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

Summary

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.

Abstract

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 α-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 °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 ≥50% inhibition of virus infectivity.

Introduction

Influenza viruses continue to cause morbidity and mortality in the human population each year. HA is the major surface glycoprotein of influenza viruses. Neutralizing antibodies targeting HA are the main immune mechanism that correlates with protection of influenza infection^1,2. Hemagglutination inhibition (HI) assays and MN assays are two methods widely used to measure antibody responses in human sera after influenza infection or vaccination³. The HI assay measures antibody inhibition of virus hemagglutination of red blood cells and is considered a surrogate assay. Unlike HI, the MN as....

Protocol

All influenza viruses should be handled according to appropriate biosafety level requirements (BSL-2 or higher) as defined in the Biosafety on Microbiological and Biomedical Laboratories (BMBL)¹².

1. Preparation of Reagents and Starting Material

1. Prepare MDCK-SIAT1 cells and sterile cell culture medium
   1. Prepare the MDCK-SIAT1 cell culture medium using 500 mL of Dulbecco's Modified Eagle Medium (DMEM) with high glucose, 10% v/v heat inactivated fetal bovine serum (FBS), 2 mM L- glutamine, 1 mM sodium pyruvate, 1 mg/mL G418 sulphate (e.g.....

Results

Determination of the infectivity of the virus stocks is the first step in the MN assay. Figure 2 illustrates the plate layout to determine the TCID₅₀ of the virus stocks. For virus stocks with unknown infectivity, the virus can be titrated from multiple pre-dilutions, for example both 10^-2 and 10^-3, in order to capture the best titration curve to calculate infectivity of the virus. The virus amount used in the MN assay should .......

Discussion

The MN assay is one of the main assays used for influenza serology to detect antibody responses following influenza infection or vaccination. Titers generated from MN assays are often used as the primary outcome of many influenza seroepidemiology studies. MN assays are also widely used for sero-diagnosis, and the evaluation of vaccine immunogenicity. International inter-lab studies have been conducted to compare MN assays performed in multiple laboratories¹⁴.

In contras.......

Materials

Name	Company	Catalog Number	Comments
Dulbecco’s Modified Eagle Medium (DMEM) with high Glucose	Life Science	11965	A critical component of Sterile Cell Culture, Virus Propagation and Virus Diluent Media
Fetal bovine serum (FBS)	Hyclone	SH30070.03
Bovine Serum Albumin (BSA) Fraction V, Protase Free	Sigma-Aldrich	3117332001
L-Glutamine	Life Science	25030-081
Sodium pyruvate	Life Science	11360-070
Geneticin G-418 disulfate salt	Sigma-Aldrich	A1720-5G
HEPES	Life Science	15630-080
Penicillin/Streptomycin	Life Science	15140-122
Acetone	VWR	67-64-1	Used at an 80% concentration
Phosphate-Citrate Buffer with Sodium Perborate	Sigma-Aldrich	SLBF2806V
O-Phenylenediamine Dihydrochloride tablet	Sigma-Aldrich	SLBQ1086V	1 tablet per 100ml of cell culture grade water
Sulfuric Acid	Fisher Scientific	A510-P500	Used 0.5M final concentration
Ethanol, Denatured, 4L	VWR	EM-AX0422-3	Used at an 70% concentration
Trypsin-EDTA	Life Science	1748048
RDE II "Seiken"	Denka Seiken	370013
Tween 20	Sigma-Aldrich	P1379-500ml
Anti-NP mouse monoclonal Ab	Millipore pool	MAB 8257 MAB 8258
Anti-mouse IgG HRP	KPL	074-1802
96-well flat-bottom plates	Thermo Scientific	3455
Plate reader	Molecular Device	Spectromax 384 plus
Cell Culture Flask 162 cm2/Vent Cap	Corning/VWR	3151