Neutralization assay with SARS-CoV-1 and SARS-CoV-2 spike pseudotyped murine leukemia virions
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Abstract
Background
Virus neutralization by antibodies is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal murine leukemia virus genome encoding firefly luciferase. This assay design is intended for use in laboratories with biocontainment level 2 and therefore circumvents the need for the biocontainment level 3 that would be required for replication-competent SARS-CoV-2 virus. To validate the pseudovirion assay, we set up comparisons with other available antibody tests including those from Abbott, Euroimmun and Siemens, using archived, known samples.
Results
11 out of 12 SARS-CoV-2-infected patient serum samples showed neutralizing activity against SARS-CoV-2-spike pseudotyped MLV viruses, with neutralizing titers-50 (NT 50 ) that ranged from 1:25 to 1:1,417. Five historical samples from patients hospitalized for severe influenza infection in 2016 tested negative in the neutralization assay (NT 50 < 25). Three serum samples with high neutralizing activity against SARS-CoV-2/MLV pseudoviruses showed no detectable neutralizing activity (NT 50 < 25) against SARS-CoV-1/MLV pseudovirions. We also compared the semiquantitative Siemens SARS-CoV-2 IgG test, which measures binding of IgG to recombinantly expressed receptor binding domain of SARS-CoV-2 spike glycoprotein with the neutralization titers obtained in the pseudovirion assay and the results show high concordance between the two tests (R 2 = 0.9344).
Conclusions
SARS-CoV-2 spike/MLV pseudovirions provide a practical means of assessing neutralizing activity of antibodies in serum or plasma from infected patients under laboratory conditions consistent with biocontainment level 2. This assay offers promise also in evaluating immunogenicity of spike glycoprotein-based candidate vaccines in the near future.
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SciScore for 10.1101/2020.07.17.207563: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Cells: HEK293FT cells, Vero E6 cells, SupT1 cells and Huh7 cells were purchased from ATCC. Vero E6suggested: RRID:CVCL_XD71)SupT1suggested: BCRC Cat# 60191, RRID:CVCL_1714)Huh7suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)HEK293FT, Vero E6 and Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, US) supplemented with 10% FBS (Gibco, US) and 2mM L-glutamine (Gibco, US) at 37°C with 5% CO2. 293ACE2 cells … SciScore for 10.1101/2020.07.17.207563: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Cells: HEK293FT cells, Vero E6 cells, SupT1 cells and Huh7 cells were purchased from ATCC. Vero E6suggested: RRID:CVCL_XD71)SupT1suggested: BCRC Cat# 60191, RRID:CVCL_1714)Huh7suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)HEK293FT, Vero E6 and Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, US) supplemented with 10% FBS (Gibco, US) and 2mM L-glutamine (Gibco, US) at 37°C with 5% CO2. 293ACE2 cells were cultured in DMEM with 10% FBS, 2mM L-glutamine and 200ug/ml hygromycin B (ThermoFisher, US). HEK293FTsuggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)293ACE2suggested: RRID:CVCL_DR94)HEK293T-hACE2 cells were a gift from Adam Bailey and Emma Winkler and were constructed as follows. HEK293T-hACE2suggested: None293T cells were then transduced with lentivirus made from this construct. 293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Software and Algorithms Sentences Resources Neutralization titers NT50 and NT80 were calculated using Prism 8 (GraphPad, US). GraphPadsuggested: (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.
- 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.
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