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NOTE: HA-specific antibodies that inhibit viral replication can generally be categorized into i) ones that bind on or adjacent of the receptor binding site on top of the globular head and ii) ones that bind distal of the receptor binding domain, which includes the lateral side of the globular head and the stalk region of the HA. Antibodies that target the receptor binding site prevent the engagement of sialic acid motifs on the surface of target cells and can be measured using a hemagglutination inhibition (HI) assay. Antibodies that are HI-negative, such as stalk-specific antibodies, can still inhibit viral replication, but can only be assessed using neutralization assays.
1. Categorizing Antibodies Based on HI Activities
<ol>
	<li>HI assay
	<ol>
		<li>In a 96-well V-bottom plate, add 25 &#181;L of 1x PBS on columns 2 to 12.</li>
		<li>Dilute the monoclonal antibody (mAb) 7B2 (head-specific), 6F12 (stalk-specific) and an isotype control to 100 &#181;g/mL in 1x PBS and aliquot 50 &#181;L of the diluted antibody preparations into column 1. Also, include a no mAb control by adding 50 &#181;L of 1x phosphate-buffered saline (PBS) (Figure 1A).</li>
		<li>Perform 2-fold serial dilutions of the antibodies by transferring 25 &#181;L from column 1 to column 2, and so forth. Discard the last 25 &#181;L from column 12 (Figure 1A). 
		NOTE: Make sure to include a row of no antibody control.</li>
		<li>Dilute the virus stock (a reassortant virus expressing the hemagglutinin (HA) and neuraminidase (NA) of A/California/04/09 with the internal segments of A/Puerto Rico/8/34) to 8 hemagglutination units/25 &#181;L diluted virus stock. Add 25 &#181;L of the diluted virus stock (8 hemagglutination) to each well (Rows A to G). 
		NOTE: The antibody and virus mixture should have a final volume of 50 &#181;L with a starting final concentration of 50 &#181;g/mL.</li>
		<li>Incubate the plate at room temperature (RT) for 45 min.</li>
		<li>For the back-titration row (H), add 50 &#181;L of 1x PBS on wells H2 to H12. Add 100 &#181;L of the 8 hemagglutination units/25 &#181;L to well H1. Serially dilute two-fold by transferring 50 &#181;L from H1 to H2, and so forth. Discard the last 50 &#181;L from well H12. Finally, add 50 &#181;L of 0.5% chicken red blood cells (RBC) to all wells of the 96-well V-bottom plate. 
		NOTE: The mAb samples in the assay should have a final volume of 100 &#181;L: mAb (25 &#181;L), virus (25 &#181;L) and RBC (50 &#181;L). The final volume of the no mAb control should contain 25 &#181;L of 1x PBS, 25 &#181;L of virus and 50 &#181;L of RBC.</li>
		<li>Incubate the plate at 4 &#176;C for 1 h.</li>
		<li>Visually read the plates for HI activity. If there is a positive readout for a particular antibody, proceed to step 2.1 to generate escape variants. If the antibody is HI-negative, proceed below to step 1.2 to assess if the antibody has neutralizing activity in a cell culture assay. 
		NOTE: A positive readout of an HI-active mAb is indicated by a dark red RBC pellet in the center of a well (Figure 1B; 7B2) that will form a tear drop when the 96-well V-bottom plate is held at a 45&#176; angle. A negative readout will not form a dark red RBC pellet in the well (Figure 1B; 6F12 and no mAb). The isotype control will also not form a dark red RBC pellet and should look identical to the stalk-specific antibody, 6F12 or the no mAb control sample (Figure 1B).</li>
	</ol>
	</li>
</ol>
2. Generation of Escape Mutant Variants
NOTE: Neutralizing antibodies that have or lack HI activity are further analyzed with the specific protocols described below.
<ol>
	<li>Protocol 1: HI-positive/Neutralization-positive antibodies (Figure 2A)&#8203;
	<ol>
		<li>Prepare a virus stock of 106 plaque forming units/milliliter (PFU/mL) in 1x PBS in a 400 &#181;L volume.</li>
		<li>Prepare four dilutions of the antibody of interest in increasing concentrations (e.g., 0, 0.5, 0.05 and 0.005 mg/mL) in 1x PBS in a volume of 100 &#181;L per dilution. 
		&#8203;NOTE: Wild-type virus should always be passaged in parallel and in the absence of antibody. The sequences of these passaged viruses will assist in distinguishing between adaptation to cell culture conditions and escape mutations.</li>
		<li>Mix 100 &#181;L of 106 PFU/mL of virus with 100 &#181;L of each antibody dilution or 100 &#181;L of 1x PBS.</li>
		<li>Incubate for 1 h in a 37 &#176;C incubator (with 5% CO2). Vortex briefly. Inject 200 &#181;L of each mixture into specific-pathogen free (SPF) embryonated chicken eggs.</li>
		<li>Incubate the eggs at 37 &#176;C (without CO2) for 40-44 h.</li>
		<li>Sacrifice the virus infected-embryonated eggs by incubating at 4 &#176;C for a minimum of 6 h.</li>
		<li>Harvest the allantoic fluid from the eggs, as previously described.</li>
		<li>Perform the hemagglutination assay, as described above. If all of the allantoic fluid preparations do not have hemagglutination titers, repeat from step 2 with antibody dilutions ranging from 0.005 mg/mL to 0.00005 mg/mL. 
		NOTE: A saturating concentration of an HI-positive antibody may neutralize all the virus particles present. Therefore, it may be necessary to decrease the amount of antibody present in the passaging.</li>
		<li>Confirm the escape variants by performing the HI assay (step 1.1). 
		NOTE: HI-active antibodies block HA engagement of sialic acid motifs on the target cells. Therefore, a virus in the presence of its cognate antibody loses the ability to agglutinate RBCs (presence of RBC pellet). Theoretically, escape variants of HI-active antibodies can still bind sialic acid motifs even in the presence of its cognate antibody and thus can agglutinate RBCs (no RBC pellet). If the HI of the antibody of interest is still detectable, repeat the protocol from step 2.1.2 with a higher starting concentration of the antibody.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Falcon 96-well clear V-bottom plate</td>
			<td>Corning, Inc.</td>
			<td>353263</td>
			<td>Assay plate use for the hemagglutination inhibition assay</td>
		</tr>
		<tr>
			<td>96-well V-bottom plate</td>
			<td>Nunc</td>
			<td>249662</td>
			<td>Assay plate used for the hemagglutination assay</td>
		</tr>
		<tr>
			<td>Chicken red blood cells</td>
			<td>Lampire Biological Laboratories</td>
			<td>7201403</td>
			<td>Used to assess the ability of influenza virus to agglutinate</td>
		</tr>
		<tr>
			<td>reassortant A/California/04/09 (H1)</td>
			<td>Palese Laboratory</td>
			<td></td>
			<td>reassortant virus expressing the HA and NA of A/California/04/09 (H1N1) with the internal segments of A/Puerto Rico/8/34 (H1N1)</td>
		</tr>
		<tr>
			<td>reassortant A/Shanghai/1/13 (H7)</td>
			<td>Palese Laboratory</td>
			<td></td>
			<td>reassortant virus expressing the HA and NA of A/Shanghai/1/13 (H7N9) with the internal segments of A/Puerto Rico/8/34 (H1N1)</td>
		</tr>
	</tbody>
</table>

generation of influenza virus escape variants against neutralizing antibodies

Source: Leon, P. E., et al. <a href="https://app.jove.com/v/56067">Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for generating escape variants of neutralizing influenza virus monoclonal antibodies. Antibody-neutralized virus suspension is injected into embryonated chicken eggs to propagate escape variants selectively. This results in a virus population predominantly of escape variants, confirmed via a Hemagglutination assay.

<img alt="Figure 1" src="/files/ftp_upload/22085/22085_Figure1.jpg" /> 
Figure 1: HI assay. (A) A schematic for the setting up a HI assay to test the activity of two mouse H1-specific mAbs 7B2 (head-specific) and 6F12 (stalk-specific) using a 96-well V-bottom plate, and (B) an example of the results of an HI assay
<img alt="Figure 2" src="/files/ftp_upload/22085/22083_Figure2.jpg" /> 
Figure 2: Generation of escape mutants. The methodology suggested will be dependent on the HI and the microneutralization activity exhibited by the antibody. The generation of escape mutants against (A) neutralizing HI-positive antibodies may require a single passage in eggs, while (B) neutralizing HI-negative antibodies may involve multiple passages with increasing antibody amounts in cell tissue culture.&#160;

Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

NOTE: HA-specific antibodies that inhibit viral replication can generally be categorized into i) ones that bind on or adjacent of the receptor binding ...

Take an influenza virus suspension, containing both wild-type and mutant strains.
A viral envelope glycoprotein termed hemagglutinin, or HA, binds to sialic acid on the host cell surface, mediating viral entry.
Add a neutralizing antibody in decreasing concentrations that binds to HA, blocking the sialic acid binding site.
Mutant strains, carrying mutations in the HA that help avoid antibody neutralization, are termed escape variants.
Inject the mixture into pathogen-free embryonated chicken eggs, delivering the viruses into the allantoic fluid, and incubate.
Antibody-neutralized viruses cannot enter the cells of the chorioallantoic membrane. Non-neutralized mutant viruses enter the cells, replicate, and are released into the allantoic fluid.
Harvest the virus population-containing allantoic fluid.
Introduce an HA-specific antibody in decreasing concentrations, which neutralizes the other strains except the escape variants.
Introduce chicken red blood cells or RBCs. Non-neutralized escape variants bind to the cells, causing the clumping of RBCs &#8212; termed hemagglutination, confirming escape variant generation.

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Nanyang Technological University

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109 Views. Source: Leon, P. E., et al. Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies. J. Vis. Exp. (2017). This video demonstrates a technique for generating escape variants of neutralizing influenza virus monoclonal antibodies. Antibody-neutralized virus suspension is injected into embryonated chicken eggs to propagate escape variants selectively. This results in a virus population predominantly of escape variants, confirmed via a Hemagglutination assay. 

Video: Generation of Influenza Virus Escape Variants against Neutralizing Antibodies - Concept

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

JoVE Journal

Immunology and Infection

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 ...

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle VLP-Based Vaccines Using a Capture Assay

Here, we present a protocol to detect neutralization epitopes on antigen-displaying virus-like particles (VLPs). Immunoprecipitation of the human immunodeficiency virus (HIV)-derived VLPs is performed using envelope glycoproteins-specific monoclonal antibodies coupled to protein G-conjugated magnetic beads. Captured VLPs are subsequently subjected to SDS-PAGE and Western blot-analysis employing viral core protein Gag-specific antibodies.

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate ...

Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

Transcript

Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

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

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay