Different Neutralization Sensitivity of SARS-CoV-2 Cell-to-Cell and Cell-Free Modes of Infection to Convalescent Sera

  1. Natalia Kruglova
  2. Andrei Siniavin
  3. Vladimir Gushchin
  4. Dmitriy Mazurov

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Abstract

The COVID-19 pandemic caused by SARS-CoV-2 has posed a global threat to human lives and economics. One of the best ways to determine protection against the infection is to quantify the neutralizing activity of serum antibodies. Multiple assays have been developed to validate SARS-CoV-2 neutralization; most of them utilized lentiviral or vesicular stomatitis virus-based particles pseudotyped with the spike (S) protein, making them safe and acceptable to work with in many labs. However, these systems are only capable of measuring infection with purified particles. This study has developed a pseudoviral assay with replication-dependent reporter vectors that can accurately quantify the level of infection directly from the virus producing cell to the permissive target cell. Comparative analysis of cell-free and cell-to-cell infection revealed that the neutralizing activity of convalescent sera was more than tenfold lower in cell cocultures than in the cell-free mode of infection. As the pseudoviral system could not properly model the mechanisms of SARS-CoV-2 transmission, similar experiments were performed with replication-competent coronavirus, which detected nearly complete SARS-CoV-2 cell-to-cell infection resistance to neutralization by convalescent sera. These findings suggest that the cell-to-cell mode of SARS-CoV-2 transmission, for which the mechanisms are largely unknown, could be of great importance for treatment and prevention of COVID-19.

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  1. Version published to 10.3390/v13061133
  2. ScreenIT

    SciScore for 10.1101/2021.05.04.442701: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Study was approved by the Local ethic committee of the Moscow First Infectious Disease Hospital (Protocol #2 dated
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: The cells have been tested negative for mycoplasma contamination.

    Table 2: Resources

    Antibodies
    SentencesResources
    Flow cytometry: To measure S protein expression on the surface of 293T pseudovirus producing cells, 3×105 live cells were incubated with the serum from a convalescent donor at the 1:100 dilution in PSB for 30 min followed by the incubation with secondary anti-human IgG antibodies conjugated with PE (1:250, # H10104, ThermoFisher Scientific) for 30 min.
    anti-human IgG
    suggested: (Thermo Fisher Scientific Cat# H10104, RRID:AB_2536546)
    ACE2 expression was assessed by staining cells with polyclonal rabbit antibodies against human ACE2 (PAB886Hu01, Cloud-Clone Corp.) followed by secondary anti-rabbit antibodies conjugated with PE (1:250, # P-2771MP, ThermoFisher Scientific).
    human ACE2
    suggested: None
    anti-rabbit
    suggested: (Molecular Probes Cat# P2771MP, RRID:AB_221651)
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: The human embryonic kidney 293T cells were obtained through NIH AIDS Research and Reference Reagent Program.
    293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    The next day, the transfected cells and 293T/ACE2 cells were detached with 1mM EDTA, mixed at the ratio of 1:1 and plated in wells of a 24-well plate with the total number of 105 cells per well.
    293T/ACE2
    suggested: RRID:CVCL_YZ65)
    The mixture was then combined with 5.4×104 uninfected Vero E6 cells in 96-well plates in the total volume of 200 µl and incubated for 5 days at 37°C.
    Vero E6
    suggested: None
    Recombinant DNA
    SentencesResources
    The HIV-1 (strain NL4-3) packaging plasmids pCMV-dR8-2 (# 12263), vector pCMV-VSV-G for expression of the protein G from vesicular stomatitis virus (# 8454) were obtained from Addgene; reporter plasmids pUCHR-inLuc-mR and pUCHR-IR-GFP were described previously 37,38.
    pCMV-VSV-G
    suggested: RRID:Addgene_8454)
    The plasmid pUCHR-hACE2 was generated by subcloning the ACE2 coding sequence from the pCG1-hACE2 plasmid obtained from Prof. Dr. Stefan Pöhlmann
    pUCHR-hACE2
    suggested: None
    pCG1-hACE2
    suggested: None
    Infection biology unit of the German Primate Center, Leibniz Institute for Primate Research) into lentiviral vector pUCHR.
    pUCHR
    suggested: RRID:Addgene_58955)
    The next day, the cells were transfected with 0.66 µg of pCMV-dR8-2, 0.88 µg of pUCHR-hACE2, and 0.22 µg pCMV-VSVG using Lipofectamine 2000 (ThermoFisher Scientific) according to the manufacturer’s instruction.
    pCMV-VSVG
    suggested: None
    The next day, the cells were transfected with 5 µg pCMV-dR8-2, 6.67 µg pUCHR-inLuc-mR or pUCHR-IR-GFP, and 3.33 µg of the S-protein coding plasmid using Lipofectamine 2000 (ThermoFisher Scientific) accordinhg to the manufacturer’s instruction.
    pUCHR-inLuc-mR
    suggested: None
    pUCHR-IR-GFP
    suggested: None
    After 24 h, the cells were transfected with 0,5 µg pCMV-GFPt and 0,3 µg pCG1-SARS-2-S, pCG1-SARS-2-SdC19 or pCG1-SARS-2-SdC19-H2 using Lipofectamine 2000 (ThermoFisher Scientific) according to the manufacturer’s instruction.
    pCMV-GFPt
    suggested: None
    pCG1-SARS-2-S
    suggested: None
    pCG1-SARS-2-SdC19
    suggested: None
    pCG1-SARS-2-SdC19-H2
    suggested: None
    The next day, the cells were transfected with 1.67 µg pCMV-dR8-2, 2.22 µg pUCHR-inLuc-mR, and 1.11 µg pCG1-SARS-2-SdFdC19 using Lipofectamine 2000 (ThermoFisher Scientific) according to the manufacturer’s instruction.
    pCMV-dR8-2
    suggested: None
    pCG1-SARS-2-SdFdC19
    suggested: None
    Software and Algorithms
    SentencesResources
    The HIV-1 (strain NL4-3) packaging plasmids pCMV-dR8-2 (# 12263), vector pCMV-VSV-G for expression of the protein G from vesicular stomatitis virus (# 8454) were obtained from Addgene; reporter plasmids pUCHR-inLuc-mR and pUCHR-IR-GFP were described previously 37,38.
    Addgene
    suggested: (Addgene, RRID:SCR_002037)
    FlowJo LLC software was used for histogram visualization.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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

    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.

  3. Version published to 10.1101/2021.05.04.442701v1 on bioRxiv