Name: a method to assess fc-mediated effector functions induced by influenza hemagglutinin specific antibodies
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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 (with 5% CO2).</li>
	<li>Transfect cells with 100 ng of DNA coding for viral hemagglutinin and 0.2 &#956;L of transfection reagent per well in a total volume of 50 &#956;L. 
	NOTE: Both plasmid and transfectant reagent are diluted in 1x&#160;Opti-Minimum Essential Media (MEM) Reduced Serum Medium.</li>
	<li>After incubating plates in a 37 &#176;C incubator with 5% CO2 for 18 h, remove transfection media.</li>
</ol>
2. Infection
<ol>
	<li>Plate A549 or Madin Darby canine kidney cells at a density of 2 x 104 cells/well in a white tissue culture treated 96-well plate and grow for at least 18 h in a 37 &#176;C incubator with 5% CO2.</li>
	<li>Dilute virus in 1x&#160;MEM and infect cells at a multiplicity of infection of 3, 1, or 0.33 for 1 h in a 37 &#176;C incubator with 5% CO2. Total volume should equal 100 &#956;L. 
	NOTE: 10x&#160;MEM is diluted with water to make a 1x&#160;MEM working stock for virus dilution mentioned above.</li>
	<li>After infection, remove the inoculum and add 100 &#956;L of 1x&#160;MEM into the plate in the absence of trypsin.</li>
	<li>Grow infected cells for 18 to 24 h in a 37 &#176;C incubator with 5% CO2.</li>
</ol>
3. Assessing the FcRg/NFAT-mediated Activation of Luciferase Activity by Monoclonal Antibodies or Sera
<ol>
	<li>First, replace growth media of transfected or infected cells with 25 &#181;L of assay media (1x&#160;RPMI 1640 supplemented with 4% (vol/vol) low IgG fetal bovine sera).</li>
	<li>In a separate 96-well plate, perform four-fold dilutions of the antibodies of interest starting at 30 &#956;g/mL using the assay media. Perform dilutions in triplicates and an example plate layout is illustrated in Figure 1A. Make sure to include a control that excludes antibody (no antibody control) as well as a background control (assay buffer alone). 
	NOTE: For both the &#8216;no antibody&#8217; and background controls, add 25 &#181;L of assay buffer instead of antibody.</li>
	<li>Transfer 25 &#956;L of the serially diluted antibodies from Step 3.2 to corresponding wells on plate of transfected cells or infected cells.</li>
	<li>Incubate plate in a 37 &#176;C incubator (with 5% CO2) for 15 min.</li>
	<li>Add 25 &#956;L of the appropriate modified effector Jurkat cell expressing murine FcgRIV or human FcgRIIIa (75,000 of cells/well). Total volume for each well should be 75&#160;&#181;L and the starting final concentration of the antibody is at 10 &#181;g/mL (Figure 1B). 
	NOTE: For the background control, add 25 &#181;L of assay buffer instead of effector cells. To examine antibody-dependent cell-mediated phagocytosis, a modified Jurkat cell expressing the human FcgRIIa can be used.</li>
	<li>Incubate plate in a 37 &#176;C incubator with 5% CO2 for 6 h.</li>
	<li>Thaw and warm luciferase substrate buffer in a 37 &#176;C water for about 1 h before use. 
	NOTE: To make the luciferase substrate buffer, reconstitute lyophilized luciferase assay substrate in assay buffer provided. The reconstituted substrate buffer can be stored at -30 &#176;C to -10 &#176;C for up to six weeks.</li>
	<li>Add 75 &#956;L/well of luciferase substrate buffer to all wells except for the background control.</li>
	<li>Read on a luminometer.</li>
	<li>Calculate fold induction with the following equation: Fold induction = (Induced value-Background value) / (No mAb value-Background value).</li>
</ol>

<ol>
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Authors declare no conflicts of interest.

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 induce Fc effector functions (data not shown), which may provide more relevant information when studying the adaptive immune response induced by infection or vaccination. We additionally speculate that the assay can be modified to measure the induction of antibodies that recognize internal viral proteins by directly coating the plates with soluble protein. However, this methodology has not been rigorously tested.
Induction of antibody-dependent effector functions can be affected by several factors including but not limited to the effector to target ratio (number of effectors cells added per well), the concentration of antibody used, the length of incubation between effector and target cells, and the concentration of low IgG serum used in the assay buffer.
Historically, the standard assay to measure the antibody-dependent cellular cytotoxicity activity of antibodies or polyclonal sera involves primary natural killer (NK) cells harvested from donors. Engagement of Fc&#947;R and activation of the intracellular pathway is typically determined by the expression of activation markers CD107a and/or IFN-&#947;19,23,24,31. Although the classical assay uses more relevant effector cells as they have not been genetically engineered, donor variation due to allelic differences in the Fc&#947;R may affect NK cell activation and result in batch to batch variation. Alternative assays used as surrogates for ADCC activity include an Fc&#947;R dimer-based ELISA or a chromium release killing assay25. Though the use of an Fc&#947;R dimer-based ELISA is high throughput, the assay may not be physiologically relevant as this assay does not use effector cells32. While the chromium release assay measures direct killing, it is not high throughput and requires primary NK cells which again are donor dependent33. One recent method describe the use of murine T-cell hybridomas stably expressing chimeric Fc&#947;R-CD3&#950; chain molecules and measures activation through the secretion of IL-2. The assay described is similar to the described method here, which makes use of a modified T-cell as a surrogate for an effector cell expressing a singular activating Fc&#947;R34. Alternatively, a cell line (NK92.05-CD16) expressing the Fc&#947;RIIIa can be used as the effector cell and upregulation of the degranulation antigen CD107a is used as an activation marker. However, the use of virions and viral peptides on ELISA plates on the latter protocol should be noted as proteins coated onto hydrophobic surfaces may not always recapitulate the physiological structure of antigens35.
The method described here is a 96-well format assay, makes use of physiologically relevant and natively expressed viral targets and can be modified for murine or human antibodies. The genetically modified Jurkat cells can be maintained in culture and cryopreserved36. Though currently the effector cells express only one activating Fc&#947;R, the many advantages of using this protocol described in this study should be taken into consideration when measuring antibody-dependent cell-mediated effector function.

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><tbody><tr><td>A549 cells&amp;nbsp;</td><td>ATCC</td><td>CCL-185</td><td>Adenocarcinomic human alveolar basal epithelial cells</td></tr><tr><td>HEK 293T cells</td><td>ATCC</td><td>CRL-3216</td><td>Human embryonic kidney cells</td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies&amp;nbsp;</td><td>11668019</td><td>Transfection reagent</td></tr><tr><td>1X Opti-MEM Reduced Serum Medium</td><td>ThermoFisher Scientific</td><td>51985-034</td><td></td></tr><tr><td>White tissue culture treated 96-well plate</td><td>Corning, Inc.&amp;nbsp;</td><td>3917</td><td>Assay plates</td></tr><tr><td>10X MEM</td><td>Gibco</td><td>11430-030</td><td></td></tr><tr><td>Jurkat cell expressing murine FcgRIV</td><td>Promega</td><td>M1201</td><td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td></tr><tr><td>Jurkat cell expressing human FcgRIIIa</td><td>Promega</td><td>G7010</td><td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td></tr><tr><td>Jurkat cell expressing human FcgRIIa</td><td>Promega</td><td>G9901</td><td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td></tr><tr><td>Luminometer</td><td>BioTek</td><td>Synergy H1 Multi-Mode reader</td><td>Luminescence plate reader</td></tr><tr><td>Bio-Glo Luciferase Assay System</td><td>Promega</td><td>G7940</td><td>Contains luciferase assay buffer and luciferase assay substrate</td></tr><tr><td>Madin Darby canine kidney cells</td><td>ATCC</td><td>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 (Figure 2)28.
To investigate if the multiplicity of infection (MOI) can affect the induction of Jurkat cells expressing the murine Fc&#947;RIV, we infected MDCK cells with X31 (a reassortant influenza A virus expressing the hemagglutinin and neuraminidase of A/Hong Kong/1/68 (H3N2) virus with the internal segments of A/Puerto Rico/8/34 (H1N1)) with an MOI of 3, 1, or 0.33. Twenty four hours later, Jurkat cells expressing murine Fc&#947;RIV and mAb 9H10 were added unto the infected MDCK cells. In Figure 3, we show that cells infected with an MOI of 3 can induce the luminescence around 3-fold higher than MDCK cells infected with an MOI of 0.33.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56256/56256fig1.jpg" /> 
Figure 1: A schematic of a sample plate layout and composition of the experimental groups. (A) The starting concentration for each antibody sample is at 10 &#181;g/mL and serially diluted across the plate (column 1 to column 12) 4-fold. Each sample is done in triplicates. Row G is reserved for the no mAb control and Row H is background. (B) Final volume for each experimental group before addition of luciferase substrate buffer is 75 &#181;L. Of note, the sample and &#8216;no mAb groups&#8217; have 25 &#181;L of effector cells added, while the background control groups have 75 &#181;L of assay media. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56256/56256fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56256/56256fig2.jpg" /> 
Figure 2: Induction of modified Jurkat cells expressing FcgR by a stalk monoclonal antibody. A549 cells were infected with A/Puerto Rico/8/34 (H1N1) with an MOI of 3. At 24 hours post infection, murine Fc&#947;RIV-expressing Jurkat cells in combination with PY102, 6F12 or a IgG control were added. Induction of Jurkat cells were assessed by luminescence signal generated by firefly luciferase under the NFAT response element. Error bars = standard deviations. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56256/56256fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56256/56256fig3.jpg" /> 
Figure 3: The multiplicity of infection affects induction of a stalk-specific antibody. MDCK cells were infected with X31 (H3N2) with an MOI of 3, 1, or 0.33. At 24 h post infection, FcgR-expressing Jurkat cells in combination with 9H10 (starting dilution of 10 &#181;g/mL) added. Induction of Jurkat cells were assessed by luminescence signal generated by firefly luciferase under the NFAT response element. Error bars = standard deviations. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56256/56256fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

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JoVE Journal

Immunology and Infection

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

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

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 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 ...

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

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

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

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

Influenza A Virus Studies in a Mouse Model of Infection

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

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

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

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

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

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

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

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 ...

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

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

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

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

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

Medicine

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

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

Source: Bailey, M. J., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56256">A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies.</a> J. Vis. Exp. (2018).
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 produced bioluminescence.

1. Expression of Influenza Virus Hemagglutinin via Transfection

	Plate Human Embryonic Kidney (HEK 293T) cells at a density of 2 x 104 cells/well in a ...

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Antibody-Based Technologies

Characterization and Functional Analysis

Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

Source: Leon, P. E., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56067">Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for generating escape variants of neutralizing influenza virus monoclonal antibodies. Antibody-neutralized virus suspension is injected into embryonated chicken eggs to propagate escape variants selectively. This results in a virus population predominantly of escape variants, confirmed via a Hemagglutination assay.

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

The Hemagglutination Inhibition Assay to Detect Serum Antibodies Against a Target Antigen

Source: Kaufmann, L. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55833">An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers.</a> J. Vis. Exp. (2017)
The video demonstrates a hemagglutination inhibition assay for detecting serum antibodies against specific antigens. Confirmation of hemagglutination inhibition in the test well and hemagglutination in the control well is achieved through distinct visual patterns. This assay plays a crucial role in assessing immune response, vaccine effectiveness, and studying viral infections in virology and immunology.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...