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

Summary

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.

Abstract

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-FcγR. Using this method, the contribution of specific Fc-FcγR interactions conferred by immunoglobulins can be determined using an in vitro assay.

Introduction

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 reach^{2,3,4,5,6,7,8}. One of the major aims of a universal influenza vaccine is to induce a strong antibody response towards the conserved regions of the influenza virus^{9,10,11,12,13,14,15,16}. Broadly reactive antibodies that recognize these conserved epitopes, composed of both neutralizing and non-neutralizing antibodies^17,18, have been shown to require Fc-FcγR interactions for optimal protection in vivo and highlight the contribution of Fc-mediated immunity to the overall immune response to influenza virus^19,20.

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 illness^1,21 – 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 detected^17,18. 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, FcγRIV. Antibody engagement of the FcγR 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 functions^{19,23,24,25,26}. First, the protocol described here can easily be adapted to incorporate different viral targets, FcγRs, and antibodies from different species (human and murine). Second, the use of a modified Jurkat cell line expressing an FcγR 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 FcγR 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 – 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 FcγR and does not express any inhibitory FcγRs, 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 contact^27,28. Here, we describe a method that assesses the ability of stalk-specific monoclonal antibodies to engage and activate the murine activating FcγRIV. Although there are exceptions^29,30, 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.

Protocol

The experiments preformed in this manuscript were done following Icahn School of Medicine’s institutional code of ethics and regulations.

1. Expression of Influenza Virus Hemagglutinin via Transfection or Infection

Transfection:

1. 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 °C incubator (with 5% CO₂).
2. Transfect cells with 100 ng of DNA coding for viral hemagglutinin and 0.2 μL of transfection reagent per well in a total volume of 50 μL.
   NOTE: Both plasmid and transfectant reagent are diluted in 1x Opti-Minimum Essential Media (MEM) Reduced Serum Medium.
3. After incubating plates in a 37 °C incubator with 5% CO₂ for 18 h, remove transfection media.

2. Infection

1. 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 °C incubator with 5% CO₂.
2. Dilute virus in 1x MEM and infect cells at a multiplicity of infection of 3, 1, or 0.33 for 1 h in a 37 °C incubator with 5% CO₂. Total volume should equal 100 μL.
   NOTE: 10x MEM is diluted with water to make a 1x MEM working stock for virus dilution mentioned above.
3. After infection, remove the inoculum and add 100 μL of 1x MEM into the plate in the absence of trypsin.
4. Grow infected cells for 18 to 24 h in a 37 °C incubator with 5% CO₂.

3. Assessing the FcRg/NFAT-mediated Activation of Luciferase Activity by Monoclonal Antibodies or Sera

1. First, replace growth media of transfected or infected cells with 25 µL of assay media (1x RPMI 1640 supplemented with 4% (vol/vol) low IgG fetal bovine sera).
2. In a separate 96-well plate, perform four-fold dilutions of the antibodies of interest starting at 30 μg/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 ‘no antibody’ and background controls, add 25 µL of assay buffer instead of antibody.
3. Transfer 25 μL of the serially diluted antibodies from Step 3.2 to corresponding wells on plate of transfected cells or infected cells.
4. Incubate plate in a 37 °C incubator (with 5% CO₂) for 15 min.
5. Add 25 μL 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 µL and the starting final concentration of the antibody is at 10 µg/mL (Figure 1B).
   NOTE: For the background control, add 25 µL 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.
6. Incubate plate in a 37 °C incubator with 5% CO₂ for 6 h.
7. Thaw and warm luciferase substrate buffer in a 37 °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 °C to -10 °C for up to six weeks.
8. Add 75 μL/well of luciferase substrate buffer to all wells except for the background control.
9. Read on a luminometer.
10. Calculate fold induction with the following equation: Fold induction = (Induced value-Background value) / (No mAb value-Background value).

Results

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 γ¹⁹. 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 FcγRIV by ~14-fold. On the other hand, the head-specific mAb, PY102 and the control IgG do not (

Discussion

The method described here allows the user to measure the ability of an HA-specific monoclonal antibody to engage the murine FcγRIV. 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 FcγRs (humans or murine). Additionally, we have used sera samples to assess the ability of antibodies in a polyclonal context to in...

Materials

Name	Company	Catalog Number	Comments
A549 cells	ATCC	CCL-185	Adenocarcinomic human alveolar basal epithelial cells
HEK 293T cells	ATCC	CRL-3216	Human embryonic kidney cells
Lipofectamine 2000	Life Technologies	11668019	Transfection reagent
1X Opti-MEM Reduced Serum Medium	ThermoFisher Scientific	51985-034
White tissue culture treated 96-well plate	Corning, Inc.	3917	Assay plates
10X MEM	Gibco	11430-030
Jurkat cell expressing murine FcgRIV	Promega	M1201	Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum
Jurkat cell expressing human FcgRIIIa	Promega	G7010	Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum
Jurkat cell expressing human FcgRIIa	Promega	G9901	Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum
Luminometer	BioTek	Synergy H1 Multi-Mode reader	Luminescence plate reader
Bio-Glo Luciferase Assay System	Promega	G7940	Contains luciferase assay buffer and luciferase assay substrate
Madin Darby canine kidney cells	ATCC	CCL-34	Canine kidney cells