tucker
  10. Rachel L. Davis
  11. Britta Flach
  12. Sarah O’Connell
  13. Shayne F. Andrew
  14. Evan Lamb
  15. Dillon R. Flebbe
  16. Saule T. Nurmukhambetova
  17. Mitzi M. Donaldson
  18. John-Paul M. Todd
  19. Alex Lee Zhu
  20. Caroline Atyeo
  21. Stephanie Fischinger
  22. Matthew J Gorman
  23. Sally Shin
  24. Venkata Viswanadh Edara
  25. Katharine Floyd
  26. Lilin Lai
  27. Alida Tylor
  28. Elizabeth McCarthy
  29. Valerie Lecouturier
  30. Sophie Ruiz
  31. Catherine Berry
  32. Timothy Tibbitts
  33. Hanne Andersen
  34. Anthony Cook
  35. Alan Dodson
  36. Laurent Pessaint
  37. Alex Van Ry
  38. Marguerite Koutsoukos
  39. Cindy Gutzeit
  40. I-Ting Teng
  41. Tongqing Zhou
  42. Dapeng Li
  43. Barton F. Haynes
  44. Peter D. Kwong
  45. Adrian McDermott
  46. Mark G. Lewis
  47. Tong Ming Fu
  48. Roman Chicz
  49. Robbert van der Most
  50. Kizzmekia S. Corbett
  51. Mehul S. Suthar
  52. Galit Alter
  53. Mario Roederer
  54. Nancy J. Sullivan
  55. Daniel C. Douek
  56. Barney S. Graham
  57. Danilo Casimiro
  58. Robert A. Seder

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Abstract

Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike trimers (preS dTM) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHP). Binding and functional neutralization assays and systems serology revealed that NHP developed AS03-dependent multi-functional humoral responses that targeted multiple spike domains and bound to a variety of antibody F C receptors mediating effector functions in vitro . Pseudovirus and live virus neutralizing IC 50 titers were on average greater than 1000 and significantly higher than a panel of human convalescent sera. NHP were challenged intranasally and intratracheally with a high dose (3×10 6 PFU) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days post-challenge, vaccinated NHP showed rapid control of viral replication in both the upper and lower airways. Notably, vaccinated NHP also had increased spike-specific IgG antibody responses in the lung as early as 2 days post challenge. Moreover, vaccine-induced IgG mediated protection from SARS-CoV-2 challenge following passive transfer to hamsters. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine are sufficient to mediate protection against SARS-CoV-2 and support the evaluation of this vaccine in human clinical trials.

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  1. ScreenIT

    SciScore for 10.1101/2021.03.02.433390: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All animal procedures were performed according to protocols approved by the Institutional Animal Care and Use Committees of the National Institute of Allergy and Infectious Diseases, National Institutes of Health and Bioqual, Inc.
    Consent: Informed consent was obtained from all participants.
    RandomizationAnimals, immunizations, challenges and sampling: Rhesus macaques were randomized into groups of 8 based on age and body weight; each group had 2 females and 6 males, except for the PBS control group, which only had 5 males.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableAnimals, immunizations, challenges and sampling: Rhesus macaques were randomized into groups of 8 based on age and body weight; each group had 2 females and 6 males, except for the PBS control group, which only had 5 males.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Total IgG antibody titers were quantitated by using the Human IgG ELISA BASIC kit: (ALP) (Mabtech) following manufactures directions.
    Total IgG
    suggested: None
    Antibody titers to measles were quantitated by using Monkey Anti-Measles IgG ELISA kit (Alpha Diagnostics International) following manufactures directions.
    Anti-Measles IgG
    suggested: None
    Plates were incubated for 1 hr with 100 µL of goat-anti-human IgG (H+L, Cat#PA1-8463) or goat-anti-monkey IgG (H+L, Cat#A18811) secondary antibody conjugated to horseradish peroxidase (HRP, Thermo Fisher) in blocking buffer at 1:10,000 or 1:4,000 dilution, respectively.
    IgG
    suggested: (Thermo Fisher Scientific Cat# A18811, RRID:AB_2535588)
    The antibody-virus mixture was added to VeroE6 cell (C1008, ATCC, #CRL-1586
    C1008
    suggested: None
    The next morning, cells were stimulated with SARS-CoV-2 spike protein (S1 peptide pools, JPT Peptide Technologies, Inc.) at a final concentration of 2 μg/ml in the presence of monensin and costimulatory antibodies anti-CD28 and -49d (clones CD28.2 and 9F10, BD Biosciences) for 6 hours.
    anti-CD28
    suggested: None
    Intracellular cytokine staining and gating for CD4, CD8 was performed as previously described86 except the following monoclonal antibodies were added: PD-1 BUV737 (clone EH12.1, BD Biosciences) in place of PD-1 BV785, TNF-FITC (clone Mab11, BD Biosciences) in place of IL-5 BB515, and CD154 (CD40L) BV785 (clone 24-31, BioLegend)
    CD4
    suggested: (BD Biosciences Cat# 564419, RRID:AB_2744419)
    PD-1 BUV737
    suggested: (BD Biosciences Cat# 565299, RRID:AB_2739167)
    BB515
    suggested: None
    CD154
    suggested: (BioLegend Cat# 310841, RRID:AB_2572186)
    CD40L
    suggested: (BioLegend Cat# 310841, RRID:AB_2572186)
    BV785
    suggested: (BioLegend Cat# 310841, RRID:AB_2572186)
    Luminex Isotype and FcR Binding Assay: To determine relative concentrations of antigen-specific antibody isotypes and Fc-receptor binding activity in the rhesus samples, a customized Luminex isotype assay was performed as previously described87.
    antigen-specific
    suggested: None
    Mouse-anti-rhesus antibody detectors were then added for each antibody isotype (IgG1, IgG2, IgG3, IgG4, IgA, NIH Nonhuman Primate Reagent Resource supported by AI126683 and OD010976).
    IgG1 , IgG2
    suggested: None
    IgG3 , IgG4
    suggested: None
    Then, tertiary anti-mouse-IgG detector antibodies conjugated to PE were added.
    anti-mouse-IgG detector
    suggested: None
    Systems serology: In order to quantify antibody functionality of plasma samples, bead-based assays were used to measure antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP) and antibody-dependent complement deposition (ADCD), as previously described88,89,90.
    antibody-dependent neutrophil phagocytosis ( ADNP
    suggested: None
    antibody-dependent complement deposition ( ADCD)
    suggested: None
    SARS-CoV-2 spike protein (kindly provided by Eric Fischer, DFCI) was coupled to fluorescent streptavidin beads (Thermo Fisher) and incubated with diluted plasma samples to allow antibody binding to occur.
    SARS-CoV-2 spike protein
    suggested: None
    After phagocytosis of immune complexes, neutrophils were stained with an anti-CD66b Pacific Blue detection antibody (Biolegend).
    anti-CD66b
    suggested: None
    For quantification of antibody-dependent NK cell activation91, diluted plasma samples were incubated in Nunc MaxiSorp plates (Thermo Fisher Scientific) coated with antigen.
    antibody-dependent NK cell activation91
    suggested: None
    antigen .
    suggested: None
    Immunohistochemistry was used to visualize SARS-CoV-2 nucleocapsid antigen (rabbit polyclonal, GeneTex), and eosinophils by staining for eosinophil peroxidase (rabbit polyclonal, Atlas Antibodies).
    SARS-CoV-2 nucleocapsid antigen
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The antibody-virus mixture was added to VeroE6 cell (C1008, ATCC, #CRL-1586
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    For ADCP, cultured human monocytes (THP-1 cell line, ATCC) were incubated with immune complexes to induce phagocytosis.
    THP-1
    suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006)
    Software and Algorithms
    SentencesResources
    The reaction was quenched by addition of 100 µL 1 N H2SO4 and absorbance was measured at a test wavelength of 450 nm and reference wavelength of 650 nm using SoftMax Pro software version 6.5 on a Spectramax Paradigm Microplate reader (Molecular Devices).
    SoftMax Pro
    suggested: None
    The FRNT-mNG50 titers were interpolated using a 4-parameter nonlinear regression in GraphPad Prism 8.4.3.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Flow cytometry: All phenotyping and ICS data were acquired on an BD FACSymphony flow cytometer and analyzed using FlowJo version 9.9.6 (Treestar, Inc., Ashland, OR).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistics: All statistical analyses were performed in Prism (version 8.4, GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    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: 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.

    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 10.1101/2021.03.02.433390 on bioRxiv