Prime-boost protein subunit vaccines against SARS-CoV-2 are highly immunogenic in mice and macaques

  1. Hyon-Xhi Tan
  2. Jennifer A Juno
  3. Wen Shi Lee
  4. Isaac Barber-Axthelm
  5. Hannah G Kelly
  6. Kathleen M Wragg
  7. Robyn Esterbauer
  8. Thakshila Amarasena
  9. Francesca L Mordant
  10. Kanta Subbarao
  11. Stephen J Kent
  12. Adam K Wheatley

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Abstract

SARS-CoV-2 vaccines are advancing into human clinical trials, with emphasis on eliciting high titres of neutralising antibodies against the viral spike (S). However, the merits of broadly targeting S versus focusing antibody onto the smaller receptor binding domain (RBD) are unclear. Here we assessed prototypic S and RBD subunit vaccines in homologous or heterologous prime-boost regimens in mice and non-human primates. We find S is highly immunogenic in mice, while the comparatively poor immunogenicity of RBD was associated with limiting germinal centre and T follicular helper cell activity. Boosting S-primed mice with either S or RBD significantly augmented neutralising titres, with RBD-focussing driving moderate improvement in serum neutralisation. In contrast, both S and RBD vaccines were comparably immunogenic in macaques, eliciting serological neutralising activity that generally exceed levels in convalescent humans. These studies confirm recombinant S proteins as promising vaccine candidates and highlight multiple pathways to achieving potent serological neutralisation.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.01.278630: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Ethics Statement: Animal studies and related experimental procedures were approved by the University of Melbourne Animal Ethics Committee (#1714193, #1914874).
    IRB: Human clinical study protocols were approved by the University of Melbourne Human Research Ethics Committee (#2056689), and all associated procedures were carried out in accordance with approved guidelines.
    Consent: All participants provided written informed consent in accordance with the Declaration of Helsinki.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableEight male macaques (Macaca nemestrina) (6-15 years old) were vaccinated with 100μg of SARS-CoV-2 spike or RBD immunogens formulated with 200μg of Monophosphoryl Lipid A (MPLA) liposomes (Polymun) (Baldrick et al., 2002) intramuscularly in the right quadriceps.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Isolated cells were stained with Aqua viability dye (Thermofisher) and Fc-blocked with a CD16/32 antibody (93; Biolegend).
    CD16/32
    suggested: None
    Cells were then surface stained with S/RBD probes and the following antibodies: B220 BUV737 (RA3-6B2), IgD BUV395 (11-26c.2a), CD45 Cy7APC (30-F11), SA BV786 (BD)
    CD45
    suggested: (BD Biosciences Cat# 564225, RRID:AB_2716861)
    Cells were then surface stained with S/RBD probes and the following antibodies: IgD AF488 (polyclonal; Southern Biotech), IgM BUV395 (G20-127), IgG BV786 (G18-145) (BD), CD14 BV510 (M5E2), CD3 BV510 (OKT3), CD8a BV510 (RPA-T8), CD16 BV510 (3G8), CD10 BV510 (HI10a)
    CD10
    suggested: (BioLegend Cat# 312220, RRID:AB_2563835)
    For intracellular transcription factor staining, cells were first stained with Aqua viability dye (Life Technologies), followed by S/RBD probes and surface antibodies: IgD AF488 (polyclonal; Southern Biotech), IgG BV786 (G18-145) (BD), CD14 BV510 (M5E2), CD3 BV510 (OKT3), CD8a BV510 (RPA-T8), CD16 BV510 (3G8), CD10 BV510 (HI10a)
    CD14
    suggested: (BioLegend Cat# 348805, RRID:AB_2889063)
    CD3
    suggested: None
    OKT3
    suggested: (BD Biosciences Cat# 566779, RRID:AB_2869862)
    CD8a
    suggested: (Meridian Life Science Cat# MAL10-078, RRID:AB_1070001)
    CD16
    suggested: None
    Flow cytometric detection of ex vivo and antigen-specific TFH: For ex vivo TFH quantification from mice, freshly isolated LN single cell suspensions were stained with the following antibodies: Live/dead Red (Life Technologies), CD3 BV510 (145-2C11; Biolegend), PD-1 BV786 (29F.1A12; Biolegend), CXCR5 BV421 (L138D7; Biolegend), CD4 BUV737 (RM4-5; BD), B220 BV605 (RA3-6B2; BD), and F4/80 PE-Dazzle 594 (T45-2342; BD).
    antigen-specific TFH
    suggested: None
    PD-1 BV786
    suggested: (BD Biosciences Cat# 563789, RRID:AB_2738425)
    B220
    suggested: (BD Biosciences Cat# 563708, RRID:AB_2738383)
    Non-human primate LN suspensions and PBMC were stained with the same protocol, using the following antibodies: Live/dead Aqua (Life Technologies), CD3 Alexa700 (SP34-2; BD), PD-1 BV421 (EH12.2H7; Biolegend), CXCR5 PE (MU5UBEE; ThermoFisher), CD4 BV605 (L200; BD), CD20 BV510 (2H7; BD), CD8 BV650 (RPA-T8; Biolegend), CD95 BUV737 (DX2; BD), ICOS PerCP-Cy5.5 (C398.4A; Biolegend)
    CXCR5
    suggested: (BD Biosciences Cat# 563105, RRID:AB_2738008)
    CD8
    suggested: (Leinco Technologies Cat# C666, RRID:AB_2829809)
    CD95
    suggested: (BD Biosciences Cat# 564710, RRID:AB_2738907)
    NHP cells were stained with the following antibodies: CD3 Alexa700 (SP34-2; BD), PD-1 BV421 (EH12.2H7; Biolegend), CXCR5 PE (MU5UBEE; ThermoFisher), CD4 BV605 (L200; BD), CD20 BV510 (2H7; BD), CD8 BV650 (RPA-T8; Biolegend), CD95 BUV737 (DX2; BD), CCR6 BV785 (G034E3; Biolegend), CXCR3 Pe-Dazzle594 (G02H57; Biolegend)
    CD4
    suggested: None
    CD20
    suggested: (BioLegend Cat# 302356, RRID:AB_2566316)
    CCR6
    suggested: (BioLegend Cat# 353422, RRID:AB_2563660)
    CXCR3
    suggested: None
    Plates were washed prior to incubation with HRP-conjugated secondary antibodies for mouse (1:10000; anti-mouse IgG; KPL), macaque (1:10000; anti-macaque IgG; Rockland) or human (1:20000; anti-human IgG; Sigma) for 1 hour at room temperature.
    anti-mouse IgG; KPL
    suggested: (SeraCare KPL Cat# 5450-0011, RRID:AB_2687537)
    anti-macaque IgG
    suggested: None
    anti-human IgG
    suggested: None
    ACE2-RBD inhibition ELISA: An ELISA to measure the ability of plasma antibodies to block interaction between recombinant human ACE2 (kindly provided by Merlin Thomas) and SARS-CoV-2 RBD was performed as previously described (Juno et al., 2020).
    ACE2
    suggested: (Thermo Fisher Scientific Cat# PA5-75453, RRID:AB_2719181)
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, the ectodomain of SARS-CoV-2 (isolate WHU1; residues 1 – 1208) with furin cleavage site removed and P986/987 stabilisation mutations36, a C-terminal T4 trimerisation domain, Avitag and His-tag, was expressed in Expi293 cells and purified by Ni-NTA size-exclusion chromatography.
    Expi293
    suggested: RRID:CVCL_D615)
    %Inhibition was plotted against plasma dilutions and the area under curve (AUC) was calculated using Graphpad Prism. Microneutralisation Assay: SARS-CoV-2 isolate CoV/Australia/VIC01/2020 (Caly et al., 2020) was passaged in Vero cells and stored at −80 °C.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Experimental Models: Organisms/Strains
    SentencesResources
    C57BL/6 or BALB/c mice were anesthetised by isoflurane inhalation prior to intramuscular injection of 50 μL vaccine in each hind quadriceps.
    BALB/c
    suggested: None
    B cell receptor sequencing and analysis: B cell receptor sequences were recovered from GC B cells (B220+IgD-GL7+) in the draining iliac lymph node of C57BL/6 mice (n=3) 14 days after a single immunisation with S.
    C57BL/6
    suggested: None
    Software and Algorithms
    SentencesResources
    %Inhibition was plotted against plasma dilutions and the area under curve (AUC) was calculated using Graphpad Prism. Microneutralisation Assay: SARS-CoV-2 isolate CoV/Australia/VIC01/2020 (Caly et al., 2020) was passaged in Vero cells and stored at −80 °C.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    All statistical analyses were performed using Prism (GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Flow cytometry data was analysed in FlowJo v9 or v10.
    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.

    About SciScore

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  2. Version published to 10.1101/2020.09.01.278630v1 on bioRxiv