Antigenic drift originating from changes to the lateral surface of the neuraminidase head of influenza A virus

Isolation of human monoclonal antibodies that recognize A(H1N1)pdm09 virus.

Peripheral blood mononuclear cells (PBMCs) were obtained from two human volunteers who were infected with A(H1N1)pdm09 virus during the 2015–2016 influenza season. The PBMCs were fused with SPYMEG cells to generate hybridomas that express a human antibody. After several rounds of screening by ELISA using purified A/California/04/2009 (CA/04/09) virus and A/Yokohama/94/2015 (YO/94/15) virus, which were isolated during the first wave of the 2009 pandemic and in the 2015–2016 season, respectively, and classified into the genetic clades 1 and 6B.1 based on their respective HA sequences, hybridomas expressing antibodies bound to YO/94/15 virus but not to CA/04/09 virus were biologically cloned. Of 701 hybridomas screened, we obtained seven clones (DA05C23, HP02C70, DA05B02, HP02B69, HP02E74, HP02E63, and HP02B24) that expressed a monoclonal antibody (mAb) specific to YO/94/15 virus. Nucleotide sequence analysis of their variable regions revealed that all seven mAbs used the same VH gene (IGHV3–15*01), although the CDR3 sequence of each heavy chain varied (Supplemental Table 1). Five mAbs used IGLV3–1*01, whereas HP02B24 and HP02E63 used IGLV3–21*01 and IGLV6–57*01, respectively.

The seven human mAbs mainly recognize the NA of A(H1N1)pdm09 virus isolated in the 2015–16 influenza season.

To determine the breadth of recognition of the seven mAbs we obtained, we performed an ELISA using A(H1N1)pre2009, A(H3N2), and YO/94/15 viruses. None of the mAbs recognized A(H1N1)pre2009 or A(H3N2) virus (Fig. 1A). A broadly reactive human anti-N1-NA mAb clone 1000-3C05 (8, 22) bound to A(H1N1)pre2009 and YO/94/15 viruses, whereas the broadly reactive human anti-HA stem mAb clone CR9114 (34) recognized all three viruses tested (Fig. 1A). A negative control, anti-B HA 1430E3/9, did not bind to any of these viruses. These results indicate that the seven mAbs isolated here are specific to A(H1N1)pdm09 virus. Next, we performed an ELISA using A(H1N1)pdm09 viruses isolated during different influenza seasons. HP02C70, DA05B02, HP02E74, HP02E63, and HP02B24 showed specific binding to YO/94/15 virus, whereas HP02B69 and DA05C23 bound preferentially to YO/94/15 virus and weakly to A/Osaka/6/2014 virus (Fig. 1B). EM-3C02, which was reported to be a strain-specific human anti-N1-NA mAb (8, 22), bound to CA/04/09 and A/Hiroshima/66/2011 viruses, whereas the broadly reactive mAb 1000-3C05 recognized all viruses tested. The anti-influenza B HA mAb clone 1430E3/9 failed to recognize any of the viruses tested. These results indicate that the seven mAbs obtained are not broadly reactive but mainly recognize YO/94/15 virus.

To determine which viral protein was recognized by these mAbs, we performed an ELISA using purified reassortant viruses possessing HA and NA derived from YO/94/15 or CA/04/09 virus. All seven mAbs bound to viruses possessing NA derived from YO/94/15 virus, whereas EM-3C02 recognized a virus possessing NA derived from CA/04/09, but not YO/94/15, virus (Fig. 1C). 1000–3C05 detected all of the viruses tested. These results demonstrate that these seven human mAbs are against the NA of A(H1N1)pdm09 virus.

We next determined the binding affinity of the seven mAbs to NA derived from YO/94/15 virus by use of a Scatchard analysis (Supplementary Fig. 1). DA05C23 and HP02C70 possessed high binding affinities (K_D = 0.86 and 0.73 nM, respectively), which were comparable to that of 1000-3C05 (K_D = 0.82 nM). However, DA05B02, HP02B69, HP02E74, HP02E63, and HP02B24 showed relatively lower binding affinities than that of 1000-3C05 (K_D = 2.57, 3.24, 4.18, 3.22, and 3.62 nM, respectively).

The seven mAbs recognize the lateral surface of the NA head.

To determine the amino acids that are important for the binding of the seven anti-NA mAbs, we compared the amino acid sequences of the NA protein from the six viruses used in Fig. 1B and identified six unique amino acids in the NA of YO/94/15 virus (Fig. 2A, 13I, 44I, 263I, 268K, 314M, and 390K; N2 numbering). We then generated mutant YO/94/15 viruses possessing single amino acid substitutions (I13V, I44L, I263V, K268N, M314I, or K390N) in their NA and examined the binding of the seven mAbs in an ELISA. HP02C70, DA05B02, HP02E74, HP02E63, and HP02B24 did not bind to virus possessing the K390N substitution in its NA but did bind to viruses possessing the I13V, I44L, I263V, K268N, or M314I substitution (Fig. 2B). DA05C23 and HP02B69 recognized all of the NA mutant viruses that possessed single amino acid substitutions, but showed decreased reactivity against the K390N mutant virus. Since these two clones weakly bound to A/Osaka/6/2014 virus (see Fig. 1B), we selected three amino acids shared only by the A/Osaka/6/2014 and YO/94/15 viruses (i.e., 38V, 321V, and 432E) for additional mutant generation. We generated mutant viruses possessing two substitutions (V38I and K390N, V321I and K390N, or E432K and K390N) in their NA and tested mAb binding. DA05C23 and HP02B69 did not bind to the virus possessing the K390N and E432K mutations in NA but still bound to the viruses possessing V38I and K390N or V321I and K390N (Fig. 2B). These results suggest that the amino acid at position 390 and the amino acids at positions 390 and 432 are important for the recognition of five and two of the monoclonal antibodies, respectively. Therefore, we mapped positions 390 (orange) and 432 (blue) together with the neuraminidase catalytic residues (cyan) on the NA head molecule (Fig. 2C). Position 390 is located at the lateral face of the NA head, whereas position 432 is located at the edge of the enzymatic center on the upper surface of NA (Fig. 2C). The distance between positions 390 and 432 based on the crystal structure of the NA head molecule is approximately 3 nm. Since an antibody footprint is typically 4–10 nm² (35, 36), both amino acids are likely to be covered by one mAb. Therefore, we suggest that the five anti-NA mAbs that recognize an epitope around position 390, mainly recognize the lateral surface of NA and that the other two anti-NA mAbs, which lose binding to NA with mutations at positions 390 and 432, recognize an epitope that spans the upper and lateral surfaces.

The seven anti-NA mAbs differ in their NI activity.

To further characterize the seven anti-NA mAbs, we assessed their ability to inhibit viral sialidase activity in an enzyme-linked lectin assay (ELLA). DA05C23, HP02C70, and DA05B02 strongly inhibited NA activity with 50% inhibition concentration (IC₅₀) values of 0.38, 0.60, and 0.73 nM, respectively (Fig. 3A). HP02B69 weakly inhibited NA activity with an IC₅₀ value of 7.52 nM, which was similar to that of 1000-3C05 (IC₅₀ = 12.05 nM). HP02E74 slightly inhibited NA activity at high concentrations (IC₅₀ = >26.67 nM), whereas HP02B24 and HP02E63 possessed no NI activity at all. To further confirm the NI activity in vitro, we performed a plaque size reduction assay. In this assay, DA05C23, HP02C70, and DA05B02 decreased viral plaque size by 50% or more at the highest concentration (Fig. 3B). The plaques that formed in the presence of HP02B69 and HP02E74 were about 30% smaller than those that formed in the absence of a mAb. In contrast, the plaque size reduction induced by HP02B24, HP02E63, and 1000-3C05 was approximately 10% at any concentration tested. Taken together, these results indicate that DA05C23, HP02C70, and DA05B02 possess relatively high NI activity, HP02B69 and HP02E74 possess relatively low NI activity, and HP02B24 and HP02E63 fail to inhibit NA activity.

The seven anti-NA mAbs activate the FcγR-mediated signaling pathway in effector cells.

We next asked whether these anti-NA mAbs elicit antibody-dependent cellular cytotoxicity (ADCC) or antibody dependent cellular phagocytosis (ADCP) by using ADCC and ADCP reporter bioassays. By using these reporter bioassays, we found that the three mAbs that possess high NI activity, HP02C70, DA05C23, and DA05B02, efficiently activated the ADCC and ADCP signaling pathways in effector cells co-cultured with both virus-infected and NA-expressing cells, except for the ADCC reporter assay with DA05C23 and NA-expressing cells, which was moderately activated (Fig. 3C). HP02B69 and HP02E74, which possessed low NI activity, moderately activated the ADCC signaling pathway and efficiently activated the ADCP signaling pathway in the effector cells co-cultured with infected and NA-expressing cells. HP02B24 and HP02E63, which had no NI activity, induced low or similar ADCC and ADCP signaling activation to that induced by the other five mAbs. 1000-3C05, which protected mice from lethal infection by activating FcγR-mediated effector cells (22), triggered ADCC and ADCP signal activation via both virus-infected and NA-expressing cells. All of the mAbs that possessed the N297Q mutation, which abolishes the interaction with FcγRs, and 1430E3/9 caused little-to-no activation in both reporter assays. These results indicate that our seven anti-NA mAbs have the potential to activate ADCC and ADCP.

The anti-NA mAbs protect mice from lethal infection via their NI and FcγR-mediated antiviral activities.

Since our mAbs possessed various levels of NI activity and activated FcγRIIIa- and FcγRIIa-mediated signaling pathways to various extents, we examined their in vivo protective efficacy against lethal influenza virus infection. For these in vivo tests, we monitored the body weight loss and survival of mice infected with 10 MLD₅₀ (50% mouse lethal dose) of a challenge virus after administration of the wild-type or mutant N297Q mAb at 30 mg/kg (results of administration at 5 or 1 mg/kg are shown in Supplementary Fig. 2). 1000-3C05, which requires FcγR-mediated effector cell activation to provide in vivo protection (22), or 1430E3/9, which is an anti-influenza B HA antibody, served as positive and negative controls, respectively. All of the wild-type anti-NA mAbs, except for DA05B02, protected all of the mice from lethal challenge infection, with the mice experiencing transient weight loss (Fig. 4). Of the mutant N297Q mAb-administrated groups, 75% of the mice that received HP02C70 or DA05B02, which possessed high NI activity, survived (Fig. 4). In particular, the N297Q mutant mAb DA05B02 showed similar in vivo protective potency to that of its wild-type counterpart. The N297Q mutants of DA05C23, HP02B69, and HP02E74, which possessed NI activity, protected 25% of the mice that experienced severe weight loss. The N297Q mutants of HP02E63 and HP02B24, which showed no NI activity, failed to protect any mice. Wild-type 1000-3C05 showed protection with transient weight loss, whereas no mice that received the N297Q mutant of 1000-3C05 survived. Mice that received 1430E3/9 experienced similar weight loss to that experienced by mice that received PBS. These results suggest that both the NI activity and FcγR-mediated antiviral activity of the anti-NA mAbs contribute to protection against influenza virus infection. Moreover, FcγR-mediated antiviral activity played central roles in the in vivo protection provided by the anti-NA mAbs that lacked NI activity.

Antibodies recognizing the lateral surface of the NA head of A(H1N1)pdm09 virus in patient sera.

To evaluate whether individuals who were infected with the A(H1N1)pdm09 virus possessed antibodies targeting the lateral surface of the NA head on the day they visited the hospital or 1 month after infection, we performed a competitive binding ELISA using a protective mAb without NI activity, HP02E63 or HP02B24, or a cocktail of our seven anti-NA mAbs (Fig. 5A). The Fc region of these mAbs was replaced with that of mouse IgG2a so as not to be recognized by the HRP-conjugated antibody. In the presence of the mAb cocktail or each mAb, we assessed antibody binding in the serum samples of patients infected with A(H1N1)pdm09 virus during the 2015–2016 influenza season to VLPs displaying YO/94/15-NA; the NA-displaying VLPs were produced by expressing Ebola virus VP40 and NA since humans do not have antibodies to VP40. All of the patients were found to possess antibodies that competed with the anti-NA mAb cocktail 30 days after infection (Fig. 5B). Serum samples from patients HP001, HP002, HP014, and HP021 or from HP002 and HP021 collected 30 days after infection contained significant amounts of antibodies that competed with antibodies HP02E63 or HP02B24, respectively (Fig. 5C and 5D). On the day that each patient visited the hospital, HP001 and HP021 had antibodies that competed with the anti-NA mAbs cocktail, HP02E63, and/or HP02B24 (Fig. 5). These results demonstrate that substantial amounts of antibodies targeting epitopes on the lateral surface of the NA head were induced upon virus infection.

NA antigenicity differs between 2009 and 2015 isolates of A(H1N1)pdm09 virus.

Influenza viruses constantly evade suppressive antibodies by acquiring mutations in the epitopes of not only HA, but also NA. Therefore, we investigated the antigenicity of CA/04/09-NA and YO/94/15-NA in an ELISA using 22 serum samples that were collected during the 2009–2010 influenza season from volunteers who had neutralization antibodies against A(H1N1)pdm09 virus (37). We found that end-point titers to YO/94/15-NA were significantly lower than those to CA/04/09-NA (Fig. 6A). These results indicate that YO/94/15-NA differed antigenically from CA/04/09-NA.

To determine which amino acid mutations are responsible for the antigenic change in NA, we compared the amino acid sequences of CA/04/09-NA and YO/94/15-NA and found 6, 4, and 3 amino acid differences around the enzymatic center of the NA head (blue; positions 199, 240, 247, 321, 372, and 432), on the lateral surface of the NA head (orange; positions 263, 268, 314, and 390), and in the stalk region (not shown in the structure; position 38, 44, and 48), respectively (Figs. 2A and 6B). Then, we performed a cell-based ELISA by using the 22 A(H1N1)pdm2009-positive sera and cells transiently expressing wild-type CA/04/09-NA or its mutants that possessed amino acid changes found in YO/94/15-NA. If the end-point titer of each serum against the mutant NA was at least 4 times lower than that against wild-type NA, we concluded that that mutation affected the antigenicity of NA. Half of the sera (11/22, blue dots) showed reduced reactivity to NA possessing mutations around the enzymatic center (Fig. 6C, left). Two of these sera (2/11) also showed decreased binding to the stalk region mutant (Fig. 6C, right). Two serum samples (2/22) mainly targeted the lateral surface of the NA head (red or orange dot in Fig. 6C, middle) and reacted with the NA mutants of the enzymatic center and stalk region with similar titers. These results indicate that the antigenic drift of NA involves mutations on the lateral surface of the NA head and that the antibodies of some individuals mainly recognize the lateral surface of the NA head.

Furthermore, we investigated whether a single mutation at position 390, which is the key residue for the binding of our mAbs, contributes to the antigenic drift on the lateral surface of the NA head by using a cell-based ELISA. Cells transiently expressing CA/04/09-NA with the N390K mutation as the antigen were incubated with the 22 A(H1N1)pdm2009-positive human sera used in Fig. 6C. We found that one sample (red dot) also showed decreased binding to the N390K mutant (Fig. 6D). These results suggest that the single N390K mutation is partially involved in the NA antigenic drift.

Ethics.

All experiments were performed in compliance with relevant ethical regulations. Human blood was collected from two volunteers by following a protocol approved by the Research Ethics Review Committee of the Institute of Medical Science, the University of Tokyo. Written informed consent was obtained from each participant. All experiments with mice were performed in accordance with the University of Tokyo’s Regulations for Animal Care and Use and were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo.

Cells.

Madin-Darby canine kidney (MDCK) cells were maintained in Eagle’s minimal essential medium (MEM) containing 5% newborn calf serum (NCS). Human lung carcinoma A549 cells were maintained in Ham’s F-12K medium containing 10% fetal calf serum (FCS). Human embryonic kidney 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS. SPYMEG cells (MBL), which are a fusion partner cell line for human hybridoma, were generated by the fusion of the mouse myeloma cell line SP2/0 with the human leukemia cell line MEG-01 and were maintained in DMEM containing 15% FCS (43). These cells were incubated at 37°C under 5% CO₂. Expi293F cells (Thermo Fisher Scientific) were maintained in Expi293 Expression Medium (Thermo Fisher Scientific) at 37°C under 8% CO₂.

Viruses.

We used the following six A(H1N1)pdm09 viruses, which have been reported previously (44): A/California/04/2009 (CA/04/09; clade 1), A/Hiroshima/66/2011 (clade 5), A/Osaka/83/2011 (clade 7), A/Osaka/33/2013 (clade 6C), A/Osaka/6/2014 (clade 6B), and A/Yokohama/94/2015 (YO/94/15; clade 6B.1). A/Brisbane/59/2007 (H1N1pre2009) and A/Hong Kong/4801/2014 (H3N2) were also used. All viruses were propagated in MDCK cells.

Plasmid-based reverse genetics.

The reassortant viruses, the NA mutant viruses, and the challenge virus were generated by plasmid-based reverse genetics, as described previously (45). For reassortant viruses, two pPol I plasmids encoding the HA and NA segments derived from the YO/94/15 or CA/04/09 viruses and six pPol I plasmids encoding the remaining segments of CA/04/09 virus were used for the plasmid transfection. For preparation of the NA mutant viruses, pPol I plasmids encoding HA and wild-type or mutated NA derived from YO/94/15 virus and six pPol I plasmids encoding the remaining segments of CA/04/09 virus were used. For preparation of the challenge virus, we used pPol I plasmids encoding the HA and NA segments of YO/94/15 virus and six pPol I plasmids encoding the remaining segments of mouse-adapted CA/04/09 (46). These 8 pPol I plasmids along with protein expression plasmids for PB2, PB1, PA, and NP derived from A/Puerto Rico/8/34 were transfected into 293T cells by use of TransIT 293 (Mirus) according to the manufacturer’s instructions. At 48 h post-transfection, the supernatants were harvested and inoculated into MDCK cells. The rescued viruses were sequenced to ensure the absence of unwanted mutations. Primer sequences are available upon request.

Hybridoma generation.

PBMCs were isolated from blood (30 ml) obtained from two volunteers, who were infected with the A(H1N1)pdm09 virus in the 2015–2016 influenza season, by using Ficoll Paque Plus (GE Healthcare). The hybridomas that were generated by fusion of PBMCs with SPYMEG cells were cultured and biologically cloned, as described previously (11). For the screening, we performed an enzyme-linked immunosorbent assay (ELISA) using purified YO/94/15 virus and CA/04/09 virus as antigens, as described below.

ELISA.

ELISA plates (96-well) were coated with each purified virus. After being blocked with 5 times-diluted Blocking One (Nakarai), these plates were incubated with the culture medium of the hybridomas or 1 μg/mL of purified mAb. A horseradish peroxidase (HRP)-conjugated goat anti-human IgG, Fcγ Fragment-specific antibody (Jackson Immuno-Research) was used as the secondary antibody, and the signal was developed using 3, 3’, 5, 5’-tetramethylbenzidine (TMB) (Thermo Fisher Scientific) as the substrate. The reaction was stopped with 2N sulfuric acid, and OD₄₅₀ values were then measured. OD₄₅₀ values of 0.1 or more were considered to indicate binding.

Expression and purification of monoclonal human IgG.

Human monoclonal antibodies were expressed by Expi293F cells and purified using a protein A column, as described previously (11, 47).

ELLA.

The inhibition of NA activity was measured by using an enzyme-linked lectin assay (ELLA), as described previously (48). Briefly, two-fold diluted antibodies (4000–7.8 ng/mL) were mixed with a predetermined amount of purified YO/94/15 virus diluted in PBS containing 1% BSA and 0.05% Tween 20 (PBS-T). The mixture was transferred to 96-well plates coated with fetuin (Sigma) and then incubated for 16 h at 37°C. The plates were washed with PBS-T and then peroxidase-conjugated peanut agglutinin (PNA; Sigma) was added to detect galactose exposed by removal of the sialic acids on fetuin. The plates were incubated at room temperature for 2 h in the dark and then washed with PBS-T before the addition of o-phenylenediamine dihydrochloride (OPD) substrate (Sigma). The reaction was stopped by the addition of 1N sulfuric acid and OD₄₉₀ values were read. The relative NA inhibition (NI) activity was calculated by dividing the OD₄₉₀ value of the test well by the OD₄₉₀ value of the “virus only” well and multiplying by 100. The 50% inhibitory concentration (IC₅₀) was determined by use of nonlinear regression analysis (GraphPad Prism software) assuming that the molecular weight of the antibody was 150 kDa.

Plaque size reduction assay.

MDCK cells were infected with 100 plaque-forming units (pfu) of YO/94/15 virus for 1 h at 37°C. After removal of the inoculum, the cells were washed with MEM containing 0.3% bovine serum albumin (BSA-MEM) and overlaid with agar containing the indicated amount of the indicated mAb and 1 μg/ml L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK)-treated trypsin. The cells were incubated at 37°C under 5% CO₂ for 2 days and then stained with crystal violet solution to visualize the plaques. The plaque images were obtained by scanning assay plates with a photo scanner (GT-X900, EPSON). The total plaque size of each well was measured using a script written in MATLAB (Mathworks), and the average plaque size was calculated by dividing by the number of plaques. Infected cells overlaid with agar that lacked mAb served as a control. The reduction in plaque size was calculated by dividing the average plaque size in the presence of each mAb by the control plaque size.

ADCC and ADCP reporter assays.

ADCC and ADCP reporter assays were performed by using ADCC and ADCP Reporter Bioassay Kits (Promega), according to the manufacturer’s instructions. ADCC and ADCP signaling activations in effector cells were measured by using two kinds of target cells: A549 cells infected with YO/94/15 virus and 293T cells expressing NA derived from YO/94/15 virus. Briefly, A549 cells were plated at a density of 10,000 cells/well in a flat-bottom white 96-well plate. After 24 h, the cells were infected with YO/94/15 virus at a multiplicity of infection (MOI) of 10. At 12 h post-infection, the cells were used as target cells. For the second kind of target cell, two days before the reporter assays were performed, 293T cells in a 6-well plate were transfected with a plasmid encoding YO/94/15-NA by using Trans-IT 293. The next day, the transfected cells were harvested and seeded at 20,000 cells/well into a white 96-well plate. Twenty-four hours later, the cells were used as target cells. In the ADCC or ADCP reporter assays, the medium of the target cells was replaced with five-fold diluted mAb (10–0.016 μg/ml) in the assay buffer (Promega). ADCC or ADCP Bioassay effector cells (a Jurkat cell line stably expressing human FcγRIIIa or FcγRIIa, human CD3γ, and an NFAT-response element driving expression of a firefly luciferase) were added to the antibody-treated target cells and incubated for 6 h at 37°C. The firefly luciferase activity was then measured using luciferase assay regents (Promega).

In vivo protection test.

Four six-week-old female BALB/c mice (Japan SLC) per group were intraperitoneally injected with PBS or each indicated antibody at 30 mg/kg in 250 μl of PBS. After 24 h, the mice were anesthetized with isoflurane and intranasally challenged with 10 MLD₅₀ (50% mouse lethal dose) of the challenge virus in 50 μl of PBS. Weight was monitored daily for 14 days and mice that lost 25% or more of their initial body weight were scored as dead and euthanized according to institutional guidelines.

Competitive binding assay.

For antigen preparation, 293T cells were transfected with pCAGGS plasmids encoding ebolavirus matrix protein VP40 and each indicated NA by using TransIT 293. After 2 days, the supernatant of the transfected cells was harvested and centrifuged at 28,000 rpm with a 20% sucrose cushion. The pellet containing VP40-induced VLPs presenting NA was resuspended in PBS. ELISA plates were coated overnight at 4°C with 5 μg/ml of the VLPs. After being blocked with 5 times-diluted Blocking One, these plates were incubated with a two-fold diluted cocktail of the seven anti-NA mAbs (DA05C23, HP02C70, DA05B02, HP02B69, HP02E74, HP02E63, and HP02B24) or with individual monoclonal antibodies (HP02E63 or HP02B24), which possessed the Fc region of mouse IgG2a, or with PBS for 1 h. The plates were then washed with PBS-T, followed by the addition of diluted serum samples. The serum samples used in this assay were collected from patients infected with A(H1N1)pdm09 virus during the 2015–2016 influenza season. After 1 h, an HRP-conjugated goat anti-human IgG, Fcγ Fragment-specific antibody was added as the secondary antibody. The plates were incubated for 1 h, and then washed with PBS-T before the addition of the TMB substrate. The reaction was stopped with 2N sulfuric acid, and OD₄₅₀ values were then measured. The wells to which PBS was added instead of monoclonal antibody served as control wells for each serum sample.

Entropy.

For each amino acid position of the aligned isolate sequences, an entropy value was computed by using the formula −ΣPi × ln(Pi), as described by Chen et al. (49). This formula follows the definition of Shannon entropy (50) that has been used to evaluate diversity. In this study, entropy was used to measure the variability of amino acids at a given position, where i = 1 to 20 represents the 20 different amino acid residues, and Pi represents the probability density of the respective residue. Entropy values range from 0 (only 1 residue present at that position) to 2.996 (all 20 residues are equally represented). The calculation was based on the NA sequences of 19,034 A(H1N1)pdm09 isolates, 4,626 A(H1N1)pre2009 isolates, and 40,516 A(H3N2) isolates obtained from the GISAID database.

Cell-based ELISA.

293T cells were transfected with FLAG-tagged wild-type or mutant NA-expression plasmids or an empty plasmid as a control using TransIT 293. At 48 h after the transfection, the cells were fixed with 4% paraformaldehyde phosphate buffer solution for 30 min at room temperature. The cells were then washed with PBS-T, and blocked with 5 times-diluted Blocking One for 1 h. The cells were then incubated for 1 h with two-fold diluted serum samples, which were collected from volunteer schoolchildren and their parents who were seropositive for A(H1N1)pdm09 virus between November 2009 and March 2010 (37), followed by incubation for 1 h with an HRP-conjugated goat anti-human IgG, Fcγ Fragment-specific antibody. The signal was developed using TMB as the substrate and stopped with 2N sulfuric acid. OD₄₅₀ values were then measured. Expression of NA was normalized with an anti-FLAG tag antibody (Wako). The mean OD₄₅₀ at each dilution was obtained by subtracting the OD₄₅₀ value for the control well (empty plasmid transfected cells). The end-point titers of serum samples were determined as the reciprocal of the highest dilution providing an OD₄₅₀ value of greater than 0.1.

K_D determination.

K_D values were determined from Scatchard plots using ELISA data (51). In the ELISA, VP40-induced VLPs presenting YO/94/15-NA were used as the antigen. After being blocked with 5-times-diluted Blocking One, antigen-coated plates were incubated with the two-fold diluted anti-NA mAb (10–0.005 μg/ml). The HRP-conjugated goat anti-human IgG, Fcγ Fragment-specific antibody was used as the secondary antibody, and the signal was developed using TMB as the substrate. The reaction was stopped with 2N sulfuric acid, and OD₄₅₀ values were then measured. Based on the OD₄₅₀ values, the concentrations of antibody bound to antigen and of free antibody were calculated (assuming that the molecular weight of the antibody was 150 kDa). When the ratio of bound to free antibody concentration was plotted against the bound antibody concentration, the negative inverse of the resulting slope was determined as the K_D.

Structural analysis.

Amino acid positions were plotted on the crystal structure of the NA protein of CA/04/09, A/Brevig Mission/1/1918 (H1N1pre2009), or A/Memphis/31/1998 (H3N2) (PDB accession code, 4B7R, 3B7E, or 4H53, respectively) by using the PyMOL molecular graphics system.

Statistical analysis.

Two-way analysis of variance (ANOVA) followed by Dunnett’s test, one-way ANOVA followed by Dunnett’s test, the log-rank test, and Wilcoxon signed rank test were performed using GraphPad Prism software. P values < 0.01 were considered significantly different. No samples were excluded from the analysis.