Prime-pull immunization of mice with a BcfA-adjuvanted vaccine elicits mucosal immunity and prevents SARS CoV-2 infection and pathology
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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
Ethics IACUC: 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 variable C57Bl/6J mice and IL-17 KO mice (male and female, older than 6 weeks old) were bred in-house. Randomization Importantly, mice were randomized and assigned to specific harvest days before the start of the experiment. Blinding A board-certified veterinary pathologist (K.N.C.) … 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
Ethics IACUC: 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 variable C57Bl/6J mice and IL-17 KO mice (male and female, older than 6 weeks old) were bred in-house. Randomization Importantly, mice were randomized and assigned to specific harvest days before the start of the experiment. Blinding A 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 Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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). CD44suggested: (BD Biosciences Cat# 560570, RRID:AB_1727486)CD62Lsuggested: (BD Biosciences Cat# 563252, RRID:AB_2738098)CD69suggested: (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). APCsuggested: (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. CD4suggested: (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-AF488suggested: (SouthernBiotech Cat# 4515-30, RRID:AB_2796029)CD44-BV421suggested: NoneCD19:APCsuggested: NonePlates 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 IgGsuggested: (Abcam Cat# ab6789, RRID:AB_955439)anti-mouse IgA HRPsuggested: NoneRat 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 mousesuggested: NoneIgG1suggested: (Thermo Fisher Scientific Cat# 88-50630-88, RRID:AB_2574915)IgG2C& IgG3-Invitrogensuggested: NoneBriefly, tissue sections were stained with a rabbit monoclonal anti-nucleocapsid antibody (Genetex # GTX635686) using standard methodology. anti-nucleocapsidsuggested: NoneExperimental Models: Cell Lines Sentences Resources 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-TMPRSS2suggested: 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/ACE2suggested: NoneBriefly, 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 E6suggested: RRID:CVCL_XD71)Experimental Models: Organisms/Strains Sentences Resources C57Bl/6J mice and IL-17 KO mice (male and female, older than 6 weeks old) were bred in-house. C57Bl/6Jsuggested: NoneSoftware and Algorithms Sentences Resources Flow cytometry antibodies were from eBioscience, BD Biosciences or R&D Systems (see Table 1). BD Biosciences or R&D Systemssuggested: None50% neutralization titers (NT50) were determined by least-squares-fit non-linear regression in GraphPad Prism5. GraphPadsuggested: (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. FlowJosuggested: (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 Prismsuggested: (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.
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