Potential of Translationally Controlled Tumor Protein-Derived Protein

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Potential of translationally controlled tumor protein-derived protein transduction domains as antigen carriers for nasal vaccine delivery Hae-duck Bae, Joohyun Lee, Xing-Hai Jin, and Kyunglim Lee Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00408 • Publication Date (Web): 25 Jul 2016 Downloaded from http://pubs.acs.org on July 28, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

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

Molecular Pharmaceutics

1

Potential of translationally controlled tumor protein-derived protein

2

transduction domains as antigen carriers for nasal vaccine delivery

3 4 5 6 7 Hae-duck Bae, Joohyun Lee, Xing-Hai Jin and Kyunglim Lee*

8 9 10

Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans

11

University, Seoul 03760, Korea

12 13 14 15 16 17 18 19

*

20

Kyunglim Lee

21

Graduate School of Pharmaceutical Sciences

22

College of Pharmacy

23

Ewha Womans University

24

Seoul 03760, Korea

25

Tel.: +82-2-3277-3024

26

Fax: +82-2-3277-2851

27

E-mail: [email protected]

Corresponding author:

1

ACS Paragon Plus Environment


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

28

ABSTRACT GRAPHIC

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 2


Page 2 of 34

Page 3 of 34

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


55

ABSTRACT

56 57

Nasal vaccination offers a promising alternative to intramuscular (i.m) vaccination,

58

because it can induce both mucosal and systemic immunity. However, its major

59

drawback is poor absorption of large antigens in the nasal epithelium. Protein

60

transduction domains (PTDs), also called cell-penetrating peptides, have been proposed

61

as vehicles for nasal delivery of therapeutic peptides and proteins. Here, we evaluated

62

the potential of a mutant PTD derived from translationally controlled tumor protein

63

(designated TCTP-PTD 13) as an antigen carrier for nasal vaccines. We first compared

64

the L- and

65

carriers. Studies in mice demonstrated that nasally administered mixtures of the model

66

antigen ovalbumin (OVA) and D-TCTP-PTD 13 induced higher plasma IgG titers and

67

secretory IgA levels in nasal washes than nasally administered OVA alone,

68

OVA/L-TCTP-PTD 13, or i.m-injected OVA. Plasma IgG subclass responses (IgG1 and

69

IgG2a) of mice nasally administered OVA/D-TCTP-PTD 13 showed that the

70

predominant IgG subclass was IgG1, indicating a Th2-biased immune response. We also

71

used synthetic CpG oligonucleotides (CpG) as a Th1 immune response-inducing

72

adjuvant. Nasally administered CpG plus OVA/D-TCTP-PTD 13 was superior in

73

eliciting systemic and mucosal immune responses compared to those induced by nasally

74

administered OVA/D-TCTP-PTD 13. Furthermore, the OVA/CpG/D-TCTP-PTD 13

75

combination skewed IgG1 and IgG2a profiles of humoral immune responses towards a

76

Th1 profile. These findings suggest that TCTP-derived PTD is a suitable vehicle to

77

efficiently carry antigens and to induce more powerful antigen-specific immune

78

responses and a more balanced Th1/Th2 response when combined with a DNA

D-forms

of TCTP-PTD 13 isomers (L- or

D-TCTP-PTD

3


13) as antigen


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

79

adjuvant.

80 81

KEYWORDS: Nasal vaccination, Protein transduction domain, Translationally

82

controlled tumor protein, TCTP-PTD, CpG

83 84

1. INTRODUCTION

85 86

Nasal vaccination offers several benefits, such as ease of self-administration,

87

low enzymatic degradation compared to the oral route, and induction of both mucosal

88

and systemic immune responses. Furthermore, a local immune response in the nasal

89

mucosa evokes antigen-specific secretory immunoglobulin A (sIgA), which may

90

provide broader cross-protection against heterologous viruses such as influenza viruses

91

1-4

92

in the nasal epithelium. This poor antigen permeability through the nasal epithelium

93

associated with nasal immunization results in lower systemic immune responses than

94

intramuscular (i.m) vaccination

95

the nasal epithelium are needed. Use of immunostimulatory adjuvants and vaccine

96

carriers would be one approach. Based on studies in animal models, toxin-based

97

adjuvants such as cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT)

98

have been suggested as possible adjuvants 8, 9. However, because toxin-based adjuvants

99

have been shown toxic to humans, less toxic mutants of CT and LT retaining adjuvant

100

activity have been generated. These are more promising candidate adjuvants for human

101

use 10-12.

. However, the major drawback of the nasal route is poor absorption of large antigens

5-7

. Therefore, methods to improve antigen uptake by

102

Another suggested approach to improving antigen uptake through the nasal

103

epithelium is to employ biodegradable and mucoadhesive polymeric carriers such as

104

chitosan, polylactide-co-glycolide, alginate, and carbopol 13. The use of mucoadhesive

105

polymers in nasal vaccine delivery systems has become the most popular approach to 4


Page 4 of 34

Page 5 of 34

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


106

improving uptake, because it offers the advantage of increasing antigen residence time

107

in the nasal cavity, thereby improving its absorption through the nasal epithelium. More

108

recently, in attempts to further improve the efficiency of nasal vaccines, co-association

109

of adjuvants, such as immunostimulatory DNA sequences (also known as CpG motifs),

110

lipopolysaccharide (LPS), a non-toxic subunit of CT, imiquimod (a Toll-like receptor 7

111

agonist), and model antigens to nanoparticle carriers has resulted in improved immune

112

responses 6, 14-16.

113

A more novel approach is the use of protein transduction domains (PTDs), also

114

called cell-penetrating peptides, as possible vehicles in nasal vaccination. PTDs have

115

been successfully applied to overcome the nasal epithelial barrier for nasal drug

116

delivery 17-21. Shinji et al. were the first to use a PTD for this purpose 22. They evaluated

117

a D-octaarginine-linked poly(N-vinylacetamide-co-acrylic acid) polymer as a carrier for

118

nasal vaccine delivery. They found that nasal co-administration of antigens (ovalbumin,

119

influenza virus HA vaccines) with the polymer enhanced the production of

120

antigen-specific IgG and sIgA in the serum and nasal cavity, respectively. This was the

121

first study to suggest that the use of PTD is an attractive strategy for nasal vaccine

122

delivery, mainly because of its ability to overcome the nasal epithelial barrier.

123

The translationally controlled tumor protein (TCTP) (also known as fortilin or

124

histamine-releasing factor) is known to play a role in human allergic response, apoptotic

125

regulation, various cancer-related functions, and hypertension

126

reported that an amino acid moiety (1-MIIYRDLISH-10) present at the N-terminus of

127

human TCTP acts as a PTD. We called it “TCTP-PTD” 28. We also found that several key

128

amino acid residues of TCTP-PTD play critical roles in facilitating cellular uptake. These

129

findings helped the design of new mutation analogs of TCTP-PTD that improve the solubility of

130

peptides and their membrane penetrating power 29. More recently, we evaluated whether nasally

131

administered insulin plus TCTP-PTD or its improved membrane penetrating ability of mutant

132

analogs can enhance nasal insulin absorption. We have shown that nasal administration of a

5


23-27

. We have previously


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

Page 6 of 34

133

TCTP-PTD analog, TCTP-PTD 13 consisting of L-amino acid (1-MIIFRALISHKK-12)

134

combined with insulin leads to enhanced nasal insulin absorption compared to nasal

135

administration of insulin alone 30.

136

In this study, we investigated whether TCTP-PTD 13 can serve as antigen

137

carriers when co-administrated nasally with a model antigen ovalbumin (OVA), and

138

improve OVA-specific immune responses in the mucosal and systemic compartments.

139

In addition, we compared the L- or

140

peptide appeared more resistant to enzymatic degradation in the nasal mucosa, which

141

may positively affect the transport of OVA through the nasal mucosa. We found that

142

nasal co-administration of OVA with the D-isomer (OVA/D-TCTP-PTD 13) resulted in

143

higher antibody responses than the nasally administered L-isomer (OVA/L-TCTP-PTD

144

13). We further investigated whether the combination of D-TCTP-PTD 13 with CpG has

145

a synergistic effect on OVA-specific immune responses, including the Th1/Th2 balance.

146

We found that the combination, CpG plus OVA/D-TCTP-PTD 13, has added beneficial

147

effects on antigen-specific antibody production in mice after nasal immunization

D-forms

of TCTP-PTD 13. The

D-form

of the

148 149

2. EXPERIMENTAL SECTION

150 151

2.1. Materials

152 153

OVA and phosphate buffered saline (PBS; pH 7.4) were purchased from

154

Sigma (St. Louis, MO). CpG ODN 2006 (5′-tcgtcgttttgtcgttttgtcgtt-3′) was purchased

155

from InvivoGen (San Diego, CA). The L- and D-forms of TCTP-PTD 13 isomers were

156

synthesized by Peptron Co., Ltd. (Daejeon, Korea). The N-termini of peptides were

157

acetylated, and the C-termini of peptides were protected by amidation. N-terminal

158

fluorescein isothiocyanate (FITC)-labeled peptides were also synthesized to evaluate

159

cellular uptake efficiency. The purity of the peptides was >90%. All of the chemicals 6


Page 7 of 34

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


160

used in this study were of analytical grade.

161 162

2.2. Cellular uptake

163 164

To evaluate FITC-labeled peptide uptake, human bronchial epithelial cells,

165

BEAS-2B (ATCC, Manassas, VA), were maintained in bronchial epithelial growth

166

medium (BEGM, Lonza, Walkersville, MD). BEAS-2B cells (8.0 × 105 per well) were

167

seeded into 6-well plates 24 h before incubation with peptides. The cells were washed

168

two times with PBS and incubated with FITC-labeled peptides (2.5 µM) in BEGM for

169

30 min at 37 °C. The cells were then treated with trypsin and washed six times with

170

ice-cold PBS in order to exclude interference from cell surface-bound FITC-labeled

171

peptides. Peptide internalization was measured by fluorescence-activated cell sorting

172

(FACS) using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) at an

173

emission of 510 nm and excitation of 530 nm. The assays were carried out twice in

174

triplicate. Flow cytometric analyses were accomplished using WinMDI version 2.8

175

software.

176 177

2.3. Animals

178 179

Female BALB/c mice were purchased from Orient Bio Co., Ltd. (Seongnam,

180

Korea). They were maintained with free access to water and a commercial rodent diet

181

(FEEDLAP Co., Ltd, Hanam, Korea). All animal studies were approved by Ewha

182

Womans University’s Institutional Animal Care and Use Committee (Approval ID:

183

2013-01-057). The photoperiod was set at a 12:12 light-dark cycle. The room

184

temperature was controlled at 23 ± 2 °C.

185 186

2.4. Nasal immunization protocol and sample collection 7



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

187 188

To evaluate the effect of L- and D-TCTP-PTD 13 as antigen carriers, they were

189

mixed with the model antigen OVA. Briefly, a 10 µM final concentration of OVA was

190

gently mixed with different concentrations of L- or D-TCTP-PTD 13 (50 µM, 150 µM,

191

or 300 µM final concentrations) in PBS (molar ratios of OVA to TCTP-PTD 13 of 1:5,

192

1:15, and 1:30). For nasal administration, female BALB/c mice (6–8 weeks old) were

193

anesthetized by intraperitoneal injection of a mixture of zoletil (50 mg/kg) and xylazine

194

(10 mg/kg) and then placed in a supine position. OVA (4.5 µg/mouse) with or without

195

PTD was nasally administered in a total volume of 10 µl on days 0 and 21. The same

196

amount of OVA was administered intramuscularly into both hind legs of mice in a total

197

volume of 100 µl. Experimental groups consisted of 5 mice.

198

To evaluate the combined effect of PTD and adjuvant, we used synthetic CpG,

199

which has a relatively low toxicity when compared with other adjuvants such as CT and

200

LT. The OVA/CpG/PTD mixtures were prepared by simple mixing. Briefly, final

201

concentrations of 10 µM of OVA and 300 µM of D-TCTP-PTD 13 were co-formulated

202

with different amounts (0.77 µg, 2.31 µg, and 4.62 µg) of CpG (for 10 µM, 30 µM, or

203

60 µM final concentrations) in PBS. Female BALB/c mice (6–8 weeks old) were

204

nasally immunized with 4.5 µg of OVA dissolved in 10 µl of PBS, either alone or

205

co-formulated with CpG and/or D-TCTP-PTD 13 two times at 3-week intervals (at 0

206

and 21 days). Experimental groups consisted of either 6 (nasal) or 5 (i.m) mice.

207

Blood samples were collected from the immunized mice at 20 days and 42 days

208

after the first immunization in heparin-containing tubes and centrifuged at 3,000 ×g for

209

25 min at 4 °C. The plasma samples were stored at −20 °C before analysis. On day 42

210

after the first immunization, all mice were sacrificed, and nasal wash samples were

211

collected using the trans-pharyngeal nasal lavaging technique 31. The nasal cavities were

212

gently washed with 0.25 ml of PBS, and the nasal wash samples were stored at −70 °C

213

until analysis. 8


Page 8 of 34

Page 9 of 34

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


214 215

2.5. Evaluation of immune response

216 217

OVA-specific

antibody

responses

were

measured

by

enzyme-linked

218

immunosorbent assay (ELISA). Briefly, 96-well plates were coated with 100 µl/well of

219

OVA (10 µg/ml) in 50 mM sodium carbonate (pH 9.6) and held overnight at 4 °C. After

220

washing three times with washing buffer (0.14 M NaCl, 50 mM Tris-HCl [pH 7.4], and

221

0.05% [v/v] Tween 20), the plates were blocked with 200 µl/well of blocking solution

222

(0.14 M NaCl, 50 mM Tris-HCl [pH 7.4], and 1% [w/v] bovine serum albumin),

223

incubated for 30 min, and washed thrice. The plates were then incubated with 2-fold

224

serially diluted plasma samples (starting at 1:50) at 100 µl/well for 60 min. The plates

225

were washed three times and incubated for 1 h with 100 µl/well of horseradish

226

peroxidase (HRP)-conjugated goat anti-mouse IgG (Bethyl, dilution 1:20,000), IgG1

227

(Bethyl, dilution 1:80,000), or IgG2a (Bethyl, dilution 1:20,000). The plates were

228

washed an additional five times. Finally, a color reaction was initiated by adding 100 µl

229

of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (50 mM sodium acetate

230

buffer at pH 5.2 containing 0.1 mg/ml TMB substrate [Sigma] with 0.02% [v/v] H2O2)

231

for 6 min. The reaction mixture was terminated by adding 100 µl of 0.2 M H2SO4, and

232

absorbances were measured at a wavelength of 450 nm with a microtiter plate reader.

233

Anti-OVA antibody titers (total IgG as well as IgG1 and IgG2a isotypes) were defined

234

as the highest plasma dilution corresponding to 20% of the ELISA signal above the

235

background. Comparisons between different experimental groups were made using

236

log-transformed titers.

237

To determine OVA-specific sIgA in nasal washes, undiluted nasal wash

238

samples were incubated on OVA-coated plates for 1 h. Bound antibodies were detected

239

using an HRP-conjugated goat anti-mouse IgA (Sigma, dilution 1:10,000) followed by

240

incubation with TMB solution for 30 min. The reaction was stopped with 0.2 M H2SO4, 9



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

241

Page 10 of 34

and absorbances were measured at a 450 nm with a microtiter plate reader.

242 243

2.6. Distribution or penetration of OVA in the nasal mucosa after nasal

244

administration of OVA alone or in combination with L- or D-TCTP-PTD 13

245 246

To determine the distribution of OVA in nasal mucosal tissues, anesthetized mice

247

were nasally administered 4.5 µg of OVA with or without L- or

248

(OVA:TCTP-PTD 13 molar ratio of 1:30). Fifteen minutes after administration, the

249

mice were perfused with PBS followed by 4% formaldehyde in PBS. Their noses were

250

further fixed in 4% formaldehyde in PBS overnight at 4 °C and then decalcified for 30 h

251

with 4 M formic acid. Paraffin sections (5 µm) prepared by standard processing

252

techniques were deparaffinized in xylene and hydrated in serial ethanol solutions. The

253

distribution of OVA in the nasal mucosa was determined by immunofluorescence.

254

Sections were subjected to antigen retrieval by treatment with 10% formic acid for 15

255

min. After washing, the sections were blocked with 2.5% normal horse serum for 30

256

min to prevent non-specific protein binding. After an additional wash, the sections were

257

incubated with a 1:200 dilution of OVA-specific rabbit antiserum (Sigma) overnight at

258

4 °C. Finally, after washing again, the sections were incubated with a 1:100 dilution of

259

Alexa 488-conjugated secondary antibody (Invitrogen, Carlsbad, CA) for 1 h at room

260

temperature and counterstained with DAPI. The distribution and penetration of OVA in

261

the nasal mucosa was determined using a fluorescence microscope.

D-TCTP-PTD

13

262 263

2.7. Assay of lactate dehydrogenase (LDH) release as an index of

264

cytotoxicity in the nasal mucosa

265 266

The degree of nasal membrane damage was determined by measuring the release

267

of LDH from the nasal mucosa. Briefly, OVA/L- or D-TCTP-PTD 13 (OVA:TCTP-PTD 10


Page 11 of 34

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


268

13 molar ratio of 1:30) mixtures were prepared as described above. Mice were treated

269

nasally with OVA/L- or

270

OVA/mouse). OVA solution without PTDs was used as a control. Mice were then

271

nasally administered either 5 % (w/v) sodium taurodeoxycholate, an anionic detergent

272

known to cause mucosal membrane damage (positive control) or PBS (negative control)

273

17, 19

274

PBS using a micropipette. The washed solutions were collected, and LDH leakage was

275

measured using a CytoTox-96 assay kit (Promega, Madison, WI). Each experimental

276

group consisted of 5 mice.

D-TCTP-PTD

13 mixtures (total volume: 10 µl, 4.5 µg

. After exposures of 15 min or 60 min, nasal cavities were washed with 400 µl of

277

To further evaluate the cytotoxicity of the three different materials (OVA,

278

OVA/L-TCTP-PTD 13, and OVA/D-TCTP-PTD 13), prepared as described above,

279

BEAS-2B cells were seeded 24 h prior to experiments into 96-well plates at a seeding

280

density of 1 × 104 per well. Cells were treated with 25 µl of OVA alone or in

281

combination with L- or D-TCTP-PTD 13 in 75 µl of BEGM for 1 h and 5 h at 37 °C.

282

PBS was used as a negative control which demonstrated 100 % cell viability. Five %

283

(w/v) sodium taurodeoxycholate was used as a positive control. After incubation, 10 µl

284

of cell counting kit-8 solution (Dojindo Laboratories, Kumamoto, Japan) was added to

285

each well, followed by incubation for 1 h at 37°C. The absorbance at 450 nm was

286

determined using a microtiter plate reader.

287 288

2.8. Electrophoretic mobility shift assay (EMSA) of TCTP-PTD 13 binding to

289

CpG DNA

290 291

CpG was incubated for 15 min at room temperature in PBS with different

292

concentrations of D-TCTP-PTD 13 corresponding to D-TCTP-PTD 13/CpG molar ratios

293

ranging between 0.5:1 and 30:1. Each well contained 2.31 µg of CpG. The preformed

294

D-TCTP-PTD

13/CpG complexes were analyzed by gel electrophoresis on a 1.2% 11



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

Page 12 of 34

295

agarose gel and stained with SYBR Safe DNA Gel Stain (Invitrogen), and visualized

296

under ultraviolet light.

297 298

2.9. Statistical Analysis

299 300

Statistical analyses were performed using GraphPadTM Prism version 5.0 for

301

Windows (GraphPad Software, San Diego, CA). All of the data were analyzed with a

302

one-way analysis of variance (ANOVA) followed by Tukey's test, with the exception of

303

antigen-specific sIgA levels in nasal secretions, which were analyzed using the

304

Kruskal-Wallis test. Error bars were expressed as the mean ± standard error of the mean

305

(SEM) from 5 to 6 mice per group. Significant differences between means were defined

306

as p < 0.05.

307 308

3. RESULTS AND DISCUSSION

309 310

3.1. Enhancement of systemic and mucosal immune responses after nasal

311

immunization with OVA/TCTP-PTD 13

312 313

Generally, peptides containing D-amino acids are more resistant to enzymatic 32-34

314

degradation than their

315

D-TCPT-PTD

316

amino acid stereochemistry may negatively affect the cell-penetrating ability of the PTD.

317

Therefore, we first confirmed whether D-TCTP-PTD 13 had the capacity to internalize

318

into cells. After N-terminal FITC labeling of peptides, the cellular uptake of

319

FITC-labeled peptides was analyzed by flow cytometry. L-TCTP-PTD 13, reported in a

320

previous study, was used as a control 29. Cellular uptake was compared after 30 min of

321

incubation with FITC-labeled peptides at a concentration of 2.5 µM. We found that

L-amino

acid counterparts

. Thus, one would expect

13 to show improved enzymatic stability. However, any alteration in

12


Page 13 of 34

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


322

D-TCTP-PTD

323

L-amino

324

stereochemistry of TCTP-PTD 13 containing

325

translocating ability.

13 was internalized approximately 1.6 times more efficiently than the

acid counterpart in BEAS-2B cells (Figure 1A). This suggests that the altered D-amino

acid did not affect its

326

Next, we evaluated whether L- or D-forms of TCTP-PTD 13 isomers could act as

327

antigen carriers, thereby enhancing the immunogenicity of the antigen. Mice were

328

nasally immunized with 4.5 µg of OVA alone or together with L- or D-TCTP-PTD 13 on

329

days 0 and 21. Three doses of TCTP-PTD 13 were selected to evaluate the dose effect.

330

As a positive control, mice were i.m-immunized with the same dose of OVA.

331

OVA-specific total IgG titers in plasma were measured by ELISA after priming

332

(Figure 1B) and boosting (Figure 1C). Following the prime immunization, nasally

333

administered OVA alone or OVA/L-TCTP-PTD 13 (1:5 molar ratio) induced low plasma

334

IgG titers in 2 out of 5 mice. In contrast, nasally administered OVA/D-TCTP-PTD 13

335

(1:5, 1:15, or 1:30) resulted in stronger IgG responses than nasal immunization with

336

OVA alone or with L-TCTP-PTD 13 (1:5, 1:15, or 1:30) added.

337

Following the boost immunization (Figure 1C), OVA/D-TCTP-PTD 13 (1:15 or

338

1:30) led to significantly higher IgG titers than nasally administered OVA alone or

339

OVA/L-TCTP-PTD 13 (1:15 or 1:30) (p < 0.05). Furthermore, increasing concentrations

340

of

341

OVA-specific IgG titer in mice immunized with OVA/D-TCTP-PTD 13 (1:15 or 1:30)

342

was higher than that in mice immunized with an OVA i.m injection, although the

343

difference was not statistically significant (p > 0.05). Nasal administration of

344

OVA/L-TCTP-PTD 13 mixtures resulted in higher mean IgG titers in all groups relative

345

to nasal immunization with OVA alone, although this difference was not significant (p >

346

0.05).

D-TCTP-PTD

13 yielded more pronounced IgG responses. The average

347

Induction of antigen-specific sIgA at the mucosal epithelium is the first line of

348

defense against pathogens following mucosal vaccination. As shown in the systemic 13



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

Page 14 of 34

349

immune responses, antigen co-administered with D-TCTP-PTD 13 may elicit stronger

350

sIgA responses than antigen alone. Therefore, we also determined OVA-specific sIgA in

351

nasal secretions 3 weeks after the boost immunization. As shown in Figure 1D, mice

352

nasally immunized OVA/D-TCTP-PTD 13 (1:15 or 1:30) mounted substantially high

353

sIgA levels in nasal secretions compared to mice immunized with other formulations.

354

There were no statistically significant differences between the immunized groups (p >

355

0.05). Taken together, these results suggest that

356

antigen carrier than L-TCTP-PTD 13.

D-TCTP-PTD

357 358 359 360 361 362 363 364

14


13 is a more potent

Page 15 of 34

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


365 366

Figure 1. (A) Cellular uptake of 2.5 µM FITC-labeled L- or D-forms of TCTP-PTD 13

367

isomers incubated BEAS-2B cells for 30 min at 37 °C. The assay was performed at least

368

two times in triplicate, and each bar represents the mean ± SD. The mean fluorescence

369

intensity of FITC in BEAS-2B cell was set as 1. *p < 0.05 versus FITC-labeled

370

L-TCTP-PTD

371

immunization. Bars represent means. (C) OVA-specific total IgG titers in plasma 3

372

weeks after the boost immunization. Vertical bars represent means ± SEM. Asterisks

373

above the bars indicate significant differences compared to nasal administration of OVA

374

alone (p < 0.05). #p < 0.05. (D) OVA-specific sIgA in nasal washes of mice 3 weeks

375

after the boost immunization. Nasal wash samples were collected from unimmunized

376

mice (control). Each bar represents the mean.

13. (B) OVA-specific total IgG titers in plasma 3 weeks after the prime

15



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

377

3.2. Plasma OVA-specific IgG subclass responses of mice nasally

378

immunized with OVA/TCTP-PTD 13

379 380

Following the second immunization, OVA-specific IgG isotypes in plasma were

381

analyzed to determine the type of Th response; a predominance of IgG2a would indicate

382

a Th1 response, while a predominance of IgG1 would indicate a Th2-type immune

383

response.

384

Figure 2A shows the plasma IgG1 and IgG2a titers generated in each of the four

385

groups. Nasal immunization with OVA alone induced a low IgG1 response in a few (3/5)

386

mice and did not induce detectable IgG2a titers (1/5 responder). As previously reported

387

35, 36

388

low IgG2a level (Figure 2A). The IgG1/IgG2a ratio after nasal immunization with

389

OVA/D-TCTP-PTD 13 (1:15 or 1:30) was not different from that in mice

390

i.m-immunized with OVA alone (Figure 2B). These results indicate that nasal

391

immunization with OVA/D-TCTP-PTD 13 elicited a Th2-type immune response, just as

392

OVA i.m administration did.

, i.m injection with OVA alone resulted in a predominantly IgG1 response with a

393 394 395 396 397 398 399 400 401 402 403 16


Page 16 of 34

Page 17 of 34

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


404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421

Figure 2. OVA-specific plasma IgG1 and IgG2a responses after nasal immunization. (A)

422

IgG1 and IgG2a titers 3 weeks after the boost immunization. The results are expressed

423

as means ± SEM. (B) IgG1/IgG2a ratios. Bars represent means.

424 425 426 427 428 429 430 17



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

431

3.3. Distribution or penetration of OVA in the nasal mucosal tissue and

432

nasal toxicity of OVA/TCTP-PTD 13

433 434

When compared to nasal co-administration of OVA with L-TCTP-PTD 13, the

435

enhanced immunogenicity of nasally administered OVA/D-TCTP-PTD 13 can be

436

ascribed to the difference in nasal delivery of OVA. To confirm this, mice were nasally

437

administered OVA alone or with L- or D-TCTP-PTD 13 added. Nasal sections were

438

stained, and the distribution or penetration of OVA in the nasal mucosa was visualized.

439

At 15 min after nasal administration, green fluorescence staining of OVA in the

440

nasal mucosal tissue sections showed that more OVA penetrated to the sub-mucosa with

441

nasally administered OVA/D-TCTP-PTD 13 than in either nasally administered OVA

442

alone or OVA/L-TCTP-PTD 13 (Figure 3A). These results confirm that D-TCTP-PTD

443

13 can facilitate the delivery of the model antigen across the nasal epithelium.

444

The nasal-associated lymphoid tissue (NALT) may serve as a prime inductive site

445

for mucosal immunity 37, 38. Although OVA was observed on the nasal epithelial surface

446

covering the NALT 15 min after nasal administration of the three different formulations,

447

the fluorescence signal of OVA in the NALT was very weak. Even though we did not

448

find any difference in the location of antigens in the NALT between the groups,

449

D-TCTP-PTD

450

sub-mucosa where OVA fluorescent signals were much stronger than those in the

451

sub-mucosa after nasal administration of OVA alone or with L-TCTP-PTD 13. This

452

difference in the transport of OVA across the nasal mucosa may correlate with the

453

differences in the antigen-specific immune responses that are shown in Figure 1.

454

However, further work is needed to confirm and elucidate these findings.

facilitated the transport of antigens across nasal epithelium to the

455

It remains to be clarified how changing the amino acid stereochemistry of

456

TCTP-PTD 13 from the L-form to the D-form positively affected OVA absorption in the

457

nasal mucosa. We speculate that improving the unnatural D-TCTP-PTD 13 stability with 18


Page 18 of 34

Page 19 of 34

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


458

a more membrane permeation power may help nasal delivery of the antigen.

459

Intermolecular binding between the PTD and its cargo and the PTD-to-cargo mixing

460

ratio are proposed as important factors in enhancing nasal drug delivery

461

understanding the potential mechanism of how the PTD increases the immunogenicity

462

of protein antigen, may require further detailed analysis of intermolecular PTD-protein

463

antigen interaction and the mechanism of cellular uptake.

30, 39

. Thus,

464

We next evaluated whether damage to nasal mucosa resulted in increased nasal

465

absorption of OVA. The degree of mucosal damage can be determined by measuring the

466

release of LDH from the nasal mucosa

467

OVA/TCTP-PTD 13 mixtures to the nasal mucosa of mice, LDH leakage from the nasal

468

mucosa was measured (Figure 3B). There was no significant difference in LDH leakage

469

from the nasal mucosa between the OVA/L- or D-TCTP-PTD 13 and PBS-treated groups

470

(negative control). In contrast, nasally administered sodium taurodeoxycholate (positive

471

control) significantly increased LDH leakage compared with that of the PBS-treated

472

group (p < 0.05). These data show that D-TCTP-PTD 13 enhanced nasal delivery of

473

OVA without causing nasal mucous membrane damage. Furthermore, OVA

474

co-incubated with L- or D-TCTP-PTD 13 in BEAS-2B cells, exhibitted no cytotoxicity

475

(Figure 3C). We also further confirmed the results of cytotoxicity assays. We found that

476

a 2-fold OVA/D-TCTP-PTD 13 doses induced observable toxicity in BEAS-2B cells

477

(results not shown). However, because the toxic concentration determined in in vitro

478

cell culture studies, may be different from toxic dose effective in in vivo situation,

479

further investigations are needed to correctly assess the possible side effects of

480

TCTP-PTD 13, although OVA/TCTP-PTD 13 mixtures did not cause any nasal toxicity

481

in a mouse model.

17, 19

. After 15 min or 60 min exposures of the

482 483 484 19



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

485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 20


Page 20 of 34

Page 21 of 34

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


512

Figure 3. Distribution of OVA in the nasal mucosal tissue and nasal toxicity of

513

OVA/TCTP-PTD 13. (A) The nasal mucosa penetration profiles of OVA (a, b, c, and d),

514

OVA/L-TCTP-PTD 13 (e, f, g, and h) and OVA/D-TCTP-PTD 13 (i, j, k, and l). Mice

515

were administered OVA (dose: 4.5 µg/mouse) on one side of the nasal cavity. The

516

undosed side was used as a negative control tissue. Nuclei are stained with DAPI.

517

NALT is indicated by arrows. (B) LDH leakage following nasal administration of OVA

518

mixed with PTDs. Sodium taurodeoxycholate (5%, w/v) was used as a positive control

519

known to be toxic. LDH values are expressed as a percentage of values from the

520

positive control. Vertical bars indicate means ± SEM. *Significantly different from the

521

PBS-treated group at p < 0.05. (C) Cytotoxicity after co-incubating OVA with PTDs in

522

BEAS-2B cells. Sodium taurodeoxycholate (5%, w/v) was used as a positive control.

523

Each bar represents the mean ± SD (n=4). *p < 0.05 compared to PBS-treated cells.

524 525

3.4. Enhancement of systemic and mucosal immune responses after nasal

526

immunization of OVA/CpG/TCTP-PTD 13

527 528

We found that nasal immunization of OVA in combination with D-TCTP-PTD

529

13 elicited a strong Th2-like systemic immune response, with a relatively weak mucosal

530

IgA response. Synthetic oligodeoxynucleotides containing CpG motifs can act as

531

Th1-promoting adjuvants and have been shown to be potent nasal adjuvants for the

532

induction of systemic and mucosal immune responses

533

investigated whether the addition of CpG to a mixture of OVA/D-TCTP-PTD 13 could

534

enhance antigen-specific immune responses, leading to the induction of Th1 immunity.

40, 41

. Therefore, we next

535

Hydrophobic and positively charged PTDs have been reported to bind

536

negatively charged nucleic acid-based molecules (DNA or siRNA) by electrostatic

537

and/or hydrophobic interactions

538

bind CpG. A constant amount of CpG was incubated with D-TCTP-PTD 13 at different

42, 43

. We first tested whether D-TCTP-PTD 13 could

21



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

539

molar ratios, and the formation of CpG/D-TCTP-PTD 13 complexes was analyzed by

540

EMSA. As shown in Figure 4A,

541

dose-dependent manner. A 1:10 molar ratio of CpG to D-TCTP-PTD 13 was mostly

542

associated with D-TCTP-PTD 13. At a CpG/D-TCTP-PTD 13 molar ratio of 1:≥15, no

543

free CpG was detected on the agarose gel.

D-TCTP-PTD

13 was able to bind CpG in a

544

To evaluate the effect of adding CpG to a mixture of OVA/D-TCTP-PTD 13,

545

mice were nasally immunized with OVA co-formulated with three different amounts of

546

CpG and D-TCTP-PTD 13 (OVA/CpG/D-TCTP-PTD 13 molar ratios of 1:1:30, 1:3:30,

547

and 1:6:30). Antigen-specific total IgG titers from plasma collected 20 days and 42 days

548

after prime immunization were measured. As expected based on previous findings, nasal

549

immunization with OVA alone induced a low IgG response in 3 out of 6 mice after the

550

prime immunization (Figure 4B) and after boosting (Figure 4C). Nasal or i.m

551

administration of all formulations resulted in significantly enhanced IgG titers after the

552

boost immunization compared to nasal administration of OVA alone (Figure 4C, p

Potential of Translationally Controlled Tumor Protein-Derived Protein

Recommend Documents