Name: measuring influenza neutralizing antibody responses to a(h3n2) viruses in human sera by microneutralization assays using mdck-siat1 cells
Uploaded: 2017-11-22T15:00:00
Description: Watch this Scientific Journal Video about Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells at JoVE.com

We thank Dr. Xiuhua Lu, Dr. Feng Liu, and Ms. Ashley Burroughs from the Influenza Division of the CDC for their critical review and assistance in preparation of&#160;this manuscript. We thank Dr. Adrian Reber from Influenza Division&#160;of the CDC for his assistance in preparing the graphics of Figure 3. Lastly, we thank Dr. M. Matrosovich, Marburg, Germany for providing the MDCK-SIAT1 cells.

All influenza viruses should be handled according to appropriate biosafety level requirements (BSL-2 or higher) as defined in the Biosafety on Microbiological and Biomedical Laboratories (BMBL)12.

1. Preparation of Reagents and Starting Material
<ol>
	<li>Prepare MDCK-SIAT1 cells&#160;and sterile cell culture medium
	<ol>
		<li>Prepare the MDCK-SIAT1 cell culture medium using 500 mL of Dulbecco&#39;s Modified Eagle Medium (DME...

<ol>
	<li>Reber, A., Katz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+assessment+of+influenza+vaccines+and+immune+correlates+of+protection.">Immunological assessment of influenza vaccines and immune correlates of protection.</a> Expert Rev of Vaccines. 12, 519-536 (2013).</li><li>Li, C. K., Rappuoli, R., Xu, X. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlates+of+protection+against+influenza+infection+in+humans--on+the+path+to+a+universal+vaccine?.">Correlates of protection against influenza infection in humans--on the path to a universal vaccine?.</a> Curr Opin in Immunol. 25, 470-476 (2013).</li><li>WHO Global Influenza Surveillance Network. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual for the labratory diagnosis and virological surveillence of influenza. , (2011).</li><li>Govorkova, E. A., Kodihalli, S., Alymova, I. V., Fanget, B., Webster, R. 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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=Neutralizing+Antibody+Responses+to+Antigenically+Drifted+Influenza+A(H3N2)+Viruses+among+Children+and+Adolescents+following+2014-2015+Inactivated+and+Live+Attenuated+Influenza+Vaccination.">Neutralizing Antibody Responses to Antigenically Drifted Influenza A(H3N2) Viruses among Children and Adolescents following 2014-2015 Inactivated and Live Attenuated Influenza Vaccination.</a> Clin Vaccine Immunol : CVI. 23, 831-839 (2016).</li><li>Lin, Y., 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=The+characteristics+and+antigenic+properties+of+recently+emerged+subclade+3C.3a+and+3C.2a+human+influenza+A(H3N2)+viruses+passaged+in+MDCK+cells.">The characteristics and antigenic properties of recently emerged subclade 3C.3a and 3C.2a human influenza A(H3N2) viruses passaged in MDCK cells.</a> Influenza Other Respir Viruses. , (2017).</li><li>Lin, Y. P., 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=Neuraminidase+receptor+binding+variants+of+human+influenza+A(H3N2)+viruses+resulting+from+substitution+of+aspartic+acid+151+in+the+catalytic+site:+a+role+in+virus+attachment?.">Neuraminidase receptor binding variants of human influenza A(H3N2) viruses resulting from substitution of aspartic acid 151 in the catalytic site: a role in virus attachment?.</a> J of Virol. 84, 6769-6781 (2010).</li><li>Skowronski, D. 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=Mutations+acquired+during+cell+culture+isolation+may+affect+antigenic+characterisation+of+influenza+A(H3N2)+clade+3C.2a+viruses.">Mutations acquired during cell culture isolation may affect antigenic characterisation of influenza A(H3N2) clade 3C.2a viruses.</a> Euro Surveillance. 21, 30112 (2016).</li><li>Matrosovich, M., Matrosovich, T., Carr, J., Roberts, N. A., Klenk, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+the+alpha-2,6-sialyltransferase+in+MDCK+cells+increases+influenza+virus+sensitivity+to+neuraminidase+inhibitors.">Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza virus sensitivity to neuraminidase inhibitors.</a> J of Virol. 77, 8418-8425 (2003).</li><li>Oh, D. Y., Barr, I. 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The MN assay is one of the main assays used for influenza serology to detect antibody responses following influenza infection or vaccination. Titers generated from MN assays are often used as the primary outcome of many influenza seroepidemiology studies. MN assays are also widely used for sero-diagnosis, and the evaluation of vaccine immunogenicity. International inter-lab studies have been conducted to compare MN assays performed in multiple laboratories14.
In contras...

Influenza viruses continue to cause morbidity and mortality in the human population each year. HA is the major surface glycoprotein of influenza viruses. Neutralizing antibodies targeting HA are the main immune mechanism that correlates with protection of influenza infection1,2. Hemagglutination inhibition (HI) assays and MN assays are two methods widely used to measure antibody responses in human sera after influenza infection or vaccination3. The HI assay measures antibody inhibition of virus hemagglutination of red blood cells and is considered a surrogate assay. Unlike HI, the MN assay can directly measure the levels of antibodies in human sera that neutralize influenza infection in cell cultures. MDCK cells are often used in influenza virus isolation and MN assays4.
Influenza viruses constantly undergo antigenic drift and shift, acquiring mutations on HA proteins that can alter the receptor binding specificity of viruses. Since 2014, new clusters of A(H3N2) viruses emerged and continued to circulate until the current season. The majority of these viruses belong to the genetic group 3C.2a and 3C.3a based on phylogenetic analysis of the HA proteins. Many of the circulating 3C.2a viruses had reduced ability to hemagglutinate red blood cells and therefore cannot be characterized by HI assays5. Neutralization assays must be used to measure the antibody responses to these viruses that do not hemagglutinate6. Furthermore, studies have shown that these contemporary A(H3N2) viruses have altered receptor binding properties compared with earlier A(H3N2) viruses and tend to accumulate culture adapted mutations and polymorphism when passaged in in vitro cell cultures7,8,9. Compared with conventional MDCK cells, MDCK-SIAT1 is a cell line developed by Matrosovich et al. through stable transfection of MDCK cells with cDNA of human &#945;2,6-sialtransferase (SIAT1). This cell line expresses increased amounts of &#945;2,6-sialic galactose moieties and decreased amounts of &#945;2,3-sialic acid moieties than the parent MDCK cells10. MDCK-SIAT1 cells have shown to improve isolation rates for A(H3N2) viruses compared to MDCK cells11. Recently, Lin et al. reported that for newly emerged 3C.2a and 3C.3a human A(H3N2) influenza viruses, more faithful virus replications and better virus infectivity were achieved when viruses were cultured in MDCK-SIAT1 cell lines compared with MDCK cells7. Thus, the MDCK-SIAT1 cells are better suited in MN assays to characterize antibody responses to the recent clusters of A(H3N2) viruses.
Here we describe a MN assay using MDCK-SIAT1 cells to measure antibody responses to contemporary 3C.2a and 3C.3a A(H3N2) viruses in human sera. Viruses grown in either eggs or cells&#160;can be used in this assay. Heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) virus to allow antibodies in the sera to bind to the virus. 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 the A(H3N2) virus to infect MDCK-SIAT1 cells and replicate. After overnight incubation, the plates are fixed and the amount of virus in each well is quantified by an ELISA using anti-influenza A NP monoclonal antibodies. The detection of NP indicates the presence of virus infection and the absence of neutralizing antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805; 50% inhibition of virus infectivity.

<table border="0" cellpadding="0" cellspacing="0" width="969">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM) with high Glucose</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">11965</td>
			<td style="width:308px;">A critical component of Sterile Cell Culture, Virus Propagation and Virus Diluent Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fetal bovine serum (FBS)</td>
			<td style="width:181px;">Hyclone</td>
			<td style="width:185px;">SH30070.03</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Bovine Serum Albumin (BSA) Fraction V, Protase Free</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">3117332001</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">L-Glutamine</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">25030-081</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Sodium pyruvate</td>
			<td style="width:181px;">Life Science</td>
			<td>11360-070</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Geneticin G-418 disulfate salt&#160;</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">A1720-5G&#160;&#160;</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">HEPES</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">15630-080</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Penicillin/Streptomycin</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">15140-122</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:295px;">Acetone</td>
			<td style="width:181px;">VWR</td>
			<td style="width:185px;">67-64-1</td>
			<td style="width:308px;">Used at an 80% concentration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Phosphate-Citrate Buffer with Sodium Perborate&#160;</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">SLBF2806V</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:295px;">O-Phenylenediamine Dihydrochloride tablet</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">SLBQ1086V</td>
			<td style="width:308px;">1 tablet per 100ml of cell culture grade water</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:295px;">Sulfuric Acid</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:185px;">A510-P500</td>
			<td style="width:308px;">Used 0.5M final concentration</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:295px;">Ethanol, Denatured, 4L</td>
			<td style="width:181px;">VWR</td>
			<td style="width:185px;">EM-AX0422-3</td>
			<td style="width:308px;">Used at an 70% concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Trypsin-EDTA</td>
			<td style="width:181px;">Life Science</td>
			<td style="width:185px;">1748048</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">RDE II &#34;Seiken&#34;</td>
			<td style="width:181px;">Denka Seiken</td>
			<td style="width:185px;">370013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Tween 20</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:185px;">P1379-500ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Anti-NP mouse monoclonal Ab</td>
			<td style="width:181px;">Millipore pool</td>
			<td style="width:185px;">MAB 8257 MAB 8258</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Anti-mouse IgG HRP&#160;</td>
			<td style="width:181px;">KPL</td>
			<td style="width:185px;">074-1802</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">96-well flat-bottom plates</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:185px;">3455</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Plate reader</td>
			<td style="width:181px;">Molecular Device</td>
			<td style="width:185px;">Spectromax 384 plus</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Cell Culture Flask 162 cm2/Vent Cap</td>
			<td style="width:181px;">Corning/VWR</td>
			<td style="width:185px;">3151</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Determination of the infectivity of the virus stocks is the first step in the MN assay. Figure 2 illustrates the plate layout to determine the TCID50 of the virus stocks. For virus stocks with unknown infectivity, the virus can be titrated from multiple pre-dilutions, for example both 10-2 and 10-3, in order to capture the best titration curve to calculate infectivity of the virus. The virus amount used in the MN assay should ...

Watch this Scientific Journal Video about Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells at JoVE.com

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.

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 ...

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