Name: measuring influenza neutralizing antibody responses to a(h3n2) viruses in human sera by microneutralization assays using mdck-siat1 cells
Uploaded: 2017-11-22T15:00:00.000+00:00
Duration: 12 min 9 s
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.

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	<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. 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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. G., Mosse, J. A., Laurie, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDCK-SIAT1+cells+show+improved+isolation+rates+for+recent+human+influenza+viruses+compared+to+conventional+MDCK+cells.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in Microbiological and Biomedical Laboratories. , (2009).</li><li>Reed, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Laboratory+Comparison+of+Influenza+Microneutralization+Assays+for+A(H1N1)pdm09,+A(H3N2),+and+A(H5N1)+Influenza+Viruses+by+CONSISE.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+Quantitative+Micro-neutralization+Assay.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ViroSpot+microneutralization+assay+for+antigenic+characterization+of+human+influenza+viruses.">ViroSpot microneutralization assay for antigenic characterization of human influenza viruses.</a> Vaccine. 35, 46-52 (2017).</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 contrast to HI, the MN assay is designed to directly measure functional antibodies that can neutralize virus infection in cell culture. There are various forms of MN assays used in field laboratories around the world. The read-out of the assays (i.e., reduction of virus infectivity in the presence of neutralizing antibodies) can be based on ELISA quantification of virus NP antibodies, HA quantification of virus, or immuno-staining of virus plaques in each well following virus infection in the presence of antibodies and incubation in cell cultures14,15. Various forms of fluorescent-based plaque reduction neutralization assays (e.g., focus reduction assays and ViroSpot assays) are often used for antigenic characterization of large numbers of influenza virus field isolates, partly due to the low virus amount required in these assays15,16. For the characterization of antibody responses to influenza in human serology, the 2-day ELISA based MN assay is recommended by World Health Organization (WHO) global influenza surveillance network for serological diagnosis of influenza3. This method is also widely used in sero-epidemiology studies and the evaluation of antibody responses following influenza vaccination. It primarily detects antibodies to influenza HA surface protein and therefore can detect functional strain-specific antibodies.
Here, we describe a 2-day ELISA-based MN assay that utilizes MDCK-SIAT1 cells. This assay is optimized to detect neutralizing antibodies to contemporary A(H3N2) viruses in human sera. Several critical steps should be considered when performing MN assays using MDCK-SIAT1 cells. First, MDCK-SIAT1 cells should be passaged in culture media containing G418 sulphate to maintain the stability of the human &#945;SIAT1 cDNA in the cell line. G418 sulphate (e.g., geneticin) is not required in the media during virus propagation and MN assays using MDCK-SIAT1 cells. Cells used in MN assays should be at log growth phase with 75 - 95% confluency. Second, good virus stocks are essential in order to carry out successful MN assays. Virus stocks should have high infectivity as measured by TCID50 titration and a minimum amount of defective particles. The minimum dilution of virus stocks to achieve 100 TCID50/well virus should be equal or greater than 1:100. Although both egg and cell passaged viruses can be used in MN assays, MDCK-SIAT1 cell line maintains better genetic stability for propagating 3C.2a and 3C.3a A(H3N2) viruses7. After virus propagation, HA and NA genes of the virus stocks should be sequenced to ensure that no egg or cell culture adapted mutations are introduced at key antigenic sites that may cause changes in the antigenicity of the virus used in the assay. Third, the amount of infectious virus used in each assay should be carefully titrated, and verified with the inclusion of a back titration for each virus in each assay. Too much virus used in the assay may lower the antibody titers detected and reduce the sensitivity of the assay. Likewise, too little virus used in the assay will cause weak VC signals and high backgrounds (CC), and false positive results. Thus, it is important to include appropriate positive and negative control sera in the assays to monitor performance. When comparing antibody responses to multiple viruses using the same sera, it is critical to ensure that back titrations of all viruses are within an acceptable range.
The MN assay using MDCK-SIAT1 cells is sensitive and specific in detecting antibody responses to contemporary 3C.2a and 3C.3a A(H3N2) influenza viruses in human sera, including antigenically drifted A(H3N2) viruses6. This assay has been used to evaluate vaccinated human sera panels to 3C.2a and 3C.3a A(H3N2) viruses following inactivated influenza vaccination5,17,18. However, as HAs of influenza viruses continue to acquire mutations that can alter antigenicity and receptor binding properties of viruses and cause antigenic drift, continuous efforts are needed to optimize existing influenza serology assays in order to maintain the sensitivity and specificity required to characterize new emerging influenza viruses.

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

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 be standardized to 100 TCID50/well (50 &#181;L). The minimum dilution of virus stocks to achieve a 100 TCID50/well is 1:100.
 
Figure 3 describes the critical steps of a MN assay using MDCK-SIAT1 cells. Heat-inactivated sera are serial-diluted in 96-well plates as illustrated in Figure 4. 100 TCID50/50 &#181;L virus is then added to each well. After 1 h incubation to allow the antibody in the sera bind to the virus, 100 &#181;L of 1.5 x 105 cells/mL of MDCK-SIAT1 cells are added to each well. For optimal results, MDCK-SIAT1 cells should be at 75 - 95% confluency (Figure 1) for both the TCID50 and MN assays. The plates are then incubated for 18 - 20 h at 37 &#176;C, 5% CO2. After overnight incubation, the amount of virus in each well is detected and quantified by an anti-influenza A NP ELISA (Figure 3).
The OD in each well represents the amount of virus infection and replication in MDCK-SIAT1 cells in the presence of serially diluted sera containing neutralizing antibodies. It can be plotted against the sera dilutions (Figure 5). The reciprocal of the highest serum dilution that achieved &#8805;50% neutralization is considered the antibody titer for the serum sample. Figure 5 contains examples of results from 2 patients with paired sera tested against A/HongKong/4801/2014 A(H3N2) 3C.2a virus pre- and post-influenza vaccination. With patient 1 pre-vaccination sera, none of the sera dilutions inhibited the virus infection (Figure 5, light blue curve) and therefore is considered negative (&#60;10). In contrast, post-vaccination sera from this patient inhibited virus replication, indicating vaccination induced neutralizing antibodies. The third dilution of this sera is the highest dilution below the 50% cut-off, thus this patient has a post-vaccination titer of 40 (Figure 5, dark blue curve). In comparison, the serum from the second patient pre-vaccination contains pre-existing neutralizing antibodies at a titer of 320. Following vaccination, the titer increased to &#62;1,280. In this case, at a 1:10 pre-dilution of serum, no antibody end titer was achieved. The serum should be re-tested at a higher pre-dilution in order to achieve end titer.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56448/56448fig1.jpg" /> 
Figure 1. MDCK-SIAT1 cell culture. MDCK-SIAT1 cells. (A) MDCK-SIAT1 confluent monolayer (&#62;100% confluency) for virus inoculation; (B) MDCK-SIAT1 cells at log phase with 75 - 95% confluency for TCID50 and MN assays.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56448/56448fig2.jpg" /> 
Figure 2. TCID plate layout. Virus infectivity is determined by TCID50. Virus stock is pre-diluted to 10-2, and then serially diluted at a &#189; log per dilution to 10-7 (column 1 - 11), at 8 replicates per dilution (row A&#160; -H). Column 12 is used as the cell only control. TCID50 of the virus stock can be calculated using the Reed-Muench method.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56448/56448fig3v3.jpg" /> 
Figure 3. Schematic of the MN assay using MDCK-SIAT1 cells. 50 &#181;L of heat-inactivated sera are serially 2-fold diluted into 96-wells plates. 50 &#181;L of 100 TCID50 of A(H3N2) virus are then added to each well. Plates are incubated at 37 &#176;C for 1 h to allow the binding of virus and antibody. Then 1.5 x 104 MDCK-SIAT1 cells are added to each well. Plates are incubated for 18 - 20 h at 37 &#176;C, 5% CO2. After overnight incubation, plates are washed and fixed, the amount of virus in each well is quantified by an ELISA using anti-influenza A NP monoclonal antibodies. Sera antibody titers are calculated based on cut-offs defined by virus controls and cell controls. <a href="https://cloudflare.jove.com/files/ftp_upload/56448/56448fig3v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56448/56448fig4.jpg" /> 
Figure 4. The MN assay plate layout. Heat-inactivated sera are pre-diluted at 1:10, then serially 2-fold diluted in 96-well plates in column 1 - 10. Virus controls (virus and cells only, no sera) and cell controls (cells only, no virus and no sera) are in column 12. Column 11 is used for back titration or control sera. <a href="https://cloudflare.jove.com/files/ftp_upload/56448/56448fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56448/56448fig5.jpg" /> 
Figure 5. The MN assay results of human sera tested against A/HongKong/4801/2014 A(H3N2) virus using MDCK-SIAT1 cells. Sera from two patients pre- and post-2016-17 seasonal influenza vaccination were tested against A/HongKong/4801/2014 A(H3N2) virus using MDCK-SIAT1 cells. Neutralization titers are the reciprocal of the highest serum dilution that achieves &#8805;50% virus neutralization, as indicated by arrows on each curve. Patient 1: pre-vaccination titer &#60;10, post-vaccination titer 40; patient 2:pre-vaccination titer 320, post-vaccination titer &#62;1,280.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

17.9K Views.  Centers for Disease Control and Prevention. 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.

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

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

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

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.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

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. ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

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 viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

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.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

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

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

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

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.

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

Medicine

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

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. Occasionally, humans also fall victim to it, which results in high mortality. Nonetheless, HPAI virus research is often hindered, considering that it must be handled within biosafety level 3 laboratories. To address this issue, pseudoviruses are adopted as an alternative to wild-type viruses in some experiments of H5 HPAI studies. Pseudoviruses prove to be the ideal tools to study neutralizing antibodies against H5 HPAI viruses. This protocol describes the procedures and critical steps of H5 HPAI pseudovirus preparations and pseudovirus neutralization assays. Also, it discusses the troubleshooting, limitation, and modifications of these assays.

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

Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. ...

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.

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

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers

Source: Gao, J., et al. <a href="https://app.jove.com/v/54573">Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay.</a> J. Vis. Exp. (2016).
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 substrate gets cleaved by the immobilized lectin-conjugated peroxidase to produce color.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Reagents and ...

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

Source: Lin, Y., et al. <a href="https://app.jove.com/v/54897">Optimization of a Quantitative Micro-neutralization Assay.</a> J. Vis. Exp. (2016)
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.

1. Virus Titration
NOTE: Depending on the number of viruses and the number of duplicates, a well plate can be set up with the following flexibilities: (1) ...