We would like to thank the Whelan lab for generously providing the VSV-S-eGFP virus used in this protocol (described in Case et al. 2020). We also thank Drs. Bill Cameron and Juthaporn Cowan (and team) for collecting the patient blood samples (REB protocol ID 20200371-01H). The authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by the generous support from the Ottawa Hospital Foundation and a grant from the Canadian Institutes of Health Research (#448323) and a Fast Grant from the Thistledown foundation for COVID-19 Science to C.S.I. T.R.J. is funded by an Ontario Graduate Scholarship and cluster Mitacs fellowship. JP is funded by a cluster Mitacs fellowship. T.A. is funded by a CIHR Banting Fellowship. We would also like to thank all the individuals who participated and donated their blood samples for this study.

1. Plating cells (Day 1) for the production and quantification of SARS-CoV-2 pseudovirus
<ol>
	<li>Preparation for tissue culture
	<ol>
		<li>Warm 1x Dulbecco&#39;s Phosphate-Buffered Saline (DPBS); Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin (optional); and 0.25% trypsin-ethylenediamine tetraacetic acid (EDTA) to 37 &#176;C in a water bath for approximately 15 min.</li>
		<li>Disinfect a tissue culture hood with 70% ethanol, and place tissue cultur...

<ol>
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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+critical+determinants+of+ACE2-SARS+CoV-2+RBD+interaction.">Characterization of critical determinants of ACE2-SARS CoV-2 RBD interaction.</a> International Journal of Molecular Sciences. 22 (5), 2268 (2021).</li><li>Azad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+S1+NanoBiT:+a+Nanoluciferase+complementation-based+biosensor+to+rapidly+probe+SARS-CoV-2+receptor+recognition.">SARS-CoV-2 S1 NanoBiT: a Nanoluciferase complementation-based biosensor to rapidly probe SARS-CoV-2 receptor recognition.</a> Biosensors and Bioelectronics. 180, 113122 (2021).</li><li>Azad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+for+SARS-CoV-2+vaccine+design:+Fusion+of+Spike+glycoprotein+transmembrane+domain+to+receptor-binding+domain+induces+trimerization.">Implications for SARS-CoV-2 vaccine design: Fusion of Spike glycoprotein transmembrane domain to receptor-binding domain induces trimerization.</a> Membranes. 10 (9), 215 (2020).</li><li>Cao, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+persistent+antibody+responses+against+the+receptor-binding+domain+of+SARS-CoV+spike+protein+in+recovered+patients.">Potent and persistent antibody responses against the receptor-binding domain of SARS-CoV spike protein in recovered patients.</a> Virology Journal. 7, 299 (2010).</li><li>To, K. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutralizing+antibody+and+soluble+ACE2+inhibition+of+a+replication-competent+VSV-SARS-CoV-2+and+a+clinical+isolate+of+SARS-CoV-2.">Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2.</a> Cell Host and Microbe. 28 (3), 475-485 (2020).</li><li>Garcia-Beltran, W. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Journal+Pre-proof+COVID-19+neutralizing+antibodies+predict+disease+severity+and+survival.">Journal Pre-proof COVID-19 neutralizing antibodies predict disease severity and survival.</a> Cell. 184 (2), 476-488 (2020).</li><li>Zeng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutralizing+antibody+against+SARS-CoV-2+spike+in+COVID-19+patients,+health+care+workers,+and+convalescent+plasma+donors.">Neutralizing antibody against SARS-CoV-2 spike in COVID-19 patients, health care workers, and convalescent plasma donors.</a> JCI insight. 5 (22), (2020).</li><li>Whitman, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+SARS-CoV-2+serology+assays+reveals+a+range+of+test+performance.">Evaluation of SARS-CoV-2 serology assays reveals a range of test performance.</a> Nature Biotechnology. 38 (10), 1174-1183 (2020).</li><li>Ainsworth, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+characteristics+of+five+immunoassays+for+SARS-CoV-2:+a+head-to-head+benchmark+comparison.">Performance characteristics of five immunoassays for SARS-CoV-2: a head-to-head benchmark comparison.</a> The Lancet Infectious Diseases. 20 (12), 1390-1400 (2020).</li><li>Sharifkashani, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin-converting+enzyme+2+(ACE2)+receptor+and+SARS-CoV-2:+Potential+therapeutic+targeting.">Angiotensin-converting enzyme 2 (ACE2) receptor and SARS-CoV-2: Potential therapeutic targeting.</a> European Journal of Pharmacology. 884, 173455 (2020).</li><li>Burki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+variants+of+SARS-CoV-2.">Understanding variants of SARS-CoV-2.</a> The Lancet. 397 (10273), 462 (2021).</li><li>Jayamohan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+pandemic:+a+review+of+molecular+diagnostic+tools+including+sample+collection+and+commercial+response+with+associated+advantages+and+limitations.">SARS-CoV-2 pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations.</a> Analytical and Bioanalytical Chemistry. 413 (1), 49-71 (2020).</li><li>Nie, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+SARS-CoV-2+neutralizing+antibody+by+a+pseudotyped+virus-based+assay.">Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay.</a> Nature Protocols. 15 (11), 3699-3715 (2020).</li><li>Crawford, K. 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The method described here may be adapted to suit varying lab environments and resources as needed. Importantly, the main limitation of this protocol is the necessity for a containment level 2 space and tissue culture hood. The application of a replicating RNA virus pseudotyped with the SARS-CoV-2 spike, such as VSV-S-eGFP, is a formidable alternative to the SARS-CoV-2 virus, which requires a containment level 3 working area, but may remain a limitation for some groups. All other steps described here are quite flexible an...

In December 2019, a novel coronavirus was identified, which we now know as SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19)1. SARS-CoV-2 is a betacoronavirus belonging to the Coronaviridae family. These enveloped viruses comprise a large positive-sense RNA genome and are responsible for respiratory and intestinal infections in both humans and animals2. As of May 2021&#160;there have been more than 157 million reported cases of COVID-19 globally and more than 3.2&#160;million deaths3. The development of an effective vaccine has become the primary goal of researchers around the globe with at least 77&#160;preclinical vaccines under investigation and 90&#160;currently undergoing clinical trials4.
Coronaviruses encode four structural proteins including the spike protein (S), nucleocapsid (N), envelope protein (E), and the membrane protein (M). Entry of SARS-CoV-2 requires interaction of the receptor-binding domain (RBD) of S with the host receptor, human angiotensin-converting enzyme 2 (hACE2), and subsequent membrane fusion following proteolytic cleavage by host cellular serine protease, transmembrane protease serine 2 (TMPRSS2)5,6,7,8,9,10. Humoral immunodominance of the S protein of SARS-CoV has been previously reported and has now been shown also for SARS-CoV-211,12,13. Indeed, neutralizing antibody responses against S have been detected in convalescent serum from SARS-CoV patients 24 months after infection14, highlighting their critical role in the long-term immune response. The S protein has been identified as a promising vaccine target and has thus become a key component of most vaccines under development15,16.
While the rapid detection of neutralizing antibodies is a critical aspect of vaccine development, it may also shed light on the rate of infection and sero-epidemiologic surveillance in impacted areas17. A replication-competent VSV pseudotyped with the SARS-CoV-2 S glycoprotein, in place of the wild-type VSV glycoprotein, to study SARS-CoV-2 infection in biosafety level 2 settings was kindly donated by Whelan and co-workers18. VSV expressing spike (VSV-S) will be utilized to determine the neutralizing antibody response against SARS-CoV-2 spike protein. As the VSV-S used here also expresses enhanced green fluorescent protein (eGFP), eGFP foci may be detected within 24 h to quantify infection, whereas plaque formation can take 48 to 72 h. Summarized here is a simple and effective protocol to determine the ability of convalescent patient serum to neutralize VSV-S-eGFP infection. This method may also be easily adapted to interrogate other potential therapeutics that aim to disrupt the host-viral interaction of SARS-CoV-2 S protein.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA (Gibco)</td>
			<td>Fisher scientific</td>
			<td>LS25200114</td>
			<td></td>
		</tr>
		<tr>
			<td>ArrayScan VTI HCS</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Automated fluorescent imager</td>
		</tr>
		<tr>
			<td>carboxymethyl cellulose</td>
			<td>Sigma</td>
			<td>C5678</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (Gibco)</td>
			<td>Fisher scientific</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (Powder) (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-800-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-Buffered Saline (DPBS)</td>
			<td>Fisher scientific</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher scientific</td>
			<td>BP-310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG Isotype Control (mouse)</td>
			<td>Thermo Fisher Scientific</td>
			<td>31903</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-2 (2019-nCoV) Spike Neutralizing Antibody, Mouse Mab</td>
			<td>SinoBiological</td>
			<td>40592-MM57</td>
			<td></td>
		</tr>
		<tr>
			<td>Vero E6 cells</td>
			<td>ATCC</td>
			<td>&#160;CRL-1586</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of sars-cov-2 neutralizing antibodies using high-throughput fluorescent imaging of pseudovirus infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

This protocol outlines a rapid and effective method for detecting neutralizing antibodies against SARS-CoV-2 S protein via inhibition of VSV-S-eGFP pseudovirus infection (quantifiable by loss of eGFP foci detected). A schematic representation of the protocol is depicted in Figure 1. It is recommended that a commercially available antibody be used as a positive control each time the assay is run to ensure the consistency of the assay. Here, we demonstrate a dilution curve using a commercially...

Watch this Scientific Journal Video about Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection at JoVE.com

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

This method provides a way of answering one of the more important questions concerning the SARS-coronavirus-2 vaccines under development. Is the vaccine able to elicit a neutralizing antibody response? The main advantage of this technique is that it can be done in a containment level two facility.It is also relatively fast and inexpensive, making it well-suited to analyzing many samples at once. Demonstrating the neutralizing acid procedure, we have Ricardo Marius, a senior technician in the lab and Taylor Jamieson, an MD PhD student. Begin by culturing Vero E6 cells in DMEM supplemented with 10%FBS at 37 degrees Celsius and 5%carbon dioxide.To passage the cells, first wash them by adding 10 milliliters of PBS and gently rocking the dish four to five times. After aspirating the PBS, add three milliliters of trypsin EDTA and rock the dish to ensure that the entire surface is covered before incubating it at 37 degrees Celsius. Once the cells have detached, deactivate the trypsin by adding seven milliliters of DMEM supplemented with 10%FBS, then resuspend the cells by pipetting up and down several times.Remove the trypsin by centrifugation and aspirate the supernatant without disturbing the cell pellet before resuspending the cells in 10 milliliters of fresh DMEM. After counting, add approximately one time 10 to the seventh cells to each 15 centimeter plate and incubate the culture for one to two days until the cells are 100%confluent. To prepare the VSV spike EGFP pseudovirus for titering, infect the cells at 0.01 multiplicity of infection with the stock virus diluted in 12 milliliters of serum-free DMEM.After adding the virus, incubate the cells for one hour at 37 degrees Celsius and 5%carbon dioxide with occasional rocking. At the end of the incubation, replace the inoculum with fresh DMEM supplemented with 2%FBS and 20 millimolar heaps, then incubate the cells at 34 degrees Celsius and 5%carbon dioxide for 24 hours. The next day, use a fluorescent microscope to visualize the EGFP expression of the infected cells.Once an extensive cytopathic effect and cell detachment are observed, collect the culture supernatant and remove the debris by centrifugation. To avoid multiple freeze-thaw cycles, aliquot the supernatant before storage at minus 80 degrees Celsius. To determine the viral titer, plate Vero E6 cells in six well plates at a seeding density of six times 10 to the fifth cells per well and incubate overnight.The next day, set up a tenfold serial dilution series of the viral stock by adding 900 microliters of medium to seven micro-centrifuge tubes and adding 100 microliters of viral stock to the first tube. After brief vortexing, transfer 100 microliters of diluted virus to each subsequent tube, vortexing between each tube. Then replace the supernatant in each well of the Vero E6 culture plate with 500 microliters of the 10 to the minus second to 10 to the minus seventh viral dilutions.After a 45 minute incubation in the cell culture incubator with gently rocking every 15 minutes, replace the inoculum with an overlay solution and incubate the cells at 34 degrees Celsius with 5%carbon dioxide for 48 hours. At the end of the incubation, to visualize the plaques by crystal violet staining, wash the cells one time with PBS before adding two milliliters of 0.1%crystal violet to each well. Place the plate on a rocker for approximately 20 minutes at room temperature before gently washing each well two times with PBS.After the last wash, allow the plates to air dry for at least one hour before using the formula as indicated to calculate the titer of the virus in each well in plaque forming units per milliliter. To plate cells for a neutralization assay, use a multi-channel pipette to add 100 microliters of Vero E6 cells to each well of a 96 well plate at a density of two times 10 to the fifth cells per milliliter, and incubate the plate for 24 hours at 37 degrees Celsius and 5%carbon dioxide. The following day, heat inactivate the patient serum samples to be tested in a 56 degrees Celsius water bath for 30 minutes.Then set up a tenfold dilution series of the patient's serum samples in an empty 96 well tissue culture plate by adding eight microliters of the serum to 72 microliters of serum-free DMEM with antibiotics in each well of row A.Then add 40 microliters of serum-free DMEM to each well of rows B to G and 80 microliters to each well of row H.Using a 12 well multichannel pipette, mix the samples in row A, then transfer 40 microliters of the sample from each well of row A to each well of row B.After mixing, repeat the dilution for each subsequent row of wells until row F.Next, add 40 microliters of VSV spike EGFP at 0.05 multiplicity of infection to each well in rows A to G, and mix by pipetting four to five times before incubating the plate for one hour at 37 degrees Celsius and 5%carbon dioxide. At the end of the incubation, carefully aspirate the supernatant from each well of the Vero E6 culture plate and transfer 60 microliters of antibody virus mixture from each well of the sample dilution plate to the corresponding well of the cell culture plate. When all the samples have been transferred, place the cell culture plate at 37 degrees Celsius and 5%carbon dioxide for one hour with rocking every 20 minutes.After the incubation is complete, add 140 microliters of carboxymethylcellulose overlay solution to each well of the cell culture plate and transfer the plate to a 34 degrees Celsius and 5%carbon dioxide incubator. After 24 hours, image the plates on an automated fluorescent imager at 488 nanometer wavelength, and use the automated counting feature of the imager to quantify the individual EGFP foci. In this representative example, a commercially available neutralizing antibody against the SARS-coronavirus-2 spike receptor binding domain was used as a positive control, alongside IgG as a negative control.The percent inhibition was calculated based on the number of EGFP foci detected via fluorescent imaging. Pseudovirus neutralization by convalescent patient samples collected approximately three months post SARS-coronavirus-2 infection showed that hospitalized patients demonstrated an increased neutralizing capacity compared to those who did not require hospitalization. This procedure can also be used to evaluate other COVID-19 prevention and therapeutic agents that aim to block viral infection.

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Immunology and Infection

4.1K vistas.  Ottawa Hospital Research Institute. Este método proporciona una forma de responder a una de las preguntas más importantes relacionadas con las vacunas contra el SARS-coronavirus-2 en desarrollo. ¿Es la vacuna capaz de provocar una respuesta de anticuerpos neutralizantes? La principal ventaja de esta técnica es que se puede hacer en una instalación de contención de nivel dos.También es relativamente rápido y económico, por lo que es muy adecuado para analizar muchas muestras a la vez. Demostrando el procedimiento...