Induction of SARS-CoV-2 protein S-specific CD8+ T cells in the lungs of gp96-Ig-S vaccinated mice

  1. Eva Fisher
  2. Laura Padula
  3. Kristin Podack
  4. Katelyn O’Neill
  5. Matthew M Seavey
  6. Padmini Jayaraman
  7. Rahul Jasuja
  8. Natasa Strbo

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Abstract

Given the aggressive spread of COVID-19-related deaths, there is an urgent public health need to support the development of vaccine candidates to rapidly improve the available control measures against SARS-CoV-2. To meet this need, we are leveraging our existing vaccine platform to target SARS-CoV-2. Here, we generated cellular heat shock chaperone protein, glycoprotein 96 (gp96), to deliver SARS-CoV-2 protein S (spike) to the immune system and to induce cell-mediated immune responses. We showed that our vaccine platform effectively stimulates a robust cellular immune response against protein S. Moreover, we confirmed that gp96-Ig, secreted from allogeneic cells expressing full-length protein S, generates powerful, protein S polyepitope-specific CD4+ and CD8+ T cell responses in both lung interstitium and airways. These findings were further strengthened by the observation that protein-S -specific CD8+ T cells were induced in human leukocyte antigen (HLA)-A2-02-01 transgenic mice thus providing encouraging translational data that the vaccine is likely to work in humans, in the context of SARS-CoV-2 antigen presentation.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.24.265090: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: The animals were housed and handled in accordance with the standards of the Association for the Assessment and Accreditation of Laboratory Animal Care International under University of Miami Institutional Animal Care & Use Committee-approved protocol.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableBoth female and male mice were used at 6–10 weeks of age.
    Cell Line AuthenticationContamination: mouse parvovirus (MPV), minute virus of mice (MVM), mycoplasma pulmonis, Mycoplasma sp., Polyoma, pneumonia virus of mice (PVM), Reovirus 3 (REO3), Sendai, Theiler’s murine encephalomyelitis virus (TMEV), and all test results were negative.

    Table 2: Resources

    Antibodies
    SentencesResources
    ELISA): Protein expression was verified by SDS-page and Western blotting using rabbit anti-SARS-CoV-2 spike glycoprotein antibody (MBS 150780) at 1/1000 dilution and secondary antibody: Peroxidase AffiniPure F(ab’)2 Fragment Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories)
    Anti-Rabbit IgG
    suggested: None
    One million cells were plated in 1 mL for 24 hours and secreted gp96-Ig production was determined by ELISA using antihuman IgG antibody for detection and human IgG1 as a standard (Figure 1b).
    antihuman IgG
    suggested: (GeneTex Cat# GTX28798, RRID:AB_374523)
    human IgG1
    suggested: None
    Rabbit anti-SARS-CoV-2 spike glycoprotein antibody (Abcam ab272504) and Donkey antirabbit IgG FITC, (BioLegend Cat# 406403) fluorescent antibody— were added in 1/50 and 1/100 dilutions of the antibodies combined in 5% BSA in PBS and/or rabbit isotype control (Abcam Ab172730 diluted 1/50), and incubated overnight at 4° C in a dark moisture chamber.
    anti-SARS-CoV-2 spike glycoprotein
    suggested: (Abcam Cat# ab272504, RRID:AB_2847845)
    antirabbit IgG
    suggested: None
    rabbit isotype control
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Generation of Vaccine Cell Lines: Human embryonic kidney (HEK)-293 cells, obtained from the American Tissue Culture Collection (ATCC, #CRL-1573) and human lung adenocarcinoma cell lines (AD100)40,41 (source: University of Miami, FL, USA) were transfected with 2 plasmids: B45 encoding gp96-Ig (source: University of Miami) and pcDNA™ 3.1(-) (Invitrogen), encoding full-length SARS-CoV-2 protein S gene (Genomic Sequence: NC_045512.2; NCBI Reference Sequence: YP_009724390.1 GenBank Reference Sequence: QHD43416).
    HEK)-293
    suggested: None
    HEK-293 and AD100 cells were simultaneously transfected with B45 and pcDNA 3.1 plasmid by lipofectamine (Invitrogen) following the manufacturers’ protocols.
    HEK-293
    suggested: None
    Equivalent number of 293-gp96-Ig-protein S and AD100-gp96-Ig-protein S cells that produce 200-ng gp96-Ig or PBS were injected via the subcutaneous (s.c.) route in C57Bl/6 and HLA-A2 transgenic mice.
    AD100-gp96-Ig-protein S
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Animals and Vaccination: Mice used in this study were colony-bred mice (C57Bl/6) and human leukocyte antigen (HLA)-A02-01 transgenic mice (C57BL/6-Mcph1Tg (HLA-A2.1)1Enge/J, Stock No: 003475) purchased from JAX Mice (the Jackson Laboratory for Genomic Medicine, Farmington, CT, USA)
    C57Bl/6
    suggested: None
    HLA)-A02-01
    suggested: RRID:IMSR_TAC:9659)
    C57BL/6-Mcph1Tg ( HLA-A2.1)1Enge/J
    suggested: None
    Software and Algorithms
    SentencesResources
    Peptide stimulated and non-stimulated cells were first labeled with live/dead detection kit (Thermo Fisher Scientific, Waltham, MA, USA) and then resuspended in BD Fc Block (clone 2.4G2) for 5 minutes at room temperature prior to staining with a surface-stain cocktail containing the following antibodies purchased from BioLegend® (San Diego, CA, USA): antigen presenting cell (APC)Cy7 CD45; Clone; AF700 CD3: Clone: 17A2; APC CD4:
    BioLegend®
    suggested: (BioLegend, RRID:SCR_001134)
    Analysis was performed using FlowJo™ software version 10.8
    FlowJo™
    suggested: (FlowJo, RRID:SCR_008520)
    Cells were acquired on SP6800 Sony instrument and data analyzed using FlowJo software version 10.8.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Comparisons of flow cytometry cell frequencies were measured by the 2-way analysis of variance (ANOVA) test with Holm-Sidak multiple-comparison test, *p<0.05, **p<0.01, and ***p<0.001, or unpaired T-tests (2-tailed) were carried out to compare the control group with each of the experimental groups (alpha level of 0.05) using the Prism software (GraphPad Software, San Diego, CA, USA).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    All statistical analysis was conducted using GraphPad Prism 8 software.
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT02117024TerminatedA Phase 2 Study of Viagenpumatucel-L (HS-110) in Patients Wi…
    NCT02439450Active, not recruitingA Study of Combination Therapies With Viagenpumatucel-L (HS-…

    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

    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 10.1101/2020.08.24.265090 on bioRxiv