Lentiviral vector-based SARS-CoV-2 pseudovirus enables analysis of neutralizing activity in COVID-19 convalescent plasma

  1. Cevriye Pamukcu
  2. Elif Celik
  3. Ebru Zeynep Ergun
  4. Zeynep Sena Karahan
  5. Gozde Turkoz
  6. Mertkaya Aras
  7. Canan Eren
  8. Uluhan Sili
  9. Huseyin Bilgin
  10. Ilke Suder
  11. Baris Can Mandaci
  12. Baran Dingiloglu
  13. Ozge Tatli
  14. Gizem Dinler Doganay
  15. Safa Baris
  16. Nesrin Ozoren
  17. Tolga Sutlu

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Abstract

As the COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to spread around the globe, effective vaccination protocols are under deployment. Alternatively, the use of convalescent plasma (CP) therapy relies on the transfer of the immunoglobulin repertoire of a donor that has recovered from the disease as a means of passive vaccination. While the lack of an effective antiviral treatment inadvertently increases the interest in CP products, initial clinical evaluation on COVID-19 patients revealed that critical factors determining the outcome of CP therapy need to be defined clearly if clinical efficacy is to be expected. Measurement of neutralizing activity against SARS-CoV-2 using wildtype virus presents a reliable functional assay but the availability of suitable BSL3 facilities for virus culture restricts its applicability. Instead, the use of pseudovirus particles containing elements from the SARS-CoV-2 virus is widely applied to determine the activity of CP or other neutralizing agents such as monoclonal antibodies.

In this study, we present our approach to optimize GFP-encoding lentiviral particles pseudotyped with the SARS-CoV-2 Spike and Membrane proteins for use in neutralization assays. Our results show the feasibility of pseudovirus production using a C-terminal truncated Spike protein which is greatly enhanced by the incorporation of the D614G mutation. Moreover, we report that the use of Sodium Butyrate during lentiviral vector production dramatically increases pseudovirus titers. Analysis of CP neutralizing activity against particles pseudotyped with wildtype or D614G mutant Spike protein in the presence or absence the M protein revealed differential activity in CP samples that did not necessarily correlate with the amount of anti-SARS-CoV-2 antibodies.

Our results indicate that the extent of neutralizing activity in CP samples depends on the quality rather than the quantity of the humoral immune responses and varies greatly between donors. Functional screening of neutralizing activity using pseudovirus-based neutralization assays must be accepted as a critical tool for choosing CP donors if clinical efficacy is to be maximized.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.28.424590: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Donors and convalescent plasma samples: This study was approved by The Turkish Ministry of Health’s Scientific Research Platform (05.05.2020) and by Marmara University Clinical Research Ethics Committee (08.05.2020/554) and by Boğaziçi University Institutional Review Board for Research with Human Subjects (FMINAREK) (04.08.2020-2020/07).
    Consent: All donors signed informed consent for the study.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The membrane was blocked with 5% skimmed milk in TBS-T and incubated overnight at 4°C with polyclonal rabbit anti human-ACE2 antibody (#4355), (Cell Signaling Technology (CST), USA).
    anti human-ACE2 antibody ( #4355) , ( Cell Signaling Technology ( CST)
    suggested: None
    Criteria to become a convalescent plasma donor involved: COVID-19 infection confirmed with a positive PCR result, 14 days past after recovery with two negative PCR results or 28 days past after recovery, and a positive SARS-CoV-2 IgG antibody result.
    SARS-CoV-2 IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cloning: For the overexpression of hACE2 protein on 293FT cells, LeGo-ACE2-iT2 and LeGo-ACE2-iT2puro vectors were constructed.
    293FT
    suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
    Pseudovirus-based neutralization assay: Sixteen hours prior to infection, 1×104 293FT-hACE2 cells were seeded in 100 μl full growth media into flat bottom 96-well plates.
    293FT-hACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    All flow cytometry data were analyzed with FlowJo v10.1 (BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Analysis of the captured images was made by Fiji distribution of Image J software 1.52p (38).
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    For the preparation of graphs and for statistical analysis, Prism v8.4.3 software (GraphPad Software) was used.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.
    • No funding statement was detected.
    • 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 10.1101/2020.12.28.424590v1 on bioRxiv