A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses

  1. Tiong Kit Tan
  2. Pramila Rijal
  3. Rolle Rahikainen
  4. Anthony H. Keeble
  5. Lisa Schimanski
  6. Saira Hussain
  7. Ruth Harvey
  8. Jack W. P. Hayes
  9. Jane C. Edwards
  10. Rebecca K. McLean
  11. Veronica Martini
  12. Miriam Pedrera
  13. Nazia Thakur
  14. Carina Conceicao
  15. Isabelle Dietrich
  16. Holly Shelton
  17. Anna Ludi
  18. Ginette Wilsden
  19. Clare Browning
  20. Adrian K. Zagrajek
  21. Dagmara Bialy
  22. Sushant Bhat
  23. Phoebe Stevenson-Leggett
  24. Philippa Hollinghurst
  25. Matthew Tully
  26. Katy Moffat
  27. Chris Chiu
  28. Ryan Waters
  29. Ashley Gray
  30. Mehreen Azhar
  31. Valerie Mioulet
  32. Joseph Newman
  33. Amin S. Asfor
  34. Alison Burman
  35. Sylvia Crossley
  36. John A. Hammond
  37. Elma Tchilian
  38. Bryan Charleston
  39. Dalan Bailey
  40. Tobias J. Tuthill
  41. Simon P. Graham
  42. Helen M. E. Duyvesteyn
  43. Tomas Malinauskas
  44. Jiandong Huo
  45. Julia A. Tree
  46. Karen R. Buttigieg
  47. Raymond J. Owens
  48. Miles W. Carroll
  49. Rodney S. Daniels
  50. John W. McCauley
  51. David I. Stuart
  52. Kuan-Ying A. Huang
  53. Mark Howarth
  54. Alain R. Townsend

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Abstract

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.

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  1. Version published to 10.1038/s41467-020-20654-7
  2. ScreenIT

    SciScore for 10.1101/2020.08.31.275701: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Mouse experiments were performed according to the UK Animals (Scientific Procedures) Act Project Licence (PBA43A2E4) and approved by the University of Oxford Local Ethical Review Body.
    IACUC: Pig immunisation and sampling: Pig studies were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and with approval from the local Animal Welfare and Ethical Review Body (AWERB) (Project Licence PP1804248).
    RandomizationNine weaned, Large White-Landrace-Hampshire cross-bred pigs of 8–10 weeks of age from a commercial rearing unit were randomly allocated to three treatment groups (5 µg RBD-SpyVLP, 50 µg RBD-SpyVLP or 100 µg spike glycoprotein) (n = 3).
    BlindingCPE was scored by researchers who were blinded to the identity of the samples.
    Power Analysisnot detected.
    Sex as a biological variableFemale C57BL/6 or BALB/c mice (∼ 5 weeks old at the time of first immunisation) were obtained from BMS Oxford or Envigo.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Plates were washed and incubated with horse radish peroxidase (HRP) conjugated goat-anti-human IgG antibody (Dako, P0447) (diluted 1:1,600 in PBS/0/1% BSA) for 1 h at RT.
    IgG
    suggested: None
    To test the reactivity of RBD-SpyVLP against a panel of anti-SARS-CoV-2 RBD antibodies, 50 μL of RBD-SpyVLP samples diluted in PBS to 0.5 μg/mL were coated on NUNC plates at 4 °C overnight.
    anti-SARS-CoV-2 RBD
    suggested: None
    To detect anti-RBD antibody in the immunised mouse sera, 50 µL purified RBD-6H
    anti-RBD
    suggested: None
    Plates were then washed with PBS and 50 μL of secondary HRP goat anti-mouse antibody (Dako P0417) diluted 1:800 in PBS/0.1% BSA was added to the wells for 1 h at RT.
    anti-mouse
    suggested: None
    Plates were washed and 50 μL of a secondary Alexa Fluor 647 goat anti-human antibody (Life Technologies A21455) (1:500 in PBS with 0.1% (w/v) BSA) were added for 1 h at RT.
    anti-human antibody ( Life Technologies A21455 )
    suggested: None
    Spike glycoprotein ELISA (Mouse and human sera): A cell-based ELISA as described previously 26 was used to determine the anti-spike glycoprotein antibody response in the mouse sera and convalescent plasma.
    anti-spike glycoprotein
    suggested: None
    For human sera, 50 μL of a secondary Alexa Fluor 647 goat-anti human antibody (1:500) (Life Technologies A21455) was used.
    human antibody
    suggested: None
    At 24 h post-infection, cells were fixed with 4% paraformaldehyde and permeabilised with 0.2% (v/v) Triton-X-100 in PBS stained to visualise virus plaques, as described previously for the neutralisation of influenza viruses 61, but using a rabbit polyclonal anti-NSP8 antibody (Antibodies Online; ABIN233792) and anti-rabbit-HRP conjugate (Bio-Rad) and detected using HRP on a TMB based substrate.
    anti-NSP8
    suggested: (Acris Antibodies GmbH Cat# AP09089SU-N, RRID:AB_2035808)
    anti-rabbit-HRP
    suggested: None
    RBD-specific antibody titres in oral and nasal swab fluids were determined by ELISA as detailed above except that the conjugated secondary antibody was replaced with either goat anti-porcine IgG HRP (Bio-Rad Antibodies) at 1:20,000 dilution in PBS with 1% (w/v) skimmed milk and 0.1% (v/v)
    anti-porcine IgG
    suggested: None
    Tween-20 or goat anti-porcine IgA HRP (Bio-Rad Antibodies) at 1:20,000 dilution in the same diluent.
    anti-porcine IgA
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    MDCK-RBD cells were generated as previously described 26, 39, 58.
    MDCK-RBD
    suggested: None
    Briefly, MDCK-Spike was produced by stably transfecting parental MDCK-SIAT1 cells with full-length SARS-CoV-2 spike glycoprotein cDNA using a lentiviral vector.
    MDCK-SIAT1
    suggested: ECACC Cat# 05071502, RRID:CVCL_Z936)
    Mouse sera was diluted as above and 50 μL was transferred to the washed plates seeded with MDCK-Spike cells for 1 h at RT.
    MDCK-Spike
    suggested: None
    Authentic SARS-CoV-2 virus neutralisation assay (PRNT): 96 well plates containing a confluent monolayer of Vero-E6 cells were incubated with 10-20 plaque forming units (PFU) of SARS CoV-2 (hCoV-19/England/02/2020, EPI_ISL_407073, kindly provided by Public Health England) and two-fold serial dilution of heat-inactivated mouse sera for 3 h at 37 °C, 5% CO2, in triplicate per serum sample.
    Vero-E6
    suggested: None
    Media in the wells seeded with Vero E6 was replaced with 100 µL DMEM with 10% (v/v) FBS and 1% Antibiotic-Antimycotic (Gibco) and 100 µL of the sera-virus mixture was added into the wells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Briefly, HEK293T cells were seeded at a density of 7.5 × 105 in 6-well plates before transfection with the following plasmids: 500 ng of SARS-CoV-2 spike, 600 ng p8.91 (HIV-1 gag-pol), 600 ng CSFLW
    HEK293T
    suggested: None
    An RBD protein with a C-terminal biotin acceptor peptide (RBD-BAP) was expressed from plasmid pOPINTTGNeo in Expi293 cells according to the manufacturer’s instructions.
    Expi293
    suggested: RRID:CVCL_D615)
    Experimental Models: Organisms/Strains
    SentencesResources
    Female C57BL/6 or BALB/c mice (∼ 5 weeks old at the time of first immunisation) were obtained from BMS Oxford or Envigo.
    C57BL/6
    suggested: None
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    Nine weaned, Large White-Landrace-Hampshire cross-bred pigs of 8–10 weeks of age from a commercial rearing unit were randomly allocated to three treatment groups (5 µg RBD-SpyVLP, 50 µg RBD-SpyVLP or 100 µg spike glycoprotein) (n = 3).
    Large White-Landrace-Hampshire cross-bred
    suggested: None
    Software and Algorithms
    SentencesResources
    The intensities of bands on each lane were quantified using ImageLab (version 5.2) software (Bio-Rad) and Fiji distribution of ImageJ (version 1.51n).
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    The intensity of the size distribution was normalised to the peak value and plotted in GraphPad Prism 8 (GraphPad Software).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Isoflurane (Abbott) lightly anaesthetised mice were immunised on day 0 and day 14 IM with 50 µL of RBD-SpyVLP at 0.1 µg or 0.5 µg or equivalent dose of unconjugated SpyTag-RBD or SpyCatcher003-mi3 VLP.
    Abbott
    suggested: (Abbott, RRID:SCR_010477)
    EPT is defined as the reciprocal of the highest serum dilution that gives a positive signal (blank+10 SD) determined using a five-parameter logistic equation calculated using GraphPad Prism 8.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Virus plaques were quantified and ND50 for sera was calculated using LabView software as described previously 61.
    LabView
    suggested: (LabView , RRID:SCR_014325)
    Cells were analysed using a BD LSRFortessa flow cytometer (BD Biosciences) and data analysed using FlowJo software (BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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.

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  3. Version published to 10.1101/2020.08.31.275701v1 on bioRxiv