Broad human and animal coronavirus neutralisation by SARS-CoV-2 S2-targeted vaccination

  1. Kevin W. Ng
  2. Nikhil Faulkner
  3. Katja Finsterbusch
  4. Mary Wu
  5. Ruth Harvey
  6. Saira Hussain
  7. Maria Greco
  8. Yafei Liu
  9. Svend Kjaer
  10. Charles Swanton
  11. Sonia Gandhi
  12. Rupert Beale
  13. Steve J. Gamblin
  14. Peter Cherepanov
  15. John McCauley
  16. Rodney Daniels
  17. Michael Howell
  18. Hisashi Arase
  19. Andeas Wack
  20. David L.V. Bauer
  21. George Kassiotis

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Abstract

Several common-cold coronaviruses (HCoVs) are endemic in humans and several variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged during the current Coronavirus disease 2019 (COVID-19) pandemic. Whilst antibody cross-reactivity with the Spike glycoproteins (S) of diverse coronaviruses has been documented, it remains unclear whether such antibody responses, typically targeting the conserved S2 subunit, contribute to or mediate protection, when induced naturally or through vaccination. Using a mouse model, we show that prior HCoV-OC43 S immunity primes neutralising antibody responses to otherwise subimmunogenic SARS-CoV-2 S exposure and promotes S2-targeting antibody responses. Moreover, mouse vaccination with SARS-CoV-2 S2 elicits antibodies that neutralise diverse animal and human alphacoronaviruses and betacoronaviruses in vitro , and protects against SARS-CoV-2 challenge in vivo . Lastly, in mice with a history of SARS-CoV-2 Wuhan-based S vaccination, further S2 vaccination induces stronger and broader neutralising antibody response than booster Wuhan S vaccination, suggesting it may prevent repertoire focusing caused by repeated homologous vaccination. The data presented here establish the protective value of an S2-targeting vaccine and support the notion that S2 vaccination may better prepare the immune system to respond to the changing nature of the S1 subunit in SARS-CoV-2 variants of concern (VOCs), as well as to unpredictable, yet inevitable future coronavirus zoonoses.

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    SciScore for 10.1101/2021.11.30.470568: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All experiments were approved by the Francis Crick Institute’s ethical committee and conducted according to local guidelines and UK Home Office regulations under the Animals Scientific Procedures Act 1986 (ASPA; project licence numbers PCD77C6D0 and P9C468066)
    Sex as a biological variablenot detected.
    RandomizationTime-points were defined based on prior experience and were not altered over the course of the study, and mice were randomly assigned to treatment groups before the start of each experiment.
    BlindingInvestigators performing live virus neutralisation and in vivo infection were blinded until after the experiment and analysis were completed.
    Power AnalysisSample sizes for all animal experiments were estimated based on prior experience to provide statistical power whilst minimizing animal use and were not altered over the course of the study.
    Cell Line AuthenticationContamination: Cell lines and plasmids: HEK293T, Vero E1, Vero E6, and SUP-T1 cells were obtained from the Francis Crick Institute’s Cell Services facility and verified as mycoplasma-free.
    Authentication: All human cell lines were further validated by DNA fingerprinting.

    Table 2: Resources

    Antibodies
    SentencesResources
    Virus plaques were visualised by immunostaining with a rabbit polyclonal anti-NSP8 antibody (ABIN233792; Antibodies Online) and an HRP-conjugated anti-rabbit antibody (1706515; Bio-Rad).
    anti-NSP8
    suggested: (Acris Antibodies GmbH Cat# AP09089SU-N, RRID:AB_2035808)
    anti-rabbit
    suggested: (Bio-Rad Cat# 170-6515, RRID:AB_11125142)
    Cells were immunostained with DAPI and Alexa488-conjugated anti-N antibody (CR3009; produced in-house) and imaged using an Opera Phenix (Perkin Elmer).
    anti-N
    suggested: None
    Performance of this high-throughput neutralisation assay has been benchmarked across laboratories participating in the establishment and validation of the WHO International Standard for SARS-CoV-2 antibody neutralisation (WHO/BS.2020.2403) (19).
    SARS-CoV-2
    suggested: None
    Peptide array: Antibody reactivity with a peptide array (12-mers overlapping by 10 amino acid residues) spanning the last 743 amino acids of SARS-CoV-2 S was carried out as previously described (28), except sera from immunised mice were used as the primary antibody and IRDye 800CW Goat anti-mouse IgG (Licor; 1:15,000 in blocking buffer) was used as a secondary antibody.
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    All viral isolates were propagated in Vero V1 cells.
    Vero V1
    suggested: None
    Cell lines and plasmids: HEK293T, Vero E1, Vero E6, and SUP-T1 cells were obtained from the Francis Crick Institute’s Cell Services facility and verified as mycoplasma-free.
    Vero E1
    suggested: None
    SUP-T1
    suggested: ICLC Cat# HTL96007, RRID:CVCL_1714)
    HEK293T cells expressing coronavirus spikes were generated by transient transfection using pcDNA3 or pCMV3-based expression plasmids and GeneJuice (EMD Millipore) 48 hours prior to use, as previously described (Fig.
    HEK293T
    suggested: None
    HCoV-NL63 S (UniProt ID: APF29071.1)
    HCoV-NL63
    suggested: RRID:CVCL_RW88)
    For PRNT assays, triplicate cultures of Vero E6 cells were incubated with SARS-CoV-2 variants and serial dilutions of heat-inactivated sera for 3 hours, the inoculum removed, and overlaid with virus growth medium containing 1.2% Avicel (FMC BioPolymer).
    Vero E6
    suggested: RRID:CVCL_XD71)
    Experimental Models: Organisms/Strains
    SentencesResources
    We tested nucleic acid and protein-based vaccine candidates targeting SARS-CoV-2 S and its constituent subunits using a prime-boost system in wild-type or K18-hACE2 transgenic mice, and tested in vitro activity of serum antibodies and in vivo protection following SARS-CoV-2 challenge.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Mice: Wild-type C57BL/6J (WT) and K18-hACE2 transgenic mice (48) on a C57BL/6J background were obtained from The Jackson Laboratory, and bred and maintained at the Francis Crick Institute’s Biological Research Facility under specific pathogen-free conditions.
    C57BL/6J
    suggested: None
    Recombinant DNA
    SentencesResources
    HEK293T cells expressing coronavirus spikes were generated by transient transfection using pcDNA3 or pCMV3-based expression plasmids and GeneJuice (EMD Millipore) 48 hours prior to use, as previously described (Fig.
    pCMV3-based
    suggested: None
    The expression vector (pME18S) encoding SARS-CoV-2 S2 was described previously (20).
    pME18S
    suggested: RRID:Addgene_52384)
    Expression vectors (pcDNA3) carrying a codon-optimised gene encoding the wild-type or D614G SARS-CoV-2 S (UniProt ID: P0DTC2) were kindly provided by Massimo Pizzato, University of Trento, Italy)
    pcDNA3
    suggested: RRID:Addgene_15475)
    Expression vectors (pCMV3) encoding HCoV-229E S (UniProt ID: APT69883.1)
    pCMV3
    suggested: RRID:Addgene_161029)
    Software and Algorithms
    SentencesResources
    Cells were incubated with sera (diluted 1:50 in PBS) for 30 min, washed with FACS buffer (PBS, 5% BSA, 0.05% sodium azide) and stained with anti-mouse IgG (Poly4053; Biolegend), IgA (Clone 11-44-2, Southern Biotech), and IgM (Clone RMM-1; Biolegend) diluted 1:200 in FACS buffer for 30 min.
    Poly4053
    suggested: None
    Cells were washed with FACS buffer and run on a Ze5 analyser (Bio-Rad) running Bio-Rad Everest software v2.4 and analysed using FlowJo v10
    Bio-Rad Everest
    suggested: None
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Plaques were quantified and IC50 values calculated using LabView or SigmaPlot software as previously described (28).
    LabView
    suggested: (LabView , RRID:SCR_014325)
    SigmaPlot
    suggested: (SigmaPlot, RRID:SCR_003210)
    Statistical analysis: Data were analysed and plotted in GraphPad Prism v8 (GraphPad Software) or SigmaPlot v14.0 (Systat Software).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 34, 35 and 36. 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

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  2. Version published to 10.1101/2021.11.30.470568 on bioRxiv