Vaksinasi Demensia

Vaccine efficacy of transcutaneous immunization with amyloid using adissolving 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: S0165-5728(13)00311-1DOI: doi: 10.1016/j.jneuroim.2013.11.002Reference: JNI 475820

To appear in: Journal of Neuroimmunology

Received date: 30 January 2013Revised date: 2 November 2013Accepted date: 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, Vac-cine ecacy of transcutaneous immunization with amyloid using a dissolving micronee-dle array in a mouse model of Alzheimers disease, Journal of Neuroimmunology (2013),doi: 10.1016/j.jneuroim.2013.11.002

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ACCEPTED MANUSCRIPTMatsuo K. et al.

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Title:

Vaccine efficacy of transcutaneous immunization with amyloid using a dissolving microneedle

array in a mouse model of Alzheimers disease

Abbreviated title:

Transcutaneous vaccination for Alzheimer's disease

Author names:

Kazuhiko Matsuo1,

, Hideaki Okamoto1, Yasuaki Kawai

1, Ying-Shu Quan

2, Fumio Kamiyama

2,

Sachiko Hirobe1, Naoki Okada

1,*, Shinsaku Nakagawa

1,*

Affiliations:

1 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

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*Corresponding author:

Naoki Okada, Ph.D.

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

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

(Phone): +81-6-6879-8176, (Fax): +81-6-6879-8176, (E-mail): [email protected]

Shinsaku Nakagawa, Ph.D.

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

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

(Phone): +81-6-6879-8175, (Fax): +81-6-6879-8179, (E-mail): [email protected]

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Short Abstract

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

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

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

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

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

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

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Abstract

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

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

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

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

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

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

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:

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Alzheimers disease, vaccine therapy, transcutaneous immunization, dissolving microneedle

array

Abbreviations

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

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

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

TBS-T, TrisHCl-buffered saline containing 0.05% Tween-20; TCI, transcutaneous

immunization; TQ, target quadrant

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1. Introduction

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

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

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

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

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).

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

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

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

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

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

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

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

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

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

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

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

A vaccine therapy for AD.

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

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

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

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

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

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.

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2. Materials and Methods

Reagents

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

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

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

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

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

-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

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

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

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

Animal Care and Use Committee of Osaka University.

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

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

(Supplementary Fig. 1).

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

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

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

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

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

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

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),

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

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.

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MH-based TCI protocol

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

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

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

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

12.8 N/200 microneedles.

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

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

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

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

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

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

measured at 450650 nm.

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

that showed 0.1 absorbance units after subtracting the background.

NORT

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

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

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

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

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

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

behavioral tests, the objects were changed each time.

MWMT

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

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

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

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

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

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

amyloid protein immunohistostain kit or a Congo red amyloid stain kit procedure. Brain

sections were observed under a stereoscopic microscope (Biozero BZ8000; Keyence, Osaka,

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

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

triplicate analyses for each mouse.

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

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

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

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

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

according to the manufacturers protocols.

Statistical analysis

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

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

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

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3. Results

Vaccine efficacy of TCI formulation using MH for AD

Anti-A142 IgG production in APPPS1 mice

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

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

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

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

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

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senile plaques from blood vessels.

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

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

on the needle length or antigen type.

Behavioral analysis

MWMT

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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)

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

with object B.

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

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

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

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

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

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

was decreased (p 0.05).

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

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

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

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

completely remove A molecule species from the brain.

Characteristics of immune responses

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

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

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

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

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

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4. Discussion

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

develop approaches to deliver antigens to LCs only.

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

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,

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

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

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for AD that is not only efficacious but also safe.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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Figure legends

Fig. 1

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

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

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

see Materials and Methods.

Fig. 2

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

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

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

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

limits. Arrowhead indicates vaccination points.

Fig. 3

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;

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

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

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

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

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

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

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

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;

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

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

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

quantitative image analysis. Data are represented as the mean SE (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

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

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

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

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

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

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

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

expressed as the mean SE of results from 38 mice.

Supplementary Fig. 1

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

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

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

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

Arrowhead indicates vaccination points.

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Graphical abstract

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Fig 1

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Fig 2

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Fig 3

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Fig 4

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Fig 5

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Fig 6

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Fig 7

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Fig 8

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May 21, 2013

Highlights

We examined vaccine efficacy of transcutaneous immunization for Alzheimers disease.

Transcutaneous immunization using MicroHyala induced anti-A142 immune responses.

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

and easy-to-use vaccine system for Alzheimers disease

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