Identification and Characterization of Novel Antibody Epitopes on the N2 Neuraminidase

Cells and viruses.A549 (ATCC CCL-185) and MDCK (ATCC CCL-34) cells were obtained from the American Type Culture Collection (ATCC) and propagated in 1× Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 1 U/ml penicillin–1 μg/ml streptomycin solution (Gibco), and 10 mM HEPES {2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid} (Gibco). Cells were kept at 37°C with 5% CO₂. A/Switzerland/9715293/2013 was obtained from the Influenza Reagent Resource (FR-1366) and grown in 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs (Charles River Laboratories) at 33°C for 3 days (33).

Antibodies.Plasmids encoding each antibody were provided by Patrick Wilson from the University of Chicago. The isolation and reactivity of each antibody has been previously described (8). Antibodies were produced by cotransfection of heavy and light chain plasmids using an ExpiFectamine 293 transfection kit according to manufacturer’s instructions (Thermo Fisher). Briefly, heavy- and light-chain plasmids were mixed with ExpiFectamine and incubated for 15 min in Opti-MEM (Gibco). HEK293F cells (Thermo Fisher) were diluted to 7 × 10⁷ cells in 30 ml of Expi293 expression medium (Thermo Fisher). The transfection mix was then added to cells, followed by incubation for 7 days at 37°C with 8% CO₂ with shaking. All antibodies were purified using gravity flow columns packed with protein G-sepharose. They were eluted into a 50-ml Falcon tube with 5 ml of 2 M Tris (pH 10) and concentrated using an Amicon Ultra 30-kDa filter unit (Millipore) (34).

Plaque assays.Virus titers were determined using a standard influenza virus plaque assay. MDCK cells were plated at 8 × 10⁵ cells per ml in a 12-well cell culture plate and incubated overnight at 37°C with 5% CO₂. The following day, allantoic fluid or cell culture supernatant was diluted 1:10 six times using 1× minimal essential medium (MEM; 10% 10× MEM [Gibco], 2 mM l-glutamine [Gibco], 0.1% sodium bicarbonate [Gibco], 10 mM HEPES, 1% 100 U/ml penicillin–100 μg/ml streptomycin solution [Gibco], and 0.2% bovine serum albumin [BSA]). The MDCK cells were washed one time using 1× phosphate-buffered saline (PBS) and then infected with 200-μl portions of each virus dilution. The cells were then incubated at 33°C with 5% CO₂ for 40 min (the plates were rocked every 10 min). After 40 min, the virus dilutions were aspirated and immediately replaced with an agarose overlay containing 2× MEM, 0.1% diethylaminoethyl (DEAE)-dextran, 1 μg/ml tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin, and 0.64% Oxoid agarose. The plates were incubated for 3 days at 33°C with 5% CO₂. To quantify virus titers, plates were fixed using 3.7% paraformaldehyde (PFA) overnight at 4°C. The overlay was then removed, and the cells were stained with a solution of 20% methanol containing 0.5% crystal violet powder.

Immunofluorescence.MDCK cells were plated in a 96-well cell culture plate at 3 × 10⁴ cells/well and incubated overnight at 37°C with 5% CO₂. The following day, the viruses were diluted to a multiplicity of infection (MOI) of 5 in 1× MEM. Cells were washed with 1× PBS and then infected with 100 μl of diluted virus. The plates were then incubated for 18 h at 33°C with 5% CO₂. The following day, the cells were fixed using 3.7% PFA at 200 μl per well. For immunofluorescence staining, the PFA was first aspirated from cells and then replaced with 200 μl of blocking solution containing 3% milk (American Bio) diluted in 1× PBS, followed by incubation for 1 h at room temperature. The blocking solution was removed and replaced with 1% milk. The primary antibodies were diluted to 300 μg in 1× PBS and then added at a 1:10 dilution to the 1% milk for a final concentration of 30 μg per well. Each plate was then incubated for 1 h at room temperature with shaking. The primary antibodies were then aspirated, and the plate was washed three times using 1× PBS. The secondary antibody Alexa Fluor 488-goat anti-human IgG(H+L) (Invitrogen) was diluted 1:500 in 1% milk and added to the plate at 100 μl/well. The plate was then incubated for 1 h at room temperature in the dark with shaking. Finally, the secondary antibody was aspirated, and the plate was washed again three times using 1× PBS. A final 50 μl of 1× PBS was added to each well to prevent the cells from drying out. We used a Celigo S adherent cell cytometer (Nexcelom Bioscience) with the two-channel “Target 1 + 2” (merge) setting to visualize the immunofluorescence. Exposure time, gain and focus (set using image-based auto focus with the 488-nm signal as the target) were automatically determined by the machine. Fluorescence was calculated using the default analysis settings, and the percent fluorescence was determined based on the wild-type signal. The images are representative of two independent immunofluorescence assays.

Escape mutagenesis.EMVs were generated using wild-type A/Switzerland/9715293/2013. First, virus was diluted to an MOI of 0.01 in 1× MEM supplemented with 1 μg/ml of TPCK-treated trypsin. Antibodies were diluted to 0.25× IC₅₀ and added to the diluted virus (for 1.5 ml/well). The virus-MAb mixture was incubated for 1 h at room temperature before being added to a confluent monolayer of MDCK cells. The cells were incubated for 3 days at 33°C with 5% CO₂. The cell culture supernatant was then collected spun at 13,000 × g for 5 min to pellet the cells. Aliquots were stored at −80°C. For subsequent passages, cell culture supernatant was diluted 1:10 in 500 μl of 1× MEM supplemented with 1 μg/ml of TPCK-treated trypsin and added to a confluent monolayer of MDCK cells. This was incubated at 33°C with 5% CO₂ for 40 min. Then an additional 1 ml of 1× MEM with 1 μg/ml of TPCK-treated trypsin and 0.5× IC₅₀ of MAb was added, and the plates were incubated at 33°C for 3 days. At each passage, the amount of MAb was doubled. Virus was passaged with a MAb up to 128× IC₅₀. Each passage was screened for the presence of EMVs using a plaque assay that contained >128× IC₅₀ of antibody in the overlay. Individual plaques were injected into 10-day-old SPF eggs and propagated as described above.

RNA extractions and deep sequencing.The E.Z.N.A viral RNA extraction kit (Omega Bio-Tek) was used for RNA extractions according to the manufacturer’s instructions. Isolated RNA was stored at −80°C until sequencing. Using the MiSeq v2, 300-cycle reagent kit (Illumina), next-generation sequencing was performed, and the genome was assembled using a pipeline developed at the Icahn School of Medicine at Mount Sinai (35). Full-length sequences were aligned to the deep sequenced segments of wild-type A/Switzerland/9715293/2013 to identify point mutations using MUSCLE in MEGA 7.0 (36).

Enzyme-linked lectin assay (ELLA).To determine NA activity for each virus, flat-bottom Immulon 4HBX microtiter plates (Thermo Scientific) were coated with 100 μl/well with 25 μg/ml of fetuin (Sigma) diluted in 1× PBS and incubated overnight at 4°C. The following day, virus was serially diluted (3-fold) in sample dilutant buffer (1× PBS with 0.9 mM CaCl₂, 0.5 mM MgCl₂ 1% bovine serum albumin (BSA), and 0.5% Tween 20) in a sterile 96-well plate. An additional volume of sample dilutant was then added at a 1:1 ratio. The diluted virus was then incubated for 1 h at room temperature, shaking. The fetuin-coated plates were then washed three times 220 μl of PBS containing 0.1% Tween 20 (PBS-T) per well using an AquaMax 3000 automated plate washer before virus dilutions were transferred to the plate. The samples were then incubated at 33°C for 18 h (overnight). The plates were washed six times with PBS-T, and then 100 μl of peroxidase-conjugated peanut agglutinin (PNA; Sigma) was added at 5 μg/ml, and the plates were incubated for 2 h at room temperature in the dark. PNA was diluted in conjugate dilutant buffer (1× PBS with 0.9 mM CaCl₂, 0.5 mM MgCl₂, and 1% BSA). The PNA was removed, and the plates were washed three times with PBS-T. SigmaFast o-phenylenediamine dihydrochloride (OPD; Sigma) was diluted in water, added at 100 μl per well, and incubated for 7 min at room temperature. Development was stopped by the addition of 50 μl of 3 M hydrochloric acid, and the absorbance at 490 nm was read using a Synergy H1 hybrid multimode microplate reader (Bio-Tek). Prism 7.0 was used to determine the effective concentration of each virus that would yield detectable NA activity. Each ELLA was performed in triplicate.

Neuraminidase inhibition assay.To determine the IC₅₀ of each MAb, flat-bottom Immulon 4HBX microtiter plates (Thermo Scientific) were again coated with 100 μl/well of 25 μg/ml fetuin (Sigma) diluted in 1× PBS, and incubated overnight at 4°C. Antibodies were diluted to 120 μg/ml in sample dilutant buffer and then serially diluted 1:3 in a sterile 96-well plate. Virus was diluted to its calculated effective concentration, added to the MAb dilutions at a 1:1 ratio, and incubated for 1 h at room temperature, shaking. Fetuin-coated plates were washed three times with PBS-T, and the virus-MAb dilutions were added. The assay was then performed according to the ELLA procedure above. IC₅₀ values were determined using Prism 7.0. NAI assays were completed in duplicates.

Plaque reduction neutralization assays.Neutralization IC₅₀ values were determined using PRNAs. First, MDCK cells were seeded at 8 × 10⁵ cells/ml onto 12-well plates. The following day, MAbs were diluted to 100 μg/ml in 300 μl of 1× MEM and then serially diluted 1:5 in a 24-well plate to a final concentration of 0.032 μg/ml in 1× MEM. Virus was diluted to 1 × 10³ PFU and added to each of the antibody dilutions (50 μl/well). The virus-MAb mixture was incubated at room temperature for 1 h, shaking. The MDCK cells were then washed one time with 1× PBS and immediately infected with 200 μl of the virus-MAb mixture and incubated at 33°C with 5% CO₂, with the plates rocked every 10 min. In the meantime, the overlay was prepared by diluting MAbs to 100 μg/ml in 625 μl of 2× MEM and then serially diluted 1:5. Then, a mixture of 1× DEAE-dextrane and 1 μg/ml TPCK-treated trypsin in sterile water for injection (Gibco) was added at 180 μl per well. After the 40 min, the inoculum was aspirated (three wells at a time) and immediately replaced by the overlay mixture containing 360 μl of 2% Oxoid agarose so that the MAb concentration within the agarose matched the same MAb concentration of the inoculum. The plates were incubated at 33°C with 5% CO₂ for 3 days and then fixed with 3.7% PFA overnight at 4°C. The overlay was removed, and the cells were stained as described above. PRNAs were conducted in duplicates.

Growth kinetics.Sterile 24-well plates were seeded with MDCK or A549 cells at 4 × 10⁵ cells/well and incubated overnight at 37°C with 5% CO₂. The following day, the viruses were diluted to an MOI of 0.01 (5 × 10³ PFU/well) in 1× MEM supplemented with 0.2 μg/ml TPCK-treated trypsin. The cells were washed with 1× PBS before being infected with virus. An aliquot of this initial virus dilution was kept and used to determine the initial titer of virus at infection. Viruses were incubated for 72 h at 33°C with 5% CO₂. Cell culture supernatant was sampled every 12 h and frozen at −20°C until virus titers were determined using plaque assays. Each experiment was performed in duplicate.