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.

a-method-to-assess-fc-mediated-effector-functions-induced-influenza

Research

JoVE Journal

Immunology and Infection

7.6K Views.  Icahn School of Medicine at Mount Sinai. 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 st...

Video: 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...

measuring the activation of fc-mediated effector functions by ha antibodies

This video demonstrates an assay measuring the antibody-mediated activation of T cell effector functions. Adding an influenza virus hemagglutinin (HA)-specific monoclonal antibody to transfected mammalian cells expressing HA results in the formation of immune complexes with HA. Upon introducing engineered T cells expressing Fc receptors and a luciferase reporter, the T cells are activated by the antibody-HA complexes, and this activation is detected by adding a luciferase substrate and...

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

Next, in a separate 96-well plate, perform 4-fold dilutions of the antibodies of interest in triplicate and fresh ADCC medium starting at 10 micrograms per milliliter according to the schematic, and taking care to include the background and no antibody...

a technique to assess the in vivo localization of tumor-specific antibodies

This video demonstrates a technique to study the in vivo localization of tumor-specific antibodies in a mouse tumor xenograft model. Upon subcutaneously injecting cancer cells into mice for tumor formation, fluorescently-labeled antibodies are injected that bind to tumor-specific receptors, enabling in vivo assessment of antibody localization via...

A Technique to Assess the In Vivo Localization of Tumor-Specific Antibodies

Incubate the mixture for two hours at 20 degrees Celsius. Then, purify the labeled conjugates by extensive dialysis against PBS. Estimate the degree of labeling by measuring the absorbance of the dye at 780 nanometers, and absorbance of the protein at 280...

an in vitro artificial activation assay for studying fc receptor activation by antibodies

This video demonstrates Fc&#947;RIIIa-driven events initiated by therapeutic antibodies in human natural killer cells. The artificial stimulation platform facilitates the exploration of downstream effector functions, encompassing cytoskeletal rearrangement, degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in...

An In Vitro Artificial Activation Assay for Studying Fc Receptor Activation by Antibodies

Add 1 microliter to each tube of cells for a final concentration of 1 microgram per milliliter, and incubate the cells on ice for 30 minutes. During incubation, prepare a solution of anti-human kappa light chain antibody in media at a concentration of 50 micrograms per milliliter. The amount of antibody solution needed will be 50 microliters per cell...

generation of escape variants of neutralizing influenza virus monoclonal antibodies

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

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...

methods to assess beta cell death mediated by cytotoxic t lymphocytes

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro.  However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

a step-by-step method to detect neutralizing antibodies against aav using a colorimetric cell-based assay

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against...

an optimized hemagglutination inhibition (hi) assay to quantify influenza-specific antibody titers

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.

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

quantitative analyses of all influenza type a viral hemagglutinins and neuraminidases using universal antibodies in simple slot blot assays

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

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses.  This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and...

a method to produce recombinant antibodies against metallopeptidase

This video demonstrates a procedure for the production of recombinant antibodies against metallopeptidase. Recombinant plasmids carrying genes for the antibody, green fluorescent protein, and antibiotic resistance are transfected into epithelial cells with lipofectamine. The transfected cells are selected and cultivated to generate recombinant...

A Method to Produce Recombinant Antibodies Against Metallopeptidase

generation of influenza virus escape variants against neutralizing antibodies

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...

Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

microbiologically induced calcite precipitation mediated by sporosarcina pasteurii

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of...

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

When the cells reach 80% to 90% confluence, dilute the pIRES2-ZsGreen1 recombinant antibodies aminopeptidases in plasmid with Opti-MEM to a final concentration of 0.1 micrograms per microliter. After five minutes, mix the 50 microliters of diluted pIRES2-ZsGreen1 recombinant antibodies aminopeptidases in plasmid with one microliter of Lipofectamine 2000 and 49 microliters of...

a technique to generate monoclonal antibodies from antigen-specific b cells

The video demonstrates a technique for generating monoclonal antibodies from antigen-specific B cells. Eukaryotic expression vectors encoding the heavy and light chains of antibodies, cloned from isolated antigen-specific B cells, are introduced into mammalian cells using polyplexes. Once inside the cells, these vectors undergo translation, and the resulting heavy and light chain peptides assemble into antibodies, which are subsequently secreted by the...

A Technique to Generate Monoclonal Antibodies from Antigen-Specific B Cells

swimming-induced paralysis (swip) assay: a method to quantify dopamine-mediated locomotion in c. elegans

Here, we introduce an assay to measure SWIP, a behavioral phenotype of&#160;C. elegans&#160;that occurs during vigorous motion like swimming.&#160; The example protocol discusses both manual and automated analysis...

Swimming-Induced Paralysis (SWIP) Assay: A Method to Quantify Dopamine-Mediated Locomotion in C. elegans

Next, dilute 0.125 micrograms of each variable heavy and light chain-expressing vectors in 10 microliters of 150 millimolar sodium chloride. Vortex all the dilutions for 10 seconds each. Then, mix the previously diluted DNA transfection reagent with 10 microliters of DNA solution. Quickly vortex the DNA transfection mixture for 15 seconds, and incubate for 15 minutes at room...

a bead-based multiplex immunoassay to identify pathogen-specific antibodies in saliva

This video demonstrates a bead-based multiplex immunoassay technique to simultaneously detect antibodies for multiple environmental pathogens in human saliva. Fluorescent beads were coupled to antigens from different pathogens, which were captured using antibodies in the human saliva sample. Upon labeling the antibodies using a fluorescent reporter, the beads were analyzed using flow cytometry to assess the presence of different pathogen-specific antibodies in the...

A Bead-Based Multiplex Immunoassay to Identify Pathogen-Specific Antibodies in Saliva

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

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, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

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

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 upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

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

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

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

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

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

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

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 versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

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

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 (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

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

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 replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

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

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

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 currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B 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 ...

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 ...

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

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

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

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

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