Polymeric caffeic acid is a safer mucosal adjuvant that augments

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Polymeric caffeic acid is a safer mucosal adjuvant that augments antigen-specific mucosal and systemic immune responses in mice Rui Tada, Daisuke Yamanaka, Miki Ogasawara, Momoko Saito, Naohito Ohno, Hiroshi Kiyono, Jun Kunisawa, and Yukihiko Aramaki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00648 • Publication Date (Web): 14 Aug 2018 Downloaded from http://pubs.acs.org on August 16, 2018

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Molecular Pharmaceutics

Tada et al.

1

Polymeric caffeic acid is a safer mucosal adjuvant

2

that augments antigen-specific mucosal and

3

systemic immune responses in mice

4 5

AUTHOR NAMES

6

Rui Tada*,†, Daisuke Yamanaka ‡, Miki Ogasawara † , Momoko Saito † , Naohito Ohno ‡,

7

Hiroshi Kiyono§, Jun Kunisawa§,⊥, and Yukihiko Aramaki†

8 9

AUTHOR ADDRESS

10

†

11

Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan

12

‡

13

University of Pharmacy and Life Sciences, Tokyo, Japan

14

§

15

Mucosal Vaccines, Department of Microbiology and Immunology, The Institute of Medical

16

Science, The University of Tokyo, Tokyo, Japan

17

⊥

18

and Nutrition (NIBIOHN), Osaka, Japan

Department of Drug Delivery and Molecular Biopharmaceutics, School of Pharmacy,

Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo

Division of Mucosal Immunology and International Research and Development Center for

Laboratory of Vaccine Materials, National Institutes of Biomedical Innovation, Health

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Tada et al. 20

ABSTRACT

21

Infections remain a major threat to human lives. To overcome the threat caused by

22

pathogens, mucosal vaccines are considered a promising strategy. However, no inactivated

23

and/or subunit mucosal vaccine has been approved for human use, largely because of the

24

lack of a safe and effective mucosal adjuvant. Here, we show that enzymatically

25

synthesized polymeric caffeic acid (pCA) can act as a potent mucosal adjuvant in mice.

26

Intranasal administration of ovalbumin (OVA) in combination with pCA resulted in the

27

induction of OVA-specific mucosal IgA and serum IgG, especially IgG1. Importantly, pCA

28

was synthesized from caffeic acid and horseradish peroxidase from coffee beans and

29

horseradish, respectively, which are commonly consumed. Therefore, pCA is believed to be

30

a highly safe material. In fact, administration of pCA did not show no distinct toxicity in

31

mice. These data indicate that pCA has merit for use as a mucosal adjuvant for nasal

32

vaccine formulations.

33 34

KEYWORDS

35

caffeic acid, functional foods, lignin, mucosal adjuvant, nasal vaccine, polyphenol

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Tada et al. 36

INTRODUCTION

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Phenolic compounds (also termed as polyphenols) are substances found

38

predominantly in plants, such as fruits, vegetables, and coffee. We ingest large amounts (as

39

high as 1 g/day/person) of polyphenols in our daily life

40

have garnered attention because of their beneficial effects on human health. For instance,

41

daily intake of polyphenols has been reported to prevent cardiovascular disease, cancer, and

42

infectious diseases

43

and their biological activities have been extensively studied. Classically, polyphenols have

44

been reported to show antioxidant activity

45

indicates that polyphenols also exert immune-modulating effects on various immune cells.

46

Namely, low-molecular weight polyphenols such as curcumin and epigallocatechin gallate

47

show anti-inflammatory properties via the inhibition of mitogen-activated protein kinases

48

(MAPKs) and nuclear factor-κB (NF-κB)

49

polyphenols, represented by lignin, which is synthesized through the enzymatic

50

polymerization of phenolic compounds, show anti-viral and anti-bacterial activities that are

51

accomplished

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low-molecular weight polyphenols, lignin-rich fractions from natural products possess

53

complex structures composed of lignins, carbohydrates, and proteins, and exhibit

54

immune-enhancing effects18. Since these polyphenols possess complex components other

1, 2

. In recent years, polyphenols

3-5

. A variety of polyphenols have been isolated from different sources,

through

immune

6-8

. Beyond antioxidant activity, recent research

9-11

enhancement

. Meanwhile, high-molecular weight

12-17

.

Unlike


the

above-mentioned


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Tada et al. 55

than polyphenols, it is difficult to conclude whether the lignin moiety itself is responsible

56

for the immune-enhancing activities. With this in mind, we have long been studying the

57

immune-modulating effects of enzymatically synthesized lignin-like polyphenols, in order

58

to investigate the role of the polyphenol moiety in the immunostimulatory activity of lignin.

59

We have used the lignin-like polymer, designated as pCA, which is enzymatically

60

synthesized in vitro using the horseradish peroxidase (HRP) from phenylpropanoids

61

including caffeic acid (CA). Unlike natural lignin, the in vitro synthesized polymer pCA

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contains neither cellulose, hemicellulose nor bacterial endotoxin. Our studies showed that

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(1) orally administrated pCA augments the cytotoxic activity of natural killer cells (NK

64

cells) in vivo, analyzed by FACS in YAC-1 target cells

65

only the production of cytokines such as interferon-γ (IFN-γ) and interleukin-2 from

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splenocytes in vitro, but also induces the production of tumor necrosis factor-α (TNF-α)

67

from bone marrow-derived dendritic cells (BMDCs) in vitro

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high-molecular weight polyphenols are capable of activating an innate immune response.

69

On the basis of these finding and the reported anti-infectious activities of polyphenols, we

70

hypothesized that polyphenols may be used as safe and effective mucosal adjuvants.

19

, and that (2) pCA induces not

20-22

, showing that lignin-like

71

Despite the significant progress resulting from modern medicine, infections are

72

primary threats to human live and at least the second leading cause of death today 23, 24. One

73

of the major reasons why control of life-threatening infectious diseases remains a great 4 ACS Paragon Plus Environment

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Tada et al. 74

challenge is the lack of mucosal vaccines. Vaccination has long been a fundamental

75

approach to preventing and/or treating infections. In general, immunizations have been

76

achieved by parenteral injections (subcutaneous and intramuscular) of vaccines containing

77

antigens and adjuvants. The vaccines induce antigen-specific immune responses in the

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systemic but not mucosal compartments, which serve as the site of entry and/or colonization

79

for most pathogens. Compared with parenteral vaccination, recently emerging mucosal

80

vaccines have been shown to elicit protective immune responses in both the systemic and

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mucosal compartments

82

against infections caused by most pathogenic microbes. However, no inactivated and/or

83

subunit mucosal vaccine is now approved for clinical use, largely because of the lack of a

84

safe and effective mucosal adjuvant. Co-administration of mucosal adjuvants is required for

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the effective induction of an antigen-specific immune response because of the inherently

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poor immunogenicity of antigenic proteins administered by the mucosal route, such as a

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nasal route

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microbe-derived substances, namely cholera toxin (CT), oligodeoxynucleotides containing

89

immunostimulatory CpG motifs (CpG ODNs), and monophosphoryl lipid A (MPL), which

90

may provoke adverse effects because of molecules derived from the pathogenic microbes

91

30-32

92

vaccines.

25-28

. Therefore, mucosal vaccines are a promising strategy to fight

29

. Mucosal adjuvants that have been reported to date are almost all

. Hence, a safe and effective mucosal adjuvant is needed for the delivery of mucosal



Tada et al. 93

Thus, the aim of this study was to develop a safe and effective mucosal vaccine

94

adjuvant from polyphenols. We synthesized polymeric caffeic acid (termed as pCA) from

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CA and HRP from coffee beans and horseradish, respectively, which are commonly

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consumed food products. Therefore, because both molecules are derived from the edible

97

ingredients, our synthetic material is expected to completely safe. To test our hypothesis

98

that polyphenols can exert adjuvant effects, we evaluated the mucosal adjuvant activity of

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pCA administered intranasally with ovalbumin (OVA) as a model antigenic protein by

100

measuring OVA-specific antibody production in both mucosal and systemic compartments

101

in mice. Moreover, we tested the in vivo safety of pCA.

102 103

Materials and methods

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Animals and materials. Female BALB/cCrSlc mice (6 weeks old) were purchased from

105

Japan SLC (Hamamatsu, Shizuoka, Japan). Animals were housed in a specific

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pathogen-free environment and used at 7–10 weeks of age. All animal experiments were

107

performed in accordance with the guidelines for laboratory animal experiments of the

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Tokyo University of Pharmacy and Life Sciences, and each experimental protocol was

109

approved by the institution’s committee for laboratory animal experiments (P15-33 and

110

P16-12). HRP was purchased from Sigma-Aldrich (St. Louis, MO, USA) and

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3-(3,4-dihydroxyphenyl)-2-propenoic acid (commonly termed as caffeic acid; CA) was 6 ACS Paragon Plus Environment

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purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Low endotoxin (less

113

than 1 EU/mg) egg white OVA and cholera toxin was obtained from Wako Pure Chemical

114

Industries (Osaka, Japan). 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) and

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3β-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol (DC-chol) were purchased

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from Avanti Polar Lipids (Alabaster, AL, USA).

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Preparation of a polymeric caffeic acid. A lignin-like polymeric caffeic acid was

119

synthesized using HRP from CA and H2O2 via oxidative polymerization, as we reported

120

previously (Figure 1) 22. Briefly, 200 mg of CA was neutralized with 1 M NaOH and diluted

121

to 10 mL with phosphate-buffered saline (PBS) containing 1 mg of HRP. Then 1.5 mol eq

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H2O2 to CA was added drop wise to the mixture of CA and HRP solution while stirring at

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25˚C for 1 h. This reaction mixture was stirred for another 2 h at room temperature and then

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heated for 20 min at 100 °C to inactivate and precipitate the HRP. After centrifugation, the

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supernatant was collected and dialyzed (MWCO 50,000) against deionized water for 2 days

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and then lyophilized to obtain the polymerized caffeic acid (pCA). This preparation was

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tested for endotoxin contaminants using an Endospecy ES-50M kit (Seikagaku Biobusiness

128

Corporation; Tokyo, Japan), which indicated that the endotoxin content in pCA was very

129

low (231.5 pg/mg). All samples were dissolved in endotoxin-free PBS (Wako Pure

130

Chemical Industries) to create a stock (10 mg/mL) that was sterilized by filtration through 7 ACS Paragon Plus Environment


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0.45-µm filter membranes (Osaka Chemical Co., Ltd., Osaka, Japan). The stock solution

132

was stored at –20°C until use.

133 134

Figure 1. Scheme for preparation and the proposed structure of enzymatically

135

polymerized caffeic acid

136 137 138 139

Preparation of liposomes DOTAP/DC-chol liposomes were prepared as follows

33, 34

. Ten micromoles of

total lipids (DOTAP/DC-chol at a 1:1 mol ratio) dissolved in chloroform were evaporated to 8 ACS Paragon Plus Environment

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dryness to obtain lipid films. The lipid films were then hydrated in 250 µL of

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phosphate-buffered saline (PBS) and vortexed for 5 min. The prepared liposomes were

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extruded 10 times by passing them through a polycarbonate membrane of appropriate pore

143

size (Advantec, Tokyo, Japan) and sterilized via filtration (0.45-µm filter membranes; Iwaki,

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Tokyo, Japan).

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Intranasal immunization of OVA plus pCA and sampling schedule. Mice were divided

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into three groups and immunized intranasally as follows under anesthesia with

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intraperitoneal injection of 0.2 mL of a mixture containing 0.75 mg/kg of medetomidine, 4

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mg/kg of midazolam, and 5 mg/kg of butorphanol tartrate. The treatment groups were the

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following: 1) PBS alone, 2) OVA alone (2.5 µg/mouse), or 3) OVA (2.5 µg/mouse) in

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combination with pCA (100 µg/mouse), and administration volumes were 6.5 µl/nostril.

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Each group of mice was immunized once weekly on days 0, 7, and 14. Blood samples were

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collected weekly via the tail vein before immunization (days 0, 7, 14, and 21). The blood

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was allowed to clot at 25°C for 30 min, followed by incubation at 4°C for 60 min, and then

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serum was separated by centrifugation at 1200 × g for 30 min. Nasal wash fluid (NW),

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bronchoalveolar lavage fluid (BALF), and vaginal wash fluid (VW) were collected in 200

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µL, 1 mL, and 100 µL of cold PBS, respectively

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until analysis by enzyme-linked immunosorbent assays (ELISAs) as described below.

33, 35, 36

. All samples were stored at –80°C



Tada et al. 159 160

ELISA for evaluating anti-OVA antibody titer in serum and mucosal fluids. For

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ELISA, 96-well Nunc MaxiSorp plates (Thermo Scientific, Waltham, MA, USA) were

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coated with 1.25 µg of OVA dissolved in 0.1 M carbonate buffer (pH 9.5) and incubated

163

overnight at 4°C. The plates were then washed with PBS containing 0.05% Tween 20

164

(PBST) and blocked with 1% bovine serum albumin (BSA; Wako Pure Chemical

165

Industries) containing PBST (BPBST) at 37°C for 60 min. The plates were washed with

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PBST, incubated with samples for 60 min at 37°C, washed again with PBST, and then

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treated with peroxidase-conjugated anti-mouse IgA, IgG, IgG1, or IgG2a secondary

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antibody (SouthernBiotech; Alabama, USA) in BPBST and developed using a

169

tetramethylbenzidine (TMB) substrate system (KPL, Maryland, USA). Color development

170

was terminated using 1 N phosphoric acid, and optical densities were measured at 450 nm

171

(reference filter 650 nm) 34. The endpoint titers were calculated as the reciprocal of the last

172

dilution reaching a cut-off value set to twice the mean optical density of a negative control

173

37, 38

.

174 175

Preparation of splenocytes for culture. Splenocytes were prepared as described

176

previously

177

sterile mesh screen using forceps, and homogenized in RPMI 1640 medium (Wako Pure

39-41

. Briefly, the spleens of BALB/c female mice were excised, placed on a


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Tada et al. 178

Chemical Industries). The obtained cell mixture was filtered through a nylon mesh,

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collected in 15-ml tubes, and centrifuged. The single-cell suspension obtained was treated

180

with ACK lysis buffer to lyse red blood cells. After centrifugation, splenocytes were

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maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine

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serum (FBS; GE Healthcare, Chicago, Il, USA), 100 µg/mL of streptomycin sulfate salt

183

(Sigma-Aldrich), and 100 U/mL of penicillin G potassium salt (Sigma-Aldrich). Cell

184

numbers were determined by the trypan blue exclusion assay using a hemocytometer. The

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cells were cultured at a density of 2 × 106 cells/well in a medium volume of 0.5 mL in

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48-well flat-bottom plates (Thermo Scientific) and restimulated with endotoxin-free OVA

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for 72 h at 37°C in an atmosphere containing 5% CO2.

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Cytokine assay. The cytokine concentrations in the samples were determined using ELISA

190

MAXTM Standard Sets (BioLegend, San Diego, CA, USA) according to the manufacturer’s

191

instructions. The data are expressed as the mean ± standard deviation from assays

192

performed in triplicate.

193 194

Total RNA extraction and quantitative real time-polymerase chain reaction (qPCR).

195

The expression of genes associated with an inflammatory response to evaluate whether

196

nasal administration of pCA promotes inflammation at the site of injection was quantified



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by qPCR experiments as follows: total RNA from nasal tissues were extracted using a

198

FavorPrep Tissue Total RNA Mini Kit (Favorgen Biotech Corporation, Ping-Tung, Taiwan)

199

followed by DNase I (Roche Life Science, Penzberg, Germany) digestion. RNA

200

concentrations in samples were quantified by spectrophotometry, and cDNAs were

201

synthesized from 0.5 µg total RNA in 10 µl reaction mixtures using ReverTra Ace qPCR

202

RT Master Mix (Toyobo, Tokyo, Japan), according to the manufacturer’s instructions, by

203

sequentially subjecting the samples to 37˚C for 15 min, 50˚C for 5 min, and 98˚C for 5 min.

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Then, qPCR was carried out according to the manufacturer’s instructions using the

205

Thunderbird SYBR qPCR Mix (Toyobo) with CFX Connect Real-Time PCR Detection

206

System (BIO-RAD, Hercules, CA, USA), by subjecting the samples to 95˚C for 1 min,

207

followed by 40 cycles of 15 sec at 95˚C and 1 min at 60˚C. The following primers were

208

synthesized

209

5′-TACAAGGAGAACCAAGCAACGAC-3′

210

5′-TGCCGTCTTTCATTACACAGGAC-3′

211

5′-TTCTGGTGCTTGTCTCACTGA-3′

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5′-CAGTATGTTCGGCTTCCCATTC-3′ (reverse). Data were analyzed with CFX manager

213

software version 3.1, and cycle threshold (Ct) values were obtained. The levels of IL-1β

214

expression in nasal tissues were normalized to the reference gene B2M. qPCR reactions on

215

each template were run in technical duplicates and specific amplification was confirmed by

by

Sigma

Genosys

(Tokyo,

Japan):

(forward) (reverse);

β2-microglobulin (forward)


IL-1β, and (B2M), and

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Tada et al. 216

melting curve analysis. Relative gene expression fold changes were calculated using the

217

comparative Ct (∆∆Ct) method.

218 219

Examination of leukocyte migration to the nasal tissue. Mice were nasally administered

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PBS (as vehicle) or pCA (100 µg/mouse). At 6 or 24 h after administration, the mice were

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sacrificed with intraperitoneal injection of sodium pentobarbital (250 mg/kg; Tokyo

222

Chemical Industry Co., Ltd., Tokyo, Japan). For the histological analysis, after the removal

223

of irrelevant tissues, skulls were fixed in 4% paraformaldehyde (PFA) in PBS for 24 h at

224

25°C. After washing with deionized water thrice, skulls were decalcified for 5 days at 4°C

225

in 10% EDTA solution (pH 7.4). After substitution with 30% sucrose in PBS, noses

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proximal to nasal-associated lymphoid tissues (NALTs) were cut and then flash-frozen in

227

OCT compound (Sakura Finetek Japan, Tokyo, Japan) on dry ice and cut into 8-µm

228

cryosections. The obtained cryosections were then stained with hematoxylin and eosin (HE)

229

and examined with a BZ-8100 fluorescent microscope (Keyence, Tokyo, Japan).

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For the flow cytometric analysis, nasal tissues were homogenized in PBS and then

231

centrifuged. Single cell suspensions were obtained by treatment with RBC lysis buffer

232

(BioLegend) to lyse red blood cells. The cells were incubated with anti-mouse CD16/CD32

233

(Tonbo Biosciences, San Diego, CA, USA) for 20 min on ice to block Fc receptors. After

234

washing with staining buffer (PBS containing 2% heat-inactivated FBS and 0.1% sodium 13 ACS Paragon Plus Environment


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azide), the cells were stained with PE/Cy7 anti-mouse CD45 (BioLegend), APC anti-mouse

236

CD11b (BioLegend), and FITC anti-mouse Ly6G (BioLegend) for neutrophils or the

237

respective isotype control. The samples were analyzed using a FACSCanto instrument (BD

238

Biosciences, Franklin Lakes, NJ, USA).

239 240

Statistical analysis. Statistical differences were assessed using the Mann–Whitney U test

241

and unpaired t-test with Welch’s correction or one-way ANOVA with post-hoc Tukey test

242

for antibody and cytokine/gene expression levels, respectively. A two-way ANOVA with

243

Bonferroni post-hoc test was used for body weight loss. P values less than 0.05 were

244

considered significant. All data were analyzed using GraphPad Prism 7 software (GraphPad

245

Software, La Jolla, CA, USA).

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RESULTS

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Evaluation of the production of antigen-specific mucosal and systemic antibodies

249

induced by intranasal immunization of OVA with pCA. We first investigated the

250

mucosal adjuvant effect of pCA when administered intranasally with antigenic protein,

251

because we recently reported that lignin-like polymerized polyphenols can activate immune

252

cells,

253

cytokines/chemokines in leukocytes, namely IL-1α, IL-6, and IL-12

including

dendritic

cells

(DCs),

and

subsequently


induce 20-22

various

. In the present

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Tada et al. 254

study, we evaluated the production of OVA-specific antibodies after intranasal

255

immunization of OVA in combination with pCA in BALB/c female mice. Our preliminary

256

investigation of dose-dependency in the mucosal adjuvant effects of pCA showed that 100

257

µg of pCA was required for the induction of maximum antigen-specific antibody responses.

258

Accordingly, in further studies, we examined the mucosal adjuvant activities of pCA at a

259

dose of 100 µg/mouse. As shown in Figure 2, intranasal immunization of pCA and OVA

260

promoted the production of OVA-specific nasal and vaginal IgA (endpoint titer with median

261

value: 180.2 and 543.9, respectively) compared to that in mice administered PBS (as a

262

vehicle) or OVA alone. In contrast, intranasal immunization with OVA and pCA induced

263

relatively low OVA-specific IgA and IgG levels in BALF (endpoint titer with median value

264

of 5.2 and 2695, respectively).

265

Additionally, pCA also induced significant OVA-specific IgG responses in serum

266

(endpoint titer with median value: 2.0 × 106) compared to those in mice administered PBS

267

(as a vehicle) or OVA alone (endpoint titer with median value: 0 and 4.9 × 105,

268

respectively) on day 21. We next examined the patterns of IgG subclasses that appeared in

269

serum to determine the types of immune responses induced by nasally administration of

270

pCA. Sera from mice immunized with OVA plus pCA showed high titers of OVA-specific

271

IgG1 (endpoint titer with median value: 4.0 × 106) and low titers of IgG2a (endpoint titer

272

with median value: 3339) on day 21, suggesting that intranasal immunization with pCA 15 ACS Paragon Plus Environment


Tada et al. 42

273

polarized immune response to a Th2 type

274

OVA-specific IgG expression in sera at different time points in mice immunized once per

275

week. On day 14 after the first immunization, OVA-specific IgG, IgG1, and IgG2a were

276

detected, with increasing antibody titers observed at day 21 (Figure 3). Cumulatively, these

277

data showed for the first time that pCA acts as mucosal adjuvant in mice.

. We then evaluated the kinetics of

278 279

Figure 2. Induction of OVA-specific mucosal IgA and IgG responses in BALB/c female

280

mice immunized intranasally with OVA plus pCA. BALB/c female mice were

281

immunized intranasally with PBS, OVA (2.5 µg/mouse) alone, or OVA (2.5 µg/mouse) plus

282

pCA (100 µg/mouse) on days 0, 7, and 14. Nasal washes, BALF, and vaginal washes were 16 ACS Paragon Plus Environment

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Tada et al. 283

collected on day 21. OVA-specific antibodies were detected by ELISA assay. The data were

284

obtained from three biologically independent experiments. PBS, n = 8; OVA, n = 8; OVA

285

plus pCA, n = 8. The box-plot shows median values with 25th–75th percentiles; error bars

286

indicate 5th–95th percentiles. Significance was assessed with the Mann–Whitney U test: *p

287

< 0.05.

288 289

Figure 3. Kinetics of the production of OVA-specific serum IgGs induced by nasally

290

administered OVA in combination with pCA in BALB/c female mice. BALB/c female

291

mice were immunized intranasally with PBS, OVA (2.5 µg/mouse) alone, or OVA (2.5

292

µg/mouse) plus pCA (100 µg/mouse) on days 0, 7, and 14. Serum samples were obtained on 17 ACS Paragon Plus Environment


Tada et al. 293

days 0, 7, 14, and 21. OVA-specific serum IgGs were evaluated by ELISA assay. The data

294

were obtained from three independent experiments and are expressed as the mean ±

295

standard error. PBS, n = 8; OVA, n = 8; OVA plus pCA, n = 8. Significance was assessed

296

with the Mann–Whitney U test: *p < 0.05.

297 298

Comparison of the mucosal adjuvant effect of pCA with an experimental mucosal

299

adjuvant, CT

300

In order to examine the efficacy of pCA as a mucosal adjuvant, we compared the

301

mucosal adjuvant effects of pCA and CT. Figure 4 shows that the mucosal adjuvant effect

302

of pCA is relatively low when compared to CT (median values were 180.2 vs. 1769, 543.9

303

vs. 713.9, and 1.7 × 106 vs. 2.9 × 107, for nasal IgA, vaginal IgA, and serum IgG,

304

respectively), especially regarding the production of OVA-specific antibodies within the

305

lung compartment (median values were 5.2 vs. 156.5 and 2695 vs. 19,604, for BALF IgA

306

and BALF IgG, respectively).


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Tada et al.

307 308

Figure 4. Comparison of the mucosal adjuvant effect of pCA with CT

309

BALB/c female mice were immunized intranasally with OVA (2.5 µg/mouse)

310

plus pCA (100 µg/mouse) or OVA (2.5 µg/mouse) plus CT (1 µg/mouse) on days 0, 7, and

311

14. Serum samples were obtained on days 0, 7, 14, and 21. OVA-specific antibody

312

responses were evaluated by ELISA assay. The data were obtained from three independent

313

experiments. OVA plus pCA, n = 8; OVA plus CT, n = 8. The box-plot shows the mean ±

314

standard error. Significance was assessed with the Mann–Whitney U test: *p < 0.05, NS:

315

not significant (p > 0.05). 19 ACS Paragon Plus Environment


Tada et al. 316

Antigen-specific Th1/Th2 cytokine production by splenocytes in vitro. Intranasal

317

immunization with OVA plus pCA preferentially induced OVA-specific serum IgG1 in

318

serum, indicating that pCA may polarize immune responses to a Th2 type. To further assess

319

the type of immune response evoked by pCA, we investigated the production of IFN-γ/Th1

320

and IL-4/Th2 by splenocytes derived from mice nasally administered pCA in combination

321

with OVA that were then re-stimulated with OVA in vitro. Splenocytes from mice

322

vaccinated with OVA and pCA showed high levels of IL-4 secretion compared to

323

splenocytes from mice vaccination with OVA alone. In addition, splenocytes from OVA

324

and pCA-vaccinated mice also secreted high levels of IFN-γ, indicating that pCA may also

325

induce a cytotoxic T lymphocyte (CTL) response when administered nasally with an

326

antigenic protein (Figure 5). These results, together with those of serum IgG levels, reveal

327

that pCA induces a mixed Th1/Th2 immune response.

328 20 ACS Paragon Plus Environment

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Tada et al. 329

Figure 5. Antigen-specific cytokine production in splenocytes from OVA- and

330

pCA-immunized BALB/c mice in vitro. Splenocytes from vaccinated mice were cultured

331

for 72 h with OVA (10 µg/mL). After culture, the culture supernatants were collected and

332

concentrations of released cytokines were determined by ELISA assay. Three independent

333

experiments were conducted, and the data are expressed as means ± standard errors. PBS, n

334

= 6; OVA, n = 6; OVA plus pCA, n = 6. Significant differences were calculated by t-test

335

with Welch’s correction: *p < 0.05.

336 337

In vivo safety assessment of mice nasally vaccinated with pCA. Finally, we assessed the

338

toxicity of pCA in vivo to confirm the safety of pCA in mice. To evaluate the toxicity

339

induced by intranasal administration of pCA, we assessed the following: 1) body weight

340

loss during the experiment, 2) expression of genes associated with inflammation, including

341

interleukin-1β (IL-1β), at the site of injection 16 h after pCA administration, and 3) the

342

infiltration of neutrophils at the site of administration. There were no differences in body

343

weights observed in pCA group when compared to PBS group (Figure 6A). Additionally,

344

intranasal administration of pCA did not induce expression of IL-1β in nasal tissue (Figure

345

6B). As polymorphonuclear leukocytes, such as neutrophils, are the major cell types

346

involved in acute inflammation, these cells are recruited to the inflammatory tissue

347

Once an inflammatory reaction occurs in the nasal tissue, infiltrated cells are seen in the 21 ACS Paragon Plus Environment

43-45

.


Tada et al. 46, 47

348

paranasal airspaces

349

mice that received pCA, similar to those that received PBS (Figure 6C). Furthermore, flow

350

cytometric analysis (Figure 6D) indicated that nasal administration of pCA led to a

351

moderate increase the number of neutrophils (3.3% versus 6.9%; PBS and pCA,

352

respectively) (Figure 6D). However, this increase was considerably less than that in

353

response to cationic liposomes, which induce robust recruitment of neutrophils (27.9%) to

354

the site of administration (unpublished results). Taken together, pCA did not show evident

355

toxicity or induce inflammation in mice.

. HE staining showed no infiltrated cells in the nasal cavities of


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Tada et al.

356



Tada et al. 357

Figure 6. In vivo safety assessment of mice nasally vaccinated with pCA. (A) BALB/c

358

female mice were immunized intranasally with PBS, OVA (2.5 µg/mouse) alone, OVA (2.5

359

µg/mouse) plus pCA (100 µg/mouse), or OVA (2.5 µg/mouse) plus CT (1 µg/mouse) once

360

per week. Body weights were recorded over 3 weeks. PBS, n = 8; pCA, n = 8; CT, n = 8.

361

Significance was assessed by two-way repeated measures ANOVA with Bonferroni

362

post-hoc test. *p < 0.05. (B) BALB/c female mice were immunized intranasally with PBS,

363

OVA (2.5 µg/mouse) alone, OVA (2.5 µg/mouse) plus pCA (100 µg/mouse), or OVA (2.5

364

µg/mouse) plus CT (1 µg/mouse). Nasal tissues were collected 16 h after administration.

365

Gene expression was quantified by qPCR. PBS, n = 4; pCA, n = 4; CT, n = 4. Significant

366

differences were calculated by one-way ANOVA with post-hoc Tukey test. *p < 0.05. (C)

367

Hematoxylin and eosin (HE) staining of representative nasal tissues from BALB/c female

368

mice 7 h after intranasal administration with PBS, pCA (100 µg/mouse), or liposomes (400

369

nmol/mouse). PBS, n = 3; pCA, n = 3, liposomes, n = 3. (D) Neutrophil infiltration into

370

nasal tissues was analyzed by flow cytometric analysis. The cells from nasal tissues in

371

female BALB/c mice 6 or 24 h after they received PBS, pCA (100 µg/mouse), or liposomes

372

(400 nmol/mouse) were stained with anti-CD45, Ly6G, and CD11b. Graphs show the

373

percentages of Ly6G+ and CD11b+ neutrophils of CD45+ leukocytes. PBS, n = 4; pCA, n =

374

4, liposomes, n = 3. The data are expressed as means ± standard deviations. Significant


Page 24 of 50

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Tada et al. 375

differences were calculated by one-way ANOVA with post-hoc Tukey test. N.S.: not

376

significant (p > 0.05), *p < 0.05.



Tada et al. 377

DISCUSSION

378

Since our previous in vitro study on enzymatically synthesized polyphenols

379

showed that these polyphenols are capable of inducing various cytokines, such as IL-1α,

380

IL-2, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and macrophage

381

inflammatory proteins-1β (MIP-1β)

382

responses 48, we hypothesized that polyphenols have potential use as mucosal adjuvants. In

383

the present study, we showed that an enzymatically synthesized polyphenol acts as a

384

mucosal adjuvant in mice with no clear adverse effects. On the basis of these findings,

385

polyphenols show the potential for safe use in the formulation of nasal vaccines against

386

pathogens.

20

, which are involved in augmenting mucosal IgA

387

The induction of a mucosal immune response is considered to be the only

388

effective strategy to neutralize microbial pathogens in the mucosal compartment of a host,

389

especially upper respiratory tract, thereby preventing the entry of microbes into the host

390

tissues. However, only a few live-attenuated mucosal vaccine are now approved for clinical

391

use, despite the presence of numerous studies that have reported many experimental

392

mucosal vaccine systems against various infectious disease in literature 49-53. The successful

393

expansion of mucosal vaccines against deadly pathogens that are currently uncontrolled will

394

depend on the development of new, safe mucosal adjuvant technologies 54. With this aim,

395

we synthesized a polyphenol from caffeic acid and HRP (coffee beans and horseradish, 26 ACS Paragon Plus Environment

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Tada et al. 396

respectively) (Figure 1), because these are commonly consumed, suggesting that our

397

synthetic material would be safe when administered to mice.

398

Herein, we showed for the first time, to our knowledge, that intranasal vaccination

399

with an antigen, OVA, plus pCA induced high levels of OVA-specific antibody in both

400

systemic and mucosal compartments of mice (Figures 2 and 3). Generally, mucosal

401

adjuvants stimulate a Th2 type of T helper response. These responses are characterized by

402

increased sIgA expression, polarized antigen-specific serum IgG1 production, and IL-4

403

production in splenocytes re-stimulated with antigen

404

immunization with pCA promoted sIgA expression (Figure 2), induced higher production of

405

antigen-specific serum IgG1 (Figure 3), and IL-4 production in splenocytes re-stimulated

406

with OVA (Figure 5). At the same time, immunization of mice with pCA led to the

407

production of IFN-γ from splenocytes, implying that pCA also induced a Th1 type of

408

immune response. In the safety tests (Figure 6), intranasal administration of pCA did not

409

affect body weight or induce the expression of inflammatory genes, and promoted only

410

modest local infiltration of neutrophils (Figures 6C & D) in nasal tissue, clearly indicating

411

that pCA was not toxic to mice, as expected. Cumulatively, these data indicate that pCA

412

acts as potent mucosal adjuvant without apparent adverse effects on mice.

55,

56

. Likewise, intranasal

413

In the present study, the precise underlying mechanism(s) of the mucosal adjuvant

414

effects of pCA were not elucidated. In general, adjuvants confer their effects through the 27 ACS Paragon Plus Environment


Page 28 of 50

Tada et al. 415

following mechanisms: (1) sustained release of antigen complexed with adjuvants at the site

416

of injection, ensuring constant release and stimulation of an immune response, which is

417

called the ‘depot effect’, and (2) activation of the innate immune system, including the

418

activation of antigen-presenting cells (APCs) for enhanced antigen uptake and upregulation

419

of cytokines/chemokines, leading to the recruitment of immune cells to the injection site 57.

420

Our earlier studies showed that pCA has immunomodulating activities. Namely, we showed

421

that orally administrated pCA activates NK cells and induces cytokine production from

422

murine splenocytes in vitro

423

recognition receptors (PRRs), including toll-like receptors (TLRs), triggering the expression

424

of cytokine/chemokine production to enhance sIgA secretion

425

about the receptors for lignin-like high molecular weight polyphenols until now. However,

426

Tsuji et al. reported that lignin-carbohydrate complexes isolated from barley husk activate

427

DCs via TLR4

428

molecular patterns (PAMPs) to the mucosal adjuvant effects of pCA. Taken together, these

429

findings indicate that the mucosal adjuvant effects of pCA may be mediated by the

430

activation of the innate immune system via signaling through host PAMPs. We are currently

431

investigating this possibility. Our preliminary experiment has shown that intranasal

432

administration of pCA results in the expression of thymic stromal lymphopoietin (TSLP),

433

which plays a crucial role in the enhancement of sIgA induced by CT at the site of injection

19-22

. Additionally, most mucosal adjuvants target patttern

58-60

. Little has been known

18

. This suggests the possible contribution of several pathogen-associated


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Tada et al. 434

61, 62

435

response to pCA.

. Thus, we are further investigating the role of TSLP in the production of sIgA in

436

In this study, we clearly showed that an enzymatically polymerized polyphenol

437

can act as effective and safe mucosal adjuvant for nasal vaccine formulations. Although the

438

precise mechanism(s) underlying the mucosal adjuvant activities of pCA when intranasally

439

co-administered with an antigenic protein has not discovered, this system can be useful for

440

nasal vaccines to defeat infectious diseases caused by pathogenic microbes. Further studies

441

are ongoing for the development of mucosal vaccines against infection by Streptococcus

442

pneumoniae using pCA as a mucosal adjuvant.

443 444

CONCLUSIONS

445

In conclusion, we offer a novel mucosal adjuvant, enzymatically synthesized pCA,

446

for use in the development of highly safe nasal vaccine systems to combat infectious

447

diseases. Intranasal administration of pCA in combination with OVA greatly enhanced

448

OVA-specific antibody responses in both mucosal and systemic compartments. Of note, we

449

did not observe toxicity in mice administered pCA. We believe that this highly safe

450

adjuvant can be utilized as a novel platform for nasal vaccines; thus, pCA can be clinically

451

applied, especially for the prevention of certain infectious diseases.



Tada et al. 452

AUTHOR INFORMATION

453

Corresponding Author

454

*

e-mail, [email protected]; phone, 81-42-676-3219; Fax, 81-42-676-3182;

455 456

ORCID

457

Rui Tada: 0000-0003-0098-9587

458 459

Author contributions

460

Conceived and designed the experiments: RT DY. Performed the experiments: RT DY MO

461

MS. Analyzed the data: RT DY JK YA. Contributed reagents/materials/analysis tools: RT

462

DY HK NO JK YA. Prepared the manuscript: RT DY JK YA.

463 464

Notes

465

The authors declare no competing financial interest.

466 467

ACKNOWLEDGEMENT

468

This study was supported in part by JSPS KAKENHI Grant Number 15K18935

469

(Grant-in-Aid for Young Scientists (B) to RT), 18K06798 (Grant-in-Aid for Scientific

470

Research (C) to RT), 15K18701 (Grant-in-Aid for Young Scientists (B) to DY), 16K08415 30 ACS Paragon Plus Environment

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Tada et al. 471

(Grant-in-Aid for Scientific Research (C) to YA and RT), 16H01373 (Grant-in-Aid for

472

Scientific Research on Innovative Areas [research in a proposed research area] to JK), and

473

Research on Development of New Drugs by the Japan Agency for Medical Research and

474

Development (AMED) (17fk0108223h0002, 17ek0410032s0102, 17fk0108207h0002,

475

17ak0101068h0001, and 17gm1010006s0101 to JK). We are grateful to Akihiro Ohshima,

476

Yuya Tanasawa, and Saeko Takahashi for their technical assistance.

477 478

ABBREVIATIONS

479

APCs, antigen-presenting cells; ATP, adenosine triphosphate; BALF, bronchoalveolar

480

lavage fluid; BPBST, 1% bovine serum albumin (BSA; Wako Pure Chemical Industries)

481

containing

482

oligodeoxynucleotides containing immunostimulatory CpG motifs; CT, cholera toxin;

483

DAMPs,

484

3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol; DCs, dendritic cells; DOTAP,

485

1,2-Dioleoyl-3-trimethylammonium-propane;

486

assays; FBS, fetal bovine serum; MAPKs, mitogen-activated protein kinases; HMGB1,

487

high-mobility group box 1; HE, hematoxylin and eosin; HRP, horseradish peroxidase;

488

IFN-γ, interferon-γ; IL-1β, interleukin-1β; IL-4, interleukin-4; MPL, monophosphoryl lipid

489

A; NW, nasal wash fluid; NK cells, natural killer cells; NF-κB, nuclear factor-κB; OVA,

PBST;

B2M,

β2-microglobulin;

damage-associated

CA,

caffeic

molecular

ELISAs,

acid;

patterns;

enzyme-linked


CpG

ODNs,

DC-chol,

immunosorbent


Tada et al. 490

ovalbumin; PAMPs, pathogen-associated molecular patterns; PBS, phosphate-buffered

491

saline; PBST, PBS containing 0.05% Tween 20; pCA, polymerized caffeic acid; PFA,

492

paraformaldehyde; qPCR, real time-polymerase chain reaction; PRRs, patttern recognition

493

receptors; TLRs, toll-like receptors; TMB, tetramethylbenzidine; TNF-α, tumor necrosis

494

factor-α; TSLP, thymic stromal lymphopoietin; VW, vaginal wash fluid;


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Tada et al. 495

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Figure 1 149x204mm (300 x 300 DPI)


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Figure 2 145x182mm (300 x 300 DPI)



Figure 3 207x231mm (300 x 300 DPI)


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Figure 4 174x155mm (300 x 300 DPI)



Figure 5 133x86mm (300 x 300 DPI)


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Figure 6 259x358mm (300 x 300 DPI)



table of contents graphic 88x26mm (300 x 300 DPI)


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Polymeric caffeic acid is a safer mucosal adjuvant that augments

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