Biochemical and Biophysical Research Communications 554 (2021) 166e172

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Nasalesubcutaneous primeeboost regimen for inactivated whole-

virus influenza vaccine efficiently protects mice against both upper
and lower respiratory tract infections
Meito Shibuya a, b, c, Shigeyuki Tamiya a, b, c, Atsushi Kawai a, b, c, Yasuo Yoshioka a, b, c, d, e, *
a
Laboratory of Nano-design for Innovative Drug Development, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka,
565-0871, Japan
b
Vaccine Creation Group, BIKEN Innovative Vaccine Research Alliance Laboratories, Institute for Open and Transdisciplinary Research Initiatives, Osaka
University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan
c
Vaccine Creation Group, BIKEN Innovative Vaccine Research Alliance Laboratories, Research Institute for Microbial Diseases, Osaka University, 3-1
Yamadaoka, Suita, Osaka, 565-0871, Japan
d
The Research Foundation for Microbial Diseases of Osaka University (BIKEN), 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan
e
Global Center for Medical Engineering and Informatics, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Although influenza vaccines are effective for reducing viral transmission and the severity of clinical
Received 17 February 2021 symptoms, influenza viruses still induce considerable morbidity and mortality worldwide. Seasonal
Accepted 18 March 2021 influenza viruses infect the upper respiratory tract initially but then often induce severe pulmonary
Available online 30 March 2021
complications in the lower respiratory tract. Therefore, influenza vaccines that prevent viral infection at
both the upper and lower respiratory tracts are highly anticipated. Here, we examined whether using
Keywords:
different vaccination routes for priming and boosting achieved protection in both regions of the respi-
IgA
ratory tract. To this end, we used inactivated whole-virion influenza vaccines to immunize mice either
IgG
Influenza virus
subcutaneously or intranasally for both priming and boosting. Regardless of the route used for boosting,
Respiratory tract the levels of virus-specific IgG in plasma were higher in mice primed subcutaneously than those in
Vaccine control mice, which received PBS only. In addition, intranasal priming followed by subcutaneous
boosting induced higher levels of virus-specific IgG in plasma than those in control mice. The levels of
virus-specific nasal IgA were higher in mice that were primed intranasally than in control mice or in mice
primed subcutaneously. Furthermore, intranasal priming but not subcutaneous priming provided pro-
tection against viral challenge in the upper respiratory tract. In addition, when coupled with subcu-
taneous boosting, both subcutaneous and intranasal priming protected against viral challenge in the
lower respiratory tract. These results indicate that intranasal priming followed by subcutaneous boosting
induces both virus-specific IgG in plasma and IgA in nasal washes and protects against virus challenge in
both the upper and lower respiratory tracts. Our results will help to develop novel vaccines against
influenza viruses and other respiratory viruses.
© 2021 Elsevier Inc. All rights reserved.

1. Introduction

Vaccines for influenza viruses are an effective way to reduce the

Abbreviations: Cal7, A/California/07/2009; HA, hemagglutinin; NA, neuramini-
dase; PBS, phosphate-buffered saline; SD, standard deviation; SV, split vaccine; Th,
CD4þ T helper; TLR, Toll-like receptor; WV, inactivated whole-virion vaccine.
* Corresponding author. Vaccine Creation Group, BIKEN Innovative Vaccine
Research Alliance Laboratories, Institute for Open and Transdisciplinary Research
Initiatives, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
E-mail address: y-yoshioka@biken.osaka-u.ac.jp (Y. Yoshioka).
severity of clinical symptoms and to limit infection and trans-
mission [1,2]. To prevent viral infection of cells and virus release
from infected cells, influenza vaccines must induce neutralizing
antibodies specific for hemagglutinin (HA) and neuraminidase
(NA), the major surface glycoproteins of influenza virus [1,2].
Several types of influenza vaccines, such as split vaccines (SVs) and
inactivated whole-virion vaccines (WVs), are available for use in

https://doi.org/10.1016/j.bbrc.2021.03.099
0006-291X/© 2021 Elsevier Inc. All rights reserved.
M. Shibuya, S. Tamiya, A. Kawai et al.
Fig. 1. Antibody responses after using different antigens for priming and boosting. Mice were immunized twice subcutaneously with SV or WV for priming and boosting.
Control mice were treated with PBS subcutaneously only. After the second immunization, levels of HA- and NA-specific total IgG in plasma were evaluated by using ELISA. We used
160- ((C), 800- (A), and 4,000- (:) foldediluted plasma samples. n ¼ 5 per group. Data are shown as means ± 1 SD. ##P < 0.01, ####P < 0.0001 vs. control group; **P < 0.01,
***P < 0.001, ****P < 0.0001 between indicated groups according to Tukey’s test. Significance of differences was analyzed for the 160-fold-diluted plasma samples only.
humans [1,2]. SVs are generated by treating purified viruses with
ether or detergents (or both) to remove the viral lipid envelope; the
HA and NA proteins are then isolated and used as SVs [2]. For WVs,
purified viruses are inactivated by treating with formalin or b-
propiolactone (or both) [2,3]. In Japan, a WV with adjuvant is
already licensed for use in adults as a vaccine against H5N1 influ-
enza virus [4]. WVs retain the original viral structure and have
single-stranded viral RNA, which is a natural ligand for Toll-like
receptor 7 (TLR7) [5,6]. Therefore, due to their stimulation of
TLR7, WVs generally induce stronger viral-specific antibody and T-
cell responses than do SVs.
Despite the development of several types of influenza vaccines,
influenza viruses still are a threat to humans and a major infectious
source of morbidity and mortality worldwide, indicating the need
for influenza vaccines with improved efficacy [1,2]. In humans, the
upper respiratory tract is the initiation site for infection by seasonal
influenza viruses. Although infection of the upper respiratory tract
induces relatively mild illness, this infection is crucial for human-
to-human transmission of virus via coughing, talking, or sneezing
[1,7]. Infection of the lower respiratory tract due to influenza virus
from the upper respiratory tract often induces severe clinical
complications, including pneumonia [1]. Therefore, one ideal
strategy is to develop vaccines that can block virus infection at both
the upper and lower respiratory tracts.
The major mechanism of defense against viral infections in the
upper respiratory tract is the production of virus-specific IgA an-
tibodies at the nasal surface [8,9]. However, most current influenza
vaccines, which are administered parenterally through subcu-
taneous or intramuscular injection, fail to stimulate the production
of virus-specific IgA in the upper respiratory tract, despite inducing
high levels of virus-specific IgG in blood [8,9]. Therefore, these
vaccines generally do not prevent viral infection at the upper res-
piratory tract, although the leakage of virus-specific IgG into the
lower respiratory tract can prevent the aggravation of illness. In
contrast, intranasal vaccination can induce not only virus-specific
IgG in blood but also virus-specific IgA in the upper respiratory
tract [8,9]. In fact, many studies have demonstrated that nasal
influenza vaccines prevent upper respiratory tract infection more
efficiently than do parenteral vaccines [10e12]. However, nasal
vaccines do not induce virus-specific IgG in blood as strongly as
parenteral vaccines [8,9]. In this regard, novel vaccination strategies
might overcome this drawback of nasal vaccines and increase the
levels of virus-specific IgG in blood.
Combining different vaccination routes, such as parenteral and
167
Biochemical and Biophysical Research Communications 554 (2021) 166e172
mucosal administration, for priming and boosting reportedly in-
duces antigen-specific antibody and T-cell responses both sys-
temically and mucosally, compared with either parenteral or
mucosal vaccination only [13e18]. Therefore, to induce strong
virus-specific antibody responses in both the systemic and nasal
compartments, combining parenteral and nasal vaccination for
priming and boosting is a rational strategy. In this study, we used an
influenza WV as an antigen to examine the efficacy of combining
different routes of immunization to induce virus-specific anti-
bodies in both the nasal cavity and in blood. We demonstrated that
the vaccination regimen combining intranasal priming with sub-
cutaneous boosting achieves antibody responses in both com-
partments and provides strong protection against virus challenge
in both the upper and lower respiratory tracts.
2. Materials and methods
2.1. Mice
Male C57BL/6J mice (age, 6e7 weeks; SLC, Kyoto, Japan) were
housed in a room with a 12:12-h light:dark cycle and had unre-
stricted access to food and water. All animal experiments were
conducted in accordance with the guidelines of Osaka University
for the ethical treatment of animals and were approved by the
Animal Care and Use Committee of the Research Institute for Mi-
crobial Diseases, Osaka University, Japan (protocol number, BIKEN-
AP-H26-11-0).
2.2. Vaccines and influenza viruses
An ether-treated HA-antigeneenriched virion-free SV and a
formalin-inactivated WV from an H1N1 influenza virus (strain: A/
California/07/2009 [Cal7]) were kindly provided by Dr. Yasuyuki
Gomi (The Research Foundation for Microbial Diseases of Osaka
University (BIKEN), Kagawa, Japan). H1N1 influenza virus (strain:
Cal7) was generously provided by Dr. Hideki Asanuma (National
Institute of Infectious Diseases, Tokyo, Japan).
2.3. Recombinant HA and NA proteins
Recombinant HA and NA proteins derived from Cal7 were
generated as described previously [19]. Briefly, the ectodomain of
HA, which carried a C-terminal hexahistidine tag and the foldon of
fibritin from bacteriophage T4, and the ectodomain of NA, which
M. Shibuya, S. Tamiya, A. Kawai et al. Biochemical and Biophysical Research Communications 554 (2021) 166e172

Fig. 2. Level of IgG in plasma according to vaccination regimen. Mice were immunized twice with WV subcutaneously (s.c.) or intranasally (i.n.). Control mice were treated with
PBS subcutaneously only. Levels of (a) WV-specific total IgG, IgG1, IgG2b, and IgG2c in plasma and (b) HA-specific total IgG in plasma were evaluated after the second vaccination.
We used (a) 4,000- ((C), 20,000- (A), and 100,000- (:) foldediluted plasma samples and (b) 160- ((C), 800- (A), and 4,000- (:) foldediluted plasma samples. n ¼ 5 per group.
Data are shown as means ± 1 SD. #P < 0.05, ##P < 0.01, ####P < 0.0001 vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 between indicated groups according to
Tukey’s test. Significance of differences was analyzed for the (a) 4,000-foldediluted and (b) 160-foldediluted plasma samples only.

had a C-terminal hexahistidine tag and an N-terminal tetrabrachion subcutaneously with phosphate-buffered saline (PBS) only. At 7
tetramerization domain from the bacterium Staphylothermus days after the second dose, samples of plasma and nasal wash were
marinus, were expressed by using the Expi293 Expression System collected and stored at 30  C until use. Nasal wash samples were
(Thermo Fisher Scientific, Hampton, NH, USA). collected by gently flushing the nasal cavity with 400 mL of PBS.

2.4. Vaccination 2.5. Antibody responses

For subcutaneous vaccination, mice were immunized at the base
of the tail by using SV (1 mg/mouse) or WV (1 mg/mouse) in a total
volume of 50 mL. For nasal vaccination, anesthetized mice were
immunized with WV (1 mg/mouse) in total volume of 5 mL (2.5 mL to
each nostril). For both routes, mice were immunized by using two
doses at a 21-day interval. Control mice were treated
168
The levels of WV-, recombinant HA-, and recombinant NA-
specific antibodies in the plasma and nasal wash samples were
analyzed by using ELISA, as described previously [20]. Briefly, 10 mg/
mL WV in PBS, 1 mg/mL recombinant HA in carbonate buffer, or
1 mg/mL recombinant NA in carbonate buffer were added to the
wells of 96-well plates, which were incubated overnight at 4  C. The
M. Shibuya, S. Tamiya, A. Kawai et al.
Fig. 3. Level of IgA in nasal wash according to vaccination regimen. Mice were immunized twice with WV subcutaneously or intranasally, as described in Fig. 2. Levels of WV-
specific IgA and HA-specific IgA in nasal wash were evaluated after the second vaccination. We used 2- (C), 10- (A), and 50- (:) foldediluted nasal wash samples. n ¼ 5 per
group. Data are shown as means ± 1 SD. #P < 0.05, ####P < 0.0001 vs. control group; *P < 0.05, ***P < 0.001, ****P < 0.0001 between indicated groups according to Tukey’s test.
Significance of differences was analyzed for the 2-foldediluted nasal wash samples only.
antigen-coated plates were then incubated with Block Ace (DS
Pharma Biomedical, Osaka, Japan), and diluted plasma and nasal
wash samples were added. The plates were then washed three
times with PBS containing 0.05% Tween 20 and incubated with
horseradish peroxidaseeconjugated goat anti-mouse IgG (Merck
Millipore, Darmstadt, Germany), IgG1 (SouthernBiotech, Birming-
ham, AL, USA), IgG2b (SouthernBiotech), or IgG2c (South-
ernBiotech) or horseradish peroxidaseeconjugated goat anti-
mouse IgA (SouthernBiotech). Plates were washed again, tetra-
methyl benzidine (Nacalai Tesque, Kyoto, Japan) was added, the
color reaction was stopped by adding 2 N H2SO4, and the absor-
bance was measured at OD450e570 in a microplate reader (Power
Wave HT, BioTek, Winooski, VT, USA).
2.6. Infection
At 10 days after the second immunization, mice were anes-
thetized and intranasally challenged with Cal7. For upper respira-
tory tract infection, mice were challenged by using 3.0  105 TCID50
of Cal7 in a total volume of 5 mL of PBS (2.5 mL to each nostril). After
3 days, nasal washes were collected by using 400 mL of PBS, and the
virus titers in nasal wash were evaluated by using MDCK cells, as
described previously [20]. For lower respiratory tract infection,
mice were challenged by using 3.0  105 TCID50 of Cal7 in 30 mL of
PBS (15 mL to each nostril). The body weights and survival of the
challenged mice were monitored for 15 days after challenge. We
defined the day on which mice weighed less than 75% of their initial
body weight as the day of death.
2.7. Statistical analyses
Statistical analyses were performed by using Prism (GraphPad
Software, San Diego, CA, USA). All data are presented as means with
standard deviations. Significant differences were determined by
Tukey’s test. Significant differences in survival rates were deter-
mined by using the log-rank test to compare KaplaneMeier curves.
A P value less than 0.05 was considered to indicate statistical
significance.
3. Results and discussion
First, we used SV or WV from H1N1 influenza A virus (strain: A/
California/07/2009 [Cal7]) as an antigen for priming and boosting
169
Biochemical and Biophysical Research Communications 554 (2021) 166e172
to assess the benefit of combining different antigens for priming
and boosting to induce antibody responses. Mice were immunized
subcutaneously with SV or WV twice, after which we measured the
levels of recombinant HA- and NA-specific total IgG in plasma
(Fig. 1). The levels of HA- and NA-specific total IgG were signifi-
cantly higher in mice primed with WV than in mice primed with SV
but did not depend on the antigen used for boosting (Fig. 1). In
particular, mice primed with WV and boosted with SV showed
significantly higher levels of HA- and NA-specific total IgG than
mice primed with SV followed by WV (Fig. 1). These results suggest
the importance of the priming antigen for the strength of immune
responses. In addition, consistent with previous results, WV is su-
perior to induce virus-specific antibody responses compared with
SV [3,21]. Therefore, we focused on WV as the antigen in subse-
quent experiments.
Next, to examine the effect of varying the administration routes
for priming and boosting on antibody responses, we allocated mice
into 4 groups. Group 1 was immunized subcutaneously for both
priming and boosting. Group 2 was immunized subcutaneously for
priming and intranasally for boosting. Group 3 was immunized
intranasally for priming and subcutaneously for boosting. Group 4
was immunized intranasally for both priming and boosting. WV
from Cal7 was used as an antigen.
Nasal vaccination in mice often involves the administration of
large volumes of fluid (e.g., 20e40 mL). However, excess vaccine
solution can flow from the upper respiratory tract to the lower
respiratory tract, thus stimulating immune responses at both sites.
Because this experimental condition might not reflect the actual
situation for nasal vaccination in humans, we instead used only 5 mL
(2.5 mL for each nostril) as the inoculation volume for intranasal
vaccination. Mice were immunized twice with WV, and we
measured the levels of WV- and HA-specific total IgG, IgG1, IgG2b,
and IgG2c in plasma (Fig. 2) and of WV- and HA-specific IgA in nasal
wash (Fig. 3). The levels of WV-specific total IgG, IgG2b, and IgG2c
and of HA-specific total IgG all showed the same tendency (Fig. 2).
Mice in group 1, group 2, and group 3 had significantly higher levels
of WV-specific total IgG, IgG2b, and IgG2c and HA-specific total IgG
than those in the control group (which received PBS subcutane-
ously only), but these antibody levels did not differ between control
mice and those in group 4 (Fig. 2). In particular, group 1 and group 3
had significantly higher levels of WV-specific total IgG, IgG2b, and
IgG2c and HA-specific total IgG than group 4 (Fig. 2). The levels of
WV-specific IgG1 in group 1 and group 2 were significantly higher
M. Shibuya, S. Tamiya, A. Kawai et al. Biochemical and Biophysical Research Communications 554 (2021) 166e172

than in controls and group 4, whereas the amounts of WV-specific
IgG1 were similar between group 3 and control mice (Fig. 2a).
In contrast, the quantities of WV- and HA-specific IgA in nasal
wash were significantly higher in mice that were primed intrana-
sally, that is, group 3 and group 4, than in control mice and those
primed subcutaneously, namely group 1 and group 2 (Fig. 3). In
particular, group 4 had the highest levels of WV- and HA-specific
IgA in nasal wash among all groups (Fig. 3). Collectively, these re-
sults suggest that the priming site plays a crucial role in defining
the strength of the IgG response in blood and of the IgA response in
nasal wash, and the vaccination regimen for group 3 induced both
virus-specific IgG in plasma and IgA in nasal wash, albeit at mod-
erate levels.
To examine the protective effects of vaccination in the upper
respiratory tract, we used a 5-mL volume of Cal7 virus as an intra-
nasal challenge dose after vaccination. In this model, we measured
the post-challenge virus titer in nasal wash as the indicator of
vaccine efficacy (Fig. 4a). The virus titers in mice primed subcuta-
neously, namely group 1 and group 2, did not differ from that in
control mice (Fig. 4a). In contrast, virus titers in nasal wash were
significantly lower in mice immunized intranasally for priming,
that is, group 3 and group 4, compared with mice in group 1, group
2, and the control group (Fig. 4a). These results suggest that the
preventive effect in the upper respiratory tract is correlated with
the ability to stimulate the production of virus-specific IgA in the
upper respiratory tract, thus indicating the usefulness of nasal
vaccine for priming, regardless of the route used for boosting.
We next investigated the protective effects of vaccination in the
lower respiratory tract, instilling 30 mL of Cal7 virus in the nares as a
post-vaccination challenge dose. In this model, we assessed body
weight loss and survival as indicators of vaccine efficacy (Fig. 4b
and c). All of the control mice died within 10 days after challenge

Fig. 4. Protective effects against influenza virus. (a) At 10 days after the second vaccination, mice were challenged with Cal7 to cause upper respiratory tract infection. At 3 days
after challenge, virus titers in nasal wash samples were evaluated. (b, c) At 10 days after the second vaccination, mice were challenged with Cal7 to cause lower respiratory tract
infection. Percentage change in (b) initial body weight and (c) survival were monitored after viral challenge. n ¼ 4 or 5 per group. Data are shown as means ± 1 SD. (a) ##P < 0.01,
###
P < 0.001 vs. control group; ***P < 0.001, ****P < 0.0001 between indicated groups according to Tukey’s test. (c) ##P < 0.01 vs. control group according to comparison of
KaplaneMeier curves by using the log-rank test.

170
M. Shibuya, S. Tamiya, A. Kawai et al.
(Fig. 4b and c). Mice in group 4 rapidly lost weight, and only 50%
survived (Fig. 4b and c). In contrast, all of the mice in group 1, group
2, and group 3 survived after viral challenge, and none of them lost
weight (Fig. 4b and c). These results imply that the preventive effect
in the lower respiratory tract is correlated with the ability to induce
virus-specific IgG in blood and that both subcutaneous and nasal
vaccination for priming followed by subcutaneous boosting protect
against virus challenge in the lower respiratory tract. Collectively,
our results indicate that intranasal administration for priming fol-
lowed by subcutaneous inoculation for boosting protects against
virus challenge in both the upper and lower respiratory tracts.
Our data showed that the protection against virus challenge in
the lower respiratory tract accomplished through nasal vaccination
for priming followed by subcutaneous vaccination (group 3) is
comparable to that of the traditional use of subcutaneous vacci-
nation for both priming and boosting (group 1) (Fig. 4b and c).
However, the levels of WV-specific total IgG and IgG1 were
significantly lower in group 3 than in group 1, despite the equiva-
lent levels of WV-specific IgG2b and IgG2c in these groups (Fig. 2).
Previous reports have shown that, in mice, virus-specific IgG2 are
more protective against influenza virus challenge than IgG1 [22].
Thus, the strong protection against virus challenge in the lower
respiratory tract of group 3 might result from virus-specific IgG2b
and IgG2c.
Although the protection achieved in the upper respiratory tract
was comparable between group 3 and group 4, group 3 had
significantly lower levels virus-specific IgA in nasal wash (Fig. 3). In
general, the magnitude of antibody response differs among
parenteral (e.g., subcutaneous, intramuscular, intradermal)
administration routes [23,24]. Therefore, modifying the parenteral
inoculation regimen for boosting after intranasal priming might
enhance the production of virus-specific IgA in the upper respira-
tory tract.
In summary, we show here that using different routes for
vaccination priming and boosting, especially intranasal priming
and subcutaneous boosting, may be an optimal regimen for pro-
tecting against viral infection in both the upper and lower respi-
ratory tracts. Because many pathogenic viruses, including influenza
viruses and SARS-CoV-2, enter through the nasal surface and
induce severe pulmonary damage in the lower respiratory tract,
preventing infection at both sites likely is an effective strategy for
combating such pathogens [25]. Therefore, the concept that we
demonstrate here will further the development of novel vaccines
against influenza viruses and other respiratory viruses.
Declaration of competing interest
Y.Y. is employed by the Research Foundation for Microbial Dis-
eases (Osaka University, Japan). The other authors have no conflicts
of interests to declare.
Acknowledgments
This study was supported by grants from the Japan Society for
the Promotion of Science (JSPS KAKENHI grant no. JP20H03404 to Y.
Yoshioka), Nagai Memorial Research Scholarship from the Phar-
maceutical Society of Japan (to S. Tamiya), and BIKEN Taniguchi
Scholarship (to A. Kawai). We thank Dr. Yasuyuki Gomi (The
Research Foundation for Microbial Diseases of Osaka University) for
providing the split vaccine and inactivated whole-virion vaccine
and Dr. Hideki Asanuma (National Institute of Infectious Diseases)
for providing influenza viruses.
171
Biochemical and Biophysical Research Communications 554 (2021) 166e172
References
[1] F. Krammer, G.J.D. Smith, R.A.M. Fouchier, M. Peiris, K. Kedzierska,
P.C. Doherty, P. Palese, M.L. Shaw, J. Treanor, R.G. Webster, A. Garcia-Sastre,
Influenza Nat. Rev. Dis. Primers 4 (2018) 3.
[2] C.J. Wei, M.C. Crank, J. Shiver, B.S. Graham, J.R. Mascola, G.J. Nabel, Next-
generation influenza vaccines: opportunities and challenges, Nat. Rev. Drug
Discov. 19 (2020) 239e252.
[3] T. Sekiya, E.J. Mifsud, M. Ohno, N. Nomura, M. Sasada, D. Fujikura, T. Daito,
M. Shingai, Y. Ohara, T. Nishimura, M. Endo, R. Mitsumata, T. Ikeda,
H. Hatanaka, H. Kitayama, K. Motokawa, T. Sobue, S. Suzuki, Y. Itoh, L.E. Brown,
K. Ogasawara, Y. Kino, H. Kida, Inactivated whole virus particle vaccine with
potent immunogenicity and limited IL-6 induction is ideal for influenza,
Vaccine 37 (2019) 2158e2166.
[4] T. Nakayama, T. Kumagai, K.J. Ishii, T. Ihara, Alum-adjuvanted H5N1 whole
virion inactivated vaccine (WIV) induced IgG1 and IgG4 antibody responses in
young children, Vaccine 30 (2012) 7662e7666.
[5] S. Koyama, T. Aoshi, T. Tanimoto, Y. Kumagai, K. Kobiyama, T. Tougan,
K. Sakurai, C. Coban, T. Horii, S. Akira, K.J. Ishii, Plasmacytoid dendritic cells
delineate immunogenicity of influenza vaccine subtypes, Sci. Transl. Med. 2
(2010) 25ra24.
[6] T. Onodera, A. Hosono, T. Odagiri, M. Tashiro, S. Kaminogawa, Y. Okuno,
T. Kurosaki, M. Ato, K. Kobayashi, Y. Takahashi, Whole-virion influenza vac-
cine recalls an early burst of high-affinity memory B cell response through TLR
signaling, J. Immunol. 196 (2016) 4172e4184.
[7] M. Richard, J.M.A. van den Brand, T.M. Bestebroer, P. Lexmond, D. de Meulder,
R.A.M. Fouchier, A.C. Lowen, S. Herfst, Influenza A viruses are transmitted via
the air from the nasal respiratory epithelium of ferrets, Nat. Commun. 11
(2020) 766.
[8] T. Azegami, Y. Yuki, H. Kiyono, Challenges in mucosal vaccines for the control
of infectious diseases, Int. Immunol. 26 (2014) 517e528.
[9] H. Yusuf, V. Kett, Current prospects and future challenges for nasal vaccine
delivery, Hum. Vaccines Immunother. 13 (2017) 34e45.
[10] A. Ainai, S. Tamura, T. Suzuki, E. van Riet, R. Ito, T. Odagiri, M. Tashiro,
T. Kurata, H. Hasegawa, Intranasal vaccination with an inactivated whole
influenza virus vaccine induces strong antibody responses in serum and nasal
mucus of healthy adults, Hum. Vaccines Immunother. 9 (2013) 1962e1970.
[11] T. Suzuki, A. Kawaguchi, A. Ainai, S. Tamura, R. Ito, P. Multihartina,
V. Setiawaty, K.N. Pangesti, T. Odagiri, M. Tashiro, H. Hasegawa, Relationship
of the quaternary structure of human secretory IgA to neutralization of
influenza virus, Proc. Natl. Acad. Sci. U. S. A. 112 (2015) 7809e7814.
[12] A. Ainai, T. Suzuki, S.I. Tamura, H. Hasegawa, Intranasal administration of
whole inactivated influenza virus vaccine as a promising influenza vaccine
candidate, Viral Immunol. 30 (2017) 451e462.
[13] H. Asanuma, F. Koide, Y. Suzuki, T. Nagamine, C. Aizawa, T. Kurata, S. Tamura,
Cross-protection against influenza virus infection in mice vaccinated by
combined nasal/subcutaneous administration, Vaccine 13 (1995) 3e5.
[14] H. Asanuma, C. Aizawa, T. Kurata, S. Tamura, IgA antibody-forming cell re-
sponses in the nasal-associated lymphoid tissue of mice vaccinated by
intranasal, intravenous and/or subcutaneous administration, Vaccine 16
(1998) 1257e1262.
[15] F. Fiorino, E. Pettini, G. Pozzi, D. Medaglini, A. Ciabattini, Prime-boost strate-
gies in mucosal immunization affect local IgA production and the type of th
response, Front. Immunol. 4 (2013) 128.
[16] A. Ciabattini, G. Prota, D. Christensen, P. Andersen, G. Pozzi, D. Medaglini,
Characterization of the antigen-specific CD4(þ) T cell response induced by
prime-boost strategies with CAF01 and CpG adjuvants administered by the
intranasal and subcutaneous routes, Front. Immunol. 6 (2015) 430.
[17] H.S. Hwang, S. Puth, W. Tan, V. Verma, K. Jeong, S.E. Lee, J.H. Rhee, More robust
gut immune responses induced by combining intranasal and sublingual
routes for prime-boost immunization, Hum. Vaccines Immunother. 14 (2018)
2194e2202.
[18] C.B. Roces, M.T. Hussain, S.T. Schmidt, D. Christensen, Y. Perrie, Investigating
prime-pull vaccination through a combination of parenteral vaccination and
intranasal boosting, Vaccines (2019) 8. Basel.
[19] S. Shirai, M. Shibuya, A. Kawai, S. Tamiya, L. Munakata, D. Omata, R. Suzuki,
T. Aoshi, Y. Yoshioka, Lipid nanoparticles potentiate CpG-
oligodeoxynucleotide-based vaccine for influenza virus, Front. Immunol. 10
(2019) 3018.
[20] M. Shibuya, T. Aoshi, E. Kuroda, Y. Yoshioka, Murine cross-reactive non-
neutralizing polyclonal IgG1 antibodies induced by influenza vaccine inhibit
the cross-protective effect of IgG2 against heterologous virus in mice, J. Virol.
(2020) 94.
[21] K. Sato, Y. Takahashi, Y. Adachi, H. Asanuma, M. Ato, M. Tashiro, S. Itamura,
Efficient protection of mice from influenza A/H1N1pdm09 virus challenge
infection via high avidity serum antibodies induced by booster immunizations
with inactivated whole virus vaccine, Heliyon 5 (2019), e01113.
[22] V.C. Huber, R.M. McKeon, M.N. Brackin, L.A. Miller, R. Keating, S.A. Brown,
N. Makarova, D.R. Perez, G.H. Macdonald, J.A. McCullers, Distinct contributions
of vaccine-induced immunoglobulin G1 (IgG1) and IgG2a antibodies to pro-
tective immunity against influenza, Clin. Vaccine Immunol. 13 (2006)
981e990.
[23] L. Zhang, W. Wang, S. Wang, Effect of vaccine administration modality on
immunogenicity and efficacy, Expert Rev. Vaccines 14 (2015) 1509e1523.
M. Shibuya, S. Tamiya, A. Kawai et al.
[24] S.T.T. Schetters, L.J.W. Kruijssen, M.H.W. Crommentuijn, H. Kalay, J.M.M. den
Haan, Y. van Kooyk, Immunological dynamics after subcutaneous immuni-
zation with a squalene-based oil-in-water adjuvant, Faseb. J. 34 (2020)
12406e12418.
[25] Z. Wang, J.C.C. Lorenzi, F. Muecksch, S. Finkin, C. Viant, C. Gaebler, M. Cipolla,
172
Biochemical and Biophysical Research Communications 554 (2021) 166e172
H.H. Hoffmann, T.Y. Oliveira, D.A. Oren, V. Ramos, L. Nogueira, E. Michailidis,
D.F. Robbiani, A. Gazumyan, C.M. Rice, T. Hatziioannou, P.D. Bieniasz,
M. Caskey, M.C. Nussenzweig, Enhanced SARS-CoV-2 neutralization by
dimeric IgA, Sci. Transl. Med. 13 (2021).