In vitro characterization of engineered red blood cells as potent viral traps against HIV-1 and SARS-CoV-2

  1. Magnus A. G. Hoffmann
  2. Collin Kieffer
  3. Pamela J. Bjorkman

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Abstract

Engineered red blood cells (RBCs) expressing viral receptors could be used therapeutically as viral traps as RBCs lack nuclei and other organelles required for viral replication. Here we show that the combination of a powerful erythroid-specific expression system and transgene codon optimization yields high expression levels of the HIV-1 receptors CD4 and CCR5, as well as a CD4-glycophorin A (CD4-GpA) fusion protein on enucleated RBCs. Engineered RBCs expressing CD4 and CCR5 were efficiently infected by HIV-1, but CD4 or CD4-GpA expression in the absence of CCR5 was sufficient to potently neutralize HIV-1 in vitro. To facilitate continuous large-scale production of engineered RBCs, we generated erythroblast cell lines stably expressing CD4-GpA or ACE2-GpA fusion proteins, which produced potent RBC viral traps against HIV-1 and SARS-CoV-2. Our results suggest that this approach warrants further investigation as a potential treatment against viral infections.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.20.423607: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    2-3 ⨯ 105 cells were collected for each condition and samples were stained with the following antibodies: APC-conjugated anti-human CD4 (Invitrogen)
    anti-human CD4
    suggested: None
    Brilliant Violet 421-conjugated anti-human CD71 antibody (BioLegend) and the nuclear stain DRAQ5 (Abcam) were diluted 1:100 and 1:1,000 in PBS+ (PBS supplemented with 2% FBS).
    anti-human CD71
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    TZM-bl reporter cells (NIH AIDS Reagents Program) were added, and luminescence was measured after 48 hours.
    TZM-bl
    suggested: None
    SARS-CoV-2 neutralization assays: Lentivirus-based SARS-CoV-2 pseudovirus was generated by transfecting HEK293T cells with a luciferase-expressing lentiviral backbone plasmid, accessory plasmids (pHDM-Hgpm2, pHDM-tat1b, pRC/CMV-rev1b), and a plasmid encoding the SARS-CoV-2 Spike protein with a 21-residue cytoplasmic tail deletion (Wuhan Hu-1 strain;
    HEK293T
    suggested: None
    1.25 ⨯ 104 293T-ACE2 cells (provided by Dr. Jesse Bloom, Fred Hutchinson Cancer Research Center) were seeded per well on poly-L-Lysine-coated 96-well plates (Corning) 18 hours before infection.
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Software and Algorithms
    SentencesResources
    Codon optimization of transgene cDNA sequences was performed using the GeneArt GeneOptimizer software (Thermo Fisher Scientific)
    GeneArt GeneOptimizer
    suggested: None
    TZM-bl reporter cells (NIH AIDS Reagents Program) were added, and luminescence was measured after 48 hours.
    AIDS Reagents Program
    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: 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 10.1101/2020.12.20.423607 on bioRxiv