Evaluation of SARS-CoV-2 neutralizing antibodies using a vesicular stomatitis virus possessing SARS-CoV-2 spike protein

  1. Hideki Tani
  2. Miyuki Kimura
  3. Long Tan
  4. Yoshihiro Yoshida
  5. Tatsuhiko Ozawa
  6. Hiroyuki Kishi
  7. Shuetsu Fukushi
  8. Masayuki Saijo
  9. Kaori Sano
  10. Tadaki Suzuki
  11. Hitoshi Kawasuji
  12. Akitoshi Ueno
  13. Yuki Miyajima
  14. Yasutaka Fukui
  15. Ippei Sakamaki
  16. Yoshihiro Yamamoto
  17. Yoshitomo Morinaga

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Abstract

Background

SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is now classified in the genus Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult due to its infectious nature.

Methods

To circumvent the need for BSL-3 laboratories, an alternative assay was developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative donors against the pseudotyped virus infection evaluated by the CRNT were compared with antibody titers determined from an enzyme-linked immunosorbent assay (ELISA) or an immunofluorescence assay (IFA).

Results

The CRNT, which used whole blood collected from hospitalized patients with COVID-19, was also examined. As a result, the inhibition of pseudotyped virus infection was specifically observed in both serum and whole blood and was also correlated with the results of the IFA.

Conclusions

In conclusion, the CRNT for COVID-19 is a convenient assay system that can be performed in a BSL-2 laboratory with high specificity and sensitivity for evaluating the occurrence of neutralizing antibodies against SARS-CoV-2.

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  1. Version published to 10.1186/s12985-021-01490-7
  2. Version published to 10.21203/rs.3.rs-86916/v1 on Research Square
  3. ScreenIT

    SciScore for 10.1101/2020.08.21.262295: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Neutralization assays with patient blood samples: The patient sera used in this study were collected from participants after obtaining informed consent.
    IRB: Ethics statement: All of the samples, protocols, and procedures were approved by the Research Ethics Committee at the University of Toyama for the use of human subjects (approval number: R2019167).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cells: Human (Huh7 and 293T), monkey (Vero), hamster (BHK and CHO), and mouse (NIH3T3) cell lines were obtained from the American Type Culture Collection (Summit Pharmaceuticals International, Tokyo, Japan).
    Huh7
    suggested: None
    NIH3T3
    suggested: KCB Cat# KCB 200645YJ, RRID:CVCL_0594)
    Coomassie Brilliant Blue (CBB) staining and Immunoblotting: Transfection of 293T cells occurred with pCAG-SARS-CoV-2 Sfull, pCAG-SARS-CoV-2 St19, or VSV-G.
    293T
    suggested: None
    To examine neutralization of the human serum or whole blood samples against pseudotyped viruses, Vero cells were treated with serially diluted sera or whole blood of convalescent patients with COVID-19 or PCR-negative donors and then inoculated with Sfullpv, St19pv, or VSVpv.
    Vero
    suggested: None
    Immunofluorescence assay (IFA): For the IFA, BHK cells transfected with pCAG-SARS-CoV-2 S-full were fixed with acetone-methanol (1:1) at 4 °C for 20 min.
    BHK
    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 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.
    • 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.

  4. Version published to 10.1101/2020.08.21.262295 on bioRxiv