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Suitability of Polymyxin B as a Mucosal Adjuvant for Intranasal Influenza and COVID 19 Vaccines
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编译时间: 2023-11-20
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Article
Suitability of Polymyxin B as a Mucosal Adjuvant for
Intranasal Influenza and COVID-19 Vaccines
Naoto Yoshino 1 , Takuya Yokoyama 2,3 , Hironori Sakai 4 , Ikumi Sugiyama 5, Takashi Odagiri 1,
Masahiro Kimura 1, Wataru Hojo 4, Tomoyuki Saino 2 and Yasushi Muraki 1,
1 Division of Infectious Diseases and Immunology, Department of Microbiology, School of Medicine,
Iwate Medical University, 1-1-1 Idaidori, Yahaba 028-3694, Iwate, Japan
2 Department of Anatomy (Cell Biology), Iwate Medical University, 1-1-1 Idaidori,
Yahaba 028-3694, Iwate, Japan
3 Laboratory of Veterinary Anatomy and Cell Biology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda,
Morioka 020-8550, Iwate, Japan
4 R&D, Cellspect Co., Ltd., 2-4-23 Kitaiioka, Morioka 020-0857, Iwate, Japan
5 Division of Advanced Pharmaceutics, Department of Clinical Pharmaceutical Science, School of Pharmacy,
Iwate Medical University, 1-1-1 Idaidori, Yahaba 028-3694, Iwate, Japan
Correspondence: ymuraki@iwate-med.ac.jp
Abstract: Polymyxin B (PMB) is an antibiotic that exhibits mucosal adjuvanticity for ovalbumin
(OVA), which enhances the immune response in the mucosal compartments of mice. Frequent
breakthrough infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants
indicate that the IgA antibody levels elicited by the mRNA vaccines in the mucosal tissues were
insufficient for the prophylaxis of this infection. It remains unknown whether PMB exhibits mucosal
adjuvanticity for antigens other than OVA. This study investigated the adjuvanticity of PMB for
the virus proteins, hemagglutinin (HA) of influenza A virus, and the S1 subunit and S protein of
SARS-CoV-2. BALB/c mice immunized either intranasally or subcutaneously with these antigens
alone or in combination with PMB were examined, and the antigen-specific antibodies were quantified.
PMB substantially increased the production of antigen-specific IgA antibodies in mucosal secretions
and IgG antibodies in plasma, indicating its adjuvanticity for both HA and S proteins. This study
Citation: Yoshino, N.; Yokoyama, T.;
also revealed that the PMB-virus antigen complex diameter is crucial for the induction of mucosal
Sakai, H.; Sugiyama, I.; Odagiri, T.;
immunity. No detrimental effects were observed on the nasal mucosa or olfactory bulb. These
Kimura, M.; Hojo, W.; Saino, T.;
Muraki, Y. Suitability of Polymyxin B findings highlight the potential of PMB as a safe candidate for intranasal vaccination to induce
as a Mucosal Adjuvant for Intranasal mucosal IgA antibodies for prophylaxis against mucosally transmitted infections.
Influenza and COVID-19 Vaccines.
Vaccines 2023, 11, 1727. https:// Keywords: polymyxin B; mucosal adjuvant; hemagglutinin; S1 subunit; S protein; influenza A virus;
doi.org/10.3390/vaccines11111727 SARS-CoV-2
Academic Editor:
Mariusz Skwarczynski
Received: 21 October 2023 1. Introduction
Revised: 15 November 2023 Polymyxin B (PMB) is a clinically used antibiotic. Using a drug-repositioning strat-
Accepted: 16 November 2023 egy [1], we observed that PMB elicits mucosal adjuvanticity; PMB evoked much higher
Published: 18 November 2023 titers of ovalbumin (OVA)-specific IgA antibodies (Abs) in the mucosal compartments
of intranasally immunized mice [2]. We also elucidated the mechanism underlying the
adjuvanticity [2]: (1) the diameter of the PMB-OVA complex formed in the immunization
Copyright: © 2023 by the authors. solution is suitable for the induction of mucosal immunity, and (2) PMB induces mast
Licensee MDPI, Basel, Switzerland. cell degranulation, leading to the activation of innate immunity and enhanced acquired
This article is an open access article immunity. However, the adjuvanticity of PMB for virus proteins remains unknown.
distributed under the terms and Coronavirus disease 2019 (COVID-19) caused by the novel severe acute respiratory
conditions of the Creative Commons syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and was declared a pandemic
Attribution (CC BY) license (https:// in March 2020. It has since become a major public health concern [3,4], with 6.7 mil-
creativecommons.org/licenses/by/ lion deaths (as of 10 March 2023) [5]. The intramuscular administration of COVID-19
4.0/). vaccines is reported to be effective, as fully vaccinated individuals experience decreased
Vaccines 2023, 11, 1727. https://doi.org/10.3390/vaccines11111727 https://www.mdpi.com/journal/vaccines
Vaccines 2023, 11, 1727 2 of 13
hospitalization and mortality compared to non-vaccinated individuals with similar risk
factors [6–9]. However, breakthrough infections in vaccinated individuals have been fre-
quently reported [10–13]. Before the COVID-19 pandemic, annual epidemics of influenza
occurred worldwide, primarily in winter, despite the global use of intramuscular or subcu-
taneous influenza vaccines (whole inactivated virus or hemagglutinin [HA] split) [14–16].
This suggests that the plasma IgG Abs induced by these vaccines do not necessarily protect
against virus infections.
A crucial step in controlling mucosally transmitted infections, such as COVID-19 and
influenza, is achieving immunoprophylaxis in mucosal tissues via mucosal immunization.
This induces pathogen-specific immunoprophylaxis primarily by inducing the production of
secretory IgA Abs in the mucosal tissues [17,18]. Particularly, intranasal vaccination is highly
potent at inducing antigen-specific IgA Abs in the respiratory tract [19]. Furthermore, mucosal
adjuvants for subunits or recombinant protein antigens are required to induce a potent immune
response; however, no mucosal adjuvants are currently available for clinical use [20].
The present study investigated whether PMB has adjuvanticity for antigens other than
OVA. We used BALB/c mice to determine the suitability of PMB as a mucosal adjuvant
for the following virus proteins: influenza A virus hemagglutinin (HA) and the S1 subunit
and S protein of SARS-CoV-2. Furthermore, we analyzed the diameter of the PMB-virus
antigen complex, which is a representative physicochemical property for efficient mucosal
adjuvanticity [21]. Additionally, we assessed the histology of the nasal cavity, including
that of the olfactory bulb, in intranasally immunized mice.
2. Materials and Methods
2.1. Ethics Statement
All animal experiments in the present study were approved by the Committee on the
Ethics of Animal Experiments (CEAE) of the Iwate Medical University (Permit No. 02-013).
The animal experiments were performed in compliance with the recommendations of the
Guidelines for Proper Conduct of Animal Experiments established by the Science Council
of Japan and the regulations established by the CEAE.
2.2. Antigens and Adjuvant
The HA split of mouse-adapted (MA)-influenza A/Iwate/1130/2009 (H1N1pdm09) [22]
was generated and provided by the Research Foundation for Microbial Diseases of Osaka
University (BIKEN), Kagawa, Japan; the MA-A/Iwate/1130/2009 virus was propagated
in MDCK cells, and the culture supernatant was purified via filtration (0.45 µm) followed
by sedimentation using a linear sucrose gradient [23]. The recombinant S1 subunit (amino
acids 251–660) of the SARS-CoV-2 Wuhan-Hu-1 strain expressed in Escherichia coli was
purchased from FAPON Biotech Inc. (Donggua, China). The recombinant S protein (amino
acid 16–1213) of the SARS-CoV-2 Wuhan-Hu-1 strain expressed in HEK239 cells was
purchased from ACROBiosystems (Newark, DE, USA). Polymyxin B sulfate (FUJIFILM
Wako Pure Chemical Co., Osaka, Japan) was used as the mucosal adjuvant.
2.3. Immunization of Mice
Five-week-old female BALB/cAJcl mice (18–21 g) were purchased from CLEA Japan
(Tokyo, Japan). The mice were acclimated to the laboratory animal facility for 1 week prior
to the experiments.
The virus antigens and PMB were dissolved in normal saline (<0.25 endotoxin unit/mL;
Otsuka Pharmaceutical Factory, Tokushima, Japan). Immunization solutions were prepared
using 1 µg of HA split, 1 µg of S1 subunit, 10 µg of S1 subunit, or 1 µg of the S protein with or
without 500 µg of PMB in 10 or 50 µL aliquots. Virus antigens were first dissolved in normal
saline at the following concentrations: 0.2 mg/mL (HA), 0.2 mg/mL (S1), 2 mg/mL (S1),
and 0.2 mg/mL (S). A 100 mg/mL PMB solution in normal saline was then prepared. Equal
volumes of the virus antigen solutions (for each of the antigens) and the PMB solution
(or normal saline for antigen alone) were mixed to prepare samples for intranasal (IN)
Vaccines 2023, 11, 1727 3 of 13
immunization. For subcutaneous (SC) immunization, the nasal immunization samples
were diluted 5-fold with normal saline. All immunization samples were prepared at the
time of use.
The mice were mildly anesthetized with ketamine (Ketarar®; Daiichi Sankyo Co.,
Ltd., Tokyo, Japan) and randomly divided into IN or SC immunization groups. For IN
immunization, mice were administered a 10 µL aliquot (5 µL/nostril) of the immunization
solution, whereas SC immunization was performed by injecting a 50 µL aliquot of the im-
munization solution into the interscapular region of the mice. Each mouse was immunized
thrice at 0, 7, and 14 days, as previously described [2,21,24].
A week after the last immunization, mucosal secretions (tracheal–bronchial lavage
[TBL], nasal washes [NWs], saliva, fecal extracts [FEs]), and plasma samples were collected,
as previously described [25,26]. Vaginal washes (VWs) were collected as per the method
reported by Wu et al. [27], with minor modifications. Briefly, 50 µL saline was injected into
the vagina of the mice and withdrawn using a 200 µL pipettor. The procedure was repeated
four times to obtain a total volume of approximately 200 µL of VW sample. The mice
were euthanized directly via cervical dislocation under anesthesia with 3.0% isoflurane and
0.5 L/min oxygen.
2.4. Enzyme-Linked Immunosorbent Assay (ELISA)
The anti-HA, anti-S1, and anti-S Abs in the samples were quantified using ELISA. The
anti-HA and anti-S Ab measurements were performed by coating the plates with the HA
and S proteins used for immunization. The experiments were performed as previously
described [28]. For quantifying the anti-S1 Ab, we employed the COVID-19 Human IgM
IgG ELISA Kit (Spike Protein) (R&D Cellspect Co., Ltd., Morioka, Japan) and coated the
wells with five times the amount of antigen (S1 subunit) used for anti-HA and anti-S
Ab measurements; the subsequent procedure followed was the same as that described
previously [28].
Mouse anti-HA monoclonal Ab (mAb) against influenza A/California/06/2009
(H1N1pdm09) (eEnzyme LLC., Gaithersburg, MD, USA) and mouse anti-S1 (Wuhan-
Hu-1; SARS-CoV-2 spike neutralizing Ab) IgG mAb (Sino Biological, Inc., Beijing, China)
were used to determine the detection limit of the ELISA systems. The detection limits
for the anti-HA, anti-S1, and anti-S Abs were found to be 6.25 ng/mL, 6.25 ng/mL, and
0.3125 ng/mL, respectively. Additionally, no nonspecific reactions were observed in the
ELISA systems using naïve BALB/c mouse plasma diluted 1:26 (×64) and NWs diluted
1:2 (×2).
2.5. Particle Diameter
The particle diameters of the virus antigen and PMB-virus antigen complex were mea-
sured using a Zetasizer Nano ZS system (Malvern Instruments Ltd., Worcestershire, UK),
as described previously [21,24].
2.6. Histopathological Analysis
A week after the last immunization, the head of each mouse was collected and fixed
in 10% neutral-buffered formalin. After washing with PBS, the specimens were decalci-
fied in 10% ethylenediamine-N,N,N’,N’-tetraacetic acid disodium (pH 7.0) for 1 week at
4 ◦C. After washing with PBS, the frontal sections were cut into 5 mm thick slices and
embedded in paraffin. Thereafter, 10 µm thick histological sections were stained with
Mayer Hematoxylin for 12 min and Eosin for 2 min. The slides were scanned using a
virtual slide scanner (NanoZoomer®-RS; Hamamatsu Photonics, Hamamatsu, Japan) at
40× magnification and visualized using a viewer software (Hamamatsu Photonics). The
thickness and morphological changes of each layer in the nasal mucosal tissue and olfactory
bulb, as well as the infiltration of leukocytes into the tissue, were analyzed. The safety of
IN immunization was evaluated compared to the SC immunization group.
Vaccines 2023, 11, 1727 4 of 13
2.7. Statistical Analysis
A one-way analysis of variance (ANOVA) was used to compare the three groups. If
the one-way ANOVA identified a significant effect in a group, a post hoc Tukey’s multiple
comparison test was performed. Differences between groups were considered significant
at a p-value of <0.05. Statistical analyses were performed with GraphPad Prism 9.5.1 (528)
(GraphPad Software, Inc., Boston, MA, USA).
3. Results and Discussion
3.1. Immunization of Mice with Influenza HA Split
In the present study, since influenza and COVID-19 vaccines are injectable vaccines,
subcutaneous (SC) inoculation was used as a control for comparison with the intranasal (IN)
vaccine. In addition, the major adverse effects of injected PMB are severe nephrotoxicity and
neurotoxicity. Owing to the apparent toxicity, SC inoculation with PMB was not performed.
Female BALB/c mice were administered influenza HA split intranasally or subcuta-
neously, and the Abs were quantified using an ELISA system. The quantity of HA-specific
IgA Abs in the HA (IN) and HA (SC) group mice was at or below the detection limit
(Figure 1). In contrast, IN administration of PMB + HA resulted in a significant increase
in the IgA levels in the TBL, NW, saliva, FE, and VW samples. Furthermore, plasma IgG
levels increased significantly in the PMB + HA (IN) and HA (SC) groups compared with
those in the HA (IN) group. However, no significant difference was observed in the amount
of plasma IgG Abs between PMB + HA (IN) and HA (SC) groups, indicating that PMB
administered intranasally with HA elicited systemic immunity to the same extent as HA
administered subcutaneously.
We previously reported that the particle diameter of the OVA-PMB complex is related
to the induction level of mucosal immunity [21]. Therefore, we measured the particle
diameters of HA split with or without PMB in the immunization solutions (Figure 2,
Table 1). The diameter of the distribution with the largest proportion of particles (dominant
diameter) in the HA and PMB + HA groups was 202.4 and 418.0 nm, respectively. This
significant difference in the dominant diameters between the HA and PMB + HA groups
(p < 0.0005, unpaired Student’s t-test) indicates that PMB formed a complex with HA in the
solution. Furthermore, the dominant diameter in the PMB + HA group (418 nm) was within
the appropriate range for eliciting mucosal immune responses (100–500 nm) [29]. Thus,
we concluded that PMB efficiently induces mucosal immunity against HA by forming a
complex with the antigen protein.
Table 1. Particle diameter distribution of virus antigens with or without polymyxin B (PMB).
Diameter Distribution by Intensity
Antigen PMB Peak 1
a Peak 2 Peak 3 PDI b
D (nm) c Int (%) d D (nm) Int (%) D (nm) Int (%)
HA – 202.4 ± 2.3 98.9 ± 1.0 39.2 ± 1.8 1.1 ± 1.0 0.207 ± 0.005
+ 418.0 ± 16.9 97.7 ± 2.0 69.1 ± 2.5 2.3 ± 2.0 N/A N/A 0.315 ± 0.025
S1 – 391.4 ± 13.1 63.4 ± 1.8 16.3 ± 1.8 36.6 ± 1.8 0.737 ± 0.081
+ 2209.3 ± 86.7 100.0 N/A N/A N/A N/A 0.161 ± 0.051
S – 246.6 ± 4.4 56.7 ± 1.9 24.5 ± 0.7 43.3 ± 1.9 N/A N/A 0.610 ± 0.132
+ 26.0 ± 1.7 48.6 ± 2.0 2.0 ± 0.0 34.3 ± 6.8 470.4 ± 79.0 17.0 ± 5.6 0.446 ± 0.139
Note. HA: hemagglutinin; S1: S1 subunit; S: S protein; PMB: polymyxin B. a The values of peak diameter are
shown as the mean ± SD of three independent experiments. b PDI; polydispersity index. c D; diameter. d Int;
intensity. N/A, not available.
 VVaacccciinneess 22002233,, 1111,, x17 F2O7R PEER REVIEW  5 5oof f1143 
TBL IgA NW IgA Saliva IgA

FE IgA VW IgA Plasma IgG
(×10,000)


IN SC IN SC IN SC
Immunization: HA split 1 ug / mouse
ELISA coating: HA split 50 ng / well  
FFiigguurree 11.. QQuuaannttiittyy ooff HHAA--ssppeecciifificc AAbb  iinn mmiiccee  iimmmmuunniizzeedd wwiitthh HHAA sspplliitt.. Miiccee wweerree ssuubbjjeecctteedd  ttoo 
iinttrranassall ((IIN)) orr ssubccuttaneeouss ((SC)) iimmuniizattiion wiitth 1 µg off HA ssplliitt ((44––6 miiccee ppeerr eexxppeerriimeenttall 
grroup)).. Muuccoossaall sseeccrreettiioonnss  ((TBLL::  ttrraccheeaall––bbrroonncchhiiaall  llaavvaaggeess; ;NW:: nassaall wassheess;; salliivaa;; FE::  fecall 
exttrracctt;; VW:: vagiinall wassh)) and pllasma were collllectted.. HA--ssppeecciifificc IIggA and IIgG Abss  iin mucosall 
sseeccrreettiioonnss ((11::22)) oorr ppllaassmaa ((11::1100,,000000)) ddiilluutteedd iinn 11% BBSSAA--PPBBSS weerree qquuaannttiififieedd uussiinngg aann EELLIISSAA ssyysstteem 
ddeessccrriibbeedd iinn tthhee MMaatteerriiaallss aanndd MMeetthhooddss sseeccttiioonn.. EEaacchh ddoott rreepprreesseennttss aa ssiinnggllee mmoouussee.. TThhee hhoorriizzoonnttaall 
ddootttteedd lliinnee iinnddiiccaatteess tthhee ddeetteeccttiioonn lliimmiitt.. SSiiggnniifificcaanntt ddifferences in OVA-specific Ab titers between 
experimental groups are indicated by asterisks:  p <  ifferences in OVA-specific Ab titers between0.005. 
experimental groups are indicated by asterisks: p < 0.005.
WMea sptrceevlilosuasrley rreeqpuoirrteedd ftohrate tfhfiec iepnatrtmicluec dosiaaml iemtemr uofn itthye [O30V]A; t-hPeMirBr ocolemipnleexli cisit irneg-
lmatuecdo tsoa lthime imnduuncittyiosnh oleuvledl boef mevuaclousaatel dim. Wmeunpirteyv [i2o1u]s.l Tyhsehroewfoerde,t whaet msuerafsau                    ct
riend( tlihpeo ppaerpttiicdlee 
dainaamloegteorfsPoMf HBA) eslpicliittswaithhigohrewr iltehvoeul toPfMIgBAinAbthperiomdmucutnioiznabtiyona cstoivluattiionngsm (F                  a
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awmaestaepr)p irno pthriea HteAfo arntrda nPsMloBc a+t iHonA ignrtoouthpes nwaassa 2l 0m2u.4c oansad, 4th1e8.I0g nAmA, bressipnemctiicveeliym. mThuins isziegd-
nwifiitchanHtA di(ffINer)enwcaes inat thore bdeolmowinathnet ddieatmecettieorns lbimetwit e(eFnig tuhree H1)A.  Tanhdis PmMaBy +b eHaAtt rgibrouutepds (tpo 
simolumtuionni.t yFaunrtdh,esrumboserqe,u  tehnet lyd,oamcqinuairnetd dimiamuetneirt yi.n  the PMB  + HA group  (418 nm) was 
within the appropriate range for eliciting mucosal immune responses (100–500 nm) [29]. 
Thus, we concluded that PMB efficiently induces mucosal immunity against HA by form-
ing a complex with the antigen protein. 
 
OD450
HA
PMB+HA
HA
HA
PMB+HA
HA
HA
PMB+HA
HA
Vaccines 2023, 11, 1727 6 of 13
 Vaccines 2023, 11, x FOR PEER REVIEW  6  of  14 
HA HA+PMB
S1 S1+PMB
S S+PMB
Figure 2. Particle diameter of PMB-virus antigenDicaommetperl e(nxm. )Influenza HA split (1 µg), SARS-CoV -2
S1 subunit (1 µg), or SARS-C oV  -2 S p  rotein  (1 µg) was mixed  with P MB (50 0 µ g) in  s alin  e. ParticleFigure 2. Particle diameter of PMB-virus antigen complex. Influenza HA split (1 µg), SARS-CoV-2 
diameters of the complexSe1 ssuwbuenriet (m1 µega)s, ourr SeAdRuS-sCinoVg-2a SZ perotatesiniz (e1 rµNg) awnaso mZixSeds ywsitteh mPM. BX (-50a0n µdg)y i-na sxaelisnei.n Pdaritcicaltee 
particle diameter and indteianmseitteyr,s roef sthpee ccotmivpelelxye.s were measured using a Zetasizer Nano ZS system. X- and y-axes indi-
cate particle diameter and intensity, respectively. 
3.2. Immunization of MTiacbelew 1.i tPhartSicAle RdiaSm-CeteorV di-s2triSbu1tioSnu obf uvinruist antigens with or without polymyxin B (PMB). 
Our findings indicate thatDPiaMmeBteer ldiicsittrisbumtiuonc obys ainltaendsjiutyv  anticity for HA split (Figure 1),
A a bpnrtoigmenp  PtiMnBg  us to examPeak 1         ine whe the r  PMB  elic
P
 i
etsaka 2d  ju  va nticity for t
PheeakS 3A  RS-CoV-2 SPDpIr   
D (nm) c Int (%) d D (nm) Int (%) D (nm)  otein.Int (%) 
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In t  + 
N/A  N/A 
2209.3 ± 86.7 
 he pre sent stud y,  th e S1 su
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N/A  N/A  0.610 ± 0.132 
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The IgA Ab level wasNboetleo. HwA:t hheemadgegltueticntiino; Sn1: lSi1m suitbuinnit;a Sl:l St phroeteeinx; aPmMBi:n peodlymyxiicne B,. wa Thhee vraeluaess Iogf pGeakA dbi-s
could be detected in thameetperl aarsem shaowonf ams thiec meeiamn ±m SDu onf itzhreede insduebpecnudetnatn exepoeurimsleyn.ts. b PDI; polydispersity index. 
c
We assumed that 1 D; dgiamofettehr. 
d
e ISnt1; isnutenbsuityn. iNt/wA, anost ianvasiulafbµ filec. ient for eliciting mucosal immunity;
therefore, we increased thMeasdt oceslalsg aere troeq1u0ireµdg foor efffithcienSt 1muscuobsaul nimitmaunidty i[m30]m; thueniri rzoeled int heleicimtinigc meui-n
the same manner. Ascosshalo iwmmnuinnityF sihgouurlde bSe2 e,vtahlueateadm. Woeu pnrtevoiofuIsglyG shAowbedin that surfactin (lipopeptide 
immunized subcutanaenoaulosgl oyf PinMcBr)e ealiscietsd a ihnigahedr 
the plasma of mice
level of IgA Ab production by 
nasal mucosa [21]. Although theo dsoem-dineapnte dniadmeentetr mof aHnAn (e20r
activating mast cells in the 
2.c4o nmm)p (aFirgeudre w2, Titahblet h1)e 
previous values (FigurweasS a1p)p(rpopIgA Abs in the variouws imth uHcAo (sINal) sweacs raet toiro bneslow tahse bdeetleoctwiont hlimeidt (eFtigeucrtei o1)n. Tlhimis mita.y be attributed to the 
The observation atbhsaentcteh of mast cell activation by PMB, resulting in the inefficient activation of innate im-munity aendS, 1susbusebquuennitltyd, aicdquniroedt  eimlimciutnmityu.  cosal immunity was confirmed
using an ELISA system in which the S protein was used as an antigen. The aliquots
mentioned in Figure S2 (from mice immunized with 10 µg of the S1 subunit) were examined.
 The IgA Ab levels in the TBL and NW were below the detection level (Figure S3). Thus,
we concluded that the S1 subunit is immunogenic in mice but unable to elicit a mucosal
immune response.
The intrinsic nature of the S1 subunit appears to be involved in eliciting inefficient
mucosal immunity (Figures S1–S3). Given that the length and width of the trimeric S protein
are 21 nm and 8.7 nm, respectively [33], the diameter of the S1 subunit was likely smaller.
Nevertheless, the dominant diameter of the S1 subunit and PMB + S1 in the immunization
solution was 391.4 nm and 2209.3 nm, respectively (Figure 2, Table 1), suggesting that
the S1 subunit forms aggregates and that these S1 aggregates form complexes with PMB
to form particles with large diameters. In the present study, we used E. coli derived-S1
with no glycans attached. Based on a previous report that glycans on bovine serum
albumin molecules suppress self-aggregation [34], we speculate that the S1 subunit is
Intensity (%)
Vaccines 2023, 11, 1727 7 of 13
readily aggregated owing to the lack of glycans, which leads to the formation of larger
particles, resulting in inefficient mucosal immunity.
3.3. Immunization of Mice with SARS-CoV-2 S Protein
The results shown in Figures S1–S3 indicate that the S1 subunit is not a viable candidate
for IN immunization in mice. Therefore, we immunized the mice with the S protein (1 µg)
Vaccines 2023, 11, x FOR PEER REVIEWw ith or without PMB, and the IgA and IgG Abs induced against the S18 souf b14u  nit were  quantified. Immunization in the PMB + S (IN) group resulted in a significant increase in
IgA Ab levels in the VW and IgG Ab levels in the plasma compared with that in the S (IN)
pglraosmupa (IgpG< A0b.0s0 (5F)i;ghuorew 1e, vloewr,etrh erigchotr rpeasnpeol)n, da isnugbsOtaDn4ti5a0l vleavleule osf wplearsemdetectTedh einl mowiceO inD thvea PluMeBs s+h So (wINn) ginroFuipg u(FrieguSr4e c3a, lnowbeera rtitgrhibt upatendel)t.o Tuhsuisn, gINd iimffmeruenn-t antigens
i(zSatpiorno wteiitnh PfoMrBi melimcitusn siyzsatetimoinc iamnmduSn1itys uagbauinsitt tfhoer SE pLrIoSteAin qaundan mtiuficcoastailo inm)m. uTnhiteyr.e  fore, we
quanTthiefi eddistrhibeuAtibosn aogf adiinasmtettheersS foprr othte iPnMinB t+h eS cEoLmISpAlexs ywsates mtri.mAondal i(qFuigoutroe f2e, aTcahbloe f the TBL,
1N). WIn, tahned PpMlaBs +m Sa ssoalmutipolne,s s(oFmigeu praerSti4c)lews eaxshuibseitdedt oa qpueakn tdifiaymtheetesr poef c4i7fi0c.4a nmti-,S wAhbicshu  sing this
iss ywstitehmin. tWhee adpeptreocpteridatae nrainngcer,e aalstehdouagmh othuins tporofpSo-rstpioenc i(fi1c7.I0g%A) wAabss niont TpBreLdoamndinNanWt.  samples
Tfhroem lowtheer mpriocpeoirntiothne oPf MPMBB+ + S (    ff        S (I
1N7.)0i%m) mcoumpared with that of PMB + HA (97.7%) may 
rgerfloeuctpth(eFidgiureere3n,cuepinptehrepOaDnevlasl)u,eisn dofi cTaBtLin 
niza tion group compared with those in the S (IN)agntdhNatWP MIgAB Apobss sbeestsweesemn PuMcoBs a+ lSa (dFjiuguvraen     ticity for3t)haenSdpPrMoBte +in H. A (Figure 1). 
TBL IgA NW IgA

Plasma IgG Plasma IgG
(×10,000) (×100)


IN SC IN SC
Immunization: S protein 1 ug / mouse
ELISA coating: S protein 50 ng / well  
FFigiguurer e3.3 A. Ammouonut notf oSf-sSp-escpifiec iAfibc Ainb minicem imicemiumnmizeudn wizietdh 1w µitgh o1f µS gproofteSinp.r Motiecien w. Mereic seuwbjecretesdu  bjected to
tion itnratrnaansaaslal( I(NIN))o orr ssuubbccuuttaanneeoouuss (S(SCC) )imimmmunuinzaiztiaotnio wniwthi 1th µ1g µogf tohfe tShAeRS-CoV-2 S protein (4–6 
mpiecre epxepr eexripmereimnteanltaglr goruopu)p.).T Thhee SS--ssppeecciifificc AAbsb sini ntheth meumcouscaol sseaclrseeticornest 
SAR S- CoV-2 S protein (4–6 micei(o1:n2s) o(r1:p2l)asomr ap (l1a:s1m0,0a0(01   :10,000 oror 1:100) diluted in 1% BSA-PBS were quantified using ELISA.  p < 0.005. 
1:100) diluted in 1% BSA-PBS were quantified using ELISA. p < 0.005.
   
 
OD450
S
PMB+S
S
S
PMB+S
S
Vaccines 2023, 11, 1727 8 of 13
Although the quantity of S-specific plasma IgG Abs in the 10,000-fold diluted plasma
sample was at or below the detection limit (Figure 3, lower left panel), IgG Abs pertain-
ing to the PMB + S (IN) and S (SC) groups were unequivocally detected in the 100-fold
diluted plasma samples (Figure 3, lower right panel). Furthermore, similar to the level
of anti-HA plasma IgG Abs (Figure 1, lower right panel), a substantial level of plasma
IgG Abs was detected in mice in the PMB + S (IN) group (Figure 3, lower right panel).
Thus, IN immunization with PMB elicits systemic immunity against the S protein and
mucosal immunity.
The distribution of diameters for the PMB + S complex was trimodal (Figure 2, Table 1).
In the PMB + S solution, some particles exhibited a peak diameter of 470.4 nm, which is
within the appropriate range, although this proportion (17.0%) was not predominant. The
lower proportion of PMB + S (17.0%) compared with that of PMB + HA (97.7%) may reflect
the difference in the OD values of TBL and NW IgA Abs between PMB + S (Figure 3) and
PMB + HA (Figure 1).
3.4. Histopathology of the Nasal Mucosa of Immunized Mice
We examined the histopathology of the immunized mice to assess the inflammatory
responses in the nasal mucosa (pseudostratified ciliated epithelium and olfactory epithe-
lium) and olfactory bulb. The mice were euthanized on day 7 after the last immunization,
and the samples were collected. We observed no apparent inflammatory lesions in the
intranasal regions where the mice were immunized with PMB + S1 or PMB + S. Similarly,
no pathological changes were detected in the olfactory bulb (Figure 4). Therefore, we
concluded that PMB + S or S did not exert detrimental effects on the nasal membrane and
olfactory bulb, at least with respect to the immunization protocol used in the present study.
The safe attainment of a sufficient IgA Ab concentration on the mucosal membrane is
important for the prophylaxis of mucosally transmitted infections. Recently, mucosal IgA
Abs associated with intramuscular mRNA vaccination has been detected [35–37]. However,
the frequent breakthrough infections of variants [10–13] indicate that the IgA Ab levels
in mucosal tissues elicited by the mRNA vaccines were insufficient for prophylaxis of the
infection. Thus, IN immunization is required to achieve sufficient levels of IgA Abs in the
nasal mucosa. The results of the present study show that PMB is a promising mucosal
adjuvant candidate for IN vaccination with an inactivated virus component.
Safety concerns have also necessitated the exploration of alternative options for IN
immunization. Bacterial toxins such as cholera toxin (CT) elicit potent mucosal adjuvan-
ticity [38]. It has been reported that the mucosal adjuvanticity of CT was approximately
1.5 times higher than that of PMB [28]. However, mice immunized intranasally with anti-
gens and CT showed severe inflammatory responses in the subepithelial nasal mucosal
tissue [28,39]. Consequently, CT is not currently used in clinical settings. In another in-
stance, a heat-labile toxin from E. coli was approved as an influenza nasal vaccine for
humans but was withdrawn owing to severe side effects such as facial palsy [40]. A live-
attenuated influenza vaccine (FluMist®) has been approved [41] and is currently available
in the USA and Europe. However, from a safety standpoint, an age limit has been imposed
for this vaccination [42]. In contrast, PMB, which is an inhalational antibiotic, has been
shown to be safe with encouraging clinical results in humans [43,44]; furthermore, the
present study has confirmed its efficacy as a mucosal adjuvant. The adverse effects of
PMB as an adjuvant are considered to be similar to those of PMB in clinical use. Thus, this
drug-repositioning strategy presented here may be a new approach for the development of
novel mucosal adjuvants.
Previous studies have demonstrated that the mechanisms of PMB as an adjuvant
are (1) complex formation with a particle diameter suitable for transfer to mucosal tis-
sues and (2) mast cell activation [21]. More recently, more potent adjuvants have been
developed by combining several compounds with immunostimulatory or antigen-delivery
properties. PMB has both immunostimulatory and antigen-delivery properties, making it a
suitable mucosal adjuvant. The mechanism by which PMB enhanced specific Ab titers is
 Vaccines 2023, 11, x FOR PEER REVIEW  9  of  14 
Vaccines 2023, 11, 1727 3.4. Histopathology of the Nasal Mucosa of Immunized Mice  9 of 13
We examined the histopathology of the immunized mice to assess the inflammatory 
responses in the nasal mucosa (pseudostratified ciliated epithelium and olfactory epithe-
liautmtri)b auntded oltfoacittsoaryd jbuuvlabn. tTichiety m, ainced wPeMreB eaulothnaenwizoeudl odnn doatyin 7d aufcteera thvier ulass-ts pimecmifiucnaizcaqtuioirne,d 
ainmdm  thuen esarmespploesn swe.erWe ecoolblescetrevde.d Wthee oabvsaeirlvaebdil intyo oafpPpMarBenat sinaflmamucmosaatol rayd jluevsiaonnts [2in2 ]t;hIeN 
inimtramnuansaizl aretigoinonws iwthhPerMe Bthpel musicaen waenrteig iemnm(iunnaiczteivda wteidthi nPflMuBen +z Sa1v oirru PsM) iBn h+i bSi. tSeidmdiliasrelays,e 
npor pogatrhesoslioognicaanl dchraendguecse dwverireu dsettietectresdin inth theer eoslpfaircatotoryry bturalbc t(Faifgteurrlee 4th).a Tl hdeorseefsooref,i wnfleu ceonnz-a
cvluirduesdc thhaallte PnMgeBi n+f eSc toior nS, cdoidm npoart eedxetortI Ndeitmrimmeunntiazla etiffoenctws iothn itnhaec tnivaasatel dminemflubernanzae vainrdu s
oalflaocnteo.rFyu brtuhlebr, maot rlee,aIsNt waditmh inreisstpraetcito ntoo  tfhPeM  imB malounneizcaatuiosned pdriosteoacsoel purosegdre  sinsi othnea npdredseeantth 
stoufdmy.i ce in all cases, as in naïve mice. Although we have not conducted experiments on
SARS-CoV-2 infection, we assume the results will be similar to those of influenza.
 
Figure 4. Histopathology of the nasal mucosa of immunized mice. Nasal mucosa (pseudostratified 
ciFliiagtuerde e4p. itHheislituompa tahnodl ooglfyacotfotrhye enpaitshaellimumuc)o asnado of lifmacmtourny ibzeudlbms oicf ei.mNmausnailzmedu cmoiscae (ipns tehued So s(tIrNa)t i-
(afi,ded,g)c,i lPiaMteBd +e Sp i(tIhNe)li u(bm,e,ahn),d oor lSfa (cStCor)y (ce,pf,iit)h gerloiuumps) wanedreo elfxaacmtoirnyedb uulsbinsgo fhiemmmatuonxiyzleind-emosicine (inHEth) e
stSai(nINin)g(.a B,da,rg; )2,0P MµmB. + S (IN) (b,e,h), or S (SC) (c,f,i) groups were examined using hematoxylin-eosin
(HE) staining. Bar; 20 µm.
The safe attainment of a sufficient IgA Ab concentration on the mucosal membrane 
is impPoMrtaBnits faonr atmhep phripohpihliyclasxtrius cotuf rmeucoconssaislltyin tgraonfsamhiyttderdo pinhfielicctihoenasd. Roefcpeonltylpy,e pmtiudceoasanld 
IgaAh yAdbros pahsosobciciattaeidl  owf aitlhk yinl ctrhaaminuss[c2u]l.aTr hmeRPNMAB- vviarcucsinaanttiiogne nhcaos mbpeelenx disetpercetseudm  [3e5d–t3o7b].e 
Hfoorwmeevderb, ythteh efrheyqdureonpt hborbeaickitnhtreoruagchti oinnfoefcttihoensh yodf rvoaprhiaonbtisc [t1a0il–o13f ]t hinedPiMcaBtew thitaht tthhee vIgirAu s
Aabn tliegveenls.  Tinh emcuocmopsalel xtihssausetsh eelvicirituesda bnyti gthene minRthNeAce vnatcecrianneds wisesruer rinosuunffidecdiebnyt fhoyrd proropphhoyb-ic
lataxiilss oof nthteh einvfeircutisonan. Ttihguens, sINid eimanmduhnyizdartoiopnh iisli rcehqeuaidresdo tno tahcehiseovlev esnutffiscidieen.t Pleavretilcsl eosf IwgAith 
Aab1s 0i0n– t5h0e0 nnamsald miaumcoetsear. Tarhee croensusildtse oref dthseu pitraebsleenfto srtuinddyu schionwg  mthuatc oPsMalBi mis ma upnroitmy iisninIgN 
mimucmosuanli azadtjiuovna[n2t9 c]a. nFduirdthaeterm foorr eIN, o vuarcpcrinevatioiouns wstiutdh iaens  uinsaincgtivsuatrefadc vtairnutss acnodmOpoVnAensut. ggest
thatStahfeetdyi acmonecteerrnosf thhaevseu arlfsaoc tnanect-eOssVitAatceodm tphele xexapffleocrtastitohne ior fa daljuvanticity [24]. Althoughwe did not co nfirm the transfe  r of P  M B-virus antige  n co mplexe
tserinnatotivmeu ocpotsiaolntsi sfsoure IsNin 
imthme upnreizsa  e
t
 n
iot n st
.uBdayc  ,
teprrial evi
toouxsinfisns  d
uicnhga  s
socnhothle  e
raretloax  ti
ionn(sChTip) ebleictwit   e
peonte  p
nat rm  tic
ulceodsaial madej  t
uervaann-d
tiacditjyuv[3a8n]t.icItithyassubgegeenstrtehpaotrtthede dthiaamt tehteermoufcaossualitaadbjluevPaMntBic-ivtyiruosf aCnTtiwgeans caopmprpolxeixmwatoeulyld 
affect vaccine efficacy.
 
Vaccines 2023, 11, 1727 10 of 13
Mucosal immunization with PMB induces specific IgA Ab responses in diverse mu-
cosal tissues. Exposure to antigens via the mucosal route leads to the generation of specific
IgA responses both locally and at remote mucosal sites [45]. However, as the mucosal
immune system exhibits compartmentalization, mucosal immunity is not always induced
equally across different mucosal tissues; for example, IN immunization primarily elicits Ab
responses in the upper respiratory tract and cervicovaginal mucosa, whereas the gut is less
likely to evoke such immune responses [46,47]. Nevertheless, our previous investigation
involving mice immunized intranasally with PMB and OVA demonstrated the presence of
OVA-specific IgA Abs in not just the NWs and VWs but in the fecal extracts and saliva as
well [2]. Similarly, in the present study, mucosal immunization resulted in the detection
of specific IgA Abs in mucosal tissues, which is typically challenging to stimulate via
IN immunization. These findings suggest that, in addition to its potential effectiveness
against respiratory tract infections, IN vaccination using PMB may be effective against gut
infections and sexually transmitted diseases.
PMB did not necessarily exhibit adjuvanticity for all three virus proteins; the focus
of the present study (influenza HA and SARS-CoV-2 S1 subunit and S protein) may not
necessarily apply to all virus proteins. Mixing antigen and PMB results in a particular
particle diameter, but why particles of that diameter are formed (the factors that deter-
mine the diameter) remains unknown. We measured particle diameters in solutions of
OVA mixed with 31 different surfactants but did not identify factors that determine the
diameter [24,48]. Thus, the inability to artificially adjust the diameter of the PMB-protein
complex is a limitation of PMB. In addition, the present study lacks an investigation into
the long-term effects and durability of the immune response induced by PMB as a mucosal
adjuvant. Further, the results obtained from the BALB/c mice model may not directly
translate to human responses. Additionally, the potential side effects or safety concerns
associated with the use of PMB as a mucosal adjuvant need further evaluation.
The present study has two innovations. First, we demonstrated that compounds with
mucosal adjuvanticity are present in clinically used and safe drugs. Second, we applied
the findings on nasal drug delivery systems to developing mucosal adjuvants. In the
present study, we demonstrated that the physicochemical properties (particle diameter)
of the antigen-adjuvant complex affect the adjuvant action. This finding will facilitate the
prediction of adjuvanticity by measuring particle diameter in vitro, thus contributing to a
reduction in the experimental animals. Furthermore, this study highlights the potential
application of drug repositioning strategies in adjuvant discovery to develop safe vaccines.
4. Conclusions
We demonstrated that PMB significantly increased the production of antigen-specific
IgA Abs in the various mucosal secretions of immunized mice, indicating the mucosal
adjuvanticity of PMB for influenza HA and SARS-CoV-2 S proteins. Furthermore, we
detected a relationship between the dominant diameter of the PMB-virus protein complex
and mucosal adjuvanticity. These findings have broadened our previous observations
regarding the mucosal adjuvanticity of PMB for OVA and the relationship between the
diameter of the PMB-OVA complex and mucosal adjuvanticity. Consequently, PMB may be
suitable for use in influenza and COVID-19 intranasal vaccinations.
Supplementary Materials: The following supporting information can be downloaded at https://
www.mdpi.com/article/10.3390/vaccines11111727/s1. Figure S1: Quantity of S1-specific antibodies
(Abs) in mice immunized with 1 µg of S1 subunit; Figure S2: Quantity of S1-specific Abs in mice
immunized with 10 µg of S1 subunit; Figure S3: Quantity of S-specific Abs in mice immunized with
10 µg of S1 subunit; Figure S4: Quantity of S1-specific Abs in mice immunized with 1 µg of S protein.
Vaccines 2023, 11, 1727 11 of 13
Author Contributions: Conceptualization: N.Y., W.H. and Y.M.; methodology: N.Y., T.Y., H.S., I.S.
and T.S.; validation: T.O., M.K. and T.S.; formal analysis: N.Y., T.Y. and T.S.; resources: W.H. and
H.S.; writing—original draft preparation: Y.M.; writing—review and editing: N.Y., T.Y. and Y.M.;
supervision and project administration: T.S. and Y.M.; funding acquisition: N.Y., W.H. and Y.M. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was supported in part by JSPS KAKENHI (Grant numbers JP21K08511 and
JP23K07948) and the 2021 Research and Development Project for COVID-19 and Other Infectious
Diseases through Industry-Academia-Government Collaboration in Iwate Prefecture. The funders
had no role in the study design, data collection, data analysis, data interpretation, or writing of
the report.
Institutional Review Board Statement: This study was conducted in accordance with the Declaration
of Helsinki and approved by the Committee on the Ethics of Animal Experiments (CEAE) of Iwate
Medical University (Permit No. 02-013).
Informed Consent Statement: Not applicable.
Data Availability Statement: The corresponding author had full access to all the data in the study.
Acknowledgments: We thank Kohei Shigyo (Research Foundation for Microbial Diseases of Osaka
University (BIKEN), Kagawa, Japan) for providing us with the influenza HA split. We thank Sumiko
Yaegashi (Iwate Medical University) for the technical assistance.
Conflicts of Interest: Authors Hironori Sakai and Wataru Hojo are employed by the company R&D,
Cellspect Co., Ltd. The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential conflict of interest.
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