Prime-pull immunization of mice with a BcfA-adjuvanted vaccine elicits mucosal immunity and prevents SARS CoV-2 infection and pathology

  1. Mohamed M. Shamseldin
  2. Ashley Zani
  3. Adam Kenney
  4. Jack Evans
  5. Cong Zeng
  6. Kaitlin A. Read
  7. Kyle Caution
  8. Jesse M. Hall
  9. Jessica M. Brown
  10. Gilian Gunsch
  11. Kara N. Corps
  12. Supranee Chaiwatpongsakorn
  13. KC Mahesh
  14. Mijia Lu
  15. Rajendar Deora
  16. Mark E. Peeples
  17. Jianrong Li
  18. Kenneth J. Oestreich
  19. Shan-Lu Liu
  20. Jacob S. Yount
  21. Purnima Dubey

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Abstract

Vaccines against SARS-CoV-2 that induce mucosal immunity capable of preventing infection and disease remain urgently needed. We show that intramuscular priming of mice with an alum and BcfA-adjuvanted Spike subunit vaccine, followed by a BcfA-adjuvanted mucosal booster, generated Th17 polarized tissue resident CD4+ T cells, and mucosal and serum antibodies. The serum antibodies efficiently neutralized SARS-CoV-2 and its Delta variant, suggesting cross-protection against a recent variant of concern (VOC). Immunization with this heterologous vaccine prevented weight loss following challenge with mouse-adapted SARS-CoV-2 and reduced viral replication in the nose and lungs. Histopathology showed a strong leukocyte and polymorphonuclear (PMN) cell infiltrate without epithelial damage in mice immunized with BcfA-containing vaccines. In contrast, viral load was not reduced in the upper respiratory tract of IL-17 knockout mice immunized with the same formulation, suggesting that the Th17 polarized T cell responses are critical for protection. We show that vaccines adjuvanted with alum and BcfA, delivered through a heterologous prime-pull regimen, protect against SARS-CoV-2 infection without causing enhanced respiratory disease.

SIGNIFICANCE

There remains a need for SARS CoV-2 booster vaccines that generate mucosal immunity and prevent transmission. We show that systemic priming followed by a mucosal booster with a BcfA-adjuvanted subunit vaccine generates neutralizing antibodies and Th17 polarized systemic and tissue-resident immune responses that provide sterilizing immunity against wildtype SARS CoV-2, and a variant of concern. Importantly, in contrast to alum alone, the addition of BcfA prevents respiratory pathology. These results suggest that a BcfA-adjuvanted mucosal booster may elicit mucosal immunity in individuals previously immunized systemically with approved vaccines. This foundational study in mice sets the stage for testing our vaccine regimen in larger animal models as a booster vaccine.

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    SciScore for 10.1101/2022.04.06.487394: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Mice: All experiments were reviewed and approved by The Ohio State University (OSU) Institutional Animal Care and Use Committee (protocol numbers 2020A00000054 and 2020A00000001).
    Euthanasia Agents: Mice were euthanized by isoflurane overdose at 2 days post infection and samples for titer (caudal right lung lobe and nasal septum) and histopathological analyses (left lung lobe) were collected.
    Sex as a biological variableC57Bl/6J mice and IL-17 KO mice (male and female, older than 6 weeks old) were bred in-house.
    RandomizationImportantly, mice were randomized and assigned to specific harvest days before the start of the experiment.
    BlindingA board-certified veterinary pathologist (K.N.C.) was blind to the experimental groups, and sections were scored qualitatively on a scale ranging from 0 to 5 for the degree of cellularity and consolidation, the thickness of the alveolar walls, degeneration and necrosis, edema, hemorrhage, infiltrating alveolar/interstitial polymorphonuclear cells (PMNs), intrabronchial PMNs, perivascular and peribronchial lymphocytes and plasma cells, and alveolar macrophages.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were then washed 2x with PBS supplemented with 1% HI-FBS (1% FBS) (FACS buffer) and resuspended in Fc Block (clone 93) (eBioscience Ref. 14-0161-86) at 4°C for 5 min before surface staining with a cocktail of the following antibodies for 20 min at 4°C: CD3 V450 (Clone 17A2) (BD Cat. 561389), CD4 BV750 (Clone H129.19) (BD Cat. 747275), CD44 PerCP Cy5.5 (Clone IM7) (BD Cat. 560570), CD62L BV605 (Clone MEL-14) (BD Cat. 563252), CD69 BV711 (Clone HI.2F3) (BD Cat. 740664), CD103 PE-CF594 (Clone M290) (BD Cat. 565849).
    CD44
    suggested: (BD Biosciences Cat# 560570, RRID:AB_1727486)
    CD62L
    suggested: (BD Biosciences Cat# 563252, RRID:AB_2738098)
    CD69
    suggested: (BD Biosciences Cat# 740664, RRID:AB_2740352)
    Following permeabilization (eBioscience Cat. 00-8333-56), intracellular staining (30 minutes at 4°C) was done using a cocktail of the following antibodies: IFNγ FITC (clone XMG1.2) (eBioscience Cat. 11-7311-82), IL-17 PE CY7 (clone eBio17B7) (eBioscience Cat. 25-7177-82), IL-5 APC (clone TRFK5) (BD Cat. 554396).
    APC
    suggested: (BD Biosciences Cat# 554396, RRID:AB_398548)
    For examination of CD8+ population, the same panel was used with the replacement of CD4 BV750 antibody with CD8 APC (clone 53-6.7) (Biolegend Cat. 100712) and probing for IFNγ only.
    CD4
    suggested: (Miltenyi Biotec Cat# 130-109-536, RRID:AB_2657974)
    For surface staining, cells were incubated for 30 minutes at 4°C with the following antibodies, as indicated: Ghost viability dye (1:400; Tonbo Biosciences Cat. 13-0870-T100); CD4-AF488 (1:300; clone GK1.5; R&D Systems Cat. FAB554G), CD44-BV421 (1:300; clone IM7; BD Biosciences Cat. 563970); CD62L-APC-eFluor780 (1:300; clone MEL-14; ThermoFisher Cat. 47-0621-82); PD-1-PE-Cy7 (1:50; clone 29F.1A12; BioLegend Cat. 135216); Cxcr5-APC (1:50; clone SPRCL5; ThermoFisher Cat. 17-7185-82); CD19:APC (1:200; clone: GL7, BioLegend Cat. 144610); GL-7: PerCP-Cy5.5 (1:200; clone: 1D3, BioLegend Cat.152410); Fas-BV421 (1:300; clone Jo2; BD Biosciences Cat. 562633).
    CD4-AF488
    suggested: (SouthernBiotech Cat# 4515-30, RRID:AB_2796029)
    CD44-BV421
    suggested: None
    CD19:APC
    suggested: None
    Plates were washed 4x with PBS-Tween 20 and incubated with secondary anti-mouse IgG HRP (Abcam Ref. ab6789) or anti-mouse IgA HRP antibodies for 1 hr at RT.
    anti-mouse IgG
    suggested: (Abcam Cat# ab6789, RRID:AB_955439)
    anti-mouse IgA HRP
    suggested: None
    Rat anti mouse antibodies specific for IgG subtypes (IgG1, IgG2b, IgG2C& IgG3-Invitrogen) (Ref. 88-50630-88 & 88-50670-22) were added (1:1000 dilution) and incubated for 1 hr at RT.
    anti mouse
    suggested: None
    IgG1
    suggested: (Thermo Fisher Scientific Cat# 88-50630-88, RRID:AB_2574915)
    IgG2C& IgG3-Invitrogen
    suggested: None
    Briefly, tissue sections were stained with a rabbit monoclonal anti-nucleocapsid antibody (Genetex # GTX635686) using standard methodology.
    anti-nucleocapsid
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    These viruses were grown in Vero-TMPRSS2 cells for another passage and sequenced again to confirm the presence of the intact furin cleavage site before being used in experiments.
    Vero-TMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
    Media (DMEM (Gibco, 11965-092) supplemented with 10% (vol/vol) fetal bovine serum (Gibco, 11965-092) and 1% (vol/vol) penicillin/streptomycin (HyClone, SV30010)) was removed from seeded HEK293T/ACE2 cells (BEI NR-52511) and replaced with the virus/serum mixture.
    HEK293T/ACE2
    suggested: None
    Briefly, right caudal lung lobes were homogenized in 1mL PBS using glass beads and serial dilutions of the clarified lung homogenates were added to a monolayer of Vero E6 cells.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Experimental Models: Organisms/Strains
    SentencesResources
    C57Bl/6J mice and IL-17 KO mice (male and female, older than 6 weeks old) were bred in-house.
    C57Bl/6J
    suggested: None
    Software and Algorithms
    SentencesResources
    Flow cytometry antibodies were from eBioscience, BD Biosciences or R&D Systems (see Table 1).
    BD Biosciences or R&D Systems
    suggested: None
    50% neutralization titers (NT50) were determined by least-squares-fit non-linear regression in GraphPad Prism5.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Analysis was performed using FlowJo software, version 10.8.0 according to the gating strategy outlined in Figures S1 and S2.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical Analysis: Serological responses, T and B cell readouts, mouse weights and viral titer data were analyzed using Graphpad Prism by the methods outlined in each figure legend.
    Graphpad Prism
    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.

    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.

  2. Version published to 10.1101/2022.04.06.487394v1 on bioRxiv