Name: a method to assess fc-mediated effector functions induced by influenza hemagglutinin specific antibodies
Uploaded: 2018-02-23T12:30:00
Description: Watch this Scientific Journal Video about A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies at JoVE.com

This project has been funded in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under CEIRS contract P01AI097092-04S1 (P.E.L.).

The experiments preformed in this manuscript were done following Icahn School of Medicine&#8217;s institutional code of ethics and regulations.
1. Expression of Influenza Virus Hemagglutinin via Transfection or Infection
Transfection:
<ol>
	<li>Plate Human Embryonic Kidney (HEK 293T) cells at a density of 2 x 104 cells/well in a white tissue culture treated 96-well plate and let it sit for 4 h in a 37 &#176;C incubator ...

<ol>
	<li>Hobson, D., Curry, R. L., Beare, A. S., Ward-Gardner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+serum+haemagglutination-inhibiting+antibody+in+protection+against+challenge+infection+with+influenza+A2+and+B+viruses.">The role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses.</a> J Hygiene. 70 (04), 767-777 (1972).</li><li>Throsby, 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=Heterosubtypic+neutralizing+monoclonal+antibodies+cross-protective+against+H5N1+and+H1N1+recovered+from+human+IgM++memory+B+cells.">Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells.</a> PloS One. 3 (12), e3942 (2008).</li><li>Ekiert, D. C., 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=Antibody+recognition+of+a+highly+conserved+influenza+virus+epitope.">Antibody recognition of a highly conserved influenza virus epitope.</a> Science. 324 (5924), 246-251 (2009).</li><li>Ekiert, D. C., 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=A+Highly+Conserved+Neutralizing+Epitope+on+Group+2+Influenza+A+Viruses.">A Highly Conserved Neutralizing Epitope on Group 2 Influenza A Viruses.</a> Science. 333 (6044), 843-850 (2011).</li><li>Tan, G. S., 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=A+pan-H1+anti-hemagglutinin+monoclonal+antibody+with+potent+broad-spectrum+efficacy+in+vivo.">A pan-H1 anti-hemagglutinin monoclonal antibody with potent broad-spectrum efficacy in vivo.</a> J Virol. 86 (11), 6179-6188 (2012).</li><li>Tan, G. S., 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=Characterization+of+a+Broadly+Neutralizing+Monoclonal+Antibody+That+Targets+the+Fusion+Domain+of+Group+2+Influenza+A+Virus+Hemagglutinin.">Characterization of a Broadly Neutralizing Monoclonal Antibody That Targets the Fusion Domain of Group 2 Influenza A Virus Hemagglutinin.</a> J Virol. 88 (23), 13580-13592 (2014).</li><li>Friesen, R. H. E., 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=A+common+solution+to+group+2+influenza+virus+neutralization.">A common solution to group 2 influenza virus neutralization.</a> PNAS. 111 (1), 445-450 (2014).</li><li>Dreyfus, C., 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=Highly+Conserved+Protective+Epitopes+on+Influenza+B+Viruses.">Highly Conserved Protective Epitopes on Influenza B Viruses.</a> Science. 337 (6100), 1343-1348 (2012).</li><li>Krammer, F., Palese, P., Steel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+Universal+Influenza+Virus+Vaccine+Design+and+Antibody+Mediated+Therapies+Based+on+Conserved+Regions+of+the+Hemagglutinin.">Advances in Universal Influenza Virus Vaccine Design and Antibody Mediated Therapies Based on Conserved Regions of the Hemagglutinin.</a> Curr Top Microb and Immunol. , (2014).</li><li>Impagliazzo, 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=A+stable+trimeric+influenza+hemagglutinin+stem+as+a+broadly+protective+immunogen.">A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen.</a> Science. , (2015).</li><li>Nachbagauer, R., Krammer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Microbiology+and+Infection.">Clinical Microbiology and Infection.</a> Clin Microb Infect. , 1-7 (2017).</li><li>Krammer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+universal+influenza+virus+vaccine+approaches.">Novel universal influenza virus vaccine approaches.</a> Current opinion in virology. 17, 95 (2016).</li><li>Steel, J., 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=Influenza+virus+vaccine+based+on+the+conserved+hemagglutinin+stalk+domain.">Influenza virus vaccine based on the conserved hemagglutinin stalk domain.</a> mBio. 1 (1), (2010).</li><li>Krammer, F., Pica, N., Hai, R., Margine, I., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+hemagglutinin+influenza+virus+vaccine+constructs+elicit+broadly+protective+stalk-specific+antibodies.">Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies.</a> J Virol. 87 (12), 6542-6550 (2013).</li><li>Krammer, F., Pica, N., Hai, R., Tan, G. S., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemagglutinin+Stalk-Reactive+Antibodies+Are+Boosted+following+Sequential+Infection+with+Seasonal+and+Pandemic+H1N1+Influenza+Virus+in+Mice.">Hemagglutinin Stalk-Reactive Antibodies Are Boosted following Sequential Infection with Seasonal and Pandemic H1N1 Influenza Virus in Mice.</a> J Virol. 86 (19), 10302-10307 (2012).</li><li>Hai, R., 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=Influenza+viruses+expressing+chimeric+hemagglutinins:+globular+head+and+stalk+domains+derived+from+different+subtypes.">Influenza viruses expressing chimeric hemagglutinins: globular head and stalk domains derived from different subtypes.</a> J Virol. 86 (10), 5774-5781 (2012).</li><li>Dunand, C. J. H., 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=Both+Neutralizing+and+Non-Neutralizing+Human+H7N9+Influenza+Vaccine-Induced+Monoclonal+Antibodies+Confer+Protection.">Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced Monoclonal Antibodies Confer Protection.</a> Cell Host and Microbe. 19 (6), 800-813 (2016).</li><li>Tan, G. S., 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=Broadly-Reactive+Neutralizing+and+Non-neutralizing+Antibodies+Directed+against+the+H7+Influenza+Virus+Hemagglutinin+Reveal+Divergent+Mechanisms+of+Protection.">Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection.</a> PLoS Path. 12 (4), e1005578 (2016).</li><li>DiLillo, D. J., Tan, G. S., Palese, P., Ravetch, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadly+neutralizing+hemagglutinin+stalk-specific+antibodies+require+Fc&#947;R+interactions+for+protection+against+influenza+virus+in+vivo.">Broadly neutralizing hemagglutinin stalk-specific antibodies require Fc&#947;R interactions for protection against influenza virus in vivo.</a> Nat Med. 20 (2), 143-151 (2014).</li><li>DiLillo, D. J., Palese, P., Wilson, P. C., Ravetch, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadly+neutralizing+anti-influenza+antibodies+require+Fc+receptor+engagement+for+in+vivo+protection.">Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection.</a> J Clin Invest. 126 (2), 605-610 (2016).</li><li>Potter, C. W., Oxford, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+immunity+to+influenza+infection+in+man.">Determinants of immunity to influenza infection in man.</a> Brit Med Bull. 35 (1), 69-75 (1979).</li><li>Memoli, M. J., 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=Evaluation+of+Antihemagglutinin+and+Antineuraminidase+Antibodies+as+Correlates+of+Protection+in+an+Influenza+A/H1N1+Virus+Healthy+Human+Challenge+Model.">Evaluation of Antihemagglutinin and Antineuraminidase Antibodies as Correlates of Protection in an Influenza A/H1N1 Virus Healthy Human Challenge Model.</a> mBio. 7 (2), e00417 (2016).</li><li>Jegaskanda, S., 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=Age-associated+cross-reactive+antibody-dependent+cellular+cytotoxicity+toward+2009+pandemic+influenza+A+virus+subtype+H1N1.">Age-associated cross-reactive antibody-dependent cellular cytotoxicity toward 2009 pandemic influenza A virus subtype H1N1.</a> J Infect Dis. 208 (7), 1051-1061 (2013).</li><li>Jegaskanda, S., 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=Cross-Reactive+Influenza-Specific+Antibody-Dependent+Cellular+Cytotoxicity+Antibodies+in+the+Absence+of+Neutralizing+Antibodies.">Cross-Reactive Influenza-Specific Antibody-Dependent Cellular Cytotoxicity Antibodies in the Absence of Neutralizing Antibodies.</a> J Immunol. 190 (4), 1837-1848 (2013).</li><li>Jegaskanda, S., Wheatley, A. K., Kent, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScienceDirectAntibody-dependent+cellular+cytotoxicity+and+influenza+virus.">ScienceDirectAntibody-dependent cellular cytotoxicity and influenza virus.</a> Curr Opin Virol. 22 (C), 89-96 (2017).</li><li>Srivastava, V., 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=Identification+of+Dominant+Antibody-Dependent+Cell-Mediated+Cytotoxicity+Epitopes+on+the+Hemagglutinin+Antigen+of+Pandemic+H1N1+Influenza+Virus.">Identification of Dominant Antibody-Dependent Cell-Mediated Cytotoxicity Epitopes on the Hemagglutinin Antigen of Pandemic H1N1 Influenza Virus.</a> J Virol. 87 (10), 5831-5840 (2013).</li><li>He, W., 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=Epitope+specificity+plays+a+critical+role+in+regulating+antibody-dependent+cell-mediated+cytotoxicity+against+influenza+A+virus.">Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus.</a> PNAS. 113 (42), 11931-11936 (2016).</li><li>Leon, P. E., 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=Optimal+activation+of+Fc-mediated+effector+functions+by+influenza+virus+hemagglutinin+antibodies+requires+two+points+of+contact.">Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact.</a> PNAS. , 201613225 (2016).</li><li>Benjamin, E., 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=A+Broadly+Neutralizing+Human+Monoclonal+Antibody+Directed+against+a+Novel+Conserved+Epitope+on+the+Influenza+Virus+H3+Hemagglutinin+Globular+Head.">A Broadly Neutralizing Human Monoclonal Antibody Directed against a Novel Conserved Epitope on the Influenza Virus H3 Hemagglutinin Globular Head.</a> J Virol. 88 (12), 6743-6750 (2014).</li><li>Ekiert, D. C., Kashyap, A. K., 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=Cross-neutralization+of+influenza+A+viruses+mediated+by+a+single+antibody+loop.">Cross-neutralization of influenza A viruses mediated by a single antibody loop.</a> Nature. 488 (7417), 526-532 (2013).</li><li>He, W., 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=Broadly+Neutralizing+Anti-Influenza+Virus+Antibodies:+Enhancement+of+Neutralizing+Potency+in+Polyclonal+Mixtures+and+IgA+Backbones.">Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones.</a> J Virol. 89 (7), 3610 (2015).</li><li>Kristensen, A. B., 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=Antibody+Responses+with+Fc-Mediated+Functions+after+Vaccination+of+HIV-Infected+Subjects+with+Trivalent+Influenza+Vaccine.">Antibody Responses with Fc-Mediated Functions after Vaccination of HIV-Infected Subjects with Trivalent Influenza Vaccine.</a> J Virol. 90 (12), 5724-5734 (2016).</li><li>Greenberg, S. B., Criswell, B. S., Six, H. R., Couch, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+cytotoxicity+to+influenza+virus-infected+cells.+II.+Requirement+for+antibody+and+non-T+lymphocytes.">Lymphocyte cytotoxicity to influenza virus-infected cells. II. Requirement for antibody and non-T lymphocytes.</a> J Immunol (Baltimore, Md. : 1950). 119 (6), 2100-2106 (1977).</li><li>Corrales-Aguilar, E., Trilling, 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=A+novel+assay+for+detecting+virus-specific+antibodies+triggering+activation+of+Fc&#947;+receptors.">A novel assay for detecting virus-specific antibodies triggering activation of Fc&#947; receptors.</a> J Immunol Meth. 387 (1-2), 21-35 (2013).</li><li>de Vries, R. D., 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=Influenza+virus-specific+antibody+dependent+cellular+cytoxicity+induced+by+vaccination+or+natural+infection.">Influenza virus-specific antibody dependent cellular cytoxicity induced by vaccination or natural infection.</a> Vaccine. 35 (2), 238-247 (2017).</li><li>Cheng, 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=Development+of+a+robust+reporter-based+ADCC+assay+with+frozen,+thaw-and-use+cells+to+measure+Fc+effector+function+of+therapeutic+antibodies.">Development of a robust reporter-based ADCC assay with frozen, thaw-and-use cells to measure Fc effector function of therapeutic antibodies.</a> J Immunol Meth. 414 (1), 69-81 (2014).</li></ol>

The method described here allows the user to measure the ability of an HA-specific monoclonal antibody to engage the murine Fc&#947;RIV. The target antigen, influenza virus hemagglutinin, is expressed on the surface of cells after infection by virus or transfection of plasmid DNA. The assay is further amenable to using different surface-expressed viral proteins in combination with other Fc&#947;Rs (humans or murine). Additionally, we have used sera samples to assess the ability of antibodies in a polyclonal context to in...

Immunity provided by the seasonal influenza vaccine has traditionally been assessed by the hemagglutination inhibition (HI) assay1, which measures the presence of antibodies that target the receptor binding site of the hemagglutinin. These HI-active antibodies can confer sterilizing immunity, but are generally narrow in their breadth of protection, providing immunity to only a select few strains of influenza virus. The isolation and characterization of broadly reactive monoclonal antibodies that recognize the hemagglutinin (HA) suggest the development of a universal influenza vaccine is within reach2,3,4,5,6,7,8. One of the major aims of a universal influenza vaccine is to induce a strong antibody response towards the conserved regions of the influenza virus9,10,11,12,13,14,15,16. Broadly reactive antibodies that recognize these conserved epitopes, composed of both neutralizing and non-neutralizing antibodies17,18, have been shown to require Fc-Fc&#947;R interactions for optimal protection in vivo and highlight the contribution of Fc-mediated immunity to the overall immune response to influenza virus19,20.
Correlates of protection are crucial in assessing immunity to infection. These metrics allow scientists and clinicians to estimate the efficacy of vaccines, the significance of the data, and the course of treatment. The only established correlate of protection against influenza virus infection is the hemagglutination inhibition assay. A titer of 1:40 is associated with a 50% reduction in the risk of illness1,21 &#8211; and reflects the presence of antibodies that inhibit agglutination by targeting the receptor binding site located on the globular head of the HA. However, it should also be noted that T-cell responses may be a better correlate of protection against influenza virus infection in the elderly. Interestingly, a recent report suggests that neuraminidase inhibition activity may be a better predictor of immunity to influenza22. Fortunately, typical in vitro assays such as neutralization or neuraminidase inhibition assays can measure HA stalk- or neuraminidase-specific antibodies. Most of these conventional in vitro assays, however, only take into account the function of the antigen binding region of the antibody and do not measure the role of the Fc region. Additionally, the contribution of non-neutralizing antibodies that protect via Fc receptor engagement in vivo are not detected17,18. In order to measure protection afforded by antibodies that induce Fc-mediated immunity such as antibody dependent cell mediated cytotoxicity (ADCC), a robust in vitro assay is needed.
The method described below assesses the ability of murine monoclonal antibodies to induce Fc-mediated functions through the use of a genetically modified Jurkat cell line expressing an activating murine type 1 FcR, Fc&#947;RIV. Antibody engagement of the Fc&#947;R transduces intracellular signaling that triggers nuclear factor of activated T-cells-mediated luciferase activity. The assay has several advantages over traditional techniques that require isolation and culture of primary effector cells and the use of flow cytometry to detect activation of Fc-mediated effector functions19,23,24,25,26. First, the protocol described here can easily be adapted to incorporate different viral targets, Fc&#947;Rs, and antibodies from different species (human and murine). Second, the use of a modified Jurkat cell line expressing an Fc&#947;R with a luciferase reporter gene under a nuclear factor of activated T-cell (NFAT) allows for a large format assay that can be easily analyzed using a plate reader measuring luminescence. Here, the traditional activation of primary effector cells is replaced with the induction of NFAT-luciferase upon antibody engagement of an Fc&#947;R on the surface of the Jurkat cell. This 96-well plate format assay allows for the measurement of up to four different samples in triplicates with seven dilutions &#8211; a number of samples that could be cumbersome using traditional techniques. There are additional points to consider with this assay that may affect the design of experiments and the interpretation of data. The viral target must be expressed on the cell surface in order for antibody recognition. To mitigate this, the target antigen (e.g. an internal viral protein) may be directly coated on the surface of a plate. However, this has not yet been rigorously tested. Additionally, the modified Jurkat cell line expresses only one type of activating Fc&#947;R and does not express any inhibitory Fc&#947;Rs, while primary cell lines express all receptors in a physiological context.
We have previously used this assay to demonstrate that epitope specificity in a polyclonal context plays a crucial role in regulating Fc-mediated effector functions and that optimal activation of antibody-dependent cell-mediated response requires two points of contact27,28. Here, we describe a method that assesses the ability of stalk-specific monoclonal antibodies to engage and activate the murine activating Fc&#947;RIV. Although there are exceptions29,30, a large number of broadly reactive antibodies target the antigenically conserved stalk region of the hemagglutinin and thus play an important role in the development of a universal influenza vaccine.

<table border="0" cellpadding="0" cellspacing="0" width="921">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">A549 cells&#160;</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CCL-185</td>
			<td style="width:353px;">Adenocarcinomic human alveolar basal epithelial cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">HEK 293T cells</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CRL-3216</td>
			<td>Human embryonic kidney cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Lipofectamine 2000</td>
			<td style="width:141px;">Life Technologies&#160;</td>
			<td style="width:184px;">11668019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X Opti-MEM Reduced Serum Medium</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">51985-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">White tissue culture treated 96-well plate</td>
			<td>Corning, Inc.&#160;</td>
			<td>3917</td>
			<td>Assay plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">10X MEM</td>
			<td style="width:141px;">Gibco</td>
			<td style="width:184px;">11430-030</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing murine FcgRIV</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">M1201</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing human FcgRIIIa</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G7010</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing human FcgRIIa</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G9901</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luminometer</td>
			<td>BioTek</td>
			<td>Synergy H1 Multi-Mode reader</td>
			<td>Luminescence plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Bio-Glo Luciferase Assay System</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G7940</td>
			<td>Contains luciferase assay buffer and luciferase assay substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Madin Darby canine kidney cells</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CCL-34</td>
			<td>Canine kidney cells</td>
		</tr>
	</tbody>
</table>

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 demonstrated that a stalk-specific mAb, 6F12, but not head-specific a head-specific mAb, PY102, was able to activate primary natural killer cells by upregulating CD107a and interferon&#160;&#947;19. To model the ability of a stalk-specific antibody to activate primary human NK cells, we show that a stalk-specific mAb, 6F12, can robustly activate the modified Jurkat cell expressing the murine Fc&#947;RIV by ~14-fold. On the other hand, the head-specific mAb, PY102 and the control IgG do not (<st...

Watch this Scientific Journal Video about A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies at JoVE.com

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

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 ...

The overall goal of this protocol is to assess the ability of an antibody or a serum sample to activate Fc-mediated effector function. This method can help answer key questions in the immunology and vaccinology fields by measuring the activation of antibody-dependent Fc-mediated immunity. The main advantage of this technique is that the experiment can be done within 24 hours from the material preparation to the data analysis.Demonstrating this procedure will be Mark Bailey, graduate student from the laboratory of Peter Palese. To induce influenza virus hemagglutinin expression via transfection, first plate human embryonic kidney 293 cells at a two times 10 to the fourth cells per well concentration in a white tissue culture treated 96-well plate. After four hours at 37 degrees Celsius and 5%CO2, transfect the cells with 100 nanograms of DNA coding for viral hemagglutinin and 0.2 microliters of transfection reagent per well and a total volume of 50 microliters of 1X optimum-reduced serum medium.Add DNA liposome complexes to each well to transfect and return the plate to the incubator for another 18 hours. To induce influenza virus hemagglutinin expression via infection, plate A549 cells at a two times 10 to the fourth cells per well concentration in a white tissue culture treated 96-well plate for an at least 18-hour incubation at 37 degrees Celsius and 5%CO2. At the end of the incubation, dilute the influenza virus to a multiplicity infection of three in 100 microliters of 1X MEM medium per well and infect the A459 cells for one hour in the cell culture incubator.At the end of the incubation, replace the inoculum with 100 microliters of fresh 1X MEM medium without virus and return the cells to the incubator for another 18 to 24 hours. To assess the ability of specific antibodies of interest to target the hemagglutinin-expressing cells, replace the supernatant of the virally-affected cell cultures with 25 microliters of Antibody-Dependent Cell-mediated Cytotoxicity or ADCC assay medium. Next, in a separate 96-well plate, perform four full dilutions of the antibodies of interest in triplicate in fresh ADCC medium starting at 10 micrograms per milliliter according to the schematic and taking care to include the background and no antibody controls.When all of the antibody has been diluted, transfer 25 microliters of the diluted antibody to the appropriate corresponding wells in the experimental cell plate and place the plate in the cell culture incubator. After 15 minutes, add 7.5 times 10 to the fourth modified effector Jurkat cells expressing the appropriate Fc receptor per well in 25 microliters of ADCC medium for a final volume of 75 microliters of ADCC medium per well. Return the plate to the cell culture incubator for six hours thawing luciferase substrate buffer in a 37 degree Celsius water bath during the last hour of the incubation.When the buffer is ready, add 75 microliters of luciferase substrate buffer to all of the wells except the background control well and read the plate on a luminometer. The fold induction of antibody-dependent cell-mediated cytotoxicity in the transfected or infected cell cultures can then be calculated. In this experiment, a stock-specific monoclonal antibody robustly activated and modified Jurkat cells expressing murine Fc gamma receptor four by approximately 14-fold compared to head-specific monoclonal and control IgG antibodies.After watching this video, you should have a good understanding of how to set up an experiment for assessing the ability of specific antibodies of interest to activate Fc-mediated immunity.

Research

JoVE Journal

Immunology and Infection

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

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 ...