Name: Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells (Video) | JoVE Journal
Uploaded: 2017-11-22T15:00:00.000+00:00
Duration: 12 min 9 s
Description: Learn microneutralization assays using MDCK-SIAT1 cells to measure neutralizing antibody responses to H3N2 influenza viruses in human sera. This method evaluates vaccine-induced immunity and protective antibody levels.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

playerTrialDesktop

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.

<ol><li>Reber, A., Katz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunological+assessment+of+influenza+vaccines+and+immune+correlates+of+protection%5BTitle%5D)+AND+%22Expert+Rev+of+Vaccines%22%5BJournal%5D)">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/pubmed?term=((Correlates+of+protection+against+influenza+infection+in+humans--on+the+path+to+a+universal+vaccine%3F%5BTitle%5D)+AND+%22Curr+Opin+in+Immunol%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=%22Manual+for+the+labratory+diagnosis+and+virological+surveillence+of+influenza%22">Manual for the labratory diagnosis and virological surveillence of influenza</a>. , 
 <a target="_blank" href="http://whqlibdoc.who.int/publications/2011/9789241548090_eng.pdf">http://whqlibdoc.who.int/publications/2011/9789241548090_eng.pdf</a> (2011).</li>
<li>Govorkova, E. A., Kodihalli, S., Alymova, I. V., Fanget, B., Webster, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growth+and+immunogenicity+of+influenza+viruses+cultivated+in+Vero+or+MDCK+cells+and+in+embryonated+chicken+eggs%5BTitle%5D)+AND+%22Dev+Biological+Stand%22%5BJournal%5D)">Growth and immunogenicity of influenza viruses cultivated in Vero or MDCK cells and in embryonated chicken eggs.</a> Dev Biological Stand. 98, discussion 73-34 39-51 (1999).</li>
<li>WHO. Recommended composition of influenza virus vaccines for use in the northern hemisphere influenza season. , 
 <a target="_blank" href="http://www.who.int/influenza/vaccines/virus/recommendations/201703_recommendation.pdf?ua=1">http://www.who.int/influenza/vaccines/virus/recommendations/201703_recommendation.pdf?ua=1</a> 2017-2018 (2017).</li>
<li>Levine, M. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutralizing+Antibody+Responses+to+Antigenically+Drifted+Influenza+A%28H3N2%29+Viruses+among+Children+and+Adolescents+following+2014-2015+Inactivated+and+Live+Attenuated+Influenza+Vaccination%5BTitle%5D)+AND+%22Clin+Vaccine+Immunol+%3A+CVI%22%5BJournal%5D)">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/pubmed?term=((The+characteristics+and+antigenic+properties+of+recently+emerged+subclade+3C.3a+and+3C.2a+human+influenza+A%28H3N2%29+viruses+passaged+in+MDCK+cells%5BTitle%5D)+AND+%22Influenza+Other+Respir+Viruses%22%5BJournal%5D)">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/pubmed?term=((Neuraminidase+receptor+binding+variants+of+human+influenza+A%28H3N2%29+viruses+resulting+from+substitution+of+aspartic+acid+151+in+the+catalytic+site%3A+a+role+in+virus+attachment%3F%5BTitle%5D)+AND+%22J+of+Virol%22%5BJournal%5D)">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/pubmed?term=((Mutations+acquired+during+cell+culture+isolation+may+affect+antigenic+characterisation+of+influenza+A%28H3N2%29+clade+3C.2a+viruses%5BTitle%5D)+AND+%22Euro+Surveillance%22%5BJournal%5D)">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/pubmed?term=((Overexpression+of+the+alpha-2%2C6-sialyltransferase+in+MDCK+cells+increases+influenza+virus+sensitivity+to+neuraminidase+inhibitors%5BTitle%5D)+AND+%22J+of+Virol%22%5BJournal%5D)">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. G., Mosse, J. A., Laurie, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MDCK-SIAT1+cells+show+improved+isolation+rates+for+recent+human+influenza+viruses+compared+to+conventional+MDCK+cells%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">MDCK-SIAT1 cells show improved isolation rates for recent human influenza viruses compared to conventional MDCK cells.</a> J Clin Microbiol. 46, 2189-2194 (2008).</li>
<li>US Department of Health and Human Services. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Biosafety+in+Microbiological+and+Biomedical+Laboratories%22">Biosafety in Microbiological and Biomedical Laboratories</a>. , (2009).</li>
<li>Reed, H. M. <a target="_blank" href="https://academic.oup.com/aje/article-abstract/27/3/493/99616/A-SIMPLE-METHOD-OF-ESTIMATING-FIFTY-PER-CENT?redirectedFrom=PDF">A simple method of estimating fifty percent endpoints.</a> The American Journal of Hygiene. 27, (1938).</li>
<li>Laurie, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((International+Laboratory+Comparison+of+Influenza+Microneutralization+Assays+for+A%28H1N1%29pdm09%2C+A%28H3N2%29%2C+and+A%28H5N1%29+Influenza+Viruses+by+CONSISE%5BTitle%5D)+AND+%22Clinical+Vaccine+Immunol+%3A+CVI%22%5BJournal%5D)">International Laboratory Comparison of Influenza Microneutralization Assays for A(H1N1)pdm09, A(H3N2), and A(H5N1) Influenza Viruses by CONSISE.</a> Clinical Vaccine Immunol : CVI. 22, 957-964 (2015).</li>
<li>Lin, Y., Gu, Y., McCauley, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+a+Quantitative+Micro-neutralization+Assay%5BTitle%5D)+AND+%22J+Vis+Exp+%3A+JoVE%22%5BJournal%5D)">Optimization of a Quantitative Micro-neutralization Assay.</a> J Vis Exp : JoVE. , (2016).</li>
<li>van Baalen, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ViroSpot+microneutralization+assay+for+antigenic+characterization+of+human+influenza+viruses%5BTitle%5D)+AND+%22Vaccine%22%5BJournal%5D)">ViroSpot microneutralization assay for antigenic characterization of human influenza viruses.</a> Vaccine. 35, 46-52 (2017).</li>
<li>WHO. Recommended composition of infleunza virus vaccines for use in the 2015-2016 northern heamisphere infleunza season. , 
 <a target="_blank" href="http://www.who.int/influenza/vaccines/virus/recommendations/2015_16_north/en">http://www.who.int/influenza/vaccines/virus/recommendations/2015_16_north/en</a> 2015-2016 (2015).</li>
<li>WHO. Recommended composition of influenza virus vaccines for use in the 2016-2017 northern hemisphere influenza season. , 
 <a target="_blank" href="http://www.who.int/influenza/vaccines/virus/recommendations/201602_recommendation.pdf?ua=1">http://www.who.int/influenza/vaccines/virus/recommendations/201602_recommendation.pdf?ua=1</a> 2016-2017 (2016).</li>
</ol>

The authors report no conflict of interest. The findings and conclusions in this publication are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention and the funding agency.

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

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 ...

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 ...

measuring-influenza-neutralizing-antibody-responses-to-h3n2-viruses

Research

JoVE Journal

Immunology and Infection

This video demonstrates Microneutralization assays to measure neutralizing antibodies using Madin-Darby Canine Kidney cells overexpressing α2,6-linked sialic acid in human sera. This assay is often used for influenza human...

A Microneutralization Assay to Measure Neutralizing Antibody Titers in Human Serum

This video demonstrates the imaging-based micro-neutralization assay to analyze the antigenic relations between influenza A and B viruses. It provides a quantitative and visual way to assess the effectiveness of antibodies or other treatments in preventing viral infection.

Analyzing the Antigenic Relationship between Viruses using an Image-Based Microneutralization Assay

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.

Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

This video demonstrates an assay measuring influenza neuraminidase (NA) inhibiting antibody titers in a serum sample. A serum sample containing antibodies against NA is added to a fetuin-coated microwell plate. Upon adding the virus, the antibodies prevent viral NA from cleaving fetuin and exposing its galactose moiety. A peroxidase-conjugated lectin is then added, which binds to the exposed galactose. The NA-inhibiting antibody titer is determined using a colorimetric test, where a peroxidase...

An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers

Source:Matthew J Murray1, Gary R McLean2,3, Matthew B Reeves11Institute of Immunity and Transplantation, University College London.2Cellular and Molecular Immunology Research Centre, London Metropolitan University.3National Heart and Lung Institute, Imperial College London.This video demonstrates an immunofluorescence assay designed to measure the neutralization of Human Cytomegalovirus (HCMV). Virus-specific antibodies are mixed with the virus and incubated with human fibroblast cells. The...

An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

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.

Optimization of a Quantitative Micro-neutralization Assay

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza A Virus Studies in a Mouse Model of Infection

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Here we describe a protocol for pseudovirus packaging and the measurement of antibody neutralizing activity.

Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity

Learn microneutralization assays using MDCK-SIAT1 cells to measure neutralizing antibody responses to H3N2 influenza viruses in human sera. This method evaluates vaccine-induced immunity and protective antibody levels.

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

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

Explore More Videos