A multiplexed high-throughput neutralization assay reveals a lack of activity against multiple variants after SARS-CoV-2 infection

  1. Craig Fenwick
  2. Priscilla Turelli
  3. Céline Pellaton
  4. Alex Farina
  5. Jérémy Campos
  6. Charlène Raclot
  7. Florence Pojer
  8. Valeria Cagno
  9. Giuseppe Pantaleo
  10. Didier Trono

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

The detection of SARS-CoV-2-specific antibodies in the serum of an individual indicates prior infection or vaccination. However, it provides limited insight into the protective nature of this immune response. Neutralizing antibodies recognizing the viral Spike are far more revealing, yet their measurement traditionally requires virus- and cell-based systems that are costly, time-consuming, poorly flexible and potentially biohazardous. Here we present a cell-free quantitative neutralization assay based on the competitive inhibition of trimeric SARS-CoV-2 Spike protein binding to the angiotensin converting enzyme 2 (ACE2) viral receptor. This high-throughput method matches the performance of the gold standard live virus infectious assay, as verified with a panel of 206 seropositive donors with varying degrees of infection severity and virus-specific IgG titers, achieving 96.7% sensitivity and 100% specificity. Furthermore, it allows for the parallel assessment of neutralizing activities against multiple SARS-CoV-2 Spike variants of concern (VOC), which is otherwise unpredictable even in individuals displaying robust neutralizing antibody responses. Profiling serum samples from 59 hospitalized COVID-19 patients, we found that although most had high activity against the 2019-nCoV Spike and to a lesser extent the B.1.1.7 variant, only 58% could efficiently neutralize a Spike derivative containing mutations present in the B.1.351 variant. In conclusion, we have developed an assay that has proven its clinical relevance in the large-scale evaluation of effective neutralizing antibody responses to VOC after natural infection and that can be applied to the characterization of vaccine-induced antibody responses and of the potency of human monoclonal antibodies.

Once sentence summary

Multiplexed cell-free neutralization assay for quantitative assessment of serum antibody responses against Spike mutations in SARS-COV-2 variants

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2021.04.08.21255150: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Study design and use of subject sera samples were approved by the Institutional Review Board of the Lausanne University Hospital and the ‘Commission d’éthique du Canton de Vaud’ (CER-VD) stated that authorization was not required.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Control wells were included on each 96-well plate that included beads alone, matching serum dilutions of a control pool of pre-COVID-19 pandemic healthy human sera (BioWest human serum AB males; VWR) and a positive control commercial anti-Spike blocking antibody (SAD-S35 from ACRO Biosciences) or recombinant produced REGN10933 neutralizing antibody discovered and marketed by Regeneron tested in a concentration response.
    anti-Spike blocking
    suggested: (Imported from the IEDB Cat# A1, RRID:AB_2848078)
    Beads were then washed on the magnetic plate washer and anti-mouse IgG-PE secondary antibody (OneLambda ThermoFisher) was added at a 1/100 dilution with 50µl per well.
    anti-mouse IgG-PE
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The virus-serum mixture was incubated at 37°C for 1 hour then 100 µl of the mixture was subsequently added to the VeroE6 cells in duplicates.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Spike-pseudotyped lentivectors were generated by co-transfecting HDM-IDTSpike-fixK, pHAGE2-CMV-Luc-ZSgreen, Hgpm2, REV1b and Tat1b (a kind gift from J.D. Bloom) plasmids into 293T cells for 24 hours with the following ratio 3/9/2/2/2 (18µg/ 56.7cm2 plate) using Fugene transfection reagent (Promega).
    293T
    suggested: None
    Following a further 1 hour incubation at 37 °C, the pseudoviruses/serum mixture was added to the 293T ACE2 cells.
    ACE2
    suggested: RRID:CVCL_DR94)
    Software and Algorithms
    SentencesResources
    Serum dilution response inhibition curves were generated with GraphPad Prism 8.3.0 using NonLinear four parameter curve fitting analysis of the log(agonist) vs. response.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Sensitivity, specificity and correlative of the tests were calculated with Excel and GraphPad prism.
    Excel
    suggested: None
    Wells were washed 3 times with water and plates were dried, scanned and analyzed for the density of live violet stained cells using ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Neutralization IC50 values were calculated as described above for the cell free neutralization assay using the GraphPad prism NonLinear four parameter curve fitting analysis.
    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 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 10.1101/2021.04.08.21255150v1 on medRxiv