Efficient production of Moloney murine leukemia virus-like particles pseudotyped with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein

  1. Sylvie Roy
  2. Karim Ghani
  3. Pedro O. de Campos-Lima
  4. Manuel Caruso

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Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak that started in China at the end of 2019 has rapidly spread to become pandemic. Several investigational vaccines that have already been tested in animals and humans were able to induce neutralizing antibodies against the SARS-CoV-2 spike (S) protein, however protection and long-term efficacy in humans remain to be demonstrated.

We have investigated if a virus-like particle (VLP) derived from Moloney murine leukemia virus (MLV) could be engineered to become a candidate SARS-CoV-2 vaccine amenable to mass production. First, we showed that a codon optimized version of the S protein could migrate efficiently to the cell membrane. However, efficient production of infectious viral particles was only achieved with stable expression of a shorter version of S in its C-terminal domain (ΔS) in 293 cells that express MLV Gag-Pol (293GP). The incorporation of ΔS was 15-times more efficient into VLPs as compared to the full-length version, and that was not due to steric interference between the S cytoplasmic tail and the MLV capsid. Indeed, a similar result was also observed with extracellular vesicles released from parental 293 and 293GP cells. The amount of ΔS incorporated into VLPs released from producer cells was robust, with an estimated 1.25 μ g/ml S2 equivalent (S is comprised of S1 and S2). Thus, a scalable platform that has the potential for production of pan-coronavirus VLP vaccines has been established. The resulting nanoparticles could potentially be used alone or as a boost for other immunization strategies for COVID-19.

IMPORTANCE

Several candidate COVID-19 vaccines have already been tested in humans, but their protective effect and long-term efficacy are uncertain. Therefore, it is necessary to continue developing new vaccine strategies that could be more potent and/or that would be easier to manufacture in large-scale. Virus-like particle (VLP) vaccines are considered highly immunogenic and have been successfully developed for human papilloma virus as well as hepatitis and influenza viruses. In this study, we report the generation of a robust Moloney murine leukemia virus platform that produces VLPs containing the spike of SARS-CoV-2. This vaccine platform that is compatible with lyophilization could simplify storage and distribution logistics immensely.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.16.298992: (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
    A human chimeric anti-S1 antibody (Genscript; 1:200 dilution) followed by an Alexa647-conjugated goat anti-human IgG (Jackson Laboratories; 1:400) were successively incubated with cells for labelling.
    anti-S1
    suggested: None
    anti-human IgG
    suggested: None
    Detached cells were labelled with a mouse anti-ACE2 antibody (R&D Systems, Minneapolis, MN1/200) followed by an Alexa488 goat anti-mouse (1:1,000; Invitrogen, Carlsbad, CA).
    anti-ACE2
    suggested: None
    anti-mouse
    suggested: None
    Immunoblotting was performed with a rabbit polyclonal antibody anti-S2 (1:400 dilution, SinoBiological, Beijing, China) and a rat monoclonal antibody anti-MLV p30 produced from the hybridoma R187 (1:2,000 dilution; American Type Culture Collection, Manassas, VA).
    anti-S2
    suggested: None
    anti-MLV
    suggested: (Creative Diagnostics Cat# DCABH-2412, RRID:AB_2476319)
    Blots were then incubated with secondary antibodies IRDylight680 goat anti-rat IgG (1:10,000; Invitrogen) and IRDye 800CW anti-rabbit IgG (1:10,000; Li-Cor Biosciences, Lincoln, NE), and analyzed with the Odyssey Infrared Imaging System (Li-Cor Biosciences).
    anti-rat IgG
    suggested: None
    anti-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell Lines: 293GP, 293 cells (ATCC, CRL-11268), and their derivatives expressing the ACE2 receptor (293-ACE2), S (293GP-S and 293-S), DeltaS (293GP-ΔS and 293-ΔS) and the Galv envelope (293GP-Galv) were cultured with Dulbecco’s modified Eagle’s medium (DMEM; Wisent, Canada) supplemented with 10% fetal calf serum (Life Technologies, Grand island, NY) and antibiotics (Wisent).
    293
    suggested: RRID:CVCL_DR94)
    Recombinant viruses from stable 293GP-S/GFP, 293GP-ΔS/GFP and 293GP-Galv/GFP cells were also produced similarly in 60-mm dishes.
    293GP-Galv/GFP
    suggested: None
    Syncytia Formation Assay: 293-ACE2 cells were mixed with 293, 293GP-S and 293GP-ΔS at a 9/1 ratio and plated at 4 × 105 cells/well in a 24-well plate.
    293-ACE2
    suggested: RRID:CVCL_DR94)
    The presence of S released in the supernatant of transiently transfected 293GP cells was analyzed by Western blot.
    293GP
    suggested: RCB Cat# RCB2354, RRID:CVCL_E072)
    Supernatants from confluent 293GP-S, 293GP-ΔS, 293-S and 293-ΔS cells were also harvested and concentrated from 60-mm dishes.
    293-ΔS
    suggested: None
    Software and Algorithms
    SentencesResources
    Plasmids: The expression plasmid pMD2ACE2iPuror containing the human angiotensin-converting enzyme (ACE2) cDNA used to generate ACE2 positive cells was constructed as follows: the ACE2 PmeI cDNA fragment obtained from the plasmid hACE2 (Addgene; #1786) was cloned in pMD2iPuror opened in EcoRV.
    Addgene
    suggested: (Addgene, RRID:SCR_002037)
    The fluorescence intensity of infected cells displayed in figure 5A were scanned using the Fiji software to evaluate the difference in viral titers (66).
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Serial dilutions of known amounts of C-terminally Fc-tagged S2 (BioVendor, Brno, Czech Republic) were used for quantification.
    BioVendor
    suggested: (BioVendor Laboratory Medicine, RRID:SCR_005143)

    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 10.1101/2020.09.16.298992 on bioRxiv