Name: an improved and high throughput respiratory syncytial virus (rsv) micro-neutralization assay
Uploaded: 2019-01-26T14:30:00.000+00:00
Duration: 9 min 14 s
Description: Watch this Scientific Journal Video about An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay at JoVE.com

The authors thank all the participants involved. We acknowledge the Victorian Government's Operational Infrastructure Support Program. PVL is a NHMRC Career Development Fellowship recipient.

NOTE: All steps have to be performed in a BSL2 hood unless stated differently. Viral titration is required in advance of a PRN assay to determine the optimal RSV concentration used in the PRN assay. It is recommended to aliquot the virus stocks in a small volume that will be thawed once and used for each NAb assay. Using the same viral stock for all NAb assays performed for all samples from one study is also recommended. Make sure culture media and phosphate-buffered saline (PBS) is warmed at 37 &#176;C before adding to cell plates.
1. RSV Viral Titration
NOTE: Depending on the number of virus stocks and the number of duplicates, the assay plate can be set up according to Figure 1. Each virus stock should be titrated in triplicate down the assay plate, starting at the highest viral concentration (i.e., 1:10). Serial titrations can be typically 1:10. A549 cell culture and maintenance as well as RSV culture procedure are done using standard procedures and are not included in this protocol.
<ol>
	<li>Seeding plates (Day 1) 
	<ol>
		<li>Resuspend A549 cells in Dulbecco&#8217;s Modified Eagles medium (DMEM) + 10% fetal calf serum (FCS) + 1000 IU penicillin/streptomycin (pen/strep) at 4 x 105/mL. Seed 96-well flat-bottom sterile plates with 100 &#956;L/well containing 4 x 104 A459 cells.</li>
		<li>Incubate plates overnight at 37 &#176;C, 5% CO2 (cells will be in the log-phase growth). 
		NOTE: Use new A549 cell vial after 23 passages. It is recommended to not start the experiments when a new cell vial is still at the first three passages.</li>
	</ol>
	</li>
	<li>Virus infection (Day 2) 
	<ol>
		<li>Preparation of virus serial dilution
		<ol>
			<li>Rapidly thaw a single frozen vial of RSV in a 37 &#176;C water bath until almost completely thawed and place immediately on ice. Prepare 3 replicates for each RSV virus stock to be assayed, use an initial virus stock dilution of 1:10 with 100 &#956;L of diluted virus/well and reserve at least one column for negative control. 
			NOTE: Figure 1 shows negative control in triplicates.</li>
			<li>Add 100 &#956;L of each diluted virus at 1:10 in triplicate of row A of a 96-well U-bottom sterile plate. Add 90 &#956;L/well of DMEM + 1000 IU pen/strep without FCS to rows B to H.</li>
			<li>Perform serial 1:10 dilutions down the plate by transferring 10 &#956;L from row A to row B. Mix solution by pipetting content up and down 5 times and continue the ten-fold dilutions until row H. Discard the final 10 &#956;L so that the final volume in each well is 90 &#956;L. 
			NOTE: It is important to use new tips for each dilution.</li>
		</ol>
		</li>
		<li>Virus inoculation to A549 cells
		<ol>
			<li>Retrieve A549 cell plate(s) prepared on the previous day. Ensure that A549 cell monolayers in the 96-well plates are ~80% confluent. Discard media by gently inverting plate and lightly blotting on sterile absorbent paper towel.</li>
			<li>Wash all wells with 100 &#956;L of PBS twice, discard excess PBS by gently inverting plate and lightly blotting on sterile absorbent paper towel after each wash. Do not allow plates to dry.</li>
			<li>Transfer the virus dilutions from the viral titration plate (Figure 1) to corresponding wells on the A549 cell plate. Incubate the plate for 1 h at 37 &#176;C, 5% CO2. After 1 h incubation, decant supernatants using a pipette (to avoid cross contamination). Take out 90 &#956;L/well.</li>
			<li>Add 100 &#956;L of 1x medium 199 (M199, see Table of Materials) + 1.5% carboxymethylcellulose sodium salt (CMC) low viscosity + 2% FCS containing pen/strep to each well. 
			NOTE: 2x M199 + 4% FCS + 2% pen/strep and 3% CMC solutions need to be prepared in advance and stored at 4 &#176;C for future use. Make sure the solution is warmed at 37 &#176;C before adding to plates.
			<ol>
				<li>Prepare 2x M199 solution: 1 sachet/500 mL distilled water + 20 mL FCS + 10 mL pen/strep (to make up 2x MI99). Filter the solution using a 0.22 &#956;m filter unit.</li>
				<li>For preparation of 3% CMC, add 15 g of CMC slowly into 500 mL of distilled water. Dissolve the CMC in water by using metallic stirrer heater at 50 &#176;C, which takes about 1 h of preparation. Mix well to make 3% CMC and autoclave the solution. 
				NOTE: The CMC solid must be added to the water in order to be dissolved; adding water to the dry solid produces a &#8220;clump&#8221; of solid that is very difficult to dissolve.</li>
				<li>Prepare 1x M199 + 1.5% CMC low viscosity + 2% FCS containing pen/strep by mixing 1:1 the 2x M199 and 3% CMC prepared in step 1.2.2.4.2.</li>
			</ol>
			</li>
			<li>Incubate plate for 3 days at 37 &#176;C, 5% CO2.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Developing assay plate and analysis (Day 5)
	<ol>
		<li>Fixation and developing assay plate
		<ol>
			<li>Discard M199 + CMC + 2% FCS containing pen/strep by gently inverting the plate. Add slowly (in order to avoid disturb the cell layer) 200 &#956;L of fixation buffer (80% acetone, 20% PBS, stored at -20 &#176;C) to fix cells. Incubate at -20 &#176;C for 20 min.</li>
			<li>Discard the fixation buffer and gently blot on absorbent paper towel. Leave plate face down to dry for 10 min. 
			NOTE: From this step onwards, assay can be performed outside the BSL2 hood.</li>
			<li>Prepare 5% milk diluent blocking solution in filtered PBS containing 0.05% polysorbate 20 (PBS-polysorbate, see Table of Materials). For one plate, calculate the volume needed for blocking based on 200 /well and 110 wells: 200/well x 110 wells = 22 mL (1.1 mL concentrated milk diluent in 20.9 mL filtered PBS-polysorbate). At this stage, also make up sufficient blocking solution for primary and secondary antibodies. For one plate, calculate the volume needed for each antibody solution based on 50 /well and 110 wells: 50 /well x 110 wells = 5.5 mL. Therefore, the total volume of milk diluent blocking solution required for a single plate assay is 33 mL.</li>
			<li>Add 200 /well of blocking solution. Incubate plate at room temperature (RT) for 30 min. Discard the solution by gently inverting the plate and blot on absorbent paper towel.</li>
			<li>Prepare 1:500 Goat X RSV antibody (mAb &#8211; primary antibody) diluted in blocking solution. Add 50 /well of the mAb solution and incubate at 37 &#176;C for 1 h. Wash plate 3 times with filtered PBS-polysorbate.</li>
			<li>Prepare 1:5,000 Alexa-Fluor donkey anti-goat IgG (secondary antibody) diluted in blocking solution. Add 50 /well of the secondary antibody solution and incubate at 37 &#176;C for 1 h. Wash plate 5 times with filtered PBS-polysorbate.</li>
			<li>Read plate on an automated spots reader using the fluorescein isothiocyanate (FITC) channel and count settings as shown in Figure 2. Wrap plate in foil and store at 4 &#176;C. Calibrate the instrument using an unused 96-well culture plate and inputting count settings before assay. 
			NOTE: Re-scanning within a few days is possible if required. The calibration and count setting can be saved and used for the future scanning if the assay uses the same type of plate used for the calibration.</li>
			<li>Check the images of wells for artefact plaques or disrupted cell monolayers (Figure 3). Exclude wells with disrupted cell monolayers. 
			NOTE: Depending on the size of the artefact plaques, manual counts may need to be performed for those wells or those wells may need to be discarded from data analysis. Criteria for a valid result include no disrupted cell monolayer or artefact plaques detected in more than one replicate well and no PFU detected in negative wells (wells without added virus).</li>
		</ol>
		</li>
		<li>Determine viral concentrations 
		<ol>
			<li>Choose the last two dilutions where spots can be counted clearly. Calculate the average number of spots from replicate wells at the same dilution. Calculate the viral titer (PFU per mL) of the stock sample using the formula below: 
			PFU/mL = Average number of plaques/(Dilution factor &#215; Volume of diluted virus added to the well) 
			NOTE: The virus concentration determined for each RSV stock can now be used in the PRN assay (step 2).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. RSV Neutralization Assay
<ol>
	<li>Seeding plates (Day 1)
	<ol>
		<li>Resuspend A549 cells in DMEM + 10% FCS + 1000 IU pen/strep at 4 x 105/mL. Seed 96-well flat-bottom sterile plates with 100 &#956;L/well containing 4 x 104 A459 cells and incubate plates overnight at 37 &#176;C, 5% CO2 (cells will be in the log-phase growth). 
		NOTE: Use new A549 cell vial after 23 passages. It is not recommended to use A549 cells before passage number 3.</li>
	</ol>
	</li>
	<li>Virus and serum preparation (Day 2) 
	NOTE: Depending on the sample type, there are required steps for pre-processing samples before the NAb assay. It is recommended to use the RSV international standard sera that are now available upon request from the National Institute for Biological Standards and Controls (NIBSC).
	<ol>
		<li>Preparation of serum dilution
		<ol>
			<li>Thaw the human RSV reference antiserum (reference serum, REF) and serum test samples at RT. Heat-inactivate serum samples and reference antiserum in a water bath at 56 &#176;C for 30 min prior to use. Prepare dilutions as per the plate template (Figure 4).</li>
			<li>Prepare 1:100 dilution of the REF. Prepare a minimum of 110 &#956;L of REF diluted in DMEM + pen/strep without FCS per assay plate (e.g., 1.5 &#956;L of REF in a total volume of 150 &#956;L). Add 110 &#956;L of 1:100 diluted REF in well A12 of a 96-well U-bottom sterile plate.</li>
			<li>Add 55 &#956;L of DMEM + pen/strep without FCS to wells B12 to H12 in column 12. Perform serial 1:2 dilutions down the plate by transferring 55 &#956;L from row A to row B, mixing solution by pipetting content up and down 5 times and continuing until row H. Discard the final 55 &#956;L of media so that the final volume in each well is 55 &#956;L. Use new tips for each dilution.</li>
			<li>Prepare 1:100 dilution of serum test samples. As all serum samples are assayed in triplicate, prepare a minimum volume of 110 &#956;L/well x 3 = 330 &#956;L per serum test sample.</li>
			<li>Add 110 &#956;L of each diluted serum test sample (1:100; S1 = serum 1, S2 = serum 2, etc.) to corresponding wells A1 to A9 and also E1 to E9 of the 96-well sterile plate according to Figure 4.</li>
			<li>Add 55 &#956;L of DMEM + pen/strep without FCS to all wells labelled X. Perform serial 1:2 dilutions as per step 2.2.1.3 until row D (for samples 1, 2, and 3). Discard the final 55 &#956;L of media so that the final volume in each well is 55 &#956;L. Similarly, perform the serial 1:2 dilutions for samples 4, 5, and 6 for rows E to H. 
			NOTE: It is important to use new tips for each dilution.</li>
			<li>Add 55 &#956;L of DMEM + pen/strep without FCS to wells in columns 10 and 11.</li>
		</ol>
		</li>
		<li>Preparation of virus
		<ol>
			<li>Rapidly thaw a single frozen vial of RSV in a 37 &#176;C water bath until almost completely thawed and place immediately on ice.&#8232;</li>
			<li>Dilute virus in DMEM + pen/strep without FCS based on the concentration of the RSV aliquot determined in step 1.3.2. Apply a dilution that gives approximately 200 PFU/well. Prepare a total volume of 5.5 mL (for 100 wells).</li>
		</ol>
		</li>
		<li>Preparation of virus-serum mixture
		<ol>
			<li>Add 55 of the diluted virus to all wells of columns 1-9 and wells of rows E-H of columns 10 and 11 (positive control) according to the plate template (Figure 4).</li>
			<li>Add 55 of DMEM + pen/strep without FCS to all wells of rows A-D of columns 10 and 11 (negative control). Incubate the plate for 1 h at 37 &#176;C, 5% CO2.</li>
		</ol>
		</li>
		<li>Virus inoculation to A549 cells
		<ol>
			<li>Prior to the completion of incubation period, retrieve A549 cell plate(s) prepared in step 2.1.1. Ensure that A549 cell monolayers in the 96-well plates are ~80% confluent.</li>
			<li>Discard media by gently inverting plate and lightly blotting on sterile absorbent paper towel. Wash all wells with 100 &#956;L of PBS twice, discard excess PBS by gently inverting plate and lightly blotting on sterile absorbent paper towel after each wash. Do not allow plates to dry.</li>
			<li>Transfer 100 /well of the virus-serum mixture to the corresponding wells on the A549 cell plate following the plate template Incubate the plate for 1 h at 37 &#176;C, 5% CO2.</li>
			<li>After 1 h, using a pipette, take out 90 /well and add 100 of prewarmed 1x M199 + 1.5% CMC + 2% FCS + pen/strep. Incubate plate for 3 days at 37 &#176;C, 5% CO2. 
			NOTE: Make sure the solution is warmed at 37 &#176;C before adding to plates.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Developing assay plate and analysis (Day 5) 
	<ol>
		<li>Develop fixation and assay plate following steps 1.3.1.1 to 1.3.1.8.</li>
		<li>Determine the 50% neutralization titer &#8232;
		<ol>
			<li>Determine the 50% neutralization titer by calculating the proportional distance between the reciprocal serum dilutions above and below 50% of the &#8216;no serum&#8217; control wells (positive virus control - column 10, rows E-H).</li>
			<li>Count the number of PFU (i.e., plaques) arising from individual viral infections in serum wells and no serum control wells. Calculate the mean number of PFU of the no serum control wells and determine the 50% value.</li>
			<li>Identify serum dilutions with counts which are immediately above and below the 50% value of the no serum control wells.Using a semi-log graph or spreadsheet template, plot the number of PFU on the x-axis (linear scale) and the reciprocal serum dilution on the y-axis (log10 scale). Draw a line between the two points and read the 50% neutralization titers of the test serum samples from this line. 
			NOTE: The RSV PRN assay worksheet (Supplementary Worksheet) is used to determine the 50% neutralization titer.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

We have developed and optimized a simple and efficient RSV micro-neutralization assay that can be readily adapted in most laboratories. This assay is able to measure viral infection ability as well as measuring the inhibition of viral infection by NAb at the cellular level using computerized image scanning. The use of an imaging-based platform and specific antibody-based systems has increased the specificity and sensitivity of spot detection compared to traditional plaque detection methods6,<...

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in the pediatric population worldwide1. Despite its high burden, there is still no vaccine or treatment available. Since 2013, the World Health Organization (WHO) has declared RSV vaccine development as a major research priority, with annual WHO consultation meetings2,3. The WHO has agreed on using RSV neutralizing antibody (NAb) measurement to monitor vaccine immunogenicity, as this is recognized as the major serological marker of protection4. NAbs have been shown to protect against severe RSV infection in a number of studies as well as clinical trials of the anti-RSV monoclonal antibody palivizumab, currently the only prophylactic strategy available4.
There are multiple NAb assay formats used by laboratories worldwide, including cell-based and molecular-based assays, which have made standardization efforts challenging5,6,7,8. However, the conventional plaque-reduction neutralization (PRN) assay that measures the number of reduced plaque forming units (PFU) by the presence of an RSV-specific antibody still remains the gold standard9. Here, we report an improved, simplified, and high-throughput PRN protocol that can be used on numerous cell lines, for different RSV strains and with increased assay throughput. This protocol has been tested using clinical samples from different settings as well as on samples from animal model experiments.

<table><tbody><tr><td>&lt;strong&gt;Cell line&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A549</td><td>ATCC</td><td>CCL-185</td><td>provided by Dr Keith Chappell, University of Queensland</td></tr><tr><td>&lt;strong&gt;Viral strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RSV A2</td><td>ATCC</td><td>VR-1540</td><td>lot number 60430286</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetone</td><td>Merck</td><td>1000142511</td><td></td></tr><tr><td>Alexa-Fluor donkey anti-goat IgG (stored at 4 &amp;deg;C)</td><td>Life Technologies</td><td>A11055</td><td></td></tr><tr><td>CMC sodium salt powder</td><td>Sigma-Aldrich</td><td>C5678-500G</td><td></td></tr><tr><td>DMEM (no serum, 3.7 g/L NaHC, P/S) (stored at 4 &amp;deg;C)</td><td>Scientific Services &amp;ndash; Tissue Culture</td><td></td><td>MCRI in house supply</td></tr><tr><td>Foetal calf Serum (stored in 50 mL aliquots at -20 &amp;deg;C)</td><td>Interpath</td><td>SFBS-F</td><td></td></tr><tr><td>Goat X RSV antibody</td><td>Merck</td><td>AB1128</td><td></td></tr><tr><td>human polyclonal antiserum to respiratory syncytial virus (RSV) (stored in 45 &amp;micro;L aliquots at -20 &amp;deg;C)</td><td>BEI Resources</td><td>NR-4022</td><td>Free order through BEI Resources upon registration. This serum belong to a panel of human antiserum and immune globulin to RSV (NR-32832)</td></tr><tr><td>M199 powder</td><td>Life Technologies</td><td>31100035</td><td></td></tr><tr><td>Milk diluent blocking solution (stored at 4 &amp;deg;C)</td><td>Australian Biosearch</td><td>50-82-01</td><td></td></tr><tr><td>Penicillin/Streptomycin (stored in 6mL aliquots at -20 &amp;deg;C)</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>s.d.H&lt;sub&gt;2&lt;/sub&gt;O from Milli-Q dispenser</td><td>Merck</td><td></td><td>In-house dispensation</td></tr><tr><td>Sterile 1x PBS for culture (stored at 4 &amp;deg;C)</td><td>Scientific Services &amp;ndash; Tissue Culture</td><td></td><td>MCRI in house supply</td></tr><tr><td>Tween 20 polysorbate</td><td>Sigma-Aldrich</td><td>9005-64-5</td><td></td></tr><tr><td>&lt;strong&gt;General Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Conical Falcon tubes (50 mL)</td><td>Invitro Technologies</td><td>FAL352070</td><td></td></tr><tr><td>Filter unit 0.22 &amp;mu;m (500 mL)</td><td>Thermo Fisher</td><td>NAL5660020</td><td></td></tr><tr><td>Sterile Eppendorf tubes (1.5 mL)</td><td>Australia PL</td><td>AM12400</td><td></td></tr><tr><td>Sterile flat-bottom plates (96-well with lid)</td><td>Interpath</td><td>655180</td><td></td></tr><tr><td>Sterile U-bottom plates (96-well with lid)</td><td>Interpath</td><td>650180</td><td></td></tr><tr><td>5 mL serological pipette</td><td>Sigma-Aldrich</td><td>CLS4487-200EA</td><td></td></tr><tr><td>10 mL serological pipette</td><td>Interpath</td><td>607180</td><td></td></tr><tr><td>25 mL serological pipette</td><td>Sigma-Aldrich</td><td>CLS4251-200EA</td><td></td></tr><tr><td>Tip Pipette 1-200 &amp;micro;L Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 96TIPS PKG960</td><td>Fisher Biotec</td><td>TF-200-L-R-S</td><td></td></tr><tr><td>Tip Pipette 5-20 &amp;micro;L Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 96TIPS PKG960</td><td>Fisher Biotec</td><td>TF-20-L-R-S</td><td></td></tr><tr><td>Tip Pipette 100-1,000 &amp;micro;LClear Maxymum Recovery Racked Pre-sterilized 10RACKS x 100TIPS PKG1000</td><td>Fisher Biotec</td><td>TF-1000-L-R-S</td><td></td></tr><tr><td>Tip Pipette 1-10 &amp;micro;L Clear Maxymum Recovery Racked Pre-sterilized 10RACKS x 100TIPS PKG1001</td><td>Fisher Biotec</td><td>TXLF-10-L-R-S</td><td></td></tr><tr><td>&lt;strong&gt;Equipments and softwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ELISpot reader system</td><td>AID iSpot, Autoimmun Diagnostika GmbH, Strasburg, Germany</td><td></td><td></td></tr><tr><td>AID ELISpot software version 5.0</td><td>AID iSpot, Autoimmun Diagnostika GmbH, Strasburg, Germany</td><td></td><td></td></tr><tr><td>Microsoft Excel 2007</td><td></td><td></td><td></td></tr></tbody></table>

an improved and high throughput respiratory syncytial virus (rsv) micro-neutralization assay

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay formats are currently in use worldwide so there is a need for an accurate and high-throughput method for measuring RSV NAbs. We describe here an imaging-based micro-neutralization assay that has been tested on RSV subgroup A and can also be adapted for RSV subgroup B and different sample types. This method is highly reproducible, with inter-assay variations for the reference antiserum being less than 10%. We believe this assay can be readily established in many laboratories worldwide at relatively low cost. Development of an improved, high-throughput assay that measures RSV NAbs represents a significant step forward for the standardization of this method internationally as well as being critical for the evaluation of novel RSV vaccine candidates in the future.

The titration of a virus stock was performed from 1:10 to 1:108 dilution to determine the virus stock concentration prior to the PRN assay (representative results shown in Figure 5). From Figure 5, PFU can be counted reliably at dilutions of 1:104 and 1:105. The average number of PFU from triplicate wells at the same dilution was calculated. Since the average number of spots at 1:105 di...

Watch this Scientific Journal Video about An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay at JoVE.com

An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay

This study describes a high throughput, imaging-based micro-neutralization assay to determine the titer of neutralizing antibodies specific for respiratory syncytial virus (RSV). This assay format has been tested on different sample types.

An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay ...

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Research

JoVE Journal

Medicine

10.7K Views.  Murdoch Children's Research Institute. This study describes a high throughput, imaging-based micro-neutralization assay to determine the titer of neutralizing antibodies specific for respiratory syncytial virus (RSV). This assay format has been tested on different sample types.

Video: An Improved and High Throughput Respiratory Syncytial Virus RSV Micro-neutralization Assay

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Immunology and Infection

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

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.

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 ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

Biology

An Assay to Measure the Neutralizing Antibody Titer Specific for Respiratory Syncytial Virus

Source: Do, L. A. H., et al., <a href="https://app.jove.com/v/59025">An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates an assay for measuring neutralizing antibodies against respiratory syncytial virus (RSV). Pre-incubation with serum containing RSV-neutralizing antibodies neutralizes the virus particles, reducing cellular infection and plaque formation. Virus-infected cells are then subjected to fixation, blocking, and fluorophore-conjugated antibody labeling for plaque visualization. This process enables the identification of the highest serum dilution that neutralizes fifty percent of RSV, representing the neutralization titer.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
NOTE:&#160;All steps must be ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum

Source: Gross, F. L. et al., <a href="https://app.jove.com/v/56448">Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells.</a>&#160;J. Vis. Exp.&#160;(2017)
This video demonstrates Microneutralization assays to measure neutralizing antibodies using Madin-Darby Canine Kidney cells overexpressing &#945;2,6-linked sialic acid in human sera. This assay is often used for influenza human serology