Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses
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Abstract
The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2–specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.
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SciScore for 10.1101/2020.06.08.140871: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: The human samples were obtained at the Rockefeller University Hospital under protocols approved by the University’s Institutional Review Board. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: All cell lines have been tested negative for contamination with mycoplasma and were obtained from the ATCC (with the exception of Huh-7.5). Table 2: Resources
Antibodies Sentences Resources Thereafter, a 55 µl aliquot of serially diluted plasma, monoclonal antibody or decoy was incubated with a 55 µl aliquot of HIV-1 (2-plasmid), HIV-1 (3-plasmid) or VSV-based SARS-CoV-2 … SciScore for 10.1101/2020.06.08.140871: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: The human samples were obtained at the Rockefeller University Hospital under protocols approved by the University’s Institutional Review Board. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: All cell lines have been tested negative for contamination with mycoplasma and were obtained from the ATCC (with the exception of Huh-7.5). Table 2: Resources
Antibodies Sentences Resources Thereafter, a 55 µl aliquot of serially diluted plasma, monoclonal antibody or decoy was incubated with a 55 µl aliquot of HIV-1 (2-plasmid), HIV-1 (3-plasmid) or VSV-based SARS-CoV-2 pseudovirus or rVSV/SARS-CoV-2/GFP containing approximately 1×103 infectious units for 1h at 37°C in a 96-well plate. HIV-1 ( 3-plasmid )suggested: NoneA rabbit polyclonal anti-SARS-CoV-2 nucleocapsid antibody (catalog no. GTX135357; GeneTex) was added to the cells at 1:500 dilution in blocking solution and incubated at 4 °C overnight. anti-SARS-CoV-2suggested: NoneAlternatively, J2, a mouse monoclonal anti-dsRNA antibody (catalog no. 10010500; Scicons) was added to the cells under similar conditions to detect virus infected cells. J2suggested: Noneanti-dsRNAsuggested: (Millipore Cat# MABE1134, RRID:AB_2819101)Goat anti-rabbit AlexaFluor 594 (catalog no. A-11012; Life Technologies) and goat anti-mouse AlexaFluor 488 (catalog no. A-11001; Life Technologies) were used as a secondary antibodies at a dilution of 1:2,000. anti-rabbitsuggested: Noneanti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources Cell lines: HEK-293T cells, HT1080 cells, Huh-7.5 hepatoma cells (H. sapiens) and VeroE6 kidney epithelial cells (Chlorocebus sabaeus) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1% nonessential amino acids (NEAA) and 10% fetal bovine serum (FBS) at 37°C and 5% CO2. HEK-293Tsuggested: NoneHuh-7.5suggested: RRID:CVCL_7927)Derivatives of 293T and HT1080 cells expressing ACE2 or ACE2* (a catalytically inactive mutant of ACE2) were generated by transducing 293T cells with CSIB(ACE2) or CSIB(ACE2*), respectively. HT1080suggested: NoneTo amplify rescued rVSVΔG/NG/NanoLuc. 5×106 293T cells were plated per 10 cm dish in 10ml in growth medium or 1.2×107 293T cells were plated in 15cm dishes. 293Tsuggested: NoneViral titers were measured on VeroE6 cells by standard plaque assay (PA). VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Software and Algorithms Sentences Resources The half maximal inhibitory concentration for plasma (NT50), antibodies (IC50) was determined using 4-parameter nonlinear regression curve fit to raw infectivity data measured as relative light units, or as the percentage of infected cells (GraphPad Prism). 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: We detected the following sentences addressing limitations in the study:A key caveat associated with the measurement of neutralizing antibody activity, whether using pseudotype assays or authentic SARS-CoV-2 virions, is that the level of neutralizing activity required to protect against SARS-CoV-2 infection in a natural situation is unknown (Kellam and Barclay, 2020). Moreover, while neutralization assays measure the ability of antibodies to inhibit viral entry, they do not capture features of the antiviral activity of antibodies such as antibody dependent cellular cytotoxicity that may be germane in vivo (Bournazos and Ravetch, 2017). Nevertheless, in vitro neutralizing activity has long been identified as a correlate of protection against infection in many viral infections (Plotkin, 2010), including coronaviruses (Kellam and Barclay, 2020). As such, we envisage that the techniques described herein might be of significant utility in curtailing the COVID19 pandemic.
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.
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