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,

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....

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

List of materials used in this article

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