A Single Amino Acid Substitution at Residue 218 of Hemagglutinin Improves the Growth of Influenza A(H7N9) Candidate Vaccine Viruses

Cells and viruses.We used MDCK cells (American Type Culture Collection, Manassas, VA) for virus growth and Vero cells (BioReliance; Invitrogen) for CVV preparation. MDCK cells were maintained in Eagle minimal essential medium (EMEM; Lonza, Basel, Switzerland) supplemented with 5% fetal bovine serum (FBS; HyClone; Thermo Scientific). Vero Working Cell Bank was thawed and maintained in Dulbecco modified Eagle medium (Gibco, Thermo Scientific, USA) supplemented with 10% FBS. Both types of cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂. For virus propagation in eggs, 10-day-old specific-pathogen-free chicken embryonated eggs (Charles River Laboratories, Malvern, MA) were used.

The viruses used in this study are listed in Table 2. Briefly, wild-type viruses were received from the indicated providers. The MDCK-adapted progeny (NL12ad) was selected through three passages in MDCK cells. The reassortant viruses were generated by the reverse genetics method (44) with the HA and NA genes of interest cotransfected with six other gene segments from the donor virus A/Puerto Rico/8/1934 (PR8) in Vero cells. The designated mutations were generated by using a QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). For rgSH2, the HA (GISAID EpiFlu, EPI439502) and NA (GISAID EpiFlu, EPI439500) genes were synthesized (GenScript USA, Inc., Piscataway, NJ). For rgGD17, the HA and NA genes from the highly pathogenic A/Guangdong/17SF003/2016 (GD17WT) were initially PCR amplified and cloned into cloning vector pGEM-T Easy (Promega, Madison, WI) for modification, with which we deleted the codon for the multibasic cleavage site RKRT from the HA gene and generated K292R (N2 numbering) mutation in NA to restore the neuraminidase inhibitor (NAi) sensitivity. The modified HA and NA genes were then cloned into the pHW2000 vector (St. Jude Children’s Research Hospital, Memphis, TN) to generate the low-pathogenic, NAi-sensitive rgGD17 reassortant virus. The rescued viruses were propagated in either MDCK cells or eggs, and the infectivity was determined by both plaque assay in MDCK cells and from the 50% egg infective dose in eggs. All of the reassortant A(H7N9) viruses were rescued and maintained in the biosafety level 3 (BSL-3) facility.

Growth kinetics.Virus replication kinetics was evaluated with multistep growth curve. Briefly, confluent monolayers of MDCK cells grown in 12-well plate were inoculated with virus at an MOI of 0.01 for 1 h, followed by washing and then incubation with UltraMDCK serum-free culture medium (Lonza, Basel, Switzerland) containing TPCK-trypsin (1.0 μg/ml; Sigma, St. Louis, MO) at 33°C for 72 h. An aliquot of culture medium from each well of infected cells was collected at 8, 24, 48, and 72 h after infection and stored at –80°C for titration by plaque assay.

Virus purification.For propagation of viruses in MDCK cells, confluent MDCK monolayers in six T-75 Corning cell culture flasks were inoculated with virus (MOI of 0.01) and incubated in UltraMDCK serum-free medium containing 1 μg/ml of TPCK-trypsin at 35°C for 72 h. The collected MDCK culture media were clarified by centrifugation (2,000 × g, 20 min, 4°C), and the final volume of the clarified culture medium for each virus preparation was 90 ml.

Each egg-grown virus stock was prepared by propagation of the virus in fifteen 10-day-old eggs at 35°C for 72 h. The harvested allantoic fluid was clarified by centrifugation (2,000 × g, 20 min at 4°C), and the final volume of the clarified allantoic fluid used for virus purification was ≥120 ml.

The MDCK-grown or egg-grown virus was pelleted by ultracentrifugation using a Beckman type 45 Ti rotor (104,350 × g, 90 min, 4°C), resuspended in phosphate-buffered saline (PBS; pH 7.2), and purified through a 30 to 60% sucrose gradient (106,500 × g, 4°C for 90 min) using a Beckman SW 32 Ti rotor and 17-ml tubes. The virus-containing fraction was collected and diluted in 15 ml of PBS, pelleted by ultracentrifugation (106,500 × g for 60 min at 4°C) using a Beckman SW 32 Ti rotor and 17-ml tubes, and resuspended in PBS. The protein content of the purified virus was determined by using a Pierce Micro BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA).

Ferret study.The ferret study was performed in a BSL-3 containment laboratory according to protocols approved by the FDA/Center for Biologics Evaluation and Research (CBER) Animal Care and Use Committee. Male ferrets, 3 to 4 months of age, were purchased from Triple F Farms (Gillett, PA) and prescreened to confirm they were seronegative to the currently circulating seasonal influenza strains or A(H7N9) viruses. Each ferret was subcutaneously implanted with an IPTT transponder for identification and temperature monitoring using a DAS-7007 R reader system (BioMedic Data Systems, Inc., Seaford, DE). For the immunogenicity study, each ferret was intranasally (i.n.) inoculated with two doses (8.7 × 10⁶ PFU) of specific CVV administered 2 weeks apart. Blood for the serum preparation was collected at 4 weeks after the initial inoculation. For the HPAI A(H7N9) challenge study, nine ferrets were randomly assigned into three groups: (i) naive ferrets; (ii) rgGD17-immunized ferrets, where each ferret was inoculated i.n. with 1.0 ml of rgGD17 (8.7 × 10⁶ PFU); and (iii) rgGD17-G218E-immunized ferrets, where each ferret was inoculated i.n. with 1.0 ml of rgGD17-G218E (8.7 × 10⁶ PFU), followed by monitoring of temperature and body weight for 2 weeks. After venous blood sample collection, as well as the baseline body weight and temperature recording, at day 14 postimmunization, each ferret was challenged i.n. with 1 ml of HPAI GD17WT (5 × 10⁷ PFU) and placed under close observation for body weight, temperature, and signs of illness for 2 weeks. A ferret would be euthanized upon more than 20% body weight loss or the occurrence of signs of a central nervous system disorder, e.g., abnormal movement and opisthotonus. The procedures used for i.n. inoculation, venous blood collection, and nasal washing were performed under light sedation using ketamine and xylazine. Blood samples were collected for antibody titration from all surviving animals on day 15 postchallenge.

Hemagglutination inhibition assay.The antigenicity of the virus was evaluated by HI assay (45). Sera from ferrets immunized with A(H7N9) viruses were pretreated with receptor-destroying enzyme (RDE; Denka Seiken Co., Ltd., Japan) and serially diluted in 2-fold increments. Then, 25-μl portions of the diluted serum sample were mixed with 25 μl of virus (4 HA units) in 96-well plates, incubated at room temperature for 30 min, and then incubated with 50 μl of 1% horse red blood cells for 1 h at room temperature. The HI titer was determined as the reciprocal of the highest serum dilution at which the agglutination was inhibited. The detection limit of this assay was a titer of 10.

Microneutralization assay.Microneutralization assay (46) was performed to evaluate the specific neutralizing antibody in the immunized ferret sera. The RDE-treated ferret sera were serially diluted with serum-free EMEM from columns 1 to 11 in a 96-well transfer plate at 25 μl/well, followed by the addition of 100 50% tissue culture infective doses (TCID₅₀) of the tested virus (100 TCID₅₀/25 μl), and then incubated at room temperature for 1 h. The virus-serum mixture was then transferred to the MDCK monolayer grown in a 96-well tissue culture plate for virus adsorption with column 12 as a negative control. After a 20-h inoculation at 37°C, the inoculum was removed and replaced with 100 μl of serum-free EMEM containing TPCK-trypsin (1 μg/ml) and then incubated at 33°C for 5 days. Subsequent to crystal violet staining, the cytopathic effect (CPE) on MDCK monolayers was examined, and the neutralizing antibody titer was determined as the reciprocal of the highest serum dilution at which no CPE was detected. The detection limit of this assay was a titer of 10.

Virus binding avidity to MDCK cells.To understand whether the MDCK adaptation was related to a change in virus binding to host cell receptors, we assessed the virus binding avidity to MDCK cells with different sialic acid (SA) densities resulting from treatment by exogenous NA. Confluent MDCK monolayer cells grown in 12-well plates were preincubated with a serial dilution (6.25 to 100 mU/ml) of NA from Clostridium perfringens (Roche, catalog no. 11585886001 [purchased from Sigma, St. Louis, MO]) for 1 h in the presence of 2 mM Ca²⁺ at 37°C, followed by a thorough PBS washing, and then inoculated with NL12WT and NL12ad viruses (MOI of 0.002) for 1 h at 37°C or preincubated with a serial dilution (0.06 to 1.0 mU/ml) of cholera filtrate NA (catalog no. 8772; Sigma, St. Louis, MO) in PBS containing 5 mM Ca²⁺ for 1 h, followed by washing with PBS, and subsequently inoculated with rgSH2WT or rgSH2-G218E (MOI of 0.002) for 1 h at 37°C. After removal of the inoculum and wash, the cells were covered with 0.8% agarose overlay containing 1 μg/ml of TPCK-trypsin and incubated at 33°C in an atmosphere with 5% CO₂ for 72 h. The virus infectivity ratio was calculated by normalizing the plaque counts of the NA-pretreated wells to those from the wells without NA pretreatment.

Biolayer interferometry.Purified whole virus binding to immobilized α2,3- and α2,6-SA receptor analogs was measured with an Octet QK^e biolayer interferometer using streptavidin biosensors (Pall ForteBio Corp., Menlo Park, CA) (34). Avian-type receptor analog Neu5Acα2-3Galβ1-4GlcNAcβ-PAA-biotin (3′SLN, catalog no. 01-077) and human-type receptor analog Neu5Acα2-6Galβ1-4GlcNAcβ-PAA-biotin (6′SLN, catalog no. 01-130) were purchased from GlycoTech Corp. (Gaithersburg, MD). The 3′SLN and 6′SLN are polyacrylamide polymers of ∼30 kDa containing 5% mol biotin and 20% mol carbohydrate.

The biotinylated SLN polymers were serially diluted in binding buffer (NaCl, 150 mM; HEPES, 10 mM; EDTA, 3 mM; 0.005% Tween 20) ranging from 0.05 to 26.7 nM for 3′SLN and 0.1 to 133 nM for 6′SLN and then immobilized on the streptavidin biosensors for 10 min, followed by virus (1.0 mg/ml) association at 25°C for 30 min and subsequent dissociation in virus-free buffer containing calcium. In the preliminary experiments, we confirmed that the NAs from all the tested viruses, including NL12 (N3), SH2 (N9), and rgGD17 (N9), were sensitive to NAi zanamivir hydrate (M1826; Moravek Biochemicals, Inc., Brea, CA). To minimize the NA-induced virus dissociation from the immobilized 3′SLN or 6′SLN, the entire association phase was recorded in the presence of 10 μM zanamivir.

For steady-state analysis, the equilibrium response, i.e., the plateau amplitude of virus bound to the immobilized 3′SLN or 6′SLN at the respective dilution, was measured at the end of a 30-min association phase. Data from two independent experiments using viruses from different preparations were analyzed by GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA), best fit to the following equation:Y=Bmax⁡⁢×[SLN]h/(Kdh+[SLN]h) where Y is the specific binding, B_max is the maximal specific binding at equilibrium (measured as nm), K_d is the SLN concentration (nM) needed to achieve a half-maximum binding at equilibrium, corresponding to the equilibrium binding constant, and h is the Hill slope, reflecting the binding cooperativity (h = 1, noncooperative binding; h > 1, positively cooperative binding; h < 1, negatively cooperative binding).

To evaluate the cohabited NA function, we measured the destroyed virus-receptor analog binding by using NAi. The 20-min associations of the same virus with 3′SLN and 6′SLN were simultaneously recorded in both NAi-containing buffer and NAi-free buffer containing 5 mM CaCl₂, and the area under the curve (AUC) for each binding curve was quantitated by using GraphPad software. Because the difference between the AUC of NAi-containing and that of NAi-free reflected the NA-destroyed binding, the apparent NA function is presented as the following AUC ratio: (AUC_{NAi-containing} – AUC_NAi-free)/AUC_{NAi-containing}.

NA activity assay.NA activity of the SH2 viruses was evaluated by the MU-NANA assay as previously described (47). Briefly, 50 μl of 20 μM 4-methylumbelliferyl-N-acetylneuraminic acid (MU-NANA; Sigma, St. Louis, MO) was added to the same volume of sample containing 32 HAU virus to incubate for 1 h at 37°C. The control (PBS [pH 7.4]) and a serially diluted standard (NA from Vibrio cholerae; Sigma, N7885) were incubated in parallel. Reactions were stopped with 0.1 M glycine (pH 10.7) in 25% ethanol, and the fluorescence was measured on an automated plate reader Victor V (Perkin-Elmer, Waltham, MA) at an excitation of 355 and an emission of 460 nm for 0.1 s per well. The viral NA activity was calculated using the standard curve of the Vibrio cholerae NA.

Scanning electronic microscopy.SEM was used to visualize virus release from the host cells. MDCK cells were grown on 12-mm poly-l-lysine-coated circular glass coverslips (Neuvitro, Vancouver, WA) in a 12-well plate at one slide per well. Cells on five slides were inoculated with the indicated virus at an MOI of 3 and then incubated at 37°C. One slide from each viral infection was washed and fixed with 2.5% glutaraldehyde at 2, 6, 8, 10, or 12 h postinfection, respectively, and postfixed with 1% OsO₄. Cells were then dehydrated and critical point dried. The slides were mounted on tubs and coated with gold-palladium for examination using MIRA3 SEM (Tescan, Brno, Czech Republic).

Transmission electron microscopy.MDCK cells (5 × 10⁵) were seeded on a preincubated Corning Transwell permeable support (polycarbonate membrane, 3.0-μm pore size; Costar, catalog no. 3402) inserted in a 12-well plate and incubated at 37°C for 16 h. After inoculation with virus (MOI of 3) from the apical side at 37°C for 2 h, the cells were washed and then incubated in UltraMDCK medium (without TPCK-trypsin) at 33°C. At 10 h postinfection, the cells were washed with PBS, fixed in 2.5% glutaraldehyde, and postfixed with 1% OsO₄. After dehydration, the polycarbonate filter was embedded in Epon12 epoxy resin at 60°C for 48 h. The samples were then sectioned, stained with uranyl acetate and lead citrate, and examined with a Zeiss Libra 120 Plus transmission electron microscope. Images were taken with a Gatan US100XP digital camera.

Statistical analysis.The quantitative experiments were repeated at least three times, and results were analyzed with a t test (two tails), a one-way analysis of variance (ANOVA) with post hoc Tukey test, or a two-way ANOVA with the Bonferroni posttest using GraphPad Prism 5.0 software. A P value of <0.05 was considered statistically significant.