Nonviral A? DNA vaccine therapy against Alzheimer’s
disease: Long-term effects and safety
Yoshio Okura*, Akira Miyakoshi*, Kuniko Kohyama*, Il-Kwon Park*, Matthias Staufenbiel†, and Yoh Matsumoto*‡
*Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Musashidai 2-6, Fuchu, Tokyo 183-8526, Japan;
and†Neuroscience Research, Novartis Institutes of Biomedical Research, CH-4002 Basel, Switzerland
Edited by Hugh O. McDevitt, Stanford University School of Medicine, Stanford, CA, and approved May 10, 2006 (received for review February 6, 2006)
It was recently demonstrated that amyloid ? (A?) peptide vacci-
mice. However, the clinical trial was halted because of the devel-
problem, anti-A? antibody therapy and other types of vaccination
nonviral A? DNA vaccines against Alzheimer’s disease. We admin-
istered these vaccines to model (APP23) mice and evaluated A?
burden reduction. Prophylactic treatments started before A? dep-
osition reduced A? burden to 15.5% and 38.5% of that found in
untreated mice at 7 and 18 months of age, respectively. Therapeu-
tic treatment started after A? deposition reduced A? burden
neither neuroinflammation nor T cell responses to A? peptide in
both APP23 and wild-type B6 mice, even after long-term vaccina-
tion. Although it is reported that other anti-A? therapies have
pharmacological and?or technical difficulties, nonviral DNA vac-
cines are highly secure and easily controllable and are promising
for the treatment of Alzheimer’s disease.
amyloid ?-peptide ? DNA vaccination
of memory and cognitive function in aged humans. The etiology
of the disease is thought to be the result of an imbalance between
amyloid ? (A?) production and clearance (amyloid cascade
hypothesis) (1, 2). On the basis of this hypothesis, Schenk et al.
(3) developed an A?-peptide vaccine, immunized amyloid pre-
cursor protein (APP)-transgenic mice with the peptide in com-
plete Freund’s adjuvant (CFA), and demonstrated a marked
amyloid reduction in the brain. Repetitive intranasal adminis-
tration of A?-peptide and adjuvant (4) and the passive transfer
of anti-A? antibodies were also effective in reducing amyloid
deposits (5). Moreover, vaccinated mice showed an improve-
ment in memory loss (6, 7). Thus, A? peptide vaccine therapy
has been shown to be effective in animal models, and human
clinical trials were started with Betabloc (AN-1792), composed
of synthetic A?1-42 and QS21 as an adjuvant (8). However, the
phase II clinical trial was halted because of the development of
acute meningoencephalitis that appeared in 18 (6%) of 298
vaccinated patients (9). Importantly, it was later demonstrated
by autopsy that there was a significant reduction of amyloid
deposition and disappearance of degenerative axons in a treated
patient (10). At the same time, T cell-dominant meningeal
encephalitis was present in the cerebral cortex. These findings
suggest that the vaccine therapy is a promising strategy for
human Alzheimer’s disease if excessive immune reactions are
minimized to avoid unwanted neuroinflammation.
Recently, it was reported that naked plasmid DNAs encoding
proteins are taken into cells and produce the proteins in a small
amount for a relatively long period when injected into the muscle
or skin (11). Then, the proteins that are released in the extra-
cellular space induce antibodies against the proteins (12, 13).
Thus, gentle and quiet immune reactions could be obtained by
DNA vaccine administration. In our and other’s laboratories,
lzheimer’s disease is a chronic neurodegenerative disorder
that is the most common cause of progressive impairment
immune therapies with DNA vaccines have been examined in
autoimmune disease models (14–17) and have been found to be
effective in preventing the diseases without the use of adjuvants.
Here, we developed nonviral A? DNA vaccines and were able
to reduce the amyloid burden in the cerebral cortex and hip-
pocampus of Alzheimer’s disease model (APP23) mice by
vaccination. Importantly, the side effects, such as T cell prolif-
eration and neuroinflammation, were absent even after long-
term administration of the vaccines in both APP23 and wild-type
Preparation and Characterization of Nonviral A? DNA Vaccines. We
prepared three types of nonviral A? DNA vaccines using a
mammalian expression vector, pTarget. The sequence of A?1-42
and additional sequences were inserted in the plasmid, as shown
in Fig. 1A. The first one contains only the A?1-42 sequence with
the Kozak sequence at the 5? end (referred to as K-A? vaccine)
(Fig. 1A1). To the second, the Ig? signal sequence of mouse Ig
was added to improve the secretion ability (IgL-A? vaccine)
(Fig. 1A2), and the third possesses the Fc portion of human Ig
at the 3? end to maintain stability (A?-Fc vaccine) (Fig. 1A3).
Before in vivo administration, these DNA vaccines were trans-
fected to HEK295T cells, and the secretion of A?1-42 peptide
into the culture supernatant was assayed with Western blotting
(Fig. 1B). The production of intracellular A?1-42 peptide was
confirmed in all three vaccines by ELISA (data not shown). It
was clearly demonstrated that the supernatants of cultured cells
that had been transfected with IgL-A? and A?-Fc vaccines
contained translated proteins (4.5 and 35 kDa, respectively),
whereas K-A?-transfected cells did not secrete the peptide into
the extracellular space. These findings indicate that the addition
of the leader sequence is important for transportation of the
protein to the extracellular space as reported in ref. 18 and that
this event is critical for the effects of DNA vaccines (see below).
Reduction of Amyloid Burden by A? DNA Vaccination. We used two
types of regimens to examine the effect of A? DNA vaccination,
i.e., prophylactic and therapeutic. For the prophylactic protocol,
vaccine administration was started at 3–4 months of age, before
the appearance of amyloid deposition. APP23 mice received 6
weekly and subsequent biweekly injections of the vaccines and
were examined at 7, 9, 12, 15, and 18 months of age (Fig. 1C1).
The paraffin-embedded sections of the brain were stained
immunohistochemically with 6F?3D against A?8-17, and the
area of amyloid depositions was quantitated as the total sum of
the pixels with NIH IMAGE software.
Conflict of interest statement: M.S. is employed by and a shareholder of Novartis Institutes
of Biomedical Research.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: A?, amyloid ?; A?-Fc, IgL-A?-pTarget, the Fc portion of immunoglobulin;
leader sequence-A?-pTarget; K-A?, Kozak-A?-pTarget; Th, T helper.
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
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In the first series of the prophylactic treatment, mice were
analyzed at 7, 9, and 12 months of age (Table 1, Exp. 1). At 7
months of age, granular amyloid depositions were recognized in
the frontal cortex in the control groups (empty-vector-
administered and untreated mice) (Fig. 2B). At this stage, A?
plaques were not detected in the hippocampus (data not shown).
In sharp contrast, cortical A? depositions in mice treated with
A?-Fc (Fig. 2A), IgL-A?, and A? (data not shown) vaccines
were significantly reduced (P ? 0.01). The A? burden was
reduced to ?15–30% of the untreated groups (Fig. 2E). At 12
months of age, amyloid depositions in untreated mice were
increased, and some of them became large (?50 ?g) in the
frontal cortices of the untreated mice (Fig. 4C, which is pub-
treatment protocol (C). (A) Three DNA vaccines were produced by using a
mammalian expression vector. DNA encoding the A?1-42 sequence was in-
serted in Xhol?Kpnl site of the plasmid (K-A? vaccine) (A1). In the second
secretive efficiency (IgL-A? vaccine) (A2). The third vaccine possesses the Fc
portion of human immunoglobulins to improve the stability of the secreted
A? proteins were detected in supernatants of cultured cells transfected with
IgL-A? and A?-Fc vaccines. (C) The protocol of vaccine treatment. To examine
the prophylactic effect of DNA vaccines, the vaccines were administrated to
APP23 mice starting from 3–4 months of age, before the appearance of
amyloid depositions. The mixture of one of the vaccines (100 ?g) and bupiv-
acaine (0.25 mg) was injected intramuscularly on a weekly basis for the first 6
weeks. Then, the vaccine without bupivacaine was injected every 2 weeks
thereafter. Mice were sampled at 7, 9, 12, 15, and 18 months of age (C1). For
from 12 months of age, after the appearance of amyloid plaques. Samplings
were performed at 15 and 18 months of age (C2). m, months of age.
Table 1. Summary of experimental data on nonviral A? DNA vaccine therapy against Alzheimer model mice
of Tx, mo
% A? burden (cortex)
% A? burden (hippocampus)
than other two vaccines. Each group consisted of 4–6 mice. Prophylactic effects were examined in Exps. 1 and 2. Therapeutic effects were examined in Exps. 3 and 4. All APP23 mice were analyzed
immunohistochemically with mAb (6F?3D) against A?7-18. Then, the reduction of amyloid plaque was quantified, as shown in Materials and Methods. In Exp. 5, DNA vaccines were administered to B6 mice to
know whether the vaccines induce neuroinflammation. Tissues from three mice in each group were immunohistochemically stained with mAbs against CD5 and Mac-3. Neg, negative finding, i.e., no
neuroinflammation; N.T., not tested; Exp., experiment; Tx, treatment; Tg, transgenic; Emp, empty vector.
www.pnas.org?cgi?doi?10.1073?pnas.0600966103Okura et al.
A? depositions were significantly reduced (P ? 0.01) to ?30–
50% of the untreated group (Fig. 4D) after A?-Fc (Fig. 4A) and
IgL-A? (Fig. 4B) vaccine treatment. A? depositions in the
hippocampus were also decreased equally (P ? 0.01) (Table 1,
Exp. 1, Group 3). It was shown that the suppressive effect of
A?-Fc vaccine was almost equal to IgL-A? vaccine. However,
K-A? vaccine was less effective than the former two (Figs. 2E
and 4D) and was not used in subsequent experiments. The
second part of the prophylactic treatment analyzed the mice at
15 and 18 months of age (Table 1, Exp. 2). At these time points,
the plaques in untreated groups had rapidly increased. Un-
treated APP23 mice showed an age-dependent increase of
amyloid plaques in the cerebral cortex (Fig. 2G, open squares)
and hippocampus (data not shown). The prophylactic protocol,
using A?-Fc vaccine, revealed that the final reduction rate of A?
burden in the cerebral cortex at 18 months of age was ?38.5%
of untreated groups (Table 1, Exp. 2 and Fig. 2G, closed
triangles). These results demonstrated that two of the three
vaccines produced in this study were effective in prophylactic
When considering the clinical applications, it is critical to
know the effects of the vaccines in therapeutic application. For
this purpose, the vaccination was started at 12 months of age, 6
months after the start of A? deposition, and the brains were
examined at 15 (Fig. 2) and 18 (Fig. 5, which is published as
supporting information on the PNAS web site) months of age. In
therapeutic treatment, amyloid plaques in the cortex were
significantly decreased (P ? 0.01) (Fig. 2F) by A?-Fc and
IgL-A? vaccination (Fig. 2D) compared with the controls (Fig.
2C). A? depositions in the hippocampus were also decreased
(P ? 0.01) (Table 1, Exp. 3). Although the therapeutic protocol
(Fig. 2G, open circles) seemed to be less effective than the
prophylactic one (Fig. 2G closed triangles), the difference was
not significant. It should be noted that APP23 mice treated with
the therapeutic protocol received DNA vaccines for only 3 and
6 months, respectively (Table 1, Exp. 3). Thus, A? DNA vaccines
had sufficient effects, even if the vaccines were administrated
after amyloid depositions appeared.
Recently, it was reported that the intracellular A? deposition
in cortical pyramidal neurons is the first neurodegenerative
event in Alzheimer’s disease development (19). Therefore, we
counted the number of neurons containing intracellular A?
depositions in the cortices of A?-Fc-vaccine-administered and
with both the prophylactic (50.2% of untreated control, P ?
0.01) and therapeutic (59.54%, P ? 0.05) treatments at 15
months of age (Fig. 6, which is published as supporting infor-
mation on the PNAS web site).
Change in the Serum A? Antibody Titer After Vaccine Administration.
The titers of serum anti-A? antibodies after the prophylactic
treatment (Table 1, Exps. 1 and 2) were determined by ELISA.
The levels of anti-A? antibodies were significantly increased (**,
P ? 0.01;*, P ? 0.05) 2- to 4-fold compared with the untreated
and empty-vector-vaccinated mice (Fig. 3A). The titers showed
an age-dependent increase in both treated and untreated mice,
because the antibody production was induced in untreated
APP23 mice by high levels of A? in the sera of aged mice. In
contrast, the anti-A? antibodies in the sera of wild-type B6 mice
were below the detection limit (data not shown). We also
analyzed the relationships between the amounts of amyloid
depositions and anti-A? titers (Fig. 3B). The amounts of amyloid
depositions were significantly smaller in mice with high antibody
titers. A significant correlation between the antibody titer and
the reduction of A? burden was observed at 7 months (r ?
?0.642) (Fig. 3B, 7 months) and 9 months (r ? ?0.38965) (Fig.
3B, 9 months) of age by the CORREL function. The difference
became less clear at a later stage (Fig. 3B, 12 months).
T Cell-Proliferation Assay After Vaccine Administration. To deter-
mine whether DNA vaccination induces the T cell activation and
proliferation that are key steps for the development of neuroin-
flammation, APP23 and B6 mice were injected with DNA
vaccines. A group of mice were also immunized with A?
peptide?CFA. Three weeks after the first injection, lymphocytes
were isolated and cultured with A? peptide (0–10 ?M) for 3
mice vaccinated with A?-Fc vaccine, amyloid plaques in the frontal cortex
were reduced after 3 months of prophylactic treatment. (B) Immunohisto-
chemical examinations revealed that granular amyloid depositions were de-
plaques were reduced in mice treated with A?-IgL vaccine. (D) At 15 months
of age amyloid plaques of variable size were detected in the frontal cortices
burden at 7 months was significantly decreased (P ? 0.01) after the prophy-
A? vaccine (31.4%) compared with those found in untreated and empty-
vector-vaccinated mice. (F) Therapeutic treatment with A?-Fc and IgL-A?
vaccines significantly reduced (P ? 0.01) cortical A? burden at 15 months. The
overall quantitative analysis is depicted in G. The amyloid deposition was first
detected in untreated mice at 7 months of age and rapidly increased after 15
months of age (open squares). Prophylactic administration of Fc-A? vaccine
prevented the A? deposition to 10–30% of that in untreated animals before
of therapeutic administration were almost the same as those of prophylactic
administration (open circles). Tx, treatment; emp, empty vector. Original
magnification, ?62 (A and B); ?24 (C and D).
Reduction of A? burden in APP23 mice after DNA vaccination. (A) In
Okura et al.
June 20, 2006 ?
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days. Incorporation of [3H]thymidine was measured by using
liquid scintillation spectrometry. T cells from APP23 mice did
not react with A? peptide after both A? DNA vaccine and A?
peptide administration (Fig. 3C). In sharp contrast, T cells from
B6 mice responded significantly to A? peptide after immuniza-
tion with A? peptide, but not after A? DNA vaccination (Fig.
3D). Notably, A? DNA vaccination did not induce T cell
3C) and 5-month treatment (data not shown). These findings
suggest that T helper (Th)1 cells in APP23 mice are in an
of long-term exposure to a high level of A?1-42.
Pathological Examination of the Brains of Vaccinated Mice. The
presence or absence of neuroinflammation in the brain was
examined immunohistochemically after the long-term adminis-
tration of DNA vaccines. Because the T cell assay demonstrated
the significant differences between APP23 and B6 mice, patho-
logical examinations were performed in both strains. Brain
sections were stained with anti-CD5 mAb against T cells and
with Mac-3 against macrophages. In the thymus (Fig. 7A, which
is published as supporting information on the PNAS web site)
and spleen (Fig. 7B), a large number of lymphocytes and
macrophages were stained positively. In sharp contrast, inflam-
matory foci in the brain parenchyma and meninges were not
detected in either APP23 (Fig. 7 C and D) or B6 (Fig. 7 E and
In this study, we developed nonviral A? DNA vaccines against
Alzheimer’s disease and demonstrated satisfactory effects in the
A? reduction in model mice. With the prophylactic and thera-
peutic protocols, treatment with both IgL-A? and A?-Fc re-
duced A? burden in the cerebral cortex to ?40–50% of the
untreated controls, although the latter was slightly more effec-
tive than the former. It should be noted that mice killed at 18
months of age received DNA vaccines for only the last 6 months.
These findings suggest that relatively short-term vaccination is
sufficient for the reduction of A? burden. Because it was
demonstrated in the other experimental setting that 50% reduc-
tion of A? burden resulted in full recovery of the cognitive
disturbance (6), the suppressive effects of DNA vaccines dem-
onstrated in this study is satisfactory. Furthermore, it was
reported that A? immunotherapy reduces not only extracellular
A? plaques but also intracellular A? accumulation and, most
triple-transgenic model of Alzheimer’s disease (20–22). Taken
together, the outcome of DNA vaccine therapy is promising
when applied to human Alzheimer’s disease.
The elevation of anti-A? antibodies was also detected after
DNA vaccination. However, the antibody elevation was mild to
moderate (?2- to 4-fold) compared with that found in mice that
had received A? peptide (10,000-fold) (3). The adjuvant in
peptide vaccines (23) may activate Th1 type T cells (10), which
induce the rapid increase of antibody titers as a result. As
demonstrated in this study, DNA vaccination was able to be
T cell proliferation in both APP23 and B6 mice (Fig. 7) and did
not cause neuroinflammation, even after long-term DNA vac-
cination (Fig. 7, which is published as supporting information on
the PNAS web site). Importantly, mild elevation of the antibody
titers induced by DNA vaccines could reduce amyloid deposits,
probably because DNA vaccination constantly induces the an-
tibody production at a low titer for a long period. Thus, the
maintenance of high anti-A? antibody titer levels is not neces-
sary for effective treatment with DNA vaccines.
To minimize excessive immune reaction in mice and patients,
we should recognize the difference in immunological reactions
clearly demonstrated here, there was no Th1 cell response to A?
peptide in APP23 mice after A? peptide?CFA injection,
whereas, in B6 mice, the same immunization protocol induced a
significant T cell response against A? peptide (Fig. 3D). These
findings strongly suggest that autoreactive Th1 cells in model
mice are in a state of immune tolerance because of a high A?
expression from an early stage of life. In contrast, Th2 cells
helping the antibody production seem to be working, as evi-
denced by the fact that vaccinated animals possessed elevated
levels of anti-A? antibodies. Similar findings were reported by
Monsonego et al. (25) also reported that the immune responses
of T and B cells of model mice are low compared with those of
wild-type mice. In contrast, a significant T cell reactivity to A?
peptide was detected in patients with Alzheimer’s disease (23).
Thus, strong immune induction is dangerous for patients with
B) Titration of anti-A? antibodies in vaccinated APP23 mice. The anti-A?
antibody titer in treated mice was significantly increased (**, P ? 0.01;*, P ?
0.05) ?2- to 4-fold compared with the untreated group. The titer levels were
increased in untreated APP23 as well as treated mice at the same age period
(A). There was significant correlation between the serum anti-A? antibody
titer and the reduction of amyloid depositions at 7 months of age (CORREL
function r ? ?0.642, t0 ? 2.648 ? t10 ? 2.228) (B). At 9 months of age, a
significant correlation was present, but the difference was less marked com-
pared with that at 7 months (r ? ?0.38965, t0 ? 3.10 ? t22 ? 2.086). A
significant difference was not noted at 12 months of age (r ? ?0.325, t0 ?
1.3309 ? t17 ? 2.110). (C and D) T cell responses in APP23 (C) and B6 (D) mice
after immunization with A? peptide?CFA or DNA vaccination. Lymphocytes
isolated from two strains were incubated with A? peptide (0–10 ?M) for 3
days. Incorporation of [3H]thymidine was measured by liquid scintillation
In contrast, A? peptide immunization, but not DNA vaccination, induced a
SD, and the representative results from three different experiments are
shown. Tx, treatment; emp, empty vector.
B and T cell responses of mice treated with A? DNA vaccines. (A and
www.pnas.org?cgi?doi?10.1073?pnas.0600966103Okura et al.
The CpG motif, which exists in plasmid DNAs, is reported to
induce Th1-type immunity and up-regulates IFN-? production
under certain circumstances (26–28). However, this phenome-
non is observed only when a relatively high dose of CpG
oligonucleotides is used (29). In contrast, empty vectors con-
taining the CpG motif ameliorated the clinical and histological
severity of the autoimmune encephalomyelitis that is thought to
be a Th1-mediated disease, as shown in previous studies by us
(30) and others (31, 32).
Recently, A? DNA vaccines were developed by using virus
vectors (33, 34). Although, these vaccines effectively decreased
A? depositions in the brains of model mice, the possibility of
viral replication could not be completely excluded. The plasmid
vector is safe and has no possibility of viral infection and
transformation because it exists as an episome without being
built into the chromosome in eukaryotic cells (12, 13). Another
important factor is related to technology. When DNA vaccines
are in clinical use, large amounts of vaccines are necessary for
treatment of a large number of patients who would be treated for
a long period. Nonviral DNA vaccines have an advantage
because they can be mass-produced with a high purity at a low
are highly effective and safe in reducing the A? burden in model
Materials and Methods
Animals. The APP23 transgenic mice used in this study express
human APP751 cDNA with Swedish double mutation under the
control of the neuron-specific mouse Thy-1 promoter (35, 36).
The Thy-1 expression cassette lacks intron 3, which contains the
elements required for expression in the thymus. RT-PCR and
Western blot analysis of APP23 mice have confirmed the lack of
detectable expression of the transgene in the thymus and spleen
(D. Abramowski, C. Sturchler-Pierrat, and M.S., unpublished
data). This finding is in agreement with a report by Moechars et
al. (37), who used the same expression cassette and also found
that the exogenous transgene with this promoter sequence is
expressed only in the brain but not in the thymus. APP23 mice
were initially established on a B6D2 background and have been
continuously backcrossed to C57BL?6J (B6). In APP23 mice,
amyloid depositions appear from 6 months of age, predomi-
nantly in the neocortex and hippocampus. A? plaques have most
of the characteristics of human Alzheimer’s disease plaques,
including fibrillary A? cores, and are surrounded by dystrophic
neuritis and activated glial cells. Region-specific amyloid-
associated neurodegeneration, including neuron loss, synapse
deficits, and cholinergic alterations, are present in these mice
(38). Wild-type B6 mice were purchased from Charles River
Breeding Laboratories (Kanagawa, Japan). All animal experi-
ments were approved by the institute committee and performed
in accordance with institutional guidelines.
The Development of DNA Vaccine. We prepared three A? DNA
vaccines using a pTarget mammalian expression system (Pro-
mega, Tokyo) (Fig. 1A). The DNA fragment, encoding A?1-42,
was made to anneal two oligonucleotides covering the entire
of the A?1-42 sequence (referred to as K-A? vaccine). In the
second vaccine, the signal sequence of mouse Ig? was added to
the 5? end of the A?1-42 sequence to improve the secretory
3? end of the A?1-42 sequence to stabilize the secreted protein
(A?-Fc vaccine). To prevent unwanted disulfide bonds, three
cystines in the sequence were substituted with serine.
Transfection of DNA Vaccines and Western Blot Analysis.ThreeDNA
vaccines were transfected to HEK293T cells and the amounts of
cells in 60–70% confluence were prepared on 6-well plates
(Costar, Cambridge, MA). The cells were first cultured in
serum-free RPMI medium 1640 for 2 h with one of three DNA
vaccines (K-A?, IgL-A?, or A?-Fc) and Lipofectamine PLUS
reagent (Invitrogen). Then, the cells were cultured overnight in
RPMI medium 1640 supplemented with 5% FBS for cellular
stabilization and growth. For the Western blot analysis, the cells
were again cultured in serum-free medium for an additional 8 h
to remove unnecessary proteins.
Culture supernatants and cell pellets were harvested and run
on NuPAGE 12% Bis-Tris gel (Invitrogen) and transferred to
the PVDF membrane (Immobilon-P; Millipore). After block-
ing with 10% nonfat milk, the blots were incubated with 6E10
(anti-human A?1–17 antibody, 1:100; Abcam, Cambridge,
U.K.) at 4°C for 1 h, followed by incubation with biotin-
conjugated anti-mouse IgG (1:1,000; Vector Laboratories) for
1 h and with ABC-HRP (VectorLaboratories) for 1 h. The
blots were developed by enhanced chemiluminescence re-
agents (Immunostar kit; Wako Biochemicals) according to the
Administration of the Vaccines. DNA vaccines (100 ?g) and bu-
pivacaine (0.25 mg) in 100 ?l was administered i.m. on a weekly
basis for 6 weeks (39). The vaccines without bupivacaine were
injected biweekly thereafter. To examine the prophylactic effect,
vaccination was started at 3–4 months of age, before amyloid
plaque appearance. The therapeutic treatment was started at 12
months of age, after amyloid plaque appearance.
Immunohistochemistry. Mice were killed under deep anesthesia,
and the brains were removed and immersion-fixed in 4% para-
formaldehyde. Paraffin-embedded sections (6 ?m) were stained
immunohistochemically with mAb (6F?3D) against A?8–17
(DAKO), anti-CD5 mAb against T lymphocytes (BD Bio-
sciences Pharmingen) and Mac-3 against mononuclear phago-
cytes (BD Biosciences Pharmingen). For 6F?3D staining, the
sections were pretreated in formic acid for 3 min. The sections
were then incubated in the primary antibody at a 1:200 dilution.
After washing, the sections were incubated with biotinylated
horse anti-mouse IgG (Vector Laboratories), followed by a
horseradish peroxidase (HRP)-labeled Vectastain Elite ABC kit
(Vector Laboratories). HRP-binding sites were detected in
0.005% diaminobenzidine and 0.01% hydrogen peroxide. CD5
(1:25) and Mac-3 (1:25) stainings were performed similarly, with
overnight incubation of the primary antibodies.
Quantitative Analysis of A? Burden. A? burdens were quantitated
in the cerebral cortex and hippocampus, according to the
method described in ref. 40. All of the procedures were per-
formed by an individual blind to the experimental condition of
the study. The images under an Olympus Vanox microscope
were captured with a 3 charge-coupled device Olympus digital
camera. The amyloid load was measured in 10 fields from the
cingulated to retrosplenial cortex in the left hemispheres of
the mice (600 ? 400 ?m each), chosen randomly. Analysis in the
hippocampus was performed on the entire hippocampus in a
similar manner. A? depositions that occupied the field were
expressed as pixels by using the NIH IMAGE software.
(Peptide Institute, Osaka) in 0.1 M sodium carbonate buffer (pH
9.5) at 4°C overnight. After washing three times, plates were
incubated for 2 h with serial dilutions of plasma samples in PBS
in 12 rows of wells starting with 4-fold-diluted plasma (the
greatest dilution tested was 1:213). The plates were washed and
Okura et al.
June 20, 2006 ?
vol. 103 ?
no. 25 ?
incubated with a 1,000-fold dilution of biotinylated anti-mouse
IgG (Vector Laboratories), followed by incubation with 2-fold
dilutions of Vectastain ABC-kit solution (Vector Laboratories).
Bound antibodies were detected by using SIGMA FAST (Sigma-
Aldrich), and the absorbance at 450 nm was read on an auto-
mated plate reader (Model 550; Bio-Rad). The antibody titer
was defined as the reciprocal of the greatest dilution of plasma
that gives half-maximal binding to A?, which was determined by
dividing the highest OD450value in the dilution range of each
sample by 2.
T Cell-Proliferation Assay. The proliferative responses of draining
lymph node cells were assayed in microtiter plates (Costar,
Cambridge, MA) by the uptake of [3H]thymidine. A?1-42
peptide (50 ?g) emulsified with CFA (twice) and Fc-A? vaccine
(three times) was injected into APP23 mice or B6 mice, and then
the drainage lymph nodes were taken 3 weeks after the first
injection. Lymph node cells (2 ? 105cells per well) were cultured
with 0.3–10 ?M A?1-42 peptide for 3 days and subsequently
pulsed for 18 h with 0.5 ?Ci (1 Ci ? 37 GBq) of [3H]thymidine
(Amersham Pharmacia Biotech). Incorporation of [3H]thymi-
dine was measured by liquid scintillation spectrometry.
Statistical Analysis. Student’s t test or Mann–Whitney’s U test was
used for the statistical analysis. Correlations between the anti-
body titer and the reduction of A? burden was estimated by the
We thank Y. Kawazoe for technical assistance. This study was supported,
in part, by Grants-in-Aid from the Ministry of Education, Japan, and a
grant from Novartis Institutes of Biomedical Research.
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