PLOS ONERESEARCH ARTICLE

An influenza HA stalk reactive polymeric IgA
antibody exhibits anti-viral function regulatedby binary interaction between HA and theantibody

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OPEN ACCESS
Citation: Sano K, Saito S, Suzuki T, Kotani O, Ainai
A, van Riet E, et al. (2021) An influenza HA stalk
reactive polymeric IgA antibody exhibits anti-viralfunction regulated by binary interaction between
HA and the antibody. PLoS ONE 16(1): e0245244.
https://doi.org/10.1371/journal.pone.0245244
Editor: Victor C. Huber, University of South Dakota,
UNITED STATES
Received: August 8, 2020
Accepted: December 23, 2020
Published: January 7, 2021
Copyright: © 2021 Sano et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: The B12 and F11
mAb sequences reported in this paper have been
deposited in the NCBI database (B12 HC variable
region, MG904946; B12 LC variable region,
MG904947; F11 HC variable region, MN218635;
F11 LC variable region, MN218636).
Funding: The work was supported in part by theJapan Agency for Medical Research andDevelopment (AMED) under Grant Numbers
JP20fk0108141 (TS), JP19fk0108051 (TS),JP19fk0108082 (TS), JP19fk0108083 (HH and

Kaori Sano1,2, Shinji Saito3, Tadaki Suzuki ID1*, Osamu Kotani4, Akira Ainai1, Elly van
Riet3, Koshiro Tabata1, Kumpei Saito1, Yoshimasa Takahashi5, Masaru Yokoyama ID4,
Hironori Sato4, Takahiro Maruno6, Kaede Usami6, Susumu Uchiyama6,7, Kiyoko OgawaGoto8, Hideki Hasegawa1,2,3,9*
1 Department of Pathology, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan, 2 Division of
Infectious Diseases Pathology, Department of Global Infectious Diseases, Tohoku Graduate School of
Medicine, Sendai, Miyagi, Japan, 3 Influenza Virus Research Center, National Institute of Infectious
Diseases, Musashimurayama, Tokyo, Japan, 4 Pathogen Genomics Center, National Institute of Infectious
Diseases, Musashimurayama, Tokyo, Japan, 5 Department of Immunology, National Institute of Infectious
Diseases, Shinjuku, Tokyo, Japan, 6 Department of Biotechnology, Graduate School of Engineering, Osaka
University, Suita, Osaka, Japan, 7 Exploratory Research Center on Life and Living Systems (ExCELLS),
National Institutes of Natural Sciences, Okazaki, Aichi, Japan, 8 Nippi Research Institute of Biomatrix, Nippi
Incorporated, Toride, Ibaraki, Japan, 9 Global Virus Network, Baltimore, MD, United States of America* tksuzuki@nih.go.jp (TS); hasegawa@nih.go.jp (HH)

Abstract
IgA antibodies, which are secreted onto the mucosal surface as secretory IgA antibodies(SIgAs), play an important role in preventing influenza virus infection. A recent study
reported that anti-hemagglutinin (HA) head-targeting antibodies increase anti-viral functions
such as hemagglutination inhibition (HI) and virus neutralization (NT), in addition to HA binding activity (reactivity) via IgA polymerization. However, the functional properties of anti-viral
IgA antibodies with mechanisms of action distinct from those of anti-HA head-targeting antibodies remain elusive. Here, we characterized the functional properties of IgG, monomeric
IgA, and polymeric IgA anti-HA stalk-binding clones F11 and FI6, and B12 (a low affinity
anti-HA stalk clone), as well as Fab-deficient (ΔFab) IgA antibodies. We found that IgA polymerization impacts the functional properties of anti-HA stalk antibodies. Unlike anti-HA head
antibodies, the anti-viral functions of anti-HA stalk antibodies were not simply enhanced by
IgA polymerization. The data suggest that two modes of binding (Fab paratope-mediatedbinding to the HA stalk, and IgA Fc glycan-mediated binding to the HA receptor binding site
(RBS)) occur during interaction between anti-stalk HA IgA antibodies and HA. In situations
where Fab paratope-mediated binding to the HA stalk exceeded IgA Fc glycan-mediatedbinding to HA RBS, IgA polymerization increased anti-viral functions. By contrast, when IgAFc glycan-mediated binding to the HA RBS was dominant, anti-viral activity will fall upon IgApolymerization. In summary, the results suggest that coordination between these two independent binding modules determines whether IgA polymerization has a negative or positiveeffect on the anti-viral functions of anti-HA stalk IgA antibodies.

PLOS ONE | https://doi.org/10.1371/journal.pone.0245244 January 7, 2021

1 / 32PLOS ONEAA), JP19fm0208002 (TS), JP19fk0108107 (SSand AA), and JP19fk0108030 (SS); and by JSPS
KAKENHI Grant Numbers JP18J11822 (KS),JP19K17947 (KS), JP20H03500 (TS) and
JP17K08386 (TS). The funders had no role in the
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing interests: The authors have declaredthat no competing interests exist.

Impact of polymerization on anti-influenza HA stalk IgA antibodies

Introduction
Influenza is a highly contagious infectious disease caused by the influenza virus. The main targets of this virus in humans are epithelial cells lining the upper respiratory tract. Therefore,
IgA antibodies, which are secreted onto the mucosal surface as secretory IgA antibodies(SIgAs), play an important role in preventing influenza virus infection [1–3]. Currently available influenza vaccines are of the injectable type, which induce systemic IgG responses but notmucosal IgA responses; such vaccines mitigate the severity of disease following virus infection
but cannot prevent infection in the first place [4]. By contrast, intranasally-administered inactivated vaccines, which are currently under development, induce secretion of SIgAs by respiratory mucosal tissues [5,6]. Thus, intranasal inactivated influenza vaccines are promisingvaccine candidates that could protect humans from infection by influenza virus [7,8].
According to a study based on human polyclonal SIgAs isolated from nasal wash samples
obtained from intranasal inactivated influenza vaccine recipients, SIgAs are secreted by theupper respiratory tract mucosa in the form of monomers, dimers, and polymers (which arelarger than dimers). Studies show that SIgA polymers exhibit higher virus-neutralizing activity
than dimers or monomers [9,10]. However, the detailed molecular mechanism(s) that underpins this phenomenon remains unclear. Previously, Saito et al. developed a method of generating polymeric SIgA in vitro, which involves co-transfecting mammalian cells with expression
plasmids encoding the four components of SIgA: immunoglobulin heavy chain (HC), lightchain (LC), joining chain (JC), and the secretory component (SC) [11]. SIgAs generated using
this method are useful in that they can be used to evaluate the effect of SIgA polymerization bycomparing the anti-viral activities of monomeric and polymeric SIgAs harboring identical variable regions. This method was used to show that anti-HA head-targeting antibodies haveincreased anti-viral activity, including HA binding activity (reactivity), hemagglutination inhibition (HI) activity, and virus neutralization (NT) activity upon IgA polymerization [11].
However, the effects of IgA polymerization on the anti-viral functions of antibodies that target
the influenza HA stalk region remain unexplained; these antibodies play a beneficial role inprotecting against influenza virus infection through mechanisms different from those of antiHA head-targeting antibodies.
The HA stalk is a relatively well-conserved region among multiple strains and subtypes ofinfluenza virus [12]. Thus, a large proportion of influenza virus broadly neutralizing antibodies(bnAbs) identified to date are anti-HA stalk antibodies [13–16]. Some studies have attempted
to induce anti-HA stalk antibodies via vaccination, and to show that these antibodies confer
protection against a broad range of influenza virus infections [17–20]. Anti-HA stalk bnAbs
inhibit conformational changes in HA molecules, a process essential for fusion of the virus
with the cell membrane following virus attachment [21–23]; by contrast, anti-HA head bnAbs
target the receptor binding domain, thereby inhibiting virus attachment to target cells [24,25].
In addition, anti-HA stalk antibodies possess neuraminidase (NA) inhibition (NI) activity[26–28]. NA digests cell surface sialic acids that bind to the HA molecule of newly budding
viruses to release viral progeny from the cell surface. By binding to the HA stalk, anti-HA stalk
antibodies physically impede access to neighboring NA molecules, a process that is required
for sialic acid digestion; this results in NI activity due to steric hindrance [26]. Thus, a detailed
understanding of the anti-viral humoral immune mechanisms that fight influenza viruses iscritical for development of more effective treatments and prophylactic strategies. However, noreports have examined anti-HA stalk antibodies in recipients of intranasal inactivated influenza vaccines, or anti-viral mechanisms that operate in the upper respiratory mucosa, which isthe first line of defense against respiratory pathogens. Therefore, to better understand themechanisms underlying mucosal humoral immunity induced by intranasal inactivated

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Impact of polymerization on anti-influenza HA stalk IgA antibodies

influenza vaccination, as well as the functional traits of mucosal IgA antibodies, it is essential
to verify the presence of anti-HA stalk antibodies in recipients of intranasal inactivated influenza vaccines and to understand the effects of SIgA polymerization on these antibodies.
In this study, we isolated an anti-HA stalk antibody from a recipient of an intranasal inactivated influenza vaccine and examined the effects of IgA polymerization on its anti-viral activity. The results suggest that IgA polymerization impacts the functions of anti-HA stalk
antibodies in various ways depending on the mode of interaction between the antibody andviral glycoproteins.

Results
Anti-viral activity of the HA stalk IgG antibody and of IgA monomers andpolymers
To examine the effect of IgA polymerization on anti-HA stalk antibody functions, we compared the anti-viral activity of anti-HA stalk IgG with that of IgA monomers and polymers.
First, we isolated the human antibody clone F11 (an anti-HA stalk antibody clone) from arecipient of an intranasal inactivated influenza vaccine. Detailed information regarding theprocedure used to screen clone F11 is provided in S1 and S2 Tables. Characterization of antibody clone F11 is described in S1 and S2 Figs and S3 Table. In addition to clone F11, we used apreviously reported anti-HA stalk bnAb, FI6 [29], for these comparisons. For each antibody
clone, we generated IgA monomers and IgA polymers comprising the two subclasses of IgA:
IgA1 and IgA2 (allotype IgA2m2). IgA polymers were produced using methods establishedpreviously [11], and successful production of IgA polymers of antibody clones F11 and FI6
was confirmed by SDS-PAGE (S3D Fig), HPLC (S3B Fig), and sedimentation velocity analytical ultracentrifugation (SV-AUC; S3E–S3I Fig) analyses.
Anti-viral activity of F11 and FI6 IgG1, and monomeric or polymeric IgA1 and IgA2m2(including NT activity, HI activity, NI activity, and binding activity), was tested against virus
strains A/California/7/2009 (H1N1, Cal7), A/New Caledonia/20/1999 (H1N1, NC20), A/
Puerto Rico/8/1934 (H1N1, PR8), A/Victoria/210/2009 (H3N2, Vic210), and A/New York/55/
2004 (H3N2, NY55). The results of these assays are summarized in Table 1.
Increased NT activity due to IgA polymerization was more evident for IgA2m2 antibodies
derived from clones FI6 and F11, both of which showed marked increases in NT titer against
Cal7, NC20, and PR8 viruses; the exception was F11 IgA2m2 against Cal7. The NT activity of
F11 and FI6 IgA1 against the PR8 virus was significantly higher after polymerization. In somecases, NT activity increased after converting the antibody backbone from IgG to an IgA1 or
IgA2m2 monomer, suggesting that not only the number of Fabs per molecule but also differences in the antibody constant region contribute to the increased NT titer. This hypothesis
was further supported by the results of NT assays using virus strains Vic210 and NY55. F11,
which did not exhibit NT activity in the IgG state, did show NT activity when converted to
IgA, clearly suggesting that anti-viral potency is derived from the IgA backbone. As describedabove, FI6 neutralized the H3 virus in both its IgG and IgA forms, although we observed amarked increase in NT activity after converting the IgG backbone to an IgA1 backbone(Table 1). These observations were consistent with those of a previous report in which
enhancement in virus neutralization activity was observed after converting the IgG backboneof an anti-HA stalk antibody to an IgA backbone [29].
To verify whether the increase in NT induced by IgA polymerization was due simply toincreased avidity, we used ELISAs to examine binding of IgA monomers and polymers to HAproteins derived from Cal7, NC20, PR8, Vic210, and NY55 viruses. However, we found no
direct association between increased IgA binding activity and NT activity after IgA

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Impact of polymerization on anti-influenza HA stalk IgA antibodies

Table 1. Anti-viral functions of IgG and IgA antibodies derived from clones F11 and FI6.
F11

FI6

H1N1
NT activity [μg/mL] Geometricmean (geometric SD)

HI activity [μg/mL] Geometricmean (geometric SD)

NI activity [ng/mL] (EC50)

Binding activity [ng/mL] (EC50)

H3N2

H1N1

H3N2

Cal7

NC20

PR8

Vic210

NY55

Cal7

NC20

PR8

Vic210

NY55

G

11.2
(1.69)

89.1(1.33)

83.3(1.79)>500>500

5.00 (1)31.5(1.43)

28.1(1.69)

2.19 (1.69)

3.48 (1.33)

A1M

2.81
(2.25)

28.1(1.69)

50.0(1.55)

1.74 (1.98) 11.1 (1.79)

3.15(1.76)

6.25 (1)39.7(2.05)

0.137(1.33)

0.69 (2.07)

A1P

4.45
(1.69)

15.8(1.43)

25.0 (1) �

1.55 (1.43) 7.81 (1.55)

1.98(1.76)

4.96(1.43)

11.1(1.33) �

0.154(2.32)

0.775(1.76)

A2M

4.45(2.25)

11.1(1.33)

89.1(1.33)

0.548(1.69)

3.1 (1.76)

2.5 (2.14)

8.84(1.46)

31.5(1.43)

0.345(1.79)

1.10 (1.98)

A2P

7.07
(1.46)

5.57(1.33) �

50.0 (1) �

0.194(1.43) �

0.977(1.55) ��

0.88(1.46) �

1.75(1.98) ��

6.25 (1) ��

0.0308(1.56) ��

0.217(1.69) ��

>500

>500

G

>500

>500

A1M

62.5 (1)62.5 (1)354 (1.46) 9.84 (1.43)

>500

>500

>500

>500

>500

>500

15.6 (1)177 (1.46)

7.81 (1)500 (1)1.95 (1)2.76 (1.46)

A1P

62.5 (1)62.5 (1)500 (1)

19.7 (1.43) 27.8 (1.33)>500

1.95 (1)

3.48 (1.33)

�

88.4(1.46) �

15.6 (1) ��

�

A2M

88.4
(1.46)

31.3 (1)500 (1)7.81 (1)7.81 (1)125 (1)

11.1(1.46)

500 (1)

3.91 (1)

3.91 (1)A2P

49.6(1.76)

7.81 (1)500 (1)

2.46 (1.43) 3.48 (1.33)

44.2(1.46) ��

11.1(1.46)

250 (1) ��

0.691(1.46) ��

0.775(1.43) ��

G

1616

2136

A1M

267

148

A1P 40.6 ����
A2M
A2P

��

��

��

3315

ND

ND

551

610

1195

96.1

64.2

301

192

125

117

106

165

126

48.9

106 ��

222

264 �

187 �

69.5 ���

48.3 ����

149

107 �

64.1

215

61.7

683

927

546

57.4

11.4

173

23.2

12.7

30.4 ����

16.2 ����

418 ��

258 ���

121 ��

114.9 ���14.02 �302 ���

6.65 ����

4.27 ���

G

800

138

422

2.51+06

8.49E+07

291

59.5

42.5

2.45E+05

1.11E+04

A1M

492

1.16E+03

3.09E+03

1.95E+04

2.01E+03

305

602

571

2.43E+03

2.48E+03

A1P

502

730 ����

1.21E+03

6.51E+03

3.43E+03

258 ����

549 �

758 ���

6.84E+03

2.58E+03

����

��

���

����

A2M 4.64E+04

1.35E+04

3.17E+04

1.90E+04

4.92E+03

1.16E+07

ND

8.45E+05

1.56E+03

647

A2P

1.56E+04

3.39E+04

7.75E+04

6.85E+03

303 ����

5.70E+04

9.39E+03

1.45E+04

3.69E+03

3.06E+03

����

���

����

����

����

����

����

����

����

NT activity, virus-neutralizing activity. Minimum virus-neutralizing concentrations are presented as the geometric mean (geometric SD) of six technical replicates. HI
activity, hemagglutination inhibition activity. Minimum HI concentrations are presented as the geometric mean (geometric SD) of six technical replicates. NI activity,
neuraminidase inhibition activity. EC50 values were calculated from three technical replicates. Binding activity: antibody binding to recombinant HA measured by
ELISA. EC50 values were calculated from three technical replicates. G, IgG1; A1M, IgA1 monomer; A1P, IgA1 polymer; A2M, IgA2m2 monomer; A2P, IgA2m2
polymer; Cal7, A/California/7/2009 (H1N1); NC20, A/New Caledonia/20/1999 (H1N1); PR8, A/Puerto Rico/8/1934 (H1N1); Vic210, A/Victoria/210/2009 (H3N2);
NY55, A/New York/55/2004 (H3N2, NY55); ND, not detected�

p < 0.05
p < 0.01

�����

p < 0.001

����p < 0.0001 (Mann–Whitney test, comparing NT and HI activity of monomers with polymers; two-way ANOVA comparing NI and binding activity curves ofmonomers with those of polymers).
https://doi.org/10.1371/journal.pone.0245244.t001

polymerization (Table 1), suggesting that the NT activity of anti-HA stalk IgA antibodies is notdefined simply by the antibodies’ binding activity, but also by their other functions. This observation is inconsistent with those of a previous report of anti-HA head antibodies [11], suggesting that the impact of IgA polymerization does not have an equal positive or negative impact
on the anti-viral functions of anti-HA stalk antibodies.

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Impact of polymerization on anti-influenza HA stalk IgA antibodies

Polymerization of anti-HA stalk IgA antibodies increases HI activity due toefficient masking of the HA receptor binding site (RBS)
To identify factors other than avidity that determine the NT titer of anti-HA stalk antibodies,
we examined other anti-viral functions of anti-HA stalk IgG, IgA monomers, and IgA polymers, starting with HI activity. HI activity is one of the main components of anti-influenza
virus immunity in vivo, along with anti-viral activity conferred by cellular immunity [30] andantibodies targeting various sites of viral components [31]; traditionally, HI activity is the correlate of protection against influenza in humans. Antibodies with HI activity block interaction
with the RBS located on the HA head [32]. Consistent with previous data, we found that F11,
an HA stalk-binding antibody with an IgG1 backbone, did not exhibit HI activity. However,HI activity emerged when the IgG backbone of clone F11 was converted to IgA1 or IgA2m2(Fig 1A and Table 1), suggesting that anti-HA stalk IgA blocks the RBS of HA. Emergence of
HI activity after conversion to IgA was confirmed by testing clone FI6 against viruses of the
H1N1 subtype. The increase in HI activity against some virus strains after IgA polymerization
was more pronounced for IgA2m2 than for IgA1 (Fig 1A and Table 1). To determine whether
the HI activity observed for anti-HA stalk IgA was due to masking of the RBS, we conductedbinding competition analyses using recombinant HA, F11 antibodies, and F045-092 IgG1 (ananti-HA RBS binding bnAb) [33]. Masking of the RBS by F11 IgG1 was quantified by measuring inhibition of F045-092 binding to HA pre-treated with F11 (Fig 1B). Binding of F045-092
IgG1 to HA after addition of F11 antibodies fell as the concentration of F11 IgG1, IgA2m2
monomer, and IgA2m2 polymer increased (Fig 1C). Notably, the RBS-masking efficiency of
F11 IgA2m2 monomers and polymers (as determined by the reciprocal of the binding capacity
for F045-092 IgG1) was significantly higher than that of IgG1. Furthermore, the RBS-masking
efficiency of anti-HA stalk IgA antibodies increased markedly after IgA polymerization (Fig1C). These results suggest that the IgA backbone blocks the RBS more efficiently than the IgG
backbone, even in the monomeric state, and that direct blockade of the RBS by the IgA Fc
region could be contributing to this phenomenon. Furthermore, increasing the molecular sizeof anti-HA stalk IgA antibodies by polymerization led to a marked increase in the efficiency of
RBS blockade, which could be the mechanism underlying increased HI activity exhibited byanti-HA stalk IgA antibodies after IgA polymerization. This increase in RBS-blocking efficiency may be caused by the large size of the HA stalk-binding polymeric IgA antibodies,
which presumably mask the RBS more efficiently than monomeric IgA antibodies. On thecontrary, in some cases, the HI activity of IgA1 fell significantly after polymerization (Table 1),
suggesting that the effect of IgA polymerization on HI activity is regulated in a complex manner not only by RBS blockade, but also by multiple mechanisms.

IgA Fc glycans facilitate the HA binding and anti-viral functions of antiHA stalk IgA antibodies, which are weakened by IgA polymerization
To verify the extent to which the IgA backbone contributes to the anti-viral activity of anti-HA
stalk antibodies, we examined the anti-viral functions of antibody clone B12, which is derived
from gene position IGHV1-69 and lacks NT activity on an IgG backbone (S1 and S2 Tables);however, it does exhibit weak binding to the HA stalk on an IgA backbone (Fig 2A). The antiviral and binding activities of B12 IgG1, monomeric or polymeric IgA1, and monomeric or
polymeric IgA2m2 were tested against Cal7, NC20, PR8, Vic210, and NY55 viruses. The resultsof these assays are summarized in Table 2. Notably, binding of monomers of B12 with IgA
backbones to HA was significantly higher than that of polymers. B12 IgA antibodies exhibited
NT activity against certain virus strains (NC20, Vic210, and NY55), although these antibodyfunctions did not increase after IgA1 polymerization and were weakened markedly by IgA2m2

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Impact of polymerization on anti-influenza HA stalk IgA antibodies

Fig 1. IgA polymers mask the hemagglutinin (HA) receptor binding site. (A) The HI titer for each IgG1, monomeric (M) or polymeric (P) IgA1 (A1), andIgA2m2 (A2m2) antibody derived from clones F11 and FI6, tested against virus strain A/New Caledonia/20/1999 (H1N1, NC20). HI activity is presented in the
scatter plots as the geometric mean, with the geometric standard deviation (SD) of six technical replicates. Y-axis values represent the HI titer, calculated as the
100,000/minimum concentration (μg/ml), which represents HI activity. Dotted lines represent the detection limit (y = 200; 500 μg/mL). �� p < 0.01, comparing
monomers with polymers (Mann–Whitney U test). ††p < 0.01 and ††††p < 0.0001, comparing IgA monomers/polymers with IgG (Kruskal–Wallis test, followed by
Dunn’s multiple comparison test). For statistical analysis, a provisional minimum HI activity value (y = 100; 1000 μg/mL) was applied to samples in which the HI
activity was below the detection limit. (B) Schematic diagram showing RBS blockade, as assessed by SPR. First, recombinant HA from virus strain A/New Caledonia/
20/1999 (H1N1) was bound to IgG1 or to monomeric or polymeric F11 IgA2m2. The amount of F045-092 IgG1 binding to F11-pre-bound HA was then measured
to evaluate RBS blocking by F11. The relative response (RU) was acquired immediately after addition of F045-092 IgG1 (binding stability). The maximum stability
value was acquired by measuring the RU immediately following binding of F045-092 IgG1 to free HA. (C) Binding of F045-092 IgG1 to F11 IgG1-, IgA2m2-, or
IgA2m2-pre-bound HA. Y-axis values represent the ratio of F045-092 IgG1 binding to the maximum stability value. �� p < 0.01, ��� p < 0.001, and ���� p < 0.0001(two-way ANOVA).
https://doi.org/10.1371/journal.pone.0245244.g001

polymerization (Fig 2B). Changes in anti-viral potency of B12 antibodies triggered by IgApolymerization tended to correspond with binding activity patterns (Table 2). This suggeststhat the impact of IgA polymerization has an equally negative impact on the various antibodyfunctions of B12 IgA antibodies.

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Impact of polymerization on anti-influenza HA stalk IgA antibodies

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Impact of polymerization on anti-influenza HA stalk IgA antibodies

Fig 2. Deglycosylation of IgA antibodies and functional analyses of deglycosylated B12 IgA antibodies. (A) Reactivity of FI6, F11, B12,and ΔFab monomeric IgA1 with the recombinant A/Brisbane/59/2007 (H1N1) HA stalk. (B) Virus neutralizing (NT) activity of IgG1,
monomeric (M), or polymeric (P) IgA1 (A1), and monomeric (M) or polymeric (P) IgA2m2 (A2m2) derived from antibody clone B12
against virus strains A/New Caledonia/20/1999 (H1N1, NC20), A/Victoria/210/2009 (H3N2, Vic210), and A/New York/55/2004 (H3N2,
NY55). NT activity is presented in the scatter plots as the geometric mean, with the geometric standard deviation (SD) from six technicalreplicates. Y-axis values represent neutralizing titers, calculated as 10,000/minimum concentration (μg/ml) of antibody that neutralized thevirus. Dotted lines represent the detection limit (y = 20; 500 μg/mL). � p < 0.05, comparing monomers with polymers (Mann–Whitney Utest). †p < 0.05, ††p < 0.01, ††††p < 0.0001, comparing IgA monomers/polymers with IgG (Kruskal–Wallis test, followed by Dunn’s
multiple comparison test). For statistical analysis, a provisional minimum NT activity value (y = 10; 1000 μg/mL) was applied to samples inwhich HI activity was below the detection limit. (C) SDS-PAGE analysis of monomeric (M) and polymeric (P) B12 IgG1 (G), IgA1 (A1),and IgA2m2 (A2m2) antibodies incubated with (+) or without (-) deglycosylation enzymes. Fetuin, the band for which shifted (i.e., areduction in molecular weight) after deglycosylation, was used as positive control. All four components of secretory IgA antibodies (heavy
chain [HC], light chain [LC], secretory component [SC], and the J chain [JC]) were observed. The heavy chain, secretory component, and J
chain bands of IgA shifted down slightly (reduced molecular weight) after deglycosylation (D) Deglycosylation of samples was confirmed in
an enzyme-linked lectin assay (ELLA). The area under the reactivity curve (AUC) was calculated from reactivity curves for lectin and eachsample was treated in the presence (+) or absence (-) of deglycosylation enzymes. Data are expressed as the mean ± SD of three technicalreplicates. Treatment with deglycosylation enzymes led to a significant increase in lectin binding in all samples, suggesting successful
deglycosylation. �� p<0.01, ��� p<0.001, and ���� p<0.0001 (Welch’s test). (E–H) Reactivity of glycosylated (-) and deglycosylated (+)
monomeric (M) and polymeric (P) B12 IgA1 (E and G) and IgA2m2 (F and H) antibodies with recombinant HA protein from the NC20 (Eand F) and Vic210 (G and H) viruses. (I–L) AUC for each glycosylated (-) and deglycosylated (+) monomeric (M) and polymeric (P) B12
IgA1 (I and K) and IgA2m2 (J and L) antibody tested against the recombinant HA protein of the NC20 (I and J) and Vic210 (K and L)
viruses. The AUC was calculated from the plots in E–H. Data are expressed as the mean ± SD of three technical replicates. Deglycosylation
reduced the reactivity of B12 monomeric IgA1 and IgA2m2. �� p < 0.01 and ���� p<0.0001 (Welch’s test). (M and N) HI titer for each
glycosylated (-) and deglycosylated (+) B12 IgG1 (G), monomeric (M), or polymeric (P) IgA1 (A1) and IgA2m2 (A2m2) antibody tested
against virus strains NC20 (M) and Vic210 (N). HI activity is presented in the scatter plots as the geometric mean, with the geometric SD ofthree technical replicates. Y-axis values represent the HI titer, calculated as the 100,000/minimum concentration (μg/ml), which represents
HI activity. Dotted lines represent the detection limit (y = 200; 500 μg/mL). �� p < 0.01, comparing monomers with polymers (Mann–
Whitney U test). For statistical analysis, a provisional minimum HI activity value (y = 100; 1000 μg/mL) was used for samples in which the
HI activity was below the detection limit.
https://doi.org/10.1371/journal.pone.0245244.g002

A recent report suggests that IgA antibodies bind non-specifically to the RBS of HA via asialylated C-terminal tail unique to IgA antibodies [34]. Therefore, we suspected that glycans
on the IgA Fc region may contribute to IgA backbone-mediated binding to HA. HPLC coupled with mass spectrometry (LC-MS) analysis of N-linked glycans released from human nasal
IgA and by recombinant F11 IgG1, IgA1, and IgA2m2 antibodies was conducted. In agreement with a previous report that used IgA isolated from human saliva, we detected terminallysialylated complex glycans in peaks with later retention times on the LC chromatogram forhuman nasal IgA. Terminally sialylated complex glycans were also detected in multiple peaks
with later retention time points on the LC chromatograms for recombinant monomeric andpolymeric IgA1 and IgA2m2 samples. By contrast, LC chromatograms for recombinant IgG1
showed fewer peaks than those for IgA samples, although several terminally sialylated glycans
were detected in peaks with later retention times (S4 Fig). These observations suggest that bothhuman nasal IgA antibodies and recombinant IgA antibodies contain sialic acids, and that sialylated complex glycans are more abundant in IgA than in IgG1 antibodies.
To verify whether the HA binding observed for B12 IgA was associated with glycans on theIgA Fc region, we treated B12 IgA1 and IgA2m2 monomers and polymers with a deglycosylating enzyme and compared the binding potency with that of untreated IgA (Fig 2C and 2D).
We found that IgA binding to HA weakened after incubation with the deglycosylation enzyme,
and that this effect was significant only for IgA monomers (Fig 2E–2L). In addition, the same
deglycosylated antibody samples were tested against NC20 and Vic210 viruses in HI assays.
B12 with an IgA backbone did not show any HI activity against the NC20 virus; this did notchange after deglycosylation (Fig 2M). Meanwhile, B12 with an IgA backbone showed mild HI
activity against the Vic210 virus, and the HI activity of B12 IgA2m2 antibodies fell after deglycosylation (Fig 2N). Taken together, these results indicate that the anti-viral activity of B12 IgA
is facilitated by Fc glycan-mediated binding of IgA to HA, and that this Fc glycan-mediatedeffect is more pronounced for monomers than for polymers.

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Impact of polymerization on anti-influenza HA stalk IgA antibodies

Table 2. Anti-viral functions of IgG and IgA antibodies derived from clones B12 and Fab-deficient (ΔFab) IgA antibodies.
ΔFab

B12
H1N1
NT activity [μg/mL] Geometricmean (Geometric SD)

H3N2

NI activity [ng/mL] (EC50)

H3N2

Cal7

NC20

PR8

Vic210

NY55

Cal7

NC20

PR8

Vic210

NY55

G

>500

>500

>500

>500

>500

n.t.

n.t.

n.t.

n.t.

n.t.

A1M

>500

>500

>500

27.8 (1.69) 158 (1.76)

n.t.

n.t.

n.t.

>500

n.t.

A1P

>500

>500

>500

24.8 (1.43)

99.2(1.43)

n.t.

n.t.

n.t.

>500

n.t.

A2M

>500

630 (1.43)>500

7.81 (1.55)

35.1(1.69)

n.t.

n.t.

n.t.

>500

n.t.

A2P

>500

500 (1)>500

22.1 (1.79)

99.2(2.32)

n.t.

n.t.

n.t.

>500

n.t.

�

HI activity [μg/mL] Geometricmean (Geometric SD)

H1N1

G

>500

>500

>500

>500

>500

n.t.

n.t.

n.t.

n.t.

n.t.

A1M

>500

500 (1)>500

125 (1)158 (1.43)

n.t.

>500

n.t.

794 (1.43)

n.t.

A1P

>500

500 (1)>500

125 (1)177 (1.46)

n.t.

>500

n.t.

>500

n.t.

A2M

>500

250 (1)>500

39.4 (1.43)

62.5 (1)

n.t.

>500

n.t.

500 (1)

n.t.

A2P

>500

500 (1) ��>500

125 (1) ��

125 (1) ��

n.t.

>500

n.t.

>500

n.t.

G

ND

9.57E+07

ND

1.62E+05

ND

n.t.

ND

n.t.

ND

n.t.

A1M

ND

1.63E+05

ND

5.03E+03

1.54E+03

n.t.

ND

n.t.

ND

n.t.
n.t.

A1P

ND

5.24E+04

ND

5.05E+03

1.64E+03

n.t.

ND

n.t.

ND

A2M

ND

8.14E+03

ND

2.70E+03

866

n.t.

ND

n.t.

ND

n.t.

A2P

ND

8.48E+04

ND

6.50E+03

ND ��

n.t.

ND

n.t.

ND

n.t.

���

Binding activity [ng/mL] (EC50)

A1M 3.80E+04

2.95E+04

1.68E+04

1.08E+05

6.28E+04

1.34E+04

2.00E+04

1.69E+04

3.65E+05

2.85E+05

A1P

4.89E+11

6.28E+10

3.11E+05

4.14E+05

6.01E+05

ND ����

1.44E+10

1.32E+08

8.92E+04

6.57E+06

����

����

���

���

��

����

A2M 2.73E+04

3.08E+04

4.08E+04

2.38E+04

2.92E+04984

1.12E+03

724

778

823

A2P

5.28E+08

3.18E+06

1.18E+07

1.45E+06

8.44E+05

2.19E+05

5.98E+05

6.72E+05

2.51E+05

1.07E+05

����

����

����

����

����

����

����

����

����

����

NT activity, virus-neutralizing activity. Minimum virus-neutralizing concentrations are presented as the geometric mean (geometric SD) of six technical replicates. HI
activity, hemagglutination inhibition activity. Minimum HI concentrations are presented as the geometric mean (geometric SD) of six technical replicates. NI activity,
neuraminidase inhibition activity. EC50 values were calculated from three technical replicates. Binding activity, antibody binding activity to recombinant HA measured
by ELISA. EC50 values were calculated from three technical replicates. G, IgG1; A1M, IgA1 monomer; A1P, IgA1 polymer; A2M, IgA2m2 monomer; A2P, IgA2m2
polymer; Cal7, A/California/7/2009 (H1N1); NC20, A/New Caledonia/20/1999 (H1N1); PR8, A/Puerto Rico/8/1934 (H1N1); Vic210, A/Victoria/210/2009 (H3N2);
NY55, A/New York/55/2004 (H3N2, NY55); ND, not detected; n.t., not tested.
�p < 0.05
p < 0.01

�����

p < 0.001

����p < 0.0001 (Mann–Whitney test comparing the NT and HI activity of monomers with polymers; two-way ANOVA comparing NI and binding activity curves ofmonomers with those of polymers).
https://doi.org/10.1371/journal.pone.0245244.t002

IgA Fc glycan-mediated binding to the HA head contributes to anti-viralfunctions in the presence of Fab paratope-mediated binding to the HA
stalk
Recent reports show that anti-HA stalk antibodies exert NI activity via steric hindrance ofneighboring NA molecules, and that this activity correlates well with the in vitro NT activity
and in vivo anti-viral activity of anti-HA stalk antibodies [26–28]. Since polymeric IgA antibodies are larger than monomers, we assumed that IgA polymers would show higher NI activity than IgG and IgA monomers. We tested this hypothesis by conducting a NI-ELLA(Enzyme-Linked Lectin Assay) using F11, FI6, B12 IgG, and IgA antibodies. In addition, we

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Impact of polymerization on anti-influenza HA stalk IgA antibodies

tested the NI activity of monomeric and polymeric forms of Fab-deficient (ΔFab) IgA1 andIgA2m2 antibodies (S3C and S3D Fig) to further verify the anti-viral functions of the IgA Fc
region. Both F11 and FI6 IgG showed moderate NI activity against H1N1 viruses (Fig 3A, 3B,
3E and 3F), whereas FI6 IgG showed even stronger NI activity against H3N2 viruses (Fig 3Gand 3H). By contrast, F11 IgG did not show NI activity against H3N2 viruses (Fig 3C and 3D).
As reported previously [26], there was a clear relationship between the NT activity and NI
activity of anti-HA stalk IgG antibodies (Table 1). Interestingly, the NI activity of F11 against
H3N2 viruses emerged after conversion of the antibody backbone from IgG to IgA (Fig 3Cand 3D). Overall, IgA antibodies showed higher NI activity than IgG antibodies (the exception
was FI6 IgA1 antibodies against H3N2 viruses) (Fig 3A–3H); in addition, the impact of IgApolymerization on NI activity differed according to the antibody clone, IgA subclass, and virus
strain. Notably, B12 IgG showed minimal NI activity against the NC20 and Vic210 viruses,
and B12 IgA antibodies also showed higher NI activity than B12 IgG, without IgA polymerization-induced functional enhancement (Fig 3I–3L). By contrast, ΔFab IgA antibodies did notshow evident NI activity despite exhibiting HA binding activity similar to that of B12 IgA antibodies (Fig 3M–3P and Table 2). The results of these assays using ΔFab and B12 IgA antibodies
suggest that the IgA Fc glycan binds to the HA RBS, leading to anti-viral activity if there is cooccurrence of Fab paratope-mediated binding to the HA stalk. Furthermore, IgA Fc glycanmediated functions tended to fall after polymerization, indicating that, unlike Fab paratopemediated HA binding and anti-viral activity, polymerization has a negative effect on anti-viral
activity in a situation where Fab paratope-mediated HA stalk-binding is relatively weak andIgA Fc glycan-mediated HA RBS binding is predominant.

In vivo passive transfer of recombinant IgG and IgA antibodies to mice
Finally, to evaluate the clinical relevance of our findings in the in vitro experiments, we conducted an in vivo passive transfer experiment in which recombinant human IgG and IgA antibodies were given to mice. Mice were infected intraperitoneally with either 200 μg of IgG1,
IgA1 monomer, or polymers of F11 or FI6 antibodies, 24 hours prior to challenge with mouseadapted A/Narita/1/2009(H1N1pdm09) virus. PBS was administered to the negative control
group. At 3 days post-infection, serum and lung wash samples were collected for antibody
measurement and virus titration (Fig 4A). The in vitro anti-viral activity of F11 and FI6 IgA1
antibodies was higher than that of their IgG forms, and the NI activity of both clones wasenhanced by IgA polymerization (Table 1 and Fig 4B). There was a significant decrease in lungwash virus titers in both groups receiving F11 and FI6 IgG1, suggesting that clone F11 exhibits
in vivo anti-viral activity (Fig 4C). In the groups receiving IgA1, virus titers in lung wash fromthe group receiving FI6 polymeric IgA1 were significantly lower than those form the control
PBS group (Fig 4C), although the concentration of FI6 IgA1 antibodies in serum and lungwash was relatively low compared with that for F11 IgA1 antibodies (Fig 4D and 4E). This suggests that higher doses of F11 IgA than FI6 IgA are required to confer anti-viral activity invivo, and that the amount of F11 IgA1 administered in the current study was insufficient toneutralize or prevent efficient propagation of the virus in mice. In addition, the virus-neutralizing activity of FI6 IgA1 polymers in vivo was significantly higher than that of FI6 IgA1 monomers (Fig 4C). Of note, the serum FI6 IgA1 concentration in the group receiving monomeric
IgA1 was significantly higher than that in the group receiving polymeric IgA1 (Fig 4D),
although there was no significant difference in IgA1 concentrations in lung wash from these
two groups (Fig 4E). This indicates that the observed difference in lung wash virus titers
between these groups was due to differences in the in vivo anti-viral activity of FI6 IgA1 monomers and polymers, rather than differences in the in vivo stability of these two structures. In

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Fig 3. Comparison of the neuraminidase inhibition activity of IgG with that of IgA monomers and polymers. Neuraminidase inhibition (NI) activity of IgG1,
monomeric (M) or polymeric (P) IgA1, and monomeric (M) or polymeric (P) IgA2m2 derived from antibody clones F11 (A–D), FI6 (E-H), and B12 (I–L), as well
as Fab-deficient (ΔFab) IgA antibodies (M–P), against virus strains A/New Caledonia/20/1999 (H1N1, NC20) (A, B, E, F, I, J, M, and N) and A/Victoria/210/2009(H3N2, Vic210) (C, D, G, H, K, L, O, and P). NI activity is presented as inhibition curves, with each point and error bar representing the mean ± SD of threetechnical replicates. Y-axis values represent percentage inhibition of neuraminidase activity. The OD values of wells incubated without antibodies were normalized
to y = 100, and the OD values of wells incubated without virus were normalized to y = 0. X-axis values represent the concentration of antibody added to each well.
Zanamivir, a neuraminidase inhibitor, was used as a positive control. � p < 0.05, �� p < 0.01, ��� p < 0.001, and ���� p < 0.0001, comparing monomers with polymers(two-way ANOVA).
https://doi.org/10.1371/journal.pone.0245244.g003

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Fig 4. In vivo passive transfer of recombinant IgG and IgA antibodies to mice. (A) Experimental schedule. Sixweek-old female BALB/c mice (six per experimental condition) were injected intraperitoneally with IgG1 (G) or IgA1
monomers (M), with polymers (P) of F11 or FI6 antibodies (200 μg/200 μl per mouse), or with PBS (200 μl per mouse)
24 hours prior to challenge with virus. Next, mice received 4×103 plaque-forming units (PFU) of mNRT virus
administered intranasally via the left nostril (20 μl per mouse) to infect the lower respiratory tracts. At 3 days postinfection, lung wash samples were collected. (B) NI activity of IgG1 and monomeric or polymeric IgA1 derived fromantibody clones F11 and FI6 against virus strain A/California/7/2009 (H1N1). NI activity is presented as inhibition
curves, with each point and error bar representing the mean ± SD of three technical replicates. Y-axis values represent
percentage inhibition of neuraminidase activity. The OD values for wells incubated without antibodies werenormalized to y = 100, and those for wells incubated without virus were normalized to y = 0. X-axis values denote theconcentration of antibody added to each well. Black line, Zanamivir; blue line, IgG1; red solid line, IgA1 monomer; reddotted line, IgA1 polymer. ��� p < 0.001 and ���� p < 0.0001, comparing monomers with polymers (two-way
ANOVA). (C) Virus titers in lung wash samples collected at 3 days post-infection. Virus titers are presented in scatterplots as the geometric mean, with the geometric SD. Y-axis values represent plaque-forming units (PFU)/mouse,
which is equal to PFU/2mL of lung wash, as calculated from the results of the plaque assay. The dotted line denotes thedetection limit (1000 PFU/mouse). �� p< 0.01, comparing monomers with polymers (Mann–Whitney test). †p< 0.05,††p< 0.01, and ††††p< 0.0001, comparing IgA monomers/polymers with IgG (Kruskal–Wallis test, followed by
Dunn’s multiple comparison test). (D, E) Antibody concentrations in serum (D) and lung wash (E) samples collected
at 3 days post-infection. Antibody concentrations are expressed on scatter plots as the geometric mean, with thegeometric SD. � p< 0.05 and �� p< 0.01, comparing monomers with polymers (Mann–Whitney test). For statisticalanalysis, a provisional antibody concentration (1 ng/mL) was applied to samples in which antibody concentrations
were below the detection limit. ns: not significant, nd: not detected. (F) Body weight changes in 6-week-old female
BALB/c mice (five or six per experimental condition) following lethal infection with mNRT virus. Mice were injected

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intraperitoneally with IgG1 harboring the N297A mutation (IgG1 N297A), gA1/IgA2m2 monomers, polymers of FI6
antibodies (200 μg/200 μl per mouse), or PBS (200 μl per mouse) 2 hours prior to challenge with virus. Next, micereceived 4 × 103 plaque-forming units (PFU) of mNRT virus intranasally via the left nostril (20 μl per mouse) to infectthe lower respiratory tract. The body weight of each mouse was normalized to that at Day 0 (the date of virus infection;y = 100). (G) Survival of mice following lethal virus infection. The survival curves for the groups receiving IgG1 N297A(left), IgA1 (middle), and IgA2m2 (right) are shown in different panels, with the survival curve of the PBS
administered group used as the negative control.
https://doi.org/10.1371/journal.pone.0245244.g004

addition, FI6 IgA1, but not F11, polymers showed in vivo anti-viral activity, although the IgA
form FI6 and F11 showed similar anti-viral activity in vitro and the IgG forms showed similar
activity in vivo, suggesting that unknown factors might regulate in vivo anti-viral activity of the
IgA forms; this is worth investigating in future studies.
Next, to measure the ability of the antibodies to protect against weight loss and mortality
after lethal challenge, we conducted long-term observations of mice receiving FI6 IgG and IgA
antibodies following virus infection. Of note, a previous study suggested that anti-HA stalk
antibodies exert anti-viral effects via Fc receptor-mediated functions [35] and that, unlike Fc
gamma receptors, mice do not express Fc alpha receptors [36]. Therefore, in this experiment,
we used IgG1 harboring a N297A mutation (IgG1 N297A) [37], which inhibits the Fc-dependent anti-viral activity of anti-HA stalk IgG1 antibodies, to enable direct comparison of protection mediated by neutralizing IgG, monomeric IgA, and polymeric IgA antibodies in theabsence of interference by Fc-dependent functions. We found that all mice succumbed tolethal virus challenge, regardless of the type of antibody administered, by Day 8 post-virus
infection; there were no significant differences in weight loss (Fig 4F) or survival (Fig 4G)
between mice receiving PBS, IgG1 N297A, or monomeric or polymeric IgA1/IgA2m2 FI6 antibodies. This suggests the importance of the Fc-dependent functions of HA stalk antibodies forprotection against lethal virus infection.

Discussion
In this study, characterization of the functional properties of IgG, monomeric IgA, and polymeric IgA antibody clones F11, FI6 (an anti-HA stalk-binding clone) [29], and B12 (a low
affinity HA stalk-binding clone), as well as ΔFab IgA antibodies, revealed the impact of IgApolymerization on the functional properties of anti-HA stalk-binding antibodies (Tables 1 and
2). The data suggest that anti-HA stalk-binding IgA antibodies interact with viral glycoproteins
via a mechanism different from that of anti-HA head-binding IgG antibodies. Fig 5 summarizes the possible modes of interaction between anti-HA stalk antibodies bearing anti-viralfunctions and viral glycoproteins, including HA and NA. Anti-HA stalk IgG antibodies bindto the HA stalk region via Fab paratopes in the absence of Fc glycan-mediated binding to the
HA RBS; they then neutralize viruses by inhibiting viral egress through “NA steric hindrance”,
resulting in NI activity (Fig 5A). By contrast, monomeric and polymeric anti-HA stalk IgA
antibodies bind to the HA stalk region via their paratopes and to the HA RBS via Fc glycans;these antibodies sterically hinder NA and mask the HA RBS, thereby inhibiting binding ofsialic acid to HA and mediating both HI activity and NI activity (Fig 5B and 5C).
SIgA plays a key role in the mechanism of action of mucosal vaccines, including intranasal
inactivated influenza vaccines; indeed, one of the most prominent features of SIgA is functional enhancement due to polymerization [9–11]. A previous study using anti-HA head bnAbclone F045-092 showed that that antibody reactivity, HI activity, and virus neutralization activity improved upon IgA polymerization. Analyses revealed that these functional enhancements
were most striking for viruses against which the original antibodies showed relatively low reactivity, although no evident functional enhancements were observed in the case of viruses

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Impact of polymerization on anti-influenza HA stalk IgA antibodies

Fig 5. Summary of the mechanisms underlying anti-viral activity mediated by anti-HA stalk antibodies. Different modes of interaction
among anti-HA stalk antibodies, NA, and HA molecules that confer anti-viral activity (including NI activity) detected in the NI-ELLA. These
interactions mediate NI activity via two binding modes between antibodies and HA: Fab paratope-mediated binding to the HA stalk and Fc
glycan-mediated binding to the HA head. (A) In the case of anti-HA stalk-binding IgG antibodies, Fab paratope-mediated binding to the HA
stalk will cause steric hindrance of neighboring NA molecules and inhibit viral egress, leading to NT activity. (B and C) In the case of HA stalktargeting IgA antibodies, Fc glycan-mediated binding will cause HI activity due to blockade of the receptor binding site on HA, thereby inhibiting
binding of sialic acid to the receptor binding site. In these interaction modes, simultaneous steric hindrance of neighboring NA molecules andblockade of the receptor binding site may also occur, leading to inhibition of both viral egress and receptor binding.
https://doi.org/10.1371/journal.pone.0245244.g005

against which the original antibodies showed relatively high reactivity [11]. A study that examined direct binding of human-derived tetrameric IgA to HA using atomic force microscopy
demonstrated that HA associates with the radial regions of the IgA architecture, and that it is

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passed continuously from one radial region to the other [9]. Another study of human polyclonal SIgA demonstrated that the dissociation rate from HA decreased as the molecularweight of SIgA increased [10]. These results suggest that polymerization of low affinity antiHA head antibodies increases their avidity for HA molecules, resulting in maximum functional activity. However, assays using B12, the low affinity anti-HA stalk antibody clone examined in the current study, suggests that polymerization of low affinity anti-HA stalk IgA
antibodies weakens their HA binding activity and anti-viral functions. The situation is thesame for ΔFab IgA antibodies, for which polymerization weakens binding to HA. Furthermore, unlike anti-HA head antibodies, no clear relationship between reactivity of anti-HA
stalk F11 and FI6 IgA antibodies and other anti-viral activities (i.e., HI, NI, and NT) was
observed. These observations suggest that the impact of IgA polymerization does not have an
equal positive or negative impact on anti-HA stalk IgA antibody reactivity and function (i.e.,
NI, HI, and NT), even when the same combination of antibody clone and virus strain are
tested. This indicates that IgA polymerization can either have a positive or negative effect, orno effect at all, on antibody function depending on the mode of interaction between the antibody and the viral glycoproteins. Furthermore, since NT activity is the ultimate outcome ofantibody binding plus HI and NI activity, it may be that the strength of NT activity reflects thediffering contributions of HA binding, HI, and NI activity. To unravel the impact of IgA polymerization on anti-viral activity, it is necessary to keep in mind that there are two types ofbinding module involved in the interaction between HA and IgA antibodies: specific binding
between the Fab paratope and the HA stalk, and IgA Fc glycan-based binding to the HA RBS(which is the mechanism by which ΔFab IgA antibodies exert HA binding activity). Thesemechanisms may operate simultaneously depending on the angle or stability of the paratope
and of binding to the HA stalk, thereby modulating the effect of IgA polymerization on antiviral activity.
For example, in cases where an antibody clone can neutralize a virus strain when it is in the
IgG state (i.e., lacking specific sialic acids on the antibody backbone), Fab paratope-mediatedbinding is dominant. In such cases, anti-viral reactivity is increased by IgA polymerization.
With respect to HI activity, binding competition analyses using SPR revealed that polymericanti-HA stalk IgA antibodies were better able to mask the RBS than monomeric anti-HA stalk
IgA antibodies (Fig 1). Regarding NI activity, it can be assumed that a larger anti-HA stalk IgA
antibody would more efficiently hinder neighboring NA molecules. By contrast, relatively
weak Fc glycan-mediated binding will occur between a low affinity anti-HA stalk antibody
clone and HA, including combinations that, at least initially, do not show NT activity in the
IgG state (as seen for clone B12 IgA, and the combination of F11 IgA plus H3N2 virus). The
characteristic of this glycan-mediated binding (and its derivative functions) is that the intensity
weakens upon IgA polymerization, possibly due to concealment of HA-targeting glycans by
the polymeric structure of SIgA. Thus, we speculate that for anti-viral functions in which
intensity patterns correlate negatively with IgA polymerization, IgA Fc glycan-mediated binding dominates over paratope-dependent binding in some cases. For example, the fall in the HI
and NI activities of F11 IgA1 against H3N2 viruses after polymerization (Table 1) indicatesthat Fc glycan-mediated binding is the main binding mode responsible for emergence of theHI, NI, and binding activity of F11 IgA1 against H3N2 viruses. NT activity was not alteredmarkedly by IgA1 polymerization, indicating that not only Fc glycan-mediated binding but
also Fab paratope-mediated binding of F11 IgA1 plays a role in neutralizing the H3N2 virus(Table 1). By contrast, the anti-viral activity of F11 IgA2m2 against H3N2 viruses clearly
increased upon IgA polymerization (Table 1), suggesting that, in the case of IgA2m2, themode of binding responsible for emergence of anti-viral functions against H3N2 viruses is Fabparatope-mediated binding.

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This difference in usage of the two types of binding module between IgA1 and IgA2m2
antibodies is probably due to structural differences. IgA antibodies are abundant in mucosal
excretions. Supposedly for this reason, IgA2 antibodies lack the 13-amino acid region recognized by the IgA1 proteases used by some bacterial species to evade immune responses [38].
Therefore, IgA2m2 antibodies possess shorter “arms” than IgA1 antibodies, resulting in
greater accessibility to the HA stalk region, which in turn may make Fab paratope-mediatedbinding more favorable than is the case for IgA1. This is consistent with results reported by astudy of anti-HA head antibodies in which a tetrameric IgA2m2 antibody showed markedly
higher reactivity against HA proteins from all viruses than did monomeric IgA2m2 or IgA1
antibodies [11]. The observation that the positive effect of IgA polymerization on antibody
reactivity is more evident for IgA2m2 antibodies supports the idea that, at least for anti-stalk
IgA2m2 antibodies, the main binding module is the paratope rather than IgA Fc glycans. To
summarize, in situations in which Fab paratope-mediated binding to the HA stalk exceeds IgA
Fc glycan-mediated binding to HA RBS, IgA polymerization increases anti-viral functions. By
contrast, when IgA Fc glycan-mediated binding to HA RBS is predominant, anti-viral activity
will fall upon IgA polymerization. The coordination between these two independent binding
modules (Fab paratope- and IgA glycan-mediated binding) will influence whether IgA polymerization has a negative or positive impact on the anti-viral functions of IgA molecules.
Our first in vivo passive transfer experiment in which mice received recombinant IgG andIgA antibodies revealed that clone F11 exhibits in vivo anti-viral activity in the IgG state, but
not in the IgA state, possibly due to the fact that anti-HA stalk antibodies exert anti-viral effects
via Fc receptor-mediated functions [35] and that, unlike Fc gamma receptors, mice do not
express Fc alpha receptors [36]. The observation that FI6 IgG1 N297A did not protect micefrom lethal virus challenge in the second in vivo passive transfer experiment further supports
this idea (Fig 4G). In the absence of Fc-dependent functions, neither IgG nor IgA anti-HA
stalk antibodies protected mice from lethal virus challenge, despite the finding that anti-HA
IgA antibodies show higher anti-viral activity in vivo than IgG antibodies (Fig 4B), and despite
the significant decrease in virus titer in lung wash from mice receiving FI6 IgA1 comparedwith the lung wash from the group receiving PBS (Fig 4C).
In conclusion, we show here that anti-HA stalk-binding IgA antibodies exert virus-neutralizing activity via two distinct modes: Fab paratope binding to the HA stalk and Fc glycan binding to the HA RBS. We also show that IgA polymerization affects the mode of antibodymediated neutralization depending upon the affinity of the HA stalk-binding antibody. Further studies utilizing more sophisticated methods (i.e., Fc alpha receptor transgenic mice) areneeded to further explore the mechanisms underlying the in vivo anti-viral activity of IgA, andthe impact of polymerization on its function.

Materials and methods
Ethics statement
This non-interventional study was performed using residual peripheral blood mononuclear
cells collected from nine vaccinated healthy adult volunteers participating in previous interventional clinical trials for an intranasal inactivated influenza vaccine (Clinical trial registry:
UMIN000008279 [39]). The study was conducted with the approval of the medical research
ethics committee of the National Institute of Infectious Diseases for the use of human subjects,
Tokyo, Japan (no. 920), and all participants provided written informed consent via an ethics
committee-approved form. The primary outcome of the interventional clinical trial is reported
separately [39]. Participants in the interventional clinical trial received three intranasal vaccinations with a monovalent WIV vaccine (45 μg HA/500 μl). Vaccines were prepared from a

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reassortant vaccine candidate, IBCDC-RG2, which is derived from A/Indonesia/5/2005(H5N1), as previously described [40]. All vaccines (lot #FPBMW1005-D) were manufactured
according to GMP guidelines and were released appropriately by the Research Foundation forMicrobial Disease of Osaka University (BIKEN, Kanonji, Kagawa, Japan). Six out of nine volunteers received vaccines containing carboxy-vinyl polymer (which was added as an excipient
to increase viscosity). Intranasal administration was performed by spraying 0.25 ml of vaccine
into each nostril (0.5 ml total) using an atomizer (Keytron Inc., Ichikawa, Chiba, Japan). For
mice experiments, 7-week-old female BALB/c mice were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan). Animal studies were performed in strict accordance with the Guidelines for Proper Conduct of Animal Experiments of the Scientific Council of Japan. All animal
experiments were conducted in strict compliance with animal husbandry and welfare regulations in handled in biosafety level two animal facilities according to the guidelines of the Animal Care and Use Committee of the National Institute of Infectious Diseases, and wereapproved by this Committee (approval no. 118088). Mice were monitored daily for clinicalsigns of morbidity and mortality up to 14 days post infection. The human endpoint was used
for mice that lost 30% or more of their initial body weight during the study. When the animals
met the criteria, they were scored dead and euthanized under excess isoflurane anesthesia
according to institutional guidelines.

Viruses and cells
Madin-Darby canine kidney (MDCK) cells (American Type Culture Collection; CCL-34) weremaintained at 37˚C/5% CO2 in minimum essential medium (MEM; Life Technologies, Carlsbad, California, U.S.A.) containing 10% fetal bovine serum (Thermo Fisher Scientific, Grand
Island, New York, U.S.A.) and pen-strep mix (100 units/ml penicillin and 100 μg/ml streptomycin; Life Technologies). Expi293F cells (Thermo Fisher Scientific) were maintained at
37˚C/8% CO2 in Erlenmeyer cell culture flasks (Corning, Tewksbury, Massachusetts, U.S.A.)
containing Expi293 Expression Medium (Thermo Fisher Scientific). A/California/7/2009(H1N1)pdm09 (X-179A), A/Puerto Rico/8/1934 (H1N1), A/New Caledonia/20/1999 (H1N1),
A/New York/55/2004 (H3N2), A/Victoria/210/2009 (H3N2), and mouse-adapted A/Narita/1/
2009 (H1N1)pdm09 viruses were grown in 10 or 11-day-old embryonated chicken eggs. A/
Narita/1/2009 (H1N1)pdm09 virus and A/Narita/1/2009 (H1N1)pdm09-derived F11 escapemutants were propagated in MDCK cells.

Analysis of WIV intranasal vaccine-induced antibody clones
Whole blood was collected from volunteers 1 week after the second vaccination, and peripheral blood mononuclear cells were isolated using Lymphoprep™ (Abbott Diagnostics Technologies AS, Oslo, Norway). Plasmablasts from nine donors were single cell sorted (gated onmarkers CD2-, CD3-, CD4-, IgD-, CD38+, CD19+, and CD27+) and antibody genes fromeach plasmablast were cloned. The variable heavy (H) and light (L) chain regions of each clone
were sequenced as described previously [41]. H and L chain sequences were subjected to analysis using IgBLAST and aligned with H and L chain variable regions of germline genes. The variable region germline genes of origin (V, D, and J for the H chain; V and J for the L chain), thelength of the three CDRs, and degree of homology with the germline gene of each antibody
clone were analyzed.

Expression and purification of IgG and IgA antibodies
Plasmids encoding the α1H, α2H, human JC, and human SC were prepared as previously
described [11]. To generate Fab-deficient (ΔFab) IgA1 and IgA2m2 antibodies, plasmids

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encoding α1H and α2H, comprising regions from CH2 to the C-terminus, were used [42].
Human SC lacking a thrombin recognition site and harboring a hexa-histidine affinity tag(His tag) at the C-terminus was synthesized and cloned into the pCXSN vector. DNA fragments encoding the variable region of the H or L chain of each clone were amplified by PCR
using cDNA obtained from single-sorted plasmablasts from vaccinated healthy adult volunteers. DNA fragments encoding the variable region of the H or L chain of antibody clone FI6
were Homo sapiens codon-optimized and synthesized by the GeneArt Strings DNA Fragments
service. Synthesized DNA fragments were cloned into α1H, α 2H, γ1HC, or κLC vectors. For
expression of the IgG1 N297A mutant, the N297A mutation was introduced into the FI6
γ1HC vector by standard site directed mutagenesis and inverse PCR. To generate IgG1,
Expi293F cells grown in Expi293 Expression Medium were diluted to 2.5 × 106 cells/ml andtransfected with 50 μg of γ1HC and 50 μg of LC per 100 ml of final culture volume using the
ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). To generate monomeric IgA,
Expi293F cells were transfected with 50 μg of αH and 50 μg of LC per 100 ml of final culture
volume. To generate polymeric SIgA, Expi293F cells were transfected with 80 μg of αH, 80 μgof LC, 40 μg of JC, and 40 μg of SC per 200 ml of final culture volume. At 7 days post-transfection, cell culture supernatants were centrifuged at 1200 × g and filtered to remove cell debris.
The supernatants were then purified using CaptureSelect IgG-Fc (Hu) (Thermo Fisher Scientific) or CaptureSelect IgA (Thermo Fisher Scientific), according to the manufacturer’s
instructions. Polymeric SIgA used for ELISA, NT, and HI assays was prepared by expressing
SIgA plus the SC with a His tag, followed by purification with CaptureSelect and separation ofpolymeric IgA from monomeric IgA by His tag affinity chromatography using a HisTrap excel
column (GE Healthcare Bio-Sciences, Uppsala, Sweden). Polymeric SIgA used for the SPR
assay was prepared by expressing IgA with the SC and His tag, followed by purification with
CaptureSelect and separation by gel filtration chromatography on a Superose6 Increase 10/300
GL column (GE Healthcare Bio-Sciences). Purified antibodies were concentrated using Amicon Ultracell (Merck, Darmstadt, Germany) centrifugation units with a cut-off of 30 kDa; thebuffer was changed to 20 mM phosphate buffer (pH 7.4) using a Zeba Spin Desalting Column(Thermo Fisher Scientific).

Assessment of monomeric and polymeric IgA antibody quality by
SDS-PAGE and HPLC analyses
To confirm successful production and purification of monomeric and polymeric IgA antibodies, purified antibodies were analyzed by SDS-PAGE on Bolt 4–12% Bis-Tris gels (Thermo
Fisher Scientific). Precision Plus Protein All Blue standards (Bio-Rad Laboratories, Inc.) wereused as molecular weight markers. The SDS-PAGE gels were stained with SimplyBlue SafeStain (Thermo Fisher Scientific). Next, IgA antibodies were analyzed using the Agilent HPLC
system equipped with a protein exclusion HPLC column (KW404-4F; 4.6 mm I.D. × 300 mm
L; SHODEX, Japan). Samples (2 μg) were injected onto the column and separated in PBS at aflow rate of 0.2 mL/min for 34 min at 22 ˚C. Detection was conducted by UV at 280 nm (reference wavelength, 360 nm). Although B11, F11, and ΔFab IgA antibodies could be analyzed by
HPLC, FI6 IgA samples, which tended to adhere to HPLC columns, could not. Thus, FI6 IgA
samples were analyzed by sedimentation velocity analytical ultracentrifugation SV-AUC.

SV-AUCSV-AUC experiments were conducted using five samples each of FI6 IgG1, IgA1 monomer/
polymer, and IgA2m2 monomer/polymer at 0.1 mg/mL and 1 mg/mL in 20 mM phosphatebuffer with 150 mM NaCl (pH 7.4). Briefly, 390 μL of each sample was loaded into the sample

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sector of a cell equipped with sapphire windows and a 12 mm double-sector charcoal-filled
epon centerpiece. Next, 400 μL of 20 mM phosphate buffer containing 150 mM NaCl was
loaded into the reference sector of each cell. Data collection was performed at 20˚C/35,000rpm using the Optima AUC (Beckman Coulter) with a UV detection system. Data were collected every 120 s at 230 nm for each 0.1 mg/mL sample and at 290 nm for each 1 mg/mL sample, with a radial increment of 10 μm. Collected data were analyzed by the program SEDFIT(version 16.2b) [43], using continuous c(s) distribution fitting for the frictional ratio, meniscus,time-invariant noise, and radial-invariant noise, and a regularization level of 0.68. Sedimentation coefficient ranges of 0–30 were evaluated at a resolution of 300. The partial specific volume of samples was used as 0.73 cm3/g. The program SEDNTERP 1.09 [44] calculated thebuffer density and viscosity as 1.0066 g/cm3 and 1.023 cP (Centipoise), respectively. Images ofc(s) distribution were generated using the program GUSSI [45].

Expression and purification of recombinant trimeric HA proteins
The mammalian cell-derived HA proteins used in this study were as follows: A/California/7/
2009 (H1N1)pdm09; A/Narita/1/2009 (H1N1)pdm09 (including wild-type virus, F11 escapemutant C1 virus, and F11 escape mutant G6 virus); A/Puerto Rico/8/1934 (H1N1); A/New
Caledonia/20/1999 (H1N1); A/New York/55/2004 (H3N2); A/Victoria/210/2009 (H3N2); andA/Indonesia/5/2005 (H5N1). These HA proteins comprise the extracellular domain of HA,
which is C-terminally fused to the thrombin site, the trimeric Foldon of T4 fibritin, and a His
tag or a Strep-tag II plus a His tag. The trimeric HA stalk of virus strain A/Brisbane/59/2007(H1N1) was expressed as described previously [19]. HA proteins were expressed using theExpi293 Expression System (Thermo Fisher Scientific), according to the manufacturer’s
instructions. At 7 days post-transfection, the medium was clarified by centrifugation at
1200 × g, filtered, and purified on a HisTrap excel column (GE Healthcare Bio-Sciences). The
purified HA proteins were concentrated using Amicon Ultracell (Merck) centrifugation units
with a cut-off of 30 kDa. The buffer was then changed to PBS (pH 7.4) using a Zeba Spin
Desalting Column (Thermo Fisher Scientific). The HA proteins were stored at -80˚C until use.

ELISA screening to identify anti-HA stalk-binding antibody clones
Half-area flat-bottomed microtiter plates (Corning) were coated overnight at 4˚C with recombinant trimeric HA proteins (50 ng/well) derived from virus strains A/California/7/2009(H1N1)pdm09, A/Narita/1/2009 (H1N1)pdm09, A/Victoria/210/2009 (H3N2), A/Indonesia/
5/2005 (H5N1), and A/Brisbane/59/2007 (H1N1)-stalk. After blocking for 1 h at 37˚C with 1%
BSA-PBS (pH 7.4), each monoclonal IgG1 antibody (250 ng/well) was added. Plates were incubated for 2 h at 37˚C and washed three times with 0.05% PBS-Tween 20. Next, an HRP-conjugated goat anti-human IgG-Fc antibody (Bethyl Laboratories, Montgomery, Texas, U.S.A.)
was diluted (1/1000) and added to each well. Plates were incubated for 1 h at 37˚C, washedthree times, and incubated with One-Step Ultra TMB ELISA HRP substrate solution (Thermo
Fisher Scientific). The HRP reaction was stopped by addition of H2SO4, and optical density at
450 nm (OD450, reference: 655 nm) was measured in an iMark™ Microplate Reader (Bio-Rad,
Hercules, California, U.S.A.).

Preparation of F11 IgG1 fabs
F11 IgG1 antibodies were digested with immobilized papain slurry (Pierce Biotechnology,
Rockford, Illinois, U.S.A.), according to the manufacturer’s instructions. Next, Fabs were purified by applying the digested antibodies to a CaptureSelect IgG-Fc (Hu) column (Thermo
Fisher Scientific) and collecting the flow-through. Fabs were concentrated using Amicon

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Ultracell (Merck, Darmstadt, Germany) centrifugation units with a cut-off of 30 kDa. The
buffer was changed to 20 mM phosphate buffer (pH 7.4) using a Zeba Spin Desalting Column(Thermo Fisher Scientific). Successful digestion and purification of Fabs were confirmed by
SDS-PAGE.

Plaque assay
A plaque assay was performed to compare the escape rate of the two mutant viruses from F11
IgG1. Briefly, 500 PFU (plaque-forming units) of wild-type, mutant C1, or mutant G6 virus
were mixed with 25 μg of F11 IgG1 (final volume, 100 μl) and incubated at 37˚C for 30 min.
Following incubation, 100 μl of 1 × MEM was added to the virus/antibody mixture, which wasthen added to the wells of a 6-well plate containing a monolayer of MDCK cells. After incubation for 1 h at 37˚C/5% CO2, each well was washed twice with PBS and overlaid with 2 ml ofagar medium containing acetylated trypsin from bovine pancreas (SIGMA-ALDRICH, SaintLouis, Missouri, U.S.A.) at a final concentration of 10 μg/ml. Plates were stained with crystal
violet following 2 days of incubation at 37˚C/5% CO2.

Generation of the F11 escape mutant virus
First, 1.0 × 106 TCID50 (50% tissue culture infectious dose) of A/Narita/1/2009 (H1N1)pdm09
virus was mixed with 690 μg or 345 μg of F11 IgG1 (total volume, 1200 μl) and incubated at
37˚C for 30 min. Next, trypsin acetylated from bovine pancreas (SIGMA-ALDRICH) was
added (final concentration, 10 μg/ml), and the virus/antibody mixture (serially diluted 2-fold)
was added to the wells of a 96-well plate containing a monolayer of MDCK cells. The plates
were incubated at 37˚C/5% CO2 for 6 or 7 days. Next, the culture medium was collected fromwells showing cytopathic effects. For plaque purification of escape mutant virus, cell culture
medium containing 100 TCID50 of virus was mixed with 5 μg of F11 IgG1 (final volume,
100 μl) and incubated at 37˚C for 30 min. Following incubation, 100 μl of 1 × MEM was added
to the virus/antibody mixture, which was then applied to the wells of a 6-well plate containing
a monolayer of MDCK cells. After incubation for 1 h at 37˚C/5% CO2, each well was washedtwice with PBS and overlaid with 2 ml of agar medium containing trypsin acetylated frombovine pancreas (SIGMA-ALDRICH) (final concentration, 10 μg/ml). After incubation for 3
days under 37˚C/5% CO2, mutant viruses were isolated from plaques formed in the wells.

Deep sequencing of the F11 escape mutant virus
Deep sequencing was performed using MiSeq (Illumina, San Diego, California, U.S.A.)),essentially as described previously [46]. Briefly, viral RNA was extracted from virus isolates
using the QIAamp Viral RNA Mini Kit (Qiagen, Venlo, Netherlands). A cDNA library was
prepared from viral RNA using a NEBNext Ultra RNA Library Prep Kit for Illumina and an
NEBNext Singleplex Oligos for Illumina (New England Biolabs, Ipswich, Massachusetts, U.S.
A.), followed by purification using Agencourt AMPure XP (Beckman Coulter, Brea, California,
U.S.A.), according to the manufacturer’s instructions. The library was sequenced with MiSeq
and MiSeq Reagent Kit v2. CLC Genomics Workbench 8 (CLC bio, Aarhus, Denmark) wasused to align sequence reads to the reference sequence of A/Narita/1/2009 (H1N1)pdm09.

ELISA to assess binding of F11 IgG1 and escape mutant HA
Half-area flat-bottomed microtiter plates (Corning) were coated (250 ng/well) overnight at
4˚C with recombinant HA proteins from A/Narita/1/2009 (H1N1)pdm09 wild-type virus, andF11 escape mutant C1 (harboring a T333K substitution) and G6 (harboring a G480D

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substitution) viruses. Plates were blocked for 1 h at 37˚C with 1% BSA-PBS (pH 7.4), and F11
IgG1 samples (serially diluted 3-fold) were added to each well. Following incubation for 2 h at
37˚C, wells were washed three times with 0.05% PBS-Tween 20. After addition of an HRP-conjugated goat anti-human IgG-Fc secondary antibody (Bethyl Laboratories), plates were incubated for a further 1 h at 37˚C, washed three times, and incubated with One-Step Ultra TMB
ELISA HRP substrate solution (Thermo Fisher Scientific). The reaction was stopped by addition of H2SO4, and optical density at 450 nm (OD450, reference: 655 nm) measured in aniMark™ Microplate Reader (Bio-Rad). Binding curves and areas under the curves (AUCs) wereconstructed using GraphPad Prism software (GraphPad Software, San Diego, California, U.S.
A.).

Molecular modeling of influenza virus HA trimer ectodomains
Three-dimensional models for the extracellular domains of HA trimers of A/Narita/1/2009(H1N1) virus and two F11 escape variants harboring a T333K substitution (C1 virus) or a
G480D substitution (G6 virus) in the ligand-free state were constructed using the homology
modeling method and Molecular Operating Environment (MOE) (Chemical Computing
Group Inc., Montreal, Quebec, Canada). The crystal structure of a HA trimer of A/California/
04/2009 (H1N1) virus at a resolution of 2.6 Å (PDB code: 3LZG) was used as the modelingtemplate. Obtained models were optimized by energy minimization using MOE. Next, an
Amber10:Extended Huckel Theory (EHT) force field was implemented in MOE, which combines Amber10 and EHT bonded parameters for energy minimization.

MD simulation of HA trimer ectodomains
HA trimer models in a ligand-free state were subjected to MD simulation, as described for HA
trimers of influenza virus [47], the envelope protein of lentiviruses [48–50], and the proteaseof feline calicivirus [51]. The simulations were performed using the pmemd.cuda.MPI modulein the Amber 16 program package [52], with the ff14SB force field [53] and the TIP3P watermodel for simulation of aqueous solutions [54]. A non-bonded cut-off of 10 Å was used. The
lengths of bonds involving hydrogen were constrained with SHAKE, a constraint algorithm
that satisfies Newtonian motion [55]. The time for all MD simulations was set to 2 fs. After
heating calculations were performed for 20 picoseconds up to 310 K using the NVT ensemble,
simulations were executed for 100 ns in 150 mM NaCl using the NPT ensemble (at 1 atm, 310
K).

Calculation of RMSF
RMSF was calculated as previously described [47,49,50] to quantify the structural dynamics ofmolecules in the MD simulations. The RMSF of Cα atoms was calculated to obtain information about atomic fluctuations of individual amino acid residues during the MD simulations.
The 15,000 snapshots, obtained from MD simulations at 70–100 ns, were used to calculate the
RMSF. The average structure obtained during 70–100 ns of MD simulation was used as the reference structure for RMSF calculation. RMSF, which quantifies differences between averagevalues and those obtained at a given time of MD simulation, was calculated using the ptraj
module in Amber, a trajectory analysis tool.

IgA binding assay
Half-area flat-bottomed microtiter plates (Corning) were coated (50 ng/well) overnight at 4˚Cwith recombinant HA proteins derived from virus strains A/California/7/2009 (H1N1)pdm09,

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A/Narita/1/2009 (H1N1)pdm09, A/Puerto Rico/8/1934 (H1N1), and A/New Caledonia/20/
1999 (H1N1). Plates were blocked for 1 h at 37˚C with 1% BSA-PBS (pH 7.4). Next, IgA samples (serially diluted 3-fold) were added to each well. Following incubation for 2 h at 37˚C,wells were washed three times with 0.05% PBS-Tween 20. After addition of an HRP-conjugated goat anti-human IgA antibody (Bethyl Laboratories) and an HRP-conjugated mouseanti-human IgA2 antibody (Abcam, Cambridge, U.K.) to detect IgA1 and IgA2m2, respectively, the plates were incubated for 1 h at 37˚C, washed three times, and incubated with OneStep Ultra TMB ELISA HRP substrate solution (Thermo Fisher Scientific). The reaction was
stopped by addition of H2SO4. Optical density at 450 nm (OD450, reference: 655 nm) was
measured in an iMark™ Microplate Reader (Bio-Rad). Binding curves and AUCs were constructed using GraphPad Prism software (GraphPad Software).

HI assay
HI titers were examined using an influenza virus microtitration method, as previously
described, with minor modifications [56,57]. Briefly, purified antibodies (serially diluted
2-fold) were mixed with an equal volume of influenza virus diluted to 4 HA units, and thenincubated for 10 min at room temperature. Next, 0.5% turkey red blood cells were added tothe mixture and activity was measured after incubation for 45 min at room temperature. HI
activity was defined as the reciprocal of the lowest concentration (μg/ml) of antibody thatcompletely inhibited virus-mediated hemagglutination of red blood cells.

Microneutralization (NT) assay
The microneutralization assay was performed using MDCK cells and 100 TCID50 of influenza
virus, essentially as previously described [58]. Briefly, each sample (serially diluted 2-fold) was
mixed with an equal volume of diluent containing influenza virus (equivalent to 100 TCID50)
and incubated for 30 min at 37˚C. This mixture was added to a monolayer of MDCK cells in
the wells of a 96-well plate. Four control wells containing virus or diluent alone were includedon each plate. The plates were incubated for 5 days at 37˚C/5% CO2. All wells were observed
for the presence or absence of cytopathic effects, fixed with 10% formalin phosphate buffer for
more than 5 min at room temperature, and then stained with Naphthol blue black. After washing and drying, cells were solubilized with 0.1 M NaOH and absorbance (A) was read at 630
nm. The average A630 value was determined from virus-only controls (Avirus) and mediumonly controls (Acell). Values >50% of the specific signal, calculated using the formula X =(Acell − Avirus)/2, were considered positive for neutralization. NT activity was defined as thereciprocal of the lowest concentration (μg/ml) of antibody at which A630 was >X.

SPR assay of F11 Fab, and of wild-type and escape mutant virus HAproteins
The SPR assay was performed using a Biacore X100 (GE Healthcare). Recombinant trimeric
HA from either A/Narita/1/2009 (H1N1)pdm09 wild-type virus, F11 escape mutant C1(T333K), or G6 (G480D) virus with a C-terminal His tag was immobilized on the surface of
Sensor Chip NTA (GE Healthcare) using the NTA reagent kit (GE Healthcare), according to
the manufacturer’s instructions. Following immobilization of trimeric HA (1 μg/ml for 180
seconds), the molecular interaction with F11 Fab (2-fold serial dilutions ranging from 0.5 nM
to 8 nM for wild-type virus HA and from 4 nM to 64 nM for mutant virus HA) was analyzed
with a contact time of 180 s and a dissociation time of 1800 s. Sensorgrams were x- and y-axis
adjusted (0 = baseline) to compare the degree of F11 Fab association and dissociation from
HA.

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Assessment of RBS blockade using SPR
Recombinant trimeric HA from virus strain A/New Caledonia/20/1999 (H1N1) harboring a
C-terminal His tag was immobilized on the surface of Sensor Chip NTA (GE Healthcare)using the NTA reagent kit (GE Healthcare). Following immobilization of trimeric HA (1 μg/
ml for 180 s), serially diluted F11 (IgG1, IgA2m2 monomer, or IgA2m2 polymer; diluted
3-fold from 0.6 μg/ml to 48 μg/ml) was added for a contact time of 180 s and a dissociation
time of 180 s. After binding of the F11 antibody to HA, F045-092 IgG1 was added at a concentration of 100 μg/ml. The contact time of F045-092 IgG1 was 180 s and the relative response(RU) at report point:binding stability (the time point immediately after the end of the F045092 IgG1 contact time) was measured as the F045-092 IgG1 binding level. The maximum stability value was acquired by measuring the RU following F045-092 IgG1 binding to HA in theabsence of F11 pre-binding. The binding ratio (%) of F045-092 IgG1 to F11 IgG1- or
IgA2m2-pre-bound HA was calculated by dividing each F045-092 IgG1 binding value by the
maximum stability value. The AUCs of the binding ratio curves were calculated by GraphPad
Prism software (GraphPad Software).

Neuraminidase inhibition NI-ELLA
NI-ELLA was performed as described previously [26,59,60], with minor changes. Briefly,
96-well clear flat bottom polystyrene high binding microplates (Corning) were coated for atleast 24 h at 4˚C with fetuin (SIGMA-ALDRICH; 25 μg/ml per 100 μl/well) in carbonate-bicarbonate buffer (pH9.6; Medicago, Uppsala, Sweden). Plates were washed three times with
PBST, followed by addition of antibodies (serially diluted 2-fold) and viruses diluted in PBST/
BSA, and then incubated for 18 h at 37˚C. An equal amount of Zanamivir, an NA inhibitor,
was used as a positive control. Following incubation, plates were washed six times with PBST.
Next, lectin from Arachis hypogaea (peanut) peroxidase conjugate (SIGMA-ALDRICH; 2 μg/
ml; 100 μl/well) diluted in PBST/BSA was added to the plates. Plates were incubated in thedark at room temperature for 2 h, washed three times, and incubated in the dark at room temperature for a further 10 min with One-Step Ultra TMB ELISA HRP substrate solution(Thermo Fisher Scientific). The reaction was stopped by addition of H2SO4. Optical density at
450 nm (OD450, reference: 655 nm) was measured in an iMark™ Microplate Reader (BioRad). Binding curves were constructed using GraphPad Prism software (GraphPad Software).

Deglycosylation of IgA antibodies
IgA antibodies were deglycosylated using Protein Deglycosylation Mix 2 (New England Biolabs), according to the non-denaturing reaction protocol distributed by the manufacturer.
Briefly, 100 μg of IgA antibody, 5 μl of 10× Deglycosylation Mix Buffer, and 5 μl of Protein
Deglycosylation Mix 2 were mixed to a total volume of 50 μl and incubated at 25˚C for 30 min,followed by an overnight incubation at 37˚C. Following deglycosylation, buffers were switched
to 20 mM phosphate buffer (PB) (pH 7.4) using a Zeba Spin Desalting Column (Thermo
Fisher Scientific). Purified antibodies were analyzed by SDS-PAGE on Bolt 4–12% Bis-Tris
gels (Thermo Fisher Scientific). Precision Plus Protein All Blue standards (Bio-Rad Laboratories, Inc.) were used as molecular weight markers for SDS-PAGE. The SDS-PAGE gels were
stained with SimplyBlue SafeStain (Thermo Fisher Scientific). Deglycosylation of samples was
confirmed in an enzyme-linked lectin assay (ELLA). Briefly, 96-well clear flat bottom polystyrene high binding microplates (Corning) were coated for at least 24 h at 4˚C with antibody
samples (2 μg/ml; 100 μl/well) or fetuin (SIGMA-ALDRICH) in carbonate-bicarbonate buffer(pH9.6; Medicago). Plates were washed six times with PBST, and lectin from Arachis hypogaea(peanut) peroxidase conjugate (SIGMA-ALDRICH; 2 μg/ml; 100 μl/well), serially diluted

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2-fold in PBST/BSA, was added to the plates. Plates were incubated in the dark at room temperature for 2 h, washed three times, and incubated in the dark at room temperature for a further 10 min with One-Step Ultra TMB ELISA HRP substrate solution (Thermo Fisher
Scientific). The reaction was stopped by addition of H2SO4. Optical density at 450 nm(OD450, reference: 655 nm) was measured in an iMark™ Microplate Reader (Bio-Rad). Binding curves were constructed and the AUCs of the binding curves were calculated using GraphPad Prism software (GraphPad Software). Deglycosylated IgA was subjected to ELISA and HI
assay, as described above.

Glycan analysis
Human nasal IgA and recombinant IgG and IgA antibodies were subjected to glycan analysis.
Human nasal IgA was purified from nasal wash concentrates using CaptureSelect IgA (Thermo
Fisher Scientific), according to the manufacturer’s instructions. Purified antibodies were concentrated using Amicon Ultracell (Merck, Darmstadt, Germany) centrifugation units with a cut-off
of 30 kDa. The human nasal wash concentrate samples used here were residual samples collectedfrom a past clinical study [60]. Antibodies were deglycosylated by PNGase F (Takara Bio, Shiga,
Japan) treatment. The released N-glycans were purified and labeled with 2-aminobenzamideusing BlotGlyco (Sumitomo Bakelite, Tokyo, Japan), according to the manufacturer’s instructions.
Liquid chromatography-mass spectrometry (LC-MS) analysis was performed by Sumitomo Bakelite using a LC-MS-IT-TOF apparatus (Shimadzu, Kyoto, Japan). N-glycan structures were predicted using the GlycoMod Tool (http://web.expasy.org/glycomod/) [61].

HPLC coupled with LC-MS
The 2-AB-labeled glycan mixture was analyzed using a Shimadzu LCMS-IT-TOF MS systemequipped with an RF-20A XS fluorescence detector (Shimadzu Corp.). Separation of 2-AB glycans by normal-phase HPLC was performed on an ACQUITY UPLC1 BEH Glycan 1.7 μm
column (2.1 mm I.D. × 150 mm L; Waters, MA, USA) with fluorescent detection at excitationand emission wavelengths of 330 and 420 nm, respectively. Samples (1 μL) were injected onto
the column, which had been equilibrated with 100% solvent B (9.9% water in acetonitrile containing 0.1% formic acid). The 2-AB glycans were separated for 50 min at 40˚C on a linear gradient of 0–100% solvent A (40% acetonitrile in water containing 0.1% formic acid) at a flowrate of 0.2 mL/min. The detailed settings of the MS device were as follows: detector voltage, 2.1
kV; CDL, 200˚C; heat block, 200˚C; Nebulizer gas, 1.5 L/min; negative ion mode auto scan, m/
z 250–2000; MS2 CID energy, 50%. According to a previous report [34] and test runs, peaks
observed at later retention time points were more likely to contain terminally sialylated complex glycans; thus peaks observed at later time points were selected to estimate glycan
composition.

In vivo passive transfer of recombinant IgG and IgA antibodies to mice
Mouse-adapted influenza virus A/ Narita/1/2009, which was obtained after 15 passages in
mice, was used as a representative A(H1N1)pdm09 virus (mNRT) for the in vivo experiment[62]. Six-week-old female BALB/c mice (n = 6 per experimental condition) were injected intraperitoneally with IgG1 or IgA1 monomers, or with polymers of F11 or FI6 antibodies (200 μg/
200 μl per mouse), or PBS (200 μl per mouse) 24 hours prior to virus challenge. Then, the mice
were received 4 × 103 PFU of mNRT virus intranasally via the left nostril (20 μl per mouse) to
infect the lower respiratory tract. At 3 days post-infection, serum and lung wash samples werecollected. Measurement of virus titers in mice lung wash samples was performed in a plaqueassay, as described by Tobita et al. [63]. In brief, serial (10-fold) dilutions of each sample were

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prepared and 200 ul aliquots of each dilution were inoculated onto MDCK cells in a 6-wellplate. After incubation for 1 hour to allow for sample absorption, each well was overlaid with 2
ml of agar medium. Plates were incubated for 2 days at 37˚C/5% CO2 and stained with crystal
violet for plaque counting. The concentrations of passively transferred IgG and IgA antibodies
in serum and lung wash samples were measured using a human IgG quantitation kit (Bethyl
Laboratories) and a human IgA quantitation kit (Bethyl Laboratories), respectively. For longterm observations following lethal challenge with virus (undertaken to evaluate the ability ofantibodies to protect against morbidity and mortality), 6-week-old female BALB/c mice (fiveor six per experimental condition) were injected intraperitoneally with IgG1 harboring the
N297A mutation, IgA1 or IgA2m2 monomers, polymers of FI6 antibodies (200 μg/200 per
mouse), or PBS (200 μl per mouse) 2 hours prior to challenge with 4 × 103 PFU of mNRT virus(administered intranasally via the left nostril (20 μl per mouse) to infect the respiratory tract.
Body weight was measured daily. Mice that lost >30% of their original body weight wereeuthanized.

Statistical analysis
All statistical analyses were performed using the Prism statistical software package (version
7.0; GraphPad Software). An unpaired Student t-test, the Mann–Whitney test, and one-way
ANOVA followed by the Kruskal–Wallis test or Welch’s test, or two-way ANOVA were used
to analyze each dataset, as indicated in the figure legends. The threshold for statistical significance was set at 5% (p < 0.05).

Supporting information
S1 Checklist. The ARRIVE guidelines 2.0: Author checklist.
(PDF)
S1 Table. Genetic characteristics of antibody clones derived from intranasally-vaccinatedhumans. Antibodies induced by intranasal inoculation with whole inactivated influenza virus(WIV) vaccine were screened for anti-HA stalk-binding clones by examining genetic characteristics, HA binding characteristics, and virus-neutralizing characteristics. A single cell sorting protocol isolated 720 plasmablasts from nine volunteers, and 452 antibody sequences wereobtained. Clones with certain genetic characteristics observed in the variable heavy (H) chainsof previously reported influenza virus bnAbs were selected: eight clones were derived from theIGHV1-69 gene, 6 clones from the IGHV1-2 gene, and 11 clones possessed long complementarity determining region (CDR)3.
(DOCX)S2 Table. Virus-neutralizing activity of antibody clones (derived from intranasally-vaccinated humans) against strains A/California/7/2009 (H1N1). Recombinant IgG1 antibodies
from 25 antibody clones were expressed in mammalian cells and purified. Binding to recombinant HA was tested in virus neutralization (NT) and enzyme-linked immunosorbent assays(ELISAs). None of the clones showed strong binding to HA from A/Victoria/210/2009(H3N2) virus (Vic210). Among the 25 antibody clones, clone F11 (derived from the IGHV169 germline gene) showed strong binding to the HA of group one influenza viruses A/California/7/2009 (H1N1)pdm09 (Cal7) and A/Narita/1/2009 (H1N1)pdm09 (NRT). In addition,clone F11 showed strong binding to the recombinant trimeric HA stalk of virus strain A/Brisbane/59/2007 (H1N1). Other antibody clones showed moderate to low binding to HA. In contrast to the results of the HA binding assays, few antibody clones showed virus-neutralizing(NT) activity. Among all 25 clones, clone F11 showed the greatest NT activity against the Cal7

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virus. Therefore, F11 was selected as a potential HA stalk-binding antibody clone induced byintranasal vaccination with an inactivated influenza vaccine.
(DOCX)S3 Table. Mutant viruses that escaped clone F11 contain mutations in the HA stalk region.
To identify the area of the HA molecule that binds F11, we generated and analyzed escapemutant viruses. Passage of NRT virus in the presence of high concentrations of F11 IgG1
yielded two escape mutants C1 and G6. The C1 virus escaped neutralization by F11 IgG1
completely. By contrast, the G6 virus showed limited infectivity in the presence of F11 IgG1.
Full sequencing of the escape virus genomes revealed that two synonymous and two non-synonymous mutations were introduced into the genome of the C1 virus. The two non-synonymous mutations were located in the nucleoprotein (NP) and HA genes. By contrast, only asingle non-synonymous mutation was identified in the genome of the G6 virus; this was
located in the HA2 region of the HA gene.
(DOCX)
S1 Fig. In vitro characterization of escape mutant viruses of anti-HA stalk antibody candidate clone F11. Two escape mutant viruses of antibody clone F11 were obtained by serial passage of A/Narita/1/2009 (H1N1)pdm09 virus in the presence of high concentrations of F11
IgG1. (A) Escape mutant C1 exhibited higher escape efficiency than escape mutant G6. (B)Recombinant trimeric HA proteins harboring each mutation (T333K (mutant C) or G480D(mutant G)) were tested in a binding assay with F11 IgG1 to examine whether the mutations in
the HA gene of escape mutant viruses affected the affinity of F11 for HA proteins. Binding of
F11 IgG1 to HA harboring the T333K mutation (C1 virus) was notably lower than that to wildtype HA. By contrast, binding of F11 IgG1 to HA harboring the G480D mutation (G6 virus) was
moderate when compared with that to wild-type HA. (C) The area under the reactivity curve(AUC) for F11 IgG1 in response to each recombinant HA (calculated from ELISA results for
F11 IgG1 and recombinant HA). Data are expressed as the mean ± SD of three technical replicates. �� p < 0.01 (Kruskal–Wallis test followed by Dunn’s multiple comparison test). (D) Surface
plasmon resonance (SPR) analysis revealed that F11 had strong affinity for wild-type HA from
A/Narita/1/2009(H1N1)pdm09, with a high association constant (ka [1/Ms] = 2.113 × 106) andan extremely low dissociation constant (kd (1/s) = below the measurement limit). An evident
decrease in association (ka [1/Ms] = 8.541 × 103) and an increase in dissociation (kd (1/s) =1.681 × 10–4) was observed in the presence of the T333K mutation (C1 virus). Although association of the G480D mutated virus (G6) decreased (ka [1/Ms] = 8.631 × 105), dissociation
remained relatively low (kd (1/s) = 1.306 × 10–5) when compared with that for wild-type HA.
(TIF)
S2 Fig. In silico characterization of escape mutant viruses of anti-HA stalk antibody candidate clone F11. To address the structural impact of these escape mutations in the HA stalkregion, we conducted molecular dynamics (MD) simulation of the HA trimer ectodomains
from the NRT, C1, and G6 viruses. (A) Flow chart showing in silico characterization of HA trimer ectodomain structures by MD simulation. (B) Structure analysis of HA from wild-type
NRT (obtained after 100 ns of MD simulation) showed that the T333K substitution occurred
near the hydrophobic groove, a target of cross-clade neutralizing antibodies, whereas the
G480D substitution arose at a site away from the groove. Three-dimensional location of escapemutations T333K and G480D (light green) in the HA trimer model of A/Narita/1/2009/H1N1.
Purple spheres represent amino acid residues comprising hydrophobic grooves on the HA
stalk. The groove of A/Narita/1/2009/H1N1 (GenBank accession number: ACR09396) comprises I389, V392, T393, and V396 in helix A, and W365 in the fusion peptide of the HA

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precursor. (C) Effect of the T333K and G480D mutations on the hydrophobic grooves. Hydrophobic surface patches on the stalk regions of HA trimers after 100 ns of MD simulation wereidentified using the protein patch analyzer in MOE and are shown in dark green. Red sticks
denote side chains on residue 333 (T333, K333, and T333 for A/Narita/1/2009/H1N1, C1, andG6, respectively). Interestingly, the T333K substitution disrupted the physical continuity of thehydrophobic surface patches in the groove, possibly due to introduction of a large positively
charged side chain (left and middle panels). By contrast, G480D mutant HA maintained thecontinuity of the hydrophobic patches (right panel). (D) Effect of the T333K and G480D mutations on the structural dynamics of HA trimers. RMSF values, which represent atomic fluctuations in the main chains of individual amino acids, were calculated using 15,000 snapshotsobtained from MD simulations of 70–100 ns. HG indicates a region between positions 389
and 396, which contains residues I389, V392, T393, and V396 in the hydrophobic groove. The
numbers on the horizontal axes indicate amino acid positions in the HA precursor of A/Narita/1/2009/H1N1. Notably, both the T333K and G480D substitutions induced marked changes
in fluctuation profiles around the hydrophobic groove. Two independent MD simulations
reproduced these results. Taken together, the results of the experimental and in silico studies
suggest that the hydrophobic groove of the HA stalk plays a key role in interaction with clone
F11. This is consistent with previous results showing that the hydrophobic groove plays a critical role in recognition of broadly neutralizing antibodies targeting the HA stalk. The data suggest that the human antibody clone F11 is an anti-HA stalk antibody with broadly neutralizing
activity.
(TIF)
S3 Fig. Structural confirmation of recombinant IgA antibodies. (A–C) Chromatograms
obtained by HPLC analysis of recombinant monomeric and polymeric IgA antibody clones
B12 (A) and F11 (B), and of Fab-deficient (ΔFab) monomeric and polymeric IgA antibodies(C). Polymeric IgA antibody samples peaked at earlier time points than monomeric IgA antibody samples, indicating differences in molecular weight. (D) SDS-PAGE analysis of recombinant monomeric (M) and polymeric (P) F11, and of ΔFab IgA1 (A1) and IgA2m2 (A2m2)
antibodies. All four components of secretory IgA antibodies (heavy chain [HC] (ΔFab HC in
ΔFab IgA samples), light chain [LC], secretory component [SC], and the J chain [JC]) wereobserved. (E–I) Sedimentation velocity analytical ultracentrifugation (SV-AUC) was performed to determine the sedimentation coefficients (s20,w) of FI6 IgG1 (E), IgA1 monomer(F), IgA2 monomer (G), IgA1 polymer (H), and IgA2 polymer (I) in solution. The main component (s20,w = 6.7 S) of IgG1 had a sedimentation coefficient typical of an antibody monomer (E). The s20,w of the main component of the IgA1 and IgA2 monomers was 6.6 S and 7.2
S, respectively, which is similar to that of the IgG1 used as a control for this study (F and G).
For the IgA1 and IgA2 polymers, multiple oligomeric species were observed (H and I). The
profiles of s20,w distribution did not change by diluting the samples from 1 mg/mL (blackline) to 0.1 mg/mL (red line). This indicates that oligomerization of IgA is not an associationdissociation equilibrium, at least within this concentration range.
(TIF)
S4 Fig. LC chromatograms of N-linked glycans from human nasal IgA antibodies andfrom recombinant IgG and IgA antibodies. LC chromatograms of N-linked glycans released
from human nasal IgA antibodies and recombinant (r) IgG/IgA antibodies. According to ourtest runs, peaks at later retention time points were more likely to contain terminally sialylated
complex glycans; thus peaks observed at later time points were selected to estimate glycan
composition. Peak patterns of IgA samples on LC chromatograms were similar, while those of
IgG samples contained fewer peaks at later time points. Human nasal IgA samples generated

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the highest number of peaks at later time points. Peaks numbered 1 to 16 (red arrows) were
estimated to contain terminally sialylated complex glycans. The estimated glycan compositions
for each peak are shown in the left panels.
(TIF)
S1 Raw images. Original images for gels. Top panel: Original image of the gel shown in Fig 2,
panel C. Bottom panel: Original gel shown in S3 Fig, panel D. Images were captured by the
LAS-3000 Imager (FUJIFILM) using a DIA illuminator with standard sensitivity. M: molecularweight marker. Numbers indicate the loading order within the gel. All lanes in the original gel
image are included in the final figure.
(TIF)

Acknowledgments
We thank Mr. T. Tanimoto, Dr. Y. Gomi, Dr. S. Manabe, Mr. T. Ishikawa, and Dr. Y. Okuno
at BIKEN for supplying the inactivated whole virus influenza vaccines; Mr. T. Miyazaki and
Mr. T. Kamishita (Toko Yakuhin Kogyo Co., Ltd.) for valuable suggestions about intranasal
vaccination procedures; Dr. H. Takahashi (Influenza Virus Research Center, National Instituteof Infectious Diseases) for valuable suggestions about escape mutant isolation; and Ms. A.
Sataka (National Institute of Infectious Diseases) for technical support with expression of antibodies and HA proteins. The calculations for the MD simulations were carried out by the
TSUBAME 3.0 supercomputer at the Tokyo Institute of Technology.

Author Contributions
Conceptualization: Kaori Sano, Tadaki Suzuki, Hideki Hasegawa.
Funding acquisition: Kaori Sano, Shinji Saito, Tadaki Suzuki, Akira Ainai, Hideki Hasegawa.
Investigation: Kaori Sano, Shinji Saito, Tadaki Suzuki, Osamu Kotani, Akira Ainai, Elly van
Riet, Koshiro Tabata, Kumpei Saito, Yoshimasa Takahashi, Masaru Yokoyama, Hironori
Sato, Takahiro Maruno, Kaede Usami, Susumu Uchiyama, Kiyoko Ogawa-Goto, Hideki
Hasegawa.
Methodology: Kaori Sano, Shinji Saito, Tadaki Suzuki, Osamu Kotani, Akira Ainai, Elly van
Riet, Koshiro Tabata, Yoshimasa Takahashi, Masaru Yokoyama, Hironori Sato, Takahiro
Maruno, Susumu Uchiyama, Kiyoko Ogawa-Goto.
Project administration: Tadaki Suzuki.
Resources: Kaori Sano, Tadaki Suzuki, Akira Ainai.
Supervision: Tadaki Suzuki, Hideki Hasegawa.
Validation: Tadaki Suzuki, Hideki Hasegawa.
Visualization: Kaori Sano.
Writing – original draft: Kaori Sano.
Writing – review & editing: Tadaki Suzuki.

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