Immunization with recombinant accessory protein-deficient SARS-CoV-2 protects against lethal challenge and viral transmission

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Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to a worldwide Coronavirus Disease 2019 (COVID-19) pandemic. Despite high efficacy of the authorized vaccines, protection against the surging variants of concern (VoC) was less robust. Live-attenuated vaccines (LAV) have been shown to elicit robust and long-term protection by induction of host innate and adaptive immune responses. We sought to develop a COVID-19 LAV by generating 3 double open reading frame (ORF)-deficient recombinant (r)SARS-CoV-2 simultaneously lacking two accessory open reading frame (ORF) proteins (ORF3a/ORF6, ORF3a/ORF7a, and ORF3a/ORF7b). Here, we report that these double ORF-deficient rSARS-CoV-2 have slower replication kinetics and reduced fitness in cultured cells as compared to their parental wild-type (WT) counterpart. Importantly, these double ORF-deficient rSARS-CoV-2 showed attenuation in both K18 hACE2 transgenic mice and golden Syrian hamsters. A single intranasal dose vaccination induced high levels of neutralizing antibodies against different SARS-CoV-2 VoC, and also activated viral component-specific T-cell responses. Notably, the double ORF-deficient rSARS-CoV-2 were able to protect, as determined by inhibition of viral replication, shedding, and transmission, against challenge with SARS-CoV-2. Collectively, our results demonstrate the feasibility to implement these double ORF-deficient rSARS-CoV-2 as safe, stable, immunogenic and protective LAV for the prevention of SARS-CoV-2 infection and associated COVID-19 disease.

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  1. SciScore for 10.1101/2022.03.13.484172: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Biosafety: All in vitro and in vivo experiments with infectious natural isolate or rSARS-CoV-2 were conducted under appropriate biosafety level 3 (BSL3) and animal BSL3 (ABSL3) laboratories, respectively, at Texas Biomedical Research Institute.
    IACUC: Biosafety (IBC) and Animal Care and Use Committees (IACUC)
    Euthanasia Agents: Animal infection was performed by intranasally inoculation after being anesthetized following gaseous sedation in an isoflurane chamber.
    Sex as a biological variableFive-week-old female K18 hACE2 transgenic mice were purchased from The Jackson Laboratory and five-week-old female golden Syrian hamsters were purchased from Charles River Laboratories.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: Multiplex cytokines and chemokines assay: Multiple cytokines and chemokines (IFN-α, IFN-γ, IL-6, IL-10, IL-17A, MCP-1, RANTES, and TNF-α) were measured using a custom 8-plex panel mouse ProcartaPlex assay (ThermoFisher Scientific, catalog# PPX-08-MXGZGFX), following the manufacturer’s instructions.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells, peptides, proteins, antibodies and viruses: African green monkey kidney epithelial cells (Vero E6, CRL-1586) were obtained from the American Type Culture Collection (ATCC, Bethesda, MD) and a Vero E6 cell line expressing hACE2 and TMPRSS2 (Vero AT) was obtained from BEI Resources (NR-54970).
    TMPRSS2
    suggested: None
    The cross-reactive mouse monoclonal antibody against SARS-CoV N protein, 1C7C7, was kindly provided by Dr. Thomas Moran at the Icahn School of Medicine at Mount Sinai.
    SARS-CoV N protein , 1C7C7
    suggested: None
    After 2 h incubation, plates were washed 3 time with PBST, then incubated with HRP-conjugated goat anti-mouse secondary antibody (ThermoFisher Scientific, catalog# 31430) at 25°C.
    anti-mouse
    suggested: (Thermo Fisher Scientific Cat# 31430, RRID:AB_228307)
    Experimental Models: Cell Lines
    SentencesResources
    After incubation at 37°C for 1 h with constant rotation, mixtures were transferred to confluent monolayers (4×105 cells/well, quadruplicates) of Vero E6 cells (Vero AT cells for o), as previously described 42.
    Vero E6
    suggested: None
    Vero AT
    suggested: None
    Multiplex cytokines and chemokines assay: Multiple cytokines and chemokines (IFN-α, IFN-γ, IL-6, IL-10, IL-17A, MCP-1, RANTES, and TNF-α) were measured using a custom 8-plex panel mouse ProcartaPlex assay (ThermoFisher Scientific, catalog# PPX-08-MXGZGFX), following the manufacturer’s instructions.
    MCP-1
    suggested: None
    Software and Algorithms
    SentencesResources
    Raw reads were quality filtered using Trimmomatic v0.39 37 and mapped to a SARS-CoV-2 USA-WA1/2020 strain reference genome (Genbank Accession No. MN985325) with Bowtie2 v2.4.1 38.
    Trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    Bowtie2
    suggested: (Bowtie 2, RRID:SCR_016368)
    We genotyped each sample for low frequency variants with LoFreq* v2.1.3.1 40 and filtered sites with less than 100x read depth or minor allele frequencies less than 1%.
    LoFreq*
    suggested: None
    Finally, we used SnpEff v4.3t 41 to identify the impact of potential variants on the protein coding regions in the SARS-CoV-2 reference genome.
    SnpEff
    suggested: (SnpEff, RRID:SCR_005191)
    Data were analyzed using FlowJo v10 (Tree Star)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Evaluation of lung pathological lesions: Macroscopic pathology scoring was evaluated using ImageJ software to determine the percentage of the total surface area of the lung (dorsal and ventral view) affected by consolidation, congestion, and atelectasis, as previously described 43.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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

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