T Cell Predominant Response to AAV-Spike Protects hACE2 Mice from SARS-CoV-2 Pneumonia

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Abstract

Prevention of COVID-19 is widely believed to depend on neutralization of SARS-CoV-2 by vaccine-induced humoral immunity 1,2 , raising concern that emerging escape variants may perpetuate the pandemic 3–6 . Here we show that a single intramuscular injection of Adeno-Associated Virus-6 (AAV6) or AAV9 encoding a modified, N-terminal domain deleted (ΔNTD) spike protein induces robust cellular immunity and provides long-term protection in k18-hACE2 transgenic mice from lethal SARS-CoV-2 challenge, associated weight loss and pneumonia independent of vaccine-induced neutralizing humoral immunity. In both mice and macaques, vaccine-induced cellular immunity results in the clearance of transduced muscle fibers coincident with macrophage and CD8+ cytotoxic T cell infiltration at the site of immunization. Additionally, mice demonstrate a strong Type-1 polarized cellular immunophenotype and equivalent ex vivo T cell reactivity to peptides of wt and alpha (B.1.1.7) variant spike. These studies demonstrate not only that AAV6 and AAV9 can function as effective vaccine platforms, but also that vaccines can provide long-term efficacy primarily through the induction of cellular immunity. The findings may provide an alternative approach to containment of the evolving COVID-19 pandemic and have broader implications for the development of variant-agnostic universal vaccines against a wider range of pathogens.

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  1. SciScore for 10.1101/2021.08.16.456441: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Mice: Murine experiments were executed in compliance with approval from the Animal Care and Use Committees of the University of Pennsylvania and University of Wisconsin at Madison. c57BL/10SnJ and B6.Cg-Tg(K18-ACE2)2Prlmn/J mice were purchased from the Jackson Laboratory.
    Euthanasia Agents: Toe pinch was performed to ensure mice were deeply anesthetized before mice were euthanized by cervical dislocation.
    Sex as a biological variablenot detected.
    RandomizationMixed gender 2-5-month-old C57BL/10 mice or 6–9-week-old k18-hACE2 mice were randomly assigned to groups and anesthetized with isoflurane before receiving an intra gastrocnemius injection of either 50 ul AAV or 50 ul formulation buffer using a custom 100 uL Hamilton syringe with a 32-gauge needle (475-41182, Lot 81008).
    BlindingAdditionally, k18-hACE2 mice were ear tagged as to keep the team performing the challenge experiments blinded to the animal’s treatment status. c57BL/10 muscle procurement, sectioning, and storage for immunohistochemistry: At pre-determined timepoints post-vaccination, mice were anesthetized with 4% isoflurane in an anesthesia chamber before being transferred to a nose cone where they were maintained on 4% isoflurane in 100% O2 at a flow rate of 1 L/minute.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The membrane was then washed 3×15 minutes in PBST and then incubated in secondary antibody solution (PBST + 5% BSA + Goat a-Rabbit-HRP 1:5000) (Abcam, ab205718) for one hour at room temperature.
    a-Rabbit-HRP
    suggested: None
    Also included in this mixture to neutralize any potential VSV-G carryover virus was 1E9F9, a mouse anti-VSV Indiana G, at a concentration of 600 ng/ml (Absolute Antibody, Ab01402-2.0).
    anti-VSV
    suggested: None
    Plates were then incubated in ddH2O for 2 minutes and then washed 5× with PBS before incubating with biotinylated anti-cytokine antibody at the manufacture’s recommended concentration in PBS + 1% BSA for 2 hr at RT.
    anti-cytokine
    suggested: None
    Plates were then incubated in ddH2O for 2 minutes and then washed 5× with PBS before incubating with ALP-conjugated anti-IFNγ antibody at the manufacture’s recommended concentration in PBS + 1% BSA for 2 hr at RT.
    anti-IFNγ
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    AAV6/9 vector generation: AAV6 and AAV9 were individually generated and purified by the University of Pennsylvania preclinical vector core using the triple transfection method in HEK293 cells as previously described12.
    HEK293
    suggested: None
    Transfection and Immunocytochemistry of C2C12 cells: 3.0E4 C2C12 cells were plated into each well of an 8 well chamber slide (Millipore, PEZGS0816) and cultured for 24 hours in growth media (DMEM high glucose supplemented with 10% FBS, 1× anti-anti, 1× GlutaMAX, 1× NEAA).
    C2C12
    suggested: None
    Neutralization Assay: Production of VSV pseudotyped with SARS-CoV-2 S: 293T cells plated 24 hours previously at 5 × 106 cells per 10 cm dish were transfected using calcium phosphate with 35 μg of pCG1 SARS-CoV-2 S D614G delta18 expression plasmid encoding a codon optimized SARS-CoV2 S gene with an 18-residue truncation in the cytoplasmic tail (kindly provided by Stefan Pohlmann).
    293T
    suggested: None
    Vero E6 cells stably expressing TMPRSS2 were seeded in 100 μl at 2.5×104 cells/well in a 96 well collagen coated plate.
    Vero E6
    suggested: None
    The serum-virus mixture was then used to replace the media on VeroE6 TMPRSS2 cells.
    VeroE6 TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: Murine experiments were executed in compliance with approval from the Animal Care and Use Committees of the University of Pennsylvania and University of Wisconsin at Madison. c57BL/10SnJ and B6.Cg-Tg(K18-ACE2)2Prlmn/J mice were purchased from the Jackson Laboratory.
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    Mixed gender 2-5-month-old C57BL/10 mice or 6–9-week-old k18-hACE2 mice were randomly assigned to groups and anesthetized with isoflurane before receiving an intra gastrocnemius injection of either 50 ul AAV or 50 ul formulation buffer using a custom 100 uL Hamilton syringe with a 32-gauge needle (475-41182, Lot 81008).
    C57BL/10
    suggested: None
    Recombinant DNA
    SentencesResources
    M8/M8B were cloned into the AAV transfer plasmid pZac2.1 along with (or flanked by) a CMV promoter and SV40 PolyA sequence.
    pZac2.1
    suggested: None
    Western Blot of transfected HEK293: M8/M8B-pZac2.1 plasmid transfected HEK293 cell pellets were lysed in RIPA lysis buffer (Santa Cruz Biotechnology, sc-24948) supplemented with protease inhibitor cocktail (Roche, 39802300).
    M8/M8B-pZac2.1
    suggested: None
    Neutralization Assay: Production of VSV pseudotyped with SARS-CoV-2 S: 293T cells plated 24 hours previously at 5 × 106 cells per 10 cm dish were transfected using calcium phosphate with 35 μg of pCG1 SARS-CoV-2 S D614G delta18 expression plasmid encoding a codon optimized SARS-CoV2 S gene with an 18-residue truncation in the cytoplasmic tail (kindly provided by Stefan Pohlmann).
    pCG1 SARS-CoV-2
    suggested: None
    Software and Algorithms
    SentencesResources
    Red blood cells were lysed with ACK lysis buffer (Quality Biological Inc.,
    Quality Biological
    suggested: None

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 3. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.


    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

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