Vaccine efficacy of transcutaneous immunization with amyloid using a
dissolving microneedle array in a mouse model of Alzheimers disease
Kazuhiko Matsuo, Hideaki Okamoto, Yasuaki Kawai, Ying-Shu Quan,
Fumio Kamiyama, Sachiko Hirobe, Naoki Okada, Shinsaku Nakagawa
PII:
DOI:
Reference:

S0165-5728(13)00311-1
doi: 10.1016/j.jneuroim.2013.11.002
JNI 475820

To appear in:

Journal of Neuroimmunology

Received date:
Revised date:
Accepted date:

30 January 2013
2 November 2013
5 November 2013

Please cite this article as: Matsuo, Kazuhiko, Okamoto, Hideaki, Kawai, Yasuaki, Quan,
Ying-Shu, Kamiyama, Fumio, Hirobe, Sachiko, Okada, Naoki, Nakagawa, Shinsaku, Vaccine ecacy of transcutaneous immunization with amyloid using a dissolving microneedle array in a mouse model of Alzheimers disease, Journal of Neuroimmunology (2013),
doi: 10.1016/j.jneuroim.2013.11.002

This is a PDF le of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its nal form. Please note that during the production process
errors may be discovered which could aect the content, and all legal disclaimers that
apply to the journal pertain.

ACCEPTED MANUSCRIPT
Matsuo K. et al.

Title:

PT

Vaccine efficacy of transcutaneous immunization with amyloid using a dissolving microneedle

SC

RI

array in a mouse model of Alzheimers disease

NU

Abbreviated title:

MA

Transcutaneous vaccination for Alzheimer's disease

Author names:

TE

Kazuhiko Matsuo1,, Hideaki Okamoto1, Yasuaki Kawai1, Ying-Shu Quan2, Fumio Kamiyama2,

Affiliations:
1

AC
CE
P

Sachiko Hirobe1, Naoki Okada1,*, Shinsaku Nakagawa1,*

Laboratory of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences,

Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan

2

CosMED Pharmaceutical Co. Ltd., 32 Higashikujokawanishi-cho, Minami-ku, Kyoto 601-8014,

Japan

Present address: Division of Chemotherapy, Kinki University Faculty of Pharmacy, 3-4-1

Kowakae, Higashi-osaka, Osaka 577-8502, Japan

1

ACCEPTED MANUSCRIPT
Matsuo K. et al.

PT

Corresponding author:

RI

Naoki Okada, Ph.D.

SC

Laboratory of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences,

NU

Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0781, Japan

MA

(Phone): +81-6-6879-8176, (Fax): +81-6-6879-8176, (E-mail): okada@phs.osaka-u.ac.jp

Shinsaku Nakagawa, Ph.D.

TE

Laboratory of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences,

AC
CE
P

Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0781, Japan

(Phone): +81-6-6879-8175, (Fax): +81-6-6879-8179, (E-mail): nakagawa@phs.osaka-u.ac.jp

ACCEPTED MANUSCRIPT
Matsuo K. et al.

Short Abstract

PT

Vaccine therapy has attracted attention for treating Alzheimers disease (AD). Injectable

RI

immunization of amyloid peptide comprising 142 amino-acid residues (A142) as antigens

SC

showed a therapeutic effect; therefore, a clinical trial was conducted. However, the trial was

NU

stopped because of meningoencephalitis caused by excess activation of Th1 cells against A142.

MA

Therefore, therapeutic approaches to induce Th2-dominant immune responses are required for
establishing an effective and safe vaccine therapy. Here, we attempted to develop easy-to-use

transcutaneous immunization using a dissolving microneedle array on the basis of the

AC
CE
P

TE

characteristic that transcutaneous immunization may induce Th2-dominant immune responses.

ACCEPTED MANUSCRIPT
Matsuo K. et al.

Abstract

PT

Vaccine therapy for Alzheimers disease (AD) based on the amyloid cascade hypothesis has

RI

recently attracted attention for treating AD. Injectable immunization using amyloid peptide

SC

(A) comprising 142 amino-acid residues (A142) as antigens showed therapeutic efficacy in

NU

mice; however, the clinical trial of this injected A142 vaccine was stopped due to the

MA

incidence of meningoencephalitis caused by excess activation of Th1 cells infiltrating the brain
as a serious adverse reaction. Because recent studies have suggested that transcutaneous

immunization (TCI) is likely to elicit Th2-dominant immune responses, TCI is expected to be

TE

effective in treating AD without inducing adverse reactions. Previously reported TCI procedures

AC
CE
P

employed complicated and impractical vaccination procedures; therefore, a simple, easy-to-use,

and novel TCI approach needs to be established. In this study, we investigated the vaccine
efficacy of an A142-containing TCI against AD using our novel dissolving microneedle array
(MicroHyala; MH). MH-based TCI induced anti-A142 immune responses by simple and
low-invasive application of A142-containing MH to the skin. Unfortunately, this TCI system
resulted in little significant improvement in cognitive function and Th2-dominant immune
responses, suggesting the need for further modification.

Keywords:
4

ACCEPTED MANUSCRIPT
Matsuo K. et al.

Alzheimers disease, vaccine therapy, transcutaneous immunization, dissolving microneedle

RI

PT

array

SC

Abbreviations

NU

A, amyloid peptide; AD, Alzheimers disease; ANOVA, analysis of variance; CC, cingulate

MA

cortex; CNS, central nervous system; CT, cholera toxin; EC, entorhinal cortex; ELISA,
enzyme-linked immunosorbent assay; HIPP, hippocampus; HRP, horseradish peroxidase; IDI,

intradermal immunization; LC, Langerhans cell; MH, MicroHyala; MWMT, Morris water maze

TrisHCl-buffered

saline

AC
CE
P

TBS-T,

TE

test; N.D., not detectable; NORT, novel object recognition test; TBS, TrisHCl-buffered saline;
containing

immunization; TQ, target quadrant

0.05%

Tween-20;

TCI,

transcutaneous

ACCEPTED MANUSCRIPT
Matsuo K. et al.

1. Introduction

PT

In Alzheimers disease (AD), gradual cerebral accumulation of amyloid peptide (A)

RI

oligomers or soluble and insoluble assemblies of A triggers a cascade of biochemical alteration

SC

(Hardy and Selkoe, 2002; Selkoe, 1991). The amyloid cascade hypothesis posits that the

NU

deposition of A in the brain parenchyma is a crucial step that ultimately leads to AD pathology

MA

such as neuronal cell death or cognitive impairment. On the basis of this hypothesis, an A
vaccine was developed in mice (Schenk et al., 1999), and human clinical trials with synthetic A

comprising 142 amino-acid residues (A142) as an antigen were initiated (Bayer et al., 2005).

TE

In the phase II study, however, approximately 6 of patients developed acute

AC
CE
P

meningoencephalitis, leading to suspension of the clinical trial (Orgogozo et al., 2003).

Examination of the brain of A142-vaccinated patients revealed the existence of
A142-reactive pro-inflammatory Th1 cell-dominant meningeal encephalitis in the cerebral
cortex, which is one of the main side effects (Ferrer et al., 2004; Nicoll et al., 2003). At the same
time, however, senile plaques composed of diverse Amolecule species were significantly
decreased in some treated patients (Nicoll et al., 2003; Hock et al., 2002, 2003). Thus, if
Th2-dominant immune responses but not Th1 immune responses can be induced, A vaccine
therapy could be an attractive approach against AD.
The skin is a well-established target organ for vaccine delivery. Skin-resident
6

ACCEPTED MANUSCRIPT
Matsuo K. et al.

antigen-presenting cells have a role in immunoregulation, including T cell stimulatory function,

PT

by producing Th2-dominant cytokines (Larregina et al., 2001; Niizeki et al., 1997). In particular,

RI

Langerhans cells (LCs), which are professional antigen-presenting cells resident in the epidermis,

SC

produce Th2-type rather than Th1-type chemokines, resulting in little induction of Th1-type

NU

immune responses (Tada et al., 2003; Fujita et al., 2005). Therefore, it has been suggested that a

MA

transcutaneous immunization (TCI) system targeting LCs may induce Th2-dominant immune
responses comparable with those induced by an injection system (Ishii et al., 2008). In contrast,

intradermal immunization (IDI) delivers antigens into the dermis, where the delivered antigens

TE

are mainly captured by dermal dendritic cells, which induce Th1- and Th2-immune responses.

AC
CE
P

A recent report by Nikolic et al. demonstrated that TCI with A142 plus cholera toxin (CT)
as an adjuvant induced anti-A142 immune responses without T cell infiltration into the brain
(Nikolic et al., 2007). A142, however, has high aggregability; therefore, efficient delivery of
A142 into the skin is difficult because of the physical barrier posed by the stratum corneum. In
the report by Nikolic et al., the skin was swabbed with acetone before vaccination to remove
surface oils and enhance penetration and then rehydrated by swabbing with 0.9 saline. The
antigen solution was then placed on the skin, enclosed by a thin layer of petroleum jelly to
prevent unnecessary leakage of the immunization solution. Although this TCI system induced
anti-A142 immune responses, it was complicated and of no practical use. A simple and
7

ACCEPTED MANUSCRIPT
Matsuo K. et al.

easy-to-use novel TCI system to induce anti-A142 immune responses is highly desirable for

PT

A vaccine therapy for AD.

RI

Previously, we developed a simple and easy-to-use TCI system using a hydrogel patch as a

SC

TCI device, which delivered antigens to LCs and induced antigen-specific Th2-dominant

NU

immune responses (Ishii et al., 2008; Matsuo et al., 2011). Unfortunately, a hydrogel patch could

MA

not achieve the delivery of aggregated molecules such as A142 to LCs.

We then developed a dissolving microneedle array (MicroHyala; MH) for use as a TCI device

(Matsuo et al., 2012a, 2012b). MH can deliver any substance, which can be encapsulated in

TE

microneedles, into the skin by simple application onto the skin without causing skin damage.

AC
CE
P

TCI using MH induced effective immune responses against various infectious diseases. In
addition, we demonstrated the safety of TCI using MH in humans (Hirobe et al., 2013).
In this study, we tested the efficacy of our TCI using MH in the transgenic APPPS1 mouse
model of AD. We used two MHs, which differed in needle length, namely MH300 (needle
length: 300 m) and MH800 (needle length: 800 m), because information on the optimal MHs
for delivery of antigens to LCs resident in the mouse epidermis was not available.

ACCEPTED MANUSCRIPT
Matsuo K. et al.

2. Materials and Methods

PT

Reagents

RI

A142, which comprised 142 amino-acid residues of A, was synthesized by GL Biochem

SC

Inc. (Shanghai, China). A135-Cys, A comprising 135 amino acid residues with Cys added

NU

to the C-terminus, was synthesized by Sigma-Aldrich Inc. (St. Louis, MO). A135-Cys

MA

increased the immunogenicity to a greater extent than A142 (Matsuda et al., 2009). CT was
purchased from BioAcademia Inc. (Osaka, Japan). Horseradish peroxidase (HRP)-labeled goat

anti-mouse IgG, IgG1, and IgG2c antibodies were purchased from SouthernBiotechnology

TE

Associates Inc. (Birmingham, AL). The amyloid protein immunohistostain kit and human

AC
CE
P

-amyloid ELISA kit were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
The Congo red amyloid stain kit was purchased from Sigma-Aldrich. The protease inhibitor
cocktail was purchased from Nacalai Tesque (Osaka, Japan). The humans amyloid oligomers
(82E1-specific) assay kit was purchased from Immuno-Biological Laboratories Co., Ltd.
(Gunma, Japan).

Animals
B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/j (APPPS1 mice) (genetic background; C57BL/6) were
obtained from The Jackson Laboratory (Bar Harbor, ME). In APPPS1 mice, A deposit levels
9

ACCEPTED MANUSCRIPT
Matsuo K. et al.

dramatically increase after 20 weeks of age and impaired spatial learning is observed between 3

PT

and 12 months of age. All animals were maintained in the experimental animal facility at Osaka

NU

SC

Animal Care and Use Committee of Osaka University.

RI

University. The experiments were conducted in accordance with the guidelines provided by the

MA

Fabrication of MHs

The antigen-containing MHs were fabricated using micromolding technologies with sodium

hyaluronate as the base material, as described previously (Matsuo et al., 2012a, 2012b). In this

AC
CE
P

(Supplementary Fig. 1).

TE

study, we used cone-shaped MHs with a needle length of 300 m (MH300) or 800 m (MH800)

Experimental design

The experimental design is shown in Fig. 1. This study was conducted in two separate
experiments, Experiment 1 and Experiment 2. APPPS1 mice began receiving each immunization
at the age of 4 months.
In Experiment 1, for the TCI group [MH800/(A142 CT)], APPPS1 mice (n 7) were
vaccinated using A142 (10 g)- and CT (0.1 g)-containing MH800 that was applied to the
skin of the back. For the IDI group, APPPS1 mice (n 8) were intradermally immunized with a
10

ACCEPTED MANUSCRIPT
Matsuo K. et al.

combination of A142 (10 g) and CT (0.1 g). In the non-immunized group, APPPS1 mice (n

PT

11) were treated with antigen and adjuvant-free MH800 (placebo).

RI

In Experiment 2, for the TCI group [MH300/(A142 CT)], APPPS1 mice (n 6) were

SC

vaccinated with A142 (10 g)- and CT (0.25 g)-containing MH300 that was applied to the

NU

skin of the back. For the TCI group [MH300/(A135-Cys CT), APPPS1 mice (n 3) were
vaccinated with A135-Cys (10 g)- and CT (0.25 g)-containing MH300 that was applied to

MA

the skin of the back. For the IDI group (A142 CT), APPPS1 mice (n 5) were intradermally

immunized with A142 (10 g) and CT (0.25 g). For the IDI group (A135-Cys CT),

TE

APPPS1 mice (n 4) were intradermally immunized with A135-Cys (10 g) and CT (0.25

AC
CE
P

g). In the non-immunized group, APPPS1 mice (n 7) were treated with an antigen and
adjuvant-free MH300 (placebo).

APPPS1 mice were vaccinated on a weekly basis for the first month, and thereafter, they were
continually immunized biweekly for the next 12 weeks. At the indicated points, serum was
collected from treated mice and the anti-A142 IgG concentration was determined by
enzyme-linked immunosorbent assay (ELISA). In addition, the novel object recognition test
(NORT), Morris water maze test (MWMT), immunostaining of brain sections, and quantitative
determination of A oligomers/fibrils, A140, or A142 in the brain were conducted at the
indicated points.
11

ACCEPTED MANUSCRIPT
Matsuo K. et al.

PT

MH-based TCI protocol

RI

The TCI procedure was described in a previous report (Matsuo et al., 2012a, 2012b).

SC

Forty-eight hours before application of each MH, the skin of the back of each mouse was

NU

exposed by shaving the fur using clippers and by removing any remaining fur using a depilatory

MA

cream without any damage. All MHs were pressed onto the skin using a handheld applicator at

12.8 N/200 microneedles.

TE

Measurement of the serum anti-A142 IgG concentration and anti-A142 IgG subclass titer

AC
CE
P

Ninety-six-well plates were coated with A142 [0.5 g/50 l in 50 mM carbonate buffer (pH
9.6)] at 4C overnight and then blocked with 200 l 5 skim milk in TrisHCl-buffered saline
(TBS) at 37C for 2 h. For measurement of the serum anti-A142 IgG concentration, a standard
curve was calculated after blocking by adding serial dilutions of murine anti-A116 IgG (clone:
6E10) in duplicate in 0.5 skim milk in TBS containing 0.05% Tween-20 (TBS-T). Serum
samples were diluted in 0.5 skim milk in TBS-T at concentrations ranging from 1:3,000 to
1:300,000, added in duplicate (50 l/well), and incubated at room temperature for 2 h. For
anti-A142 IgG subclass analysis, after blocking, serial dilutions of the serum samples were
added to the plates (50 l/well) and incubated at room temperature for 2 h. The plates were
12

ACCEPTED MANUSCRIPT
Matsuo K. et al.

washed three times with TBS-T. HRP-labeled goat anti-mouse IgG, IgG1, or IgG2c antibodies

PT

diluted 1/5000 were then added (50 l/well). After 2-h incubation at room temperature, the plates

RI

were washed three times with TBS-T. The reaction was developed using a tetramethylbenzidine

SC

solution, and color development was terminated by adding 2 N H2SO4. Optical densities were

NU

measured at 450650 nm.

MA

The concentration of anti-A142 IgG was examined using the standard curve. End-point
titers of the anti-A142 IgG subclass were expressed as the reciprocal log2 of the last dilution

AC
CE
P

NORT

TE

that showed 0.1 absorbance units after subtracting the background.

The test procedure comprised three sessions: habituation, training, and retention. Each mouse
was individually habituated to the box (30 30 30 cm), with 1 h of exploration in the absence
of objects (habituation session). During the training session conducted 1 day after the habituation
session, two objects were placed in the back corners of the box. A mouse was then placed
midway to the front of the box, and the total time spent exploring the two objects was recorded
for 20 min. During the retention session, animals were placed back into the same box 1 h after
the training session, and one of the objects used during training (familiar object) was replaced
with a novel object. The animals were then allowed to explore freely for 10 min, and the time
13

ACCEPTED MANUSCRIPT
Matsuo K. et al.

spent exploring each object was recorded. Throughout the experiments, the objects were used in

PT

a counterbalanced manner in terms of their physical complexity and emotional neutrality. A

RI

preference index, the ratio of the amount of time spent exploring any one of the two objects

SC

(training session) or the novel object (retention session) over the total time spent exploring both

NU

objects, was used as a measure of cognitive function. To eliminate the influence of the previous

MA

behavioral tests, the objects were changed each time.

MWMT

TE

This test was conducted in a circular pool 1 m in diameter and filled with water at a

AC
CE
P

temperature of 25 1C. A hidden platform (7 cm in diameter), 1 cm below the surface of the

water, was used. The mice were given two trials (one block) for 5 consecutive days, during
which the platform was left in the same position. The time taken to locate the escape platform
(escape latency) was determined in each trial. One hour after the last training trial, the mice were
subjected to a transfer test without the platform and allowed to search the pool for 60 s (probe
test). In the probe test, the time spent in the training quadrant, swimming distance in the training
quadrant, and number of times a mouse crossed the platform location were measured.

Immunostaining of brain sections

14

ACCEPTED MANUSCRIPT
Matsuo K. et al.

The left hemisphere was harvested, fixed with 4% paraformaldehyde in 0.1 M phosphate

PT

buffer (pH 7.4) for 6 h at room temperature, and embedded in O.C.T. compound. Frozen sections

RI

(6-m thick) were prepared. Immunostaining of senile plaques composed of diverse A molecule

SC

species with anti-A140 antibody, anti-A142 antibody, or Congo red was performed using an

NU

amyloid protein immunohistostain kit or a Congo red amyloid stain kit procedure. Brain
sections were observed under a stereoscopic microscope (Biozero BZ8000; Keyence, Osaka,

MA

Japan), and the number of plaques with a minor axis of >3 m was determined using the image

AC
CE
P

TE

triplicate analyses for each mouse.

analysis system BZ Analyzer II (Keyence). Data are expressed as average values based on

Quantitative determination of A oligomers/fibrils, A140, or A142 in the brain

The right hemisphere was dissected into three different regions of the brain, namely the
cingulate cortex (CC), hippocampus (HIPP), and entorhinal cortex (EC) sections, and the weight
of each tissue was then measured. Each sample was homogenized with 60 times its volume of
TBS containing protease inhibitor cocktail and centrifuged at 15,000 rpm for 15 min. The
supernatant comprised the soluble extraction fraction of the brain. The pellets were homogenized
in TBS containing 5 M guanidine and protease inhibitor cocktail, incubated for 4 h at room
temperature, and centrifuged at 15,000 rpm for 15 min at 4C. The supernatant comprised the
15

ACCEPTED MANUSCRIPT
Matsuo K. et al.

insoluble extraction fraction of the brain. The amount of A140 or A142 in the soluble and

PT

insoluble fractions and A oligomers/fibrils in the soluble fraction was measured using the

RI

human -amyloid ELISA kit (Wako Pure Chemical Industries, Ltd.) and humans amyloid

SC

oligomers (82E1-specific) assay kit (Immuno-Biological Laboratories Co., Ltd.), respectively,

MA

NU

according to the manufacturers protocols.

Statistical analysis

Statistical significance was evaluated using one-way analysis of variance (ANOVA) followed

TE

by Tukeys test for multiple comparisons in MWMT, NORT, and quantitative measurement of

AC
CE
P

A accumulation in the brain. (*; p 0.05, **; p 0.01)

16

ACCEPTED MANUSCRIPT
Matsuo K. et al.

3. Results

PT

Vaccine efficacy of TCI formulation using MH for AD

RI

Anti-A142 IgG production in APPPS1 mice

SC

Our preliminary data revealed that TCI using MH800 induced anti-A142 IgG in C57BL/6

NU

mice (Supplementary Fig. 2). Thus, we investigated whether TCI using our MH also induced

MA

anti-A142 IgG antibody production in APPPS1 mice. TCI using MH800 or MH300 containing
any antigens induced anti-A142 IgG in serum of APPPS1 mice after a single vaccination (Fig.

2A, B). In each TCI group, the serum concentration of anti-A142 IgG reached the peak 4

TE

weeks after the vaccination for four times. After the administration interval was changed, the

AC
CE
P

serum concentration of anti-A142 IgG decreased but was maintained at approximately

100150 g/ml during the vaccination period. The anti-A142 IgG concentration of the TCI
group using MH300 containing A135-Cys and CT was higher than that of the TCI group using
MH300 containing A142 and CT. Each TCI group induced effective anti-A142 IgG
production in comparison with each IDI group.
In both experiments, the anti-A142 IgG concentration decreased after the vaccination period
was changed. The precise reasons remain unclear; however, immune tolerance to A142 may
have been induced because A142 was a self-antigen or anti-A142 IgG may have been
distributed to various organs, particularly the central nervous system (CNS), where it recognized
17

ACCEPTED MANUSCRIPT
Matsuo K. et al.

senile plaques from blood vessels.

PT

Taken together, TCI using MH could deliver A142 or A135-Cys into the skin and induce

RI

anti-A142 IgG production with just a few vaccinations, although the results varied depending

NU

SC

on the needle length or antigen type.

MA

Behavioral analysis
MWMT

We conducted MWMT as a behavior test for assessing cognitive function.

TE

In the experiments using both MH800 and MH300, there were little significant differences

AC
CE
P

between littermate wild-type mice and placebo-treated APPPS1 mice in the time to reach the
platform on training days (Fig. 3A, F), suggesting that the present MWMT did not perform well.
In probe tests, however, placebo-treated APPPS1 mice showed a slight declining trend in the
time to cross the platform location, the time spent in the target quadrant (TQ), and the number of
times the platform location was crossed compared with littermate wild-type mice (Fig. 3BE,
GI). Because placebo-treated APPPS1 mice seemed to show a slight memory deficit, we
evaluated the cognitive function in each vaccinated APPPS1 mouse.
In both experiments using MH800 and MH300, the time to reach the platform decreased with
training; however, the extent of the decrease was not significantly different among all groups
18

ACCEPTED MANUSCRIPT
Matsuo K. et al.

(Fig. 3B, G). In the probe test of experiment using MH800, the crossing index (i.e., the number

PT

of times a mouse crossed the platform location in the probe test) tended to increase in APPPS1

RI

mice in the TCI group compared with that in placebo-treated APPPS1 mice (p 0.05) and was

SC

almost identical to that in littermate wild-type mice (Fig. 3E). However, the time spent in the

NU

training quadrant and the swimming distance in the training quadrant were not significantly

MA

different among groups (Fig. 3C, D). The IDI APPPS1 mice achieved values equivalent to those
of the placebo-treated group. In the probe test of APPPS1 mice treated with TCI using MH300

containing A135-Cys and CT and IDI using A135-Cys and CT, the mice showed a slight

TE

increase in the tendency to cross the platform location sooner and to spend more time in TQ than

AC
CE
P

placebo-treated APPPS1 mice, and these mice performed the same as the littermate wild-type
mice, although no statistically significant differences were observed (Fig. 3H-J). In contrast,
there were no differences among the groups receiving TCI using MH300 containing A142 and
CT, IDI of A142 and CT, and the placebo-treated APPPS1 mice group.

NORT
In experiments using MH300, the recognition memory of the mice was also evaluated using
NORT, in which the motor activity of the mice has little influence on the experimental results.
During training, the mice were presented with two objects, A and B. Both littermate wild-type
19

ACCEPTED MANUSCRIPT
Matsuo K. et al.

mice and placebo-treated APPPS1 mice spent approximately equal time investigating each object

PT

(Fig. 4A). The total number of times the mice approached or sniffed the objects during training

RI

was similar between littermate wild-type mice and placebo-treated APPPS1 mice, indicating that

SC

they had comparable attention, motivation, and visual perception. Following training, one of the

NU

original objects was replaced with a new object. MH300/(A1-35-Cys and CT)-treated APPPS1

MA

mice and MH300/(A142 and CT)-treated APPPS1 mice spent approximately 20 more time
with the novel object C than with the already known object B, similar to littermate wild-type

mice, whereas placebo-treated APPPS1 mice spent equal time with both objects (less than 5)

AC
CE
P

with object B.

TE

(Fig. 4B). Each of the IDI control groups spent approximately 10 more time with object C than

From behavioral analysis data, MH-based TCI showed the potential to improve cognitive
function; however, the effect was small and slight. Therefore, further investigation will be
required to obtain more effective improvement in cognitive function.

Quantitative analysis of accumulation of various A molecule species in the brain

We analyzed brain sections for senile plaque deposits. Senile plaque aggregates comprise both
A140 and A142. Observation of brain sections stained with anti-A140, anti-A142, or
Congo red revealed a significant decrease in senile plaques in TCI groups using either MH300 or
20

ACCEPTED MANUSCRIPT
Matsuo K. et al.

MH800 compared with those in placebo-treated and IDI groups (Fig. 5A, D).

PT

We analyzed the number of senile plaques and the ratio of plaque area to total brain area from

RI

stained brain sections. In APPPS1 mice vaccinated with MH800 or MH300, the number and area

SC

of spots stained with anti-A142 or Congo red were smaller than those in the placebo-treated

NU

group (p 0.01 or 0.05) (Fig. 5B, C, E, F). In experiment using MH800, although the number of
senile plaques stained with anti-A140 was the same as that in the other groups, the area ratio

MA

was decreased (p 0.05).

Next, we determined the amount of A140 or A142 in soluble or insoluble forms and of

TE

A oligomers/fibrils in CC, HIPP, and EC sections, in which marked lesions are observed in AD

AC
CE
P

patients. The amount of each A molecule species in the brain of mice in the TCI group using
MH800 and MH300 was almost the same as that in the placebo-treated control group, indicating
no reduction in any A molecule species (Fig. 6AE, HL). The amount of each type of A
molecule species in the serum of mice in the MH800-based TCI group was higher than that in the
placebo-treated control group (p 0.05) (Fig. 6F, G). Each A molecule species in the serum of
mice in the MH300-based TCI groups was almost the same as that in the placebo-treated group;
however, in APPPS1 mice intradermally injected with A135-Cys and CT, there was an
obvious increase in both A140 and A142 in serum (p 0.05) (Fig. 6M, N).
These results suggest that anti-A142 IgG induced by MH-based TCI may prevent A
21

ACCEPTED MANUSCRIPT
Matsuo K. et al.

aggregation or cause A disaggregation throughout the immunization period; however, it cannot

RI

PT

completely remove A molecule species from the brain.

SC

Characteristics of immune responses

NU

We evaluated the characteristics of the immune responses induced by our TCI system using

MA

MH300 or MH800. Among the IgG subclasses in C57BL/6 mice, IgG1 and IgG2c are classified
as Th2-dependent and Th1-dependent, respectively, because IL-4 (a Th2-type cytokine) and

IFN- (a Th1-type cytokine) induce a class switch to IgG1 and IgG2a/c, respectively

TE

(Bergstedt-Lindqvist et al., 1988; Finkelman et al., 1988). We analyzed the IgG subclass to

AC
CE
P

evaluate the Th1/Th2 immune balance induced by our TCI system.

The IgG subclass in APPPS1 mice vaccinated by MH800 containing A142 and CT showed
a pattern similar to that of the IDI group (Fig. 7A, B). In TCI using MH300 containing A142
and CT, the IgG subclass pattern was almost same as that of the IDI group with A142 and CT
(Fig. 7C, D). In mice vaccinated with MH300 containing A135-Cys and CT, the ratio of IgG1
to IgG2c was much lower than that in IDI-treated mice (Fig. 7D).
These data indicate that TCI using both MH300 and MH800 induced similar immune
characteristics as the IDI system, suggesting that TCI using MH may induce acute
meningoencephalitis as an adverse side effect. This is a significant problem that requires
22

ACCEPTED MANUSCRIPT
Matsuo K. et al.

AC
CE
P

TE

MA

NU

SC

RI

PT

attention, and additional modification of our system to reduce adverse effects is desirable.

23

ACCEPTED MANUSCRIPT
Matsuo K. et al.

4. Discussion

PT

The purpose of the present study was to provide a simple and easy-to-use TCI system for AD

RI

vaccine therapy. Our MH-based TCI induced anti-A142 IgG following simple and damageless

SC

skin application and this effect was more than that of the IDI groups. This result suggested that

NU

almost the entire A142 encapsulated in each MH was efficiently delivered into the skin. In

MA

addition, because skin has many immunocompetent cells such as LCs, dermal dendritic cells, and
keratinocytes, MH-based TCI system targeting the skin immune system may induce efficient

immune responses compared with IDI groups. Previously-developed TCI procedure needed the

TE

complicated method to induce anti-A142 immune responses because of poor skin permeability

AC
CE
P

of A142 (Nikolic et al., 2007). Therefore our MH-based TCI system had an advantage in terms
of efficient delivery of antigens into the skin and immune induction in a simple, easy-to-use, and
low-invasive method.

Unfortunately, we did not obtain a significant improvement in cognitive function and

reduction of A molecular species in APPPS1 mice vaccinated with the MH-based TCI system.
In NORT, however, APPPS1 mice of TCI using MH300 seemed to show recovery of cognitive
function. The effect of TCI (MH300)/(A135-Cys and CT) group was better than that of TCI
(MH300)/(A142 and CT) group. The anti-A142 IgG level of TCI (MH300)/(A135-Cys
and CT) group was also higher than that of TCI (MH300)/(A142 and CT) group. A135-Cys
24

ACCEPTED MANUSCRIPT
Matsuo K. et al.

increased the immunogenicity to a greater extent than A142 (Matsuda et al., 2009) in injection

PT

system. These results suggested that A135-Cys may be applicable to our TCI system and that

RI

the level of anti-A142 IgG may be important for improvement of cognitive function in

SC

MH-based TCI system. Considering other reports, the effective concentration of anti-A142

NU

IgG in serum to clear senile plaques and improve cognitive function may be approximately

MA

100150 g/ml (Li et al., 2011; Movsesyan et al., 2010), which was almost same value as our
TCI system. On the other hand, recent studies have suggested that not only the concentration of

anti-A142 IgG but also the epitopes or isotypes of anti-A142 IgG are important for

TE

removing senile plaques and improving cognitive function (Bard et al., 2000; DeMattos et al.,

AC
CE
P

2001; McLaurin et al., 2002). This is reason why MH-based TCI induced anti-A142 IgG
production but did not significantly achieve reduction of A molecular species and improvement
in cognitive function. In future examination, it will be necessary to investigate epitope mapping
of anti-A142 IgG induced by TCI method using MH800 or MH300.
In addition, our TCI system did not always induce Th2-dominant immune responses. LCs
resident in the epidermal layer are suggested to play an important role in the induction of
Th2-dominant immune responses. Because both MH300 and MH800 were long enough to
deliver antigens into not only the epidermis but also into the dermis in mice, MH300 and MH800
delivered antigens not only to LCs but also to dermal dendritic cells, which induced Th1- and
25

ACCEPTED MANUSCRIPT
Matsuo K. et al.

Th2-type immune responses. Therefore, IgG subclass analysis revealed that TCI using MH300 or

PT

MH800 induced Th1- and Th2-type immune responses. Our preliminary results suggested that

RI

MH300 and MH800 delivered antigens into both the epidermis and dermis. Thus, we need to

SC

develop approaches to deliver antigens to LCs only.

NU

At present, we are pursuing several approaches using our MH-based TCI system for AD to

MA

control Th2-dominant immune responses and elicit effective improvement in cognitive function.
One approach is the fabrication of MH with a needle length that will only reach the epidermis in

which LCs are resident. Another approach is to combine antigens with appropriate adjuvants,

TE

which enhance the induction of immune responses and alteration of immune balances (Ishii and

AC
CE
P

Akira, 2007). Numerous studies have investigated the development of adjuvants (Boeglin et al.,
2011; Sariol et al., 2011), and we conducted in vivo screening for the purpose of adjuvant
development. We also prepared novel antigens that have the ability to target LCs involved in the
induction of Th2-dominant immune responses such as fusion antigens with targeting agents for
specific LC cell markers. In addition, we are attempting to investigate the efficacy of novel TCI
approaches using microneedle puncture methods. After puncturing the skin with MH, a hydrogel
patch formulation containing A142 is applied to the skin. This method is expected to
efficiently deliver antigens to LCs even if the antigens are aggregated and induce Th2-dominant
immune responses. By combining these approaches, we should be able to devise a TCI system
26

ACCEPTED MANUSCRIPT
Matsuo K. et al.

for AD that is not only efficacious but also safe.

PT

Several immune therapies for AD treatment have been reported, and some clinical trials are in

RI

progress. In particular, several treatments based on the administration of anti-A antibody drugs

SC

have been applied in clinical trials (Blennow et al., 2012; Farlow et al., 2012; Winblad et al.,

NU

2012). However, the problems related to injection remain. In addition, vaccination procedures

MA

that have been developed without injection systems include a TCI system that uses a gene gun
with plasmid DNA encoding A (Lambracht-Washington et al., 2009) and oral administration

using recombinant adeno-associated viral vector carrying A cDNA (Hara et al., 2004; Mouri et

TE

al., 2007) or plant-based vaccine containing A (Ishii-Katsuno et al., 2010; Nojima et al., 2011).

AC
CE
P

These vaccine therapies induced Th2-dominant immune responses and improved cognitive
function; however, there are some difficulties in formulating technology for practical use. In
contrast, our MH-based TCI system can efficiently immunize by simple application and MH is a
safe TCI device in human (Hirobe et al., 2013), which suggests a possible patient-led approach
that is unique and highly innovative. In addition, MH-based TCI is not associated with pain and
may increase the compliance of AD patients.
Vaccines are established as effective preventive approaches against infectious disease.
Recently, vaccine therapies intended to treat patients with cancer or autoimmune diseases have
been developed and are under investigation in clinical trials. The TCI system has the advantages
27

ACCEPTED MANUSCRIPT
Matsuo K. et al.

of being simple and painless, contributing to increased compliance of patients. The TCI system is

PT

also attracting attention as a promising procedure not only for the prevention of infectious

RI

disease but also for the treatment of cancer or autoimmune diseases. We are attempting to expand

SC

the application of MH-based TCI such as cancer or autoimmune disease treatments.

NU

In this study, we demonstrated that each MH-based TCI system efficiently induced

MA

anti-A142 immune responses via simple and low-invasive application to the skin. Our TCI
system has significant advantages because it is a simple, easy-to-use, painless, and minimally

invasive vaccination method. In future investigations, we will make modifications to overcome

TE

the issues noted here and continue to attempt to develop a simple, easy-to-use, minimally

AC
CE
P

invasive, effective, and safe TCI system for AD.

Acknowledgments:

This study was supported by the Advanced Research for Medical Products Mining Programme
of the National Institute of Biomedical Innovation and by Grant-in-Aid for Young Scientists (B)
(24790154) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
We thank Drs. Kazuhiro Takuma, Norihito Shintani, and Yukio Ago at Osaka University for their
advice regarding behavior analysis procedure. The authors declare no competing financial
interests.
28

ACCEPTED MANUSCRIPT
Matsuo K. et al.

References
Bard, F., Cannon, C., Barbour, R., Burke, R.L., Games, D., Grajeda, H., Guido, T., Hu, K.,

PT

RI

Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Lieberburg, I., Motter, R.,

SC

Nguyen, M., Soriano, F., Vasquez, N., Weiss, K., Welch, B., Seubert, P., Schenk, D.,

NU

Yednock, T. 2000. Peripherally administered antibodies against amyloid beta-peptide enter

MA

the central nervous system and reduce pathology in a mouse model of Alzheimer disease.
Nat. Med. 6, 916919.

S.B.,

Bayer, A.J., Bullock, R., Jones, R.W., Wilkinson, D., Paterson, K.R., Jenkins, L., Millais,
Donoghue, S. 2005. Evaluation of the safety and immunogenicity of synthetic

TE

AC
CE
P

Abeta42 (AN1792) in patients with AD. Neurology 64, 94101.

Bergstedt-Lindqvist, S., Moon, H.B., Persson, U., Mller, G., Heusser, C., Severinson, E.
1988. Interleukin 4 instructs uncommitted B lymphocytes to switch to IgG1 and IgE. Eur. J.
Immunol. 18, 10731077.

Blennow, K., Zetterberg, H., Rinne, J.O., Salloway, S., Wei, J., Black, R., Grundman, M.,
Liu, E.; AAB-001 201/202 Investigators. 2012. Effect of immunotherapy with
bapineuzumab on cerebrospinal fluid biomarker levels in patients with mild to moderate
Alzheimer disease. Arch. Neurol. 69, 10021010.

Boeglin, E., Smulski, C.R., Brun, S., Milosevic, S., Schneider, P., Fournel, S. 2011. Toll-like
29

ACCEPTED MANUSCRIPT
Matsuo K. et al.

receptor agonists synergize with CD40L to induce either proliferation or plasma cell

DeMattos, R.B., Bales, K.R., Cummins, D.J., Dodart, J.C., Paul, S.M., Holtzman, D.M.

RI

PT

differentiation of mouse B cells. PLoS One 6, e25542.

SC

2001. Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases

NU

brain A beta burden in a mouse model of Alzheimer's disease. Proc. Natl. Acad. Sci. U.S.A

MA

98, 88508855.

Farlow, M., Arnold, S.E., van Dyck, C.H., Aisen, P.S., Snider, B.J., Porsteinsson, A.P.,

Friedrich, S., Dean, R.A., Gonzales, C., Sethuraman, G., DeMattos, R.B., Mohs, R., Paul,

TE

S.M., Siemers, E.R. 2012. Safety and biomarker effects of solanezumab in patients with

AC
CE
P

Alzheimer's disease. Alzheimers Dement. 8, 261271.

Ferrer, I., Boada Rovira, M., Sanchez Guerra, M.L., Rey, M.J., Costa-Jussa, F. 2004.
Neuropathology and pathogenesis of encephalitis following amyloid-beta immunization in
Alzheimer's disease. Brain Pathol. 14, 1120.

Finkelman, F.D., Katona, I.M., Mosmann, T.R., Coffman, R.L. 1988. IFN- regulates the
isotypes of Ig secreted during in vivo humoral immune responses. J. Immunol. 140,
10221027.

Fujita, H., Asahina, A., Sugaya, M., Nakamura, K., Gao, P., Fujiwara, H., Tamaki, K. 2005.
Differential production of Th1- and Th2-type chemokines by mouse Langerhans cells and
30

ACCEPTED MANUSCRIPT
Matsuo K. et al.

splenic dendritic cells. J. Invest. Dermatol. 124, 343350.

Hara, H., Monsonego, A., Yuasa, K., Adachi, K., Xiao, X., Takeda, S., Takahashi, K., Weiner,

PT

RI

H.L., Tabira, T. 2004. Development of a safe oral Abeta vaccine using recombinant

Hardy, J., Selkoe, D.J. 2002. The amyloid hypothesis of Alzheimer's disease: progress and

NU

SC

adeno-associated virus vector for Alzheimer's disease. J. Alzheimers Dis. 6, 483488.

MA

problems on the road to therapeutics. Science 297, 353356.

Hirobe, S., Azukizawa, H., Matsuo, K., Zhai, Y., Quan, Y.S., Kamiyama, F., Suzuki, H.,

Katayama, I., Okada, N., Nakagawa, S. 2013. Development and Clinical study of a

AC
CE
P

26642674.

TE

self-dissolving microneedle patch for transcutaneous immunization device. Pharm. Res. 30,

Hock, C., Konietzko, U., Papassotiropoulos, A., Wollmer, A., Streffer, J., von Rotz, R.C.,
Davey, G., Moritz, E., Nitsch, R.M. 2002. Generation of antibodies specific for beta-amyloid
by vaccination of patients with Alzheimer disease. Nat. Med. 8, 12701275.

Hock, C., Konietzko, U., Streffer, J.R., Tracy, J., Signorell, A., Muller-Tillmanns, B., Lemke,
U., Henke, K., Moritz, E., Garcia, E., Wollmer, M.A., Umbricht, D., de Quervain, D.J.,
Hofmann, M., Maddalena, A., Papassotiropoulos, A., Nitsch, R.M. 2003. Antibodies against
beta-amyloid slow cognitive decline in Alzheimer's disease. Neuron 38, 547554.

Ishii-Katsuno, R., Nakajima, A., Katsuno, T., Nojima, J., Futai, E., Sasagawa, N., Yoshida, T.,
31

ACCEPTED MANUSCRIPT
Matsuo K. et al.

Watanabe, Y., Ishiura, S. 2010. Reduction of amyloid beta-peptide accumulation in Tg2576

Ishii, K.J., Akira, S. 2007. Toll or toll-free adjuvant path toward the optimal vaccine

RI

PT

transgenic mice by oral vaccination. Biochem. Biophys. Res. Commun. 399, 593599.

Ishii, Y., Nakae, T., Sakamoto, F., Matsuo, K., Matsuo, K., Quan, Y.S., Kamiyama, F., Fujita,

NU

SC

development. J. Clin. Immunol. 27, 363371.

MA

T., Yamamoto, A., Nakagawa, S., Okada, N. 2008. A transcutaneous vaccination system
using a hydrogel patch for viral and bacterial infection. J. Control. Release 131, 113120.
Lambracht-Washington, D., Qu, B.X., Fu, M., Eagar, T.N., Stve, O., Rosenberg, R.N. 2009.

TE

DNA beta-amyloid(1-42) trimer immunization for Alzheimer disease in a wild-type mouse

AC
CE
P

model. JAMA. 302, 17961802.

Larregina, A.T., Morelli, A.E., Spencer, L.A., Logar, A.J., Watkins, S.C., Thomson, A.W.,
Falo, L.D. Jr 2001. Dermal-resident CD14+ cells differentiate into Langerhans cells. Nat.
Immunol. 2, 11511158.

Li, Y., Ma, Y., Zong, L.X., Xing, X.N., Sha, S., Cao, Y.P. 2011. Intranasal inoculation with
an adenovirus vaccine encoding ten repeats of A3-10 induces Th2 immune response against
amyloid- in wild-type mouse. Neurosci. Lett. 505, 128133.

Matsuda, J., Kaminaka, K., Nozaki, C. 2009. Amyloid beta peptides with an additional
cysteine residue can enhance immunogenicity and reduce the amyloid beta burden in an
32

ACCEPTED MANUSCRIPT
Matsuo K. et al.

Alzheimer's disease mouse model. Biochem. Biophys. Res. Commun. 382, 149152.
Matsuo, K., Hirobe, S., Yokota, Y., Ayabe, Y., Seto, M., Quan, Y.S., Kamiyama, F., Tougan,

PT

RI

T., Horii, T., Mukai, Y., Okada, N., Nakagawa, S. 2012. Transcutaneous immunization using

SC

a dissolving microneedle array protects against tetanus, diphtheria, malaria, and influenza. J.

Matsuo, K., Ishii, Y., Quan, Y.S., Kamiyama, F., Mukai, Y., Yoshioka, Y., Okada, N.,

MA

NU

Control. Release 160, 495501

Nakagawa, S. 2011. Transcutaneous vaccination using a hydrogel patch induces effective

immune responses to tetanus and diphtheria toxoid in hairless rat. J. Control. Release 149,

Matsuo, K., Yokota, Y., Zhai, Y., Quan, Y.S., Kamiyama, F., Mukai, Y., Okada, N.,

AC
CE
P

TE

1520.

Nakagawa, S. 2012. A low-invasive and effective transcutaneous immunization system using

a novel dissolving microneedle array for soluble and particulate antigens. J. Control. Release
161, 1017.

McLaurin, J., Cecal, R., Kierstead, M.E., Tian, X., Phinney, A.L., Manea, M., French, J.E.,
Lambermon, M.H., Darabie, A.A., Brown, M.E., Janus, C., Chishti, M.A., Horne, P.,
Westaway, D., Fraser, P.E., Mount, H.T., Przybylski, M., St George-Hyslop, P. 2002.
Therapeutically effective antibodies against amyloid-beta peptide target amyloid-beta
residues 4-10 and inhibit cytotoxicity and fibrillogenesis. Nat. Med. 8, 12631269.
33

ACCEPTED MANUSCRIPT
Matsuo K. et al.

Movsesyan, N., Davtyan, H., Mkrtichyan, M., Petrushina, I., Tiraturyan, T., Ross, T.,

PT

Agadjanyan, M.G., Ghochikyan, A., Cribbs, D.H. 2010. Low concentrations of anti-A

RI

antibodies generated in Tg2576 mice by DNA epitope vaccine fused with 3C3d molecular

Mouri, A., Noda, Y., Hara, H., Mizoguchi, H., Tabira, T., Nabeshima, T. 2007. Oral

NU

SC

adjuvant do not affect AD pathology. Hum. Gene Ther. 21, 128133.

MA

vaccination with a viral vector containing Abeta cDNA attenuates age-related Abeta
accumulation and memory deficits without causing inflammation in a mouse Alzheimer

Nicoll, J.A., Wilkinson, D., Holmes, C., Steart, P., Markham, H., Weller, R.O. 2003.

TE

model. FASEB J. 21, 21352148.

AC
CE
P

Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide:

a case report. Nat. Med. 9, 448452.

Niizeki, H., Streilein, J.W. 1997. Hapten-specific tolerance induced by acute, low-dose
ultraviolet B radiation of skin is mediated via interleukin-10. J. Invest. Dermatol. 109,
2530.

Nikolic, W.V., Bai, Y., Obregon, D., Hou, H., Mori, T., Zeng, J., Ehrhart, J., Shytle, R.D.,
Giunta, B., Morgan, D., Town, T., Tan, J. 2007. Transcutaneous beta-amyloid immunization
reduces cerebral beta-amyloid deposits without T cell infiltration and microhemorrhage.
Proc. Natl. Acad. Sci. U.S.A. 104, 25072512.
34

ACCEPTED MANUSCRIPT
Matsuo K. et al.

Nojima, J., Maeda, A., Aoki, S., Suo, S., Yanagihara, D., Watanabe, Y., Yoshida, T., Ishiura,

PT

S. 2011. Effect of rice-expressed amyloid in the Tg2576 Alzheimer's disease transgenic

Orgogozo, J.M., Gilman, S., Dartigues, J.F., Laurent, B., Puel, M., Kirby, L.C., Jouanny, P.,

SC

RI

mouse model. Vaccine 29, 62526258.

NU

Dubois, B., Eisner, L., Flitman, S., Michel, B.F., Boada, M., Frank, A., Hock, C. 2003.

MA

Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization.

Neurology 61, 4654.

Sariol, C.A., Martnez, M.I., Rivera, F., Rodrguez, I.V., Pantoja, P., Abel, K., Arana, T.,

TE

Giavedoni, L., Hodara, V., White, L.J., Angler, Y.I., Montaner, L.J., Kraiselburd, E.N. 2011.

AC
CE
P

Decreased dengue replication and an increased anti-viral humoral response with the use of
combined Toll-like receptor 3 and 7/8 agonists in macaques. PLoS One 6, e19323.

Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu, K., Huang, J.,
Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Liao, Z., Lieberburg, I., Motter, R.,
Mutter, L., Soriano, F., Shopp, G., Vasquez, N., Vandevert, C., Walker, S., Wogulis, M.,
Yednock, T., Games, D., Seubert, P. 1999. Immunization with amyloid-beta attenuates
Alzheimer-disease-like pathology in the PDAPP mouse. Nature 400, 173177.

Selkoe, D.J. 1991. The molecular pathology of Alzheimer's disease. Neuron 6, 487498.

Tada, Y., Asahina, A., Fujita, H., Sugaya, M., Tamaki, K. 2003. Langerhans cells do not
35

ACCEPTED MANUSCRIPT
Matsuo K. et al.

produce interferon-. J. Invest. Dermatol. 120, 891892.

PT

Winblad, B., Andreasen, N., Minthon, L., Floesser, A., Imbert, G., Dumortier, T., Maguire,

RI

R.P., Blennow, K., Lundmark, J., Staufenbiel, M., Orgogozo, J.M., Graf, A. 2012. Safety,

SC

tolerability, and antibody response of active A immunotherapy with CAD106 in patients

NU

with Alzheimer's disease: randomised, double-blind, placebo-controlled, first-in-human

TE

MA

study. Lancet Neurol. 11, 597604.

AC
CE
P

36

ACCEPTED MANUSCRIPT
Matsuo K. et al.

Figure legends

PT

Fig. 1

RI

Experimental design. This study was conducted as two separate experiments, Experiment 1 and

SC

Experiment 2. APPPS1 mice started receiving immunizations at the age of 4 months. (A)

NU

Experimental schedule. (B) Experimental groups in Experiment 1 and Experiment 2. For details,

MA

see Materials and Methods.

Fig. 2

TE

Anti-A142 antibody production after transcutaneous vaccination of APPPS1 mice. (A;

AC
CE
P

experiment using MH800) A142 (10 g)- and CT (0.1 g)-containing MH800 () was
applied to the skin of the back of APPPS1 mice for 6 h. As a control IDI group, APPPS1 mice
were intradermally injected with A142 (10 g) and CT (0.1 g) (). (B; experiment using
MH300) A142 (10 g)- and CT (0.25 g)-containing MH300 () or A135-Cys (10 g)and CT (0.25 g)-containing MH300 () was applied to the skin of the back of APPPS1 mice
for 6 h. As a control IDI group, APPPS1 mice were intradermally injected with A142 (10 g)
and CT (0.25 g) () or A135-Cys (1 g) and CT (0.25 g) (). These procedures were
conducted on a weekly basis for the first month. Thereafter, these mice were continually
immunized biweekly for the next 12 weeks. At the indicated points, sera collected from these
37

ACCEPTED MANUSCRIPT
Matsuo K. et al.

mice were assayed for anti-A142 IgG antibody concentration by ELISA. Data are expressed as

PT

the mean SE of results from 38 mice. Placebo-treated APPPS1 mice: below the detection

SC

RI

limits. Arrowhead indicates vaccination points.

NU

Fig. 3

MA

Effect of transcutaneous vaccination on cognitive function in APPPS1 mice and littermate

wild-type mice based on MWMT. Cognitive function in immunized APPPS1 mice and

littermate wild-type mice was evaluated using MWMT (A-E; experiment using MH800, F-J;

TE

experiment using MH300). (A, F) Escape latency during a 60-s session with littermate wild-type

AC
CE
P

mice and placebo-treated APPPS1 mice in the water maze test. (B, G) Escape latency during a
60-s session with immunized APPPS1 mice in the water maze test. (C, H) Time spent in the TQ
in probe trials. (D,I) Distance spent in TQ in probe trials. (E,J) Crossing index at the platform
location in probe trials. Values indicate the mean SE (littermate wild-type mice, n = 7 or 12;
immunized APPPS1 mice, n = 38; placebo-treated APPPS1 mice, n = 7 or 11). Statistical
significance was evaluated by one-way ANOVA followed by Tukeys test for multiple
comparisons.: *; p 0.05 versus littermate wild-type mice.

Fig. 4
38

ACCEPTED MANUSCRIPT
Matsuo K. et al.

Effect of transcutaneous vaccination on cognitive function in APPPS1 mice and littermate

PT

mice based on NORT. Cognitive function in APPPS1 mice and littermate wild-type mice was

RI

evaluated using NORT (experiment using MH300). Each mouse was individually habituated to

SC

the box with 1 h of exploration in the absence of objects (habituation session). For details, see

NU

Materials and Methods. A preference index, the ratio of the amount of time spent exploring any

MA

one of the two objects over the total time spent exploring both objects in the training session (A)
or the retention session (B), was used as a measure of cognitive function. Values indicate the

mean SE (littermate wild-type mice, n = 12; immunized APPPS1 mice, n = 36;

TE

placebo-treated APPPS1 mice, n = 7). Statistical significance was evaluated using one-way

AC
CE
P

ANOVA followed by Tukeys test for multiple comparisons.: *; p 0.05 versus littermate
wild-type mice.

Fig. 5

Observation of senile plaques deposited on brain sections. The brain was harvested, and brain
sagittal sections from HIPP regions were prepared. Sections were stained with anti-A140
antibody, anti-A142 antibody, or Congo red. (A-C; experiment using MH800, D-F; experiment
using MH300) (A,D) The stained sections were photographed under a stereoscopic microscope.
Scale bar 300 m. Arrows indicate the stained plaques. (B,E) Number of stained plaques and
39

ACCEPTED MANUSCRIPT
Matsuo K. et al.

(C,F) area percentage of the brain sections occupied by stained plaques were calculated by

PT

quantitative image analysis. Data are represented as the mean SE (immunized APPPS1 mice, n

RI

= 38; placebo-treated APPPS1 mice, n = 7 or 11). Statistical significance was evaluated by

SC

one-way ANOVA followed by Tukeys test for multiple comparisons.: *; p 0.05 versus

MA

NU

placebo-treated group, **; p 0.01 versus placebo-treated group.

Fig. 6

Quantitative analysis of amount of A molecule species in the brain. The brain and serum

TE

were collected from vaccinated APPPS1 mice, and soluble or insoluble homogenate fractions of

AC
CE
P

the CC, HIPP, or EC were prepared. A140, A142, or A oligomers/fibrils levels in soluble
or insoluble fractions (A-E; experiment using MH800, H-L; experiment using MH300) or serum
(F,G; experiment using MH800, M,N; experiment using MH300) were determined by ELISA.
Values are represented as the mean SE (immunized APPPS1 mice, n = 38; placebo-treated
APPPS1 mice, n = 7 or 11). N.D., not detectable. Statistical significance was evaluated using
one-way ANOVA followed by Tukeys test for multiple comparisons. (*; p 0.05 versus
placebo-treated group, **; p 0.01 versus placebo-treated group)

Fig. 7
40

ACCEPTED MANUSCRIPT
Matsuo K. et al.

Analysis of anti-A142 IgG subclass. A,B; experiment using MH800, C,D; experiment using

PT

MH300. For details of vaccination protocol, see Materials and Methods. (A,C) Sera collected

RI

from these mice were assayed for anti-A142 IgG subclass (IgG1, IgG2c) titer by ELISA.

MA

NU

expressed as the mean SE of results from 38 mice.

SC

(B,D) The ratio of anti-A142 IgG1 titer to anti-A142 IgG2c titer was calculated. Data are

Supplementary Fig. 1

Dissolving microneedle array (MicroHyala; MH). (A) Bright-field micrograph of whole MH.

TE

(B) Bright-field micrograph of microneedles on a cone-shaped MH before or after insertion into

AC
CE
P

the skin. MH was applied to the skin on the back of BALB/c mice. One hour later, each MH was
removed and photographed under a stereoscopic microscope.

Supplementary Fig. 2

Anti-A142 antibody production after transcutaneous vaccination of C57BL/6 mice.

A142 (67 g)- and CT (0.1 g)-containing MH800 was applied to the skin of the back of
C57BL/6 mice for 6 h. As a control IDI group, APPPS1 mice were intradermally injected with
A142 (67 g) and CT (0.1 g). These procedures were conducted on weeks 0, 1, 2, 3, 4, and 6.
At the indicated points, sera collected from these mice were assayed for anti-A142 IgG
41

ACCEPTED MANUSCRIPT
Matsuo K. et al.

antibody concentration by ELISA. Data are expressed as the mean SE of results from 5 mice.

AC
CE
P

TE

MA

NU

SC

RI

PT

Arrowhead indicates vaccination points.

42

ACCEPTED MANUSCRIPT

SC

RI

PT

Matsuo K. et al.

AC
CE
P

TE

MA

NU

Graphical abstract

43

ACCEPTED MANUSCRIPT

Fig 1

AC
CE
P

TE

MA

NU

SC

RI

PT

Matsuo K. et al.

44

ACCEPTED MANUSCRIPT

AC
CE
P

Fig 2

TE

MA

NU

SC

RI

PT

Matsuo K. et al.

45

ACCEPTED MANUSCRIPT

AC
CE
P

TE

MA

NU

SC

RI

PT

Matsuo K. et al.

Fig 3

46

ACCEPTED MANUSCRIPT

AC
CE
P

TE

MA

NU

SC

RI

PT

Matsuo K. et al.

Fig 4

47

ACCEPTED MANUSCRIPT

AC
CE
P

TE

MA

NU

SC

RI

PT

Matsuo K. et al.

Fig 5

48

ACCEPTED MANUSCRIPT

AC
CE
P

TE

MA

NU

SC

RI

PT

Matsuo K. et al.

49

ACCEPTED MANUSCRIPT
Matsuo K. et al.

AC
CE
P

TE

MA

NU

SC

RI

PT

Fig 6

50

ACCEPTED MANUSCRIPT

NU

SC

RI

PT

Matsuo K. et al.

AC
CE
P

TE

MA

Fig 7

51

ACCEPTED MANUSCRIPT

AC
CE
P

TE

MA

NU

SC

RI

PT

Matsuo K. et al.

Fig 8

52

ACCEPTED MANUSCRIPT
Matsuo K. et al.

May 21, 2013

Highlights

PT

We examined vaccine efficacy of transcutaneous immunization for Alzheimers disease.

RI

Transcutaneous immunization using MicroHyala induced anti-A142 immune responses.

SC

MicroHyala-based transcutaneous immunization will contribute to the development of simple

AC
CE
P

TE

MA

NU

and easy-to-use vaccine system for Alzheimers disease

53