Mucosal Immunity against Neuraminidase Prevents Influenza B Virus Transmission in Guinea Pigs

Ethics statement.All animal procedures in this study were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines and have been approved by the Icahn School of Medicine at Mount Sinai IACUC.

Cells and viruses.Sf9 cells (CRL-1711, ATCC) for baculovirus rescue were grown in Trichoplusia Ni medium-formulation Hink insect cell medium (Gemini Bioproducts) supplemented with 10% fetal bovine serum (FBS; Sigma) and penicillin (100 U/ml)-streptomycin (100 μg/ml) solution (Gibco). BTI-TN-5B1-4 (High Five) cells for protein expression were grown in serum-free SFX medium (HyClone) supplemented with penicillin (100 U/ml)-streptomycin (100 μg/ml) solution. Madin-Darby canine kidney (MDCK) cells were grown in Dulbecco modified Eagle medium supplemented with 5% FBS and penicillin (100 U/ml)-streptomycin (100 μg/ml) solution. The influenza B virus strains B/Malaysia/2506/2004, B/Florida/04/2006, B/Brisbane/60/2008, and B/Lee/1940 were grown in 10-day-old embryonated chicken eggs (Charles River) for 72 h at 33°C. The eggs were then cooled overnight at 4°C before harvesting the allantoic fluid. Harvested allantoic fluid was centrifuged at 4,000 × g for 10 min at 4°C to remove debris. Viruses were then aliquoted and stored at –80°C prior to determining stock titers via plaque assay. To purify viruses for enzyme linked immunosorbent assays (ELISAs), virus stocks were grown in eggs (as above) and harvested 72 h later. Virus was then purified over a 30% sucrose cushion in 1× NTE buffer (0.5 mM NaCl, 10 mM Tris-HCl [pH 7.5], 5 mM EDTA), and the purified virus concentration was determined using Bradford’s reagent.

Recombinant proteins.Soluble HA and NA proteins containing a T4 foldon trimerization domain or a vasodilator stimulated phosphoprotein tetramerization domain, respectively, were generated using the baculovirus expression system as previously described (9, 20, 39). The HA and NA recombinant proteins expressed C-terminal and N-terminal hexahistidine tags, respectively, for purification purposes.

Guinea pig vaccination.Five- to six-week-old female guinea pigs were purchased from Charles River Laboratory and randomly assigned to different vaccination groups. Guinea pigs were either primed i.m. or i.n. with 10 μ of A/Shanghai/1/2013 H7 HA (as an irrelevant protein control), B/Malaysia/2506/2004 NA, or B/Florida/04/2006 NA recombinant protein adjuvanted with 10 μ of poly(I⋅C) (Invivogen). A boost via the i.n or i.m. routes with 10 μ of poly(I⋅C)-adjuvanted recombinant protein was given 28 days later. At 4 weeks after the boost, the vaccinated guinea pigs were used as vaccinated donors or recipients in transmission experiments.

Transmission experiments.Guinea pig transmission experiments were performed in an environmentally controlled chamber (model 6030; Caron Products & Services, Inc.), as previously described (40). Donor guinea pigs were anaesthetized with ketamine (30 mg/kg) and xylazine (5 mg/kg) before being challenged i.n. with 10⁴ PFU of B/Malaysia/2506/2004 or B/Florida/04/2006 in 300 μ of phosphate-buffered saline (PBS). The following day, donor and recipient transmission pairs were set up in cages that precluded physical contact but allowed lateral airflow (airborne transmission) or were cocaged (contact transmission). On days 2, 4, 6, 8, and 10 from the initial donor challenge, nasal washes were collected from anaesthetized donor and recipient guinea pigs. Blood was collected via venipuncture of the lateral saphenous vein at the prime, challenge, and euthanasia time points for the determination of serum antibody responses and NA inhibition (NI). Nasal washes were also collected at prime and challenge time points for the determination of antibody responses and NI. On days 18 to 21 postchallenge, guinea pigs were anaesthetized with ketamine (44 mg/kg) and xylazine (5 mg/kg) and terminally bled.

For B/Malaysia/2506/2004 transmission experiments, guinea pigs were housed at 20°C and 20% relative humidity (RH). The B/Florida/04/2006 transmission experiments occurred at 5°C and 20% RH. These environmental settings have been previously shown to be optimal for these influenza B virus strains to transmit efficiently among guinea pigs (41).

Virus titers.Virus titers were determined by plaque assay on MDCK cell monolayers. Virus stocks and nasal washes were diluted 10-fold in PBS and incubated on MDCK cells for 1 h before the addition of an agarose overlay containing a final concentration of 0.64% agarose (Oxoid), 1× minimum essential medium (MEM) (10% 10× minimal essential medium [Gibco], 2 mM l-glutamine, 0.1% of sodium bicarbonate [wt/vol; Gibco], 10 mM 4-HEPES, 100 U/ml penicillin–100 μ/ml streptomycin, and 0.2% bovine serum albumin (BSA), 1 μ/ml TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone)-treated trypsin, and 0.1% (wt/vol) DEAE (diethylaminoethyl)-dextran was added to the cells. The cells were then incubated for 72 h at 33°C, and visible plaques were counted after fixing them with 3.7% formaldehyde and visualization with a crystal violet counterstain (Sigma-Aldrich). All virus titers are presented as the log₁₀ PFU/ml. The limit of detection for these assays was 10 PFU/ml. The area under the curve (AUC) values for virus titers over the time course of infection were calculated using the log PFU/ml, and the AUC value was calculated using GraphPad Prism 7 software.

Enzyme linked immunosorbent assay.Immulon plates (Immulon 4HBX; Thermo Scientific) were coated with 2 μg/ml of purified virus (50 μl/well) in coating buffer (KPL coating solution; Sera Care) at 4°C overnight. The following day, the plates were washed three times with PBS containing 0.1% Tween 20 (PBS-T) and blocked in blocking solution (3% goat serum and 0.5% milk in PBS-T) for 1 h at room temperature. After blocking, prediluted serum was added to the first well to a final concentration of 1:100 in blocking solution. For assessing antibody responses in the nasal washes, a neat nasal wash was added to the first well. Serum and nasal washes were then serially diluted and incubated at room temperature for 2 h. Plates were then washed three times with PBS-T before the addition of donkey anti-guinea pig IgG-horseradish peroxidase (IgG-HRP; EMD Millipore) in blocking solution for 1 h at room temperature. Plates were then washed four times with PBS-T with shaking and then the O-phenylenediamine dihydrochloride (OPD) substrate (SigmaFast OPD; Sigma-Aldrich) was added. After 10 min of incubation at room temperature, the reaction was stopped by adding 50 μl of 3 M HCl to the mixture. The optical density (OD) was measured at 490 nm on a Synergy 4 plate reader (BioTek). A cutoff value of the average of the OD values of blank wells plus three standard deviations was established for each plate and used for calculating the AUC values of the nasal washes and the sera, which were the readout for this assay.

Enzyme-linked lectin assay to determine NI.Immulon plates (Immulon 4HBX; Thermo Scientific) were coated with 50 μ/μ of fetuin (150 μl/well) in coating buffer (KPL coating solution; Sera Care) at 4°C overnight. The following day, the plates were washed six times with PBS-T and blocked in blocking solution (5% BSA in PBS) for 2 h at room temperature. While the plates were being blocked, virus was serially diluted 1:2 on a separate 96-well plate in PBS containing 1% BSA. After this blocking step, 100 μ of serially diluted virus was transferred to the fetuin-coated plates, and the plates were incubated at 33°C for 2 h. The plates were then washed, and a secondary solution of peanut agglutinin (PNA) conjugated to HRP (5 μ/ml) was added to the plates. After a 2-h incubation in the dark, the plates were washed six times and developed with 100 μ of SigmaFast OPD. After developing for 7 min, 50 μ of 3 M HCl was added, and the OD was measured at 490 nm on a Synergy 4 plate reader (BioTek).

To perform NI assays, Immulon plates were coated and blocked as described above. While the plates were being blocked, guinea pig serum and nasal washes were serially diluted 1:2 in a separate 96-well plate in PBS. A starting dilution of 1:50 was used for serum, while we started with a neat nasal wash. A 75-μ portion of virus diluted to a 2× 50% effective concentration was added to each well of the serially diluted serum plate, and the plates were shaken at room temperature for 2 h. Before the shaking incubation time expired, the fetuin-coated plates were washed six times with PBS-T, and 100 μ of the virus/serum mixture was transferred to the fetuin-coated plates, followed by incubation for 2 h at 33°C. The remainder of the NI assay was performed as described above. The values obtained from the plate reader were divided by the average of the virus-only well subtracted from the no-virus control well and multiplied by 100 to obtain the NA activity. The percent inhibition was calculated by subtracting the NA activity from 100.

Statistics.Grouped data (except in Fig. 2C and D) are expressed as individual dot plots, and means are represented as geometric mean (GEM). Data in Fig. 2C and D are presented as grouped data, and means are expressed as standard errors of the mean (SEM). Statistical differences between two groups were determined by Student paired t test. Statistical differences between three or more groups were determined by two-way analysis of variance, followed by a Bonferroni multiple-comparison test. All statistical analyses were performed using GraphPad Prism 7 for Windows. In all cases, probability levels of <0.05 were indicative of statistical significance (*, P < 0.05; ***, P < 0.001).