Hepatitis B core VLP-based mis-disordered tau vaccine elicits strong immune response and alleviates cognitive deficits and neuropathology progression in Tau.P301S mouse model of Alzheimer’s disease and frontotemporal dementia
Ji et al. Alzheimer's Research & Therapy
Hepatitis B core VLP-based mis-disordered tau vaccine elicits strong immune response and alleviates cognitive deficits and neuropathology progression in Tau. P301S mouse model of Alzheimer's disease and frontotemporal dementia
Mei Ji 0 2
Xi-xiu Xie 0
Dong-qun Liu 0 2
Xiao-lin Yu 0
Yue Zhang 0 1
Ling-Xiao Zhang 0 2
Shao-wei Wang 0
Ya-ru Huang 0 2
Rui-tian Liu 0
0 National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences , Haidian District, Beijing 100190 , China
1 Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University , Taian 271018 , China
2 University of Chinese Academy of Sciences , Beijing 100049 , China
Background: Truncated mis-disordered tau protein plays an important role in the pathogenesis of Alzheimer's disease (AD) and frontotemporal dementia (FTD). Tau294-305, an epitope in the truncated tau, is essential for pathological tau-tau interaction and aggregation. A tau294-305-targeted approach may have beneficial effects in the treatment of AD and FTD. Methods: In this study, we genetically fused tau294-305 epitope to the hepatitis B virus core protein (HBc) major immunodominant region (MIR) (with the resultant protein termed T294-HBc), and we subcutaneously immunized a Tau.P301S transgenic mouse model of FTD and AD with T294-HBc four times. The levels and characteristics of antibodies induced by T294-HBc were determined by enzyme-linked immunosorbent assay. The effect of T294-HBc on the cognitive deficits of Tau.P301S mice was tested using the Morris water maze test, novel object recognition, and a Y-maze test. Western blot analysis and IHC were applied to measure the effect of T294-HBc on tau pathologies and neuroinflammation in the mouse brains. Results: The results showed that T294-HBc self-assembled into HBc chimeric virus-like particles (VLPs) with tau294-305 displayed on the surface and that it induced high antibody titers specifically against the mis-disordered truncated tau. Further investigation showed that these antibodies simultaneously bound to microtubule-binding regions 1-4 (MTBR1-4) [tau263-274, tau294-305, tau325-336, tau357-368 and tau294-305(P301S)]. Moreover, T294-HBc VLP vaccination significantly ameliorated memory and cognitive decline; reduced the levels of AT8-positive tau, truncated tau monomer, and oligomer; attenuated microgliosis and astrogliosis; and rescued synaptic deficits in Tau.P301S transgenic mice. (Continued on next page)
(Continued from previous page)
Conclusions: T294-HBc VLP vaccine elicited strong immune response and alleviated cognitive deficits and neuropathology
progression in Tau.P301S mice, indicating that the T294-HBc VLP vaccine has promising therapeutic potential for the
treatment of AD and FTD.
Alzheimer’s disease (AD) is an age-related
neurodegenerative disorder characterized by progressive memory loss
], intracellular neurofibrillary tangles (NFTs) composed
of microtubule-associated protein tau, and extracellular
amyloid plaques formed by amyloid-β (Aβ) aggregates in
the brain [
]. Unlike Aβ plaques, the amount and extent
of NFT pathology positively correlate with the severity of
the cognitive deficit of AD [
]. Tau inclusions are also
found in other tauopathies that lack Aβ pathology, such as
corticobasal degeneration, Pick’s disease, and progressive
supranuclear palsy . Notably, mutations in the tau gene
(such as P301S, P301L) cause some forms of
frontotemporal dementia (FTD), indicating that tau dysfunction
alone is sufficient to cause neurodegeneration [
pharmacological treatment of AD is based on
cholinesterase inhibitors and memantine. However, this treatment
could not halt the disease’s progress [
Previous reports suggest that cerebrospinal fluid tau in
patients with AD and patients with mild cognitive
impairment comprises primarily truncated forms of tau (151–
391/2N4R, sequence corresponding to full-length 2N4R
tau), which are conformationally different from normal
healthy tau and essential for pathologic tau-tau interaction
]. During the development of AD, the mis-disordered
truncated tau aggregates to tau tangles and oligomers and
drives AD-like neurofibrillary degeneration accompanied
by microglial and astroglial activation in the brain [
Therefore, therapy targeting mis-disordered truncated tau
may be an effective treatment strategy . In the past few
years, the field of anti-tau immunotherapies has been
galvanized by hundreds of studies trying to optimize the
approach for treating AD. Tau-targeted immunotherapies
have shown potential for AD treatment, some of which
have paved the way to clinical trials [
with passive immunotherapy, active immunotherapy can
provide persistent effects because it uses the immune
system to produce long-lasting antibody. Because active
immunization with full-length Aβ induced meningitis in
clinical trials by a cell-mediated type 1 helper T cell (Th1)
immune response [
], the second generation of AD
vaccines was developed by conjugating a B-cell epitope of
tau or Aβ with a carrier [
]. Previous studies
demonstrated that tau294–305 is a structural determinant of the
truncated tau protein for the pathological tau-tau
interaction, and it contains a motif, “HXPGGG,” which
localizes not only in tau299–304 (within microtubule-binding
region 2 [MTBR2]) but also in tau268–273 (within MTBR1),
tau330–335 (within MTBR3), and tau362–367 (within MTBR4).
A tau294–305-targeting approach reduced tau pathology and
associated behavioral deficits in transgenic tau rats .
Virus-like particles (VLPs) are multimeric nanoparticles
that are assembled from viral structural proteins and are
biosafe because they lack a viral genome [
contain repetitive high-density viral surface proteins and
are an excellent platform from which to present
heterologous antigens. Tau294–305 is a short peptide with low
immunogenicity. To develop a vaccine with tau294–305 as
an immunogen to induce high antibody titers, tau294–305
should be combined with a carrier such as keyhole limpet
hemocyanin. Hepatitis B virus core protein (HBc) VLPs
are promising carriers because of their high capacity for
foreign insertions, high-level generation, and efficient
self-assembly in virtually all known homologous and
heterologous expression systems, including bacteria and
]. The major immunodominant region (MIR)
in HBc is generally used for the insertion of foreign B-cell
epitopes to maximally expose these epitopes on the VLP
surface and consequently provide the most efficient
immunogenic activity. In this study, we fused tau294–305 to
HBc MIR and assessed its effect on tauopathies in
Tau.P301S transgenic mice.
Plasmid construction and protein expression
A truncated HBc gene (coding for amino acids 1 to
149) was cloned into pBR327 vector, which was a
kind gift from Professor Andris Kazaks (Latvian
Biomedical Research and Study Center, Latvia) [
The tau294–305 gene was inserted into the site
between the codons for Asp78 and Pro79 in the
immunodominant loop region (Fig. 1a). The
resultant vector T294-HBc-pBR and HBc-pBR was
transformed into Escherichia coli BL21 (DE3)-competent
cells (Takara, Dalian, China). Cells were cultivated
in M9 salt medium supplemented with 1% casamino
acids (BD Biosciences, San Jose, CA, USA), 0.2%
glucose (Amersco, Solon, OH, USA), 50 μg/ml
ampicillin without additional induction of Ptrp for
20–24 hours at 37 °C [
For the purification of the VLPs, 2 g of wet cells were
resuspended in 40 ml of lysis buffer [50 mM Tris-HCl,
150 mM NaCl, pH 8.0, 5 mM ethylenediaminetetraacetic
acid (EDTA), 100 μg/ml phenylmethylsulfonyl fluoride]
and then ultrasonicated for 30 minutes at 50% power at
3-second intervals in an ice bath. After centrifugation at
12,000 rpm for 15 minutes, ammonium sulfate was added
to the supernatant until 33% saturation was reached. The
sample prepared by ammonium sulfate precipitation was
subjected to sucrose discontinuous gradient centrifugation
at 112,000 × g for 16 hours at 4 °C. The collected sucrose
solution was then loaded onto a hydroxyapatite column
(Bio-Rad Laboratories, Hercules, CA, USA), and the
flow-through was collected [
]. Purified protein was
concentrated and determined by using a bicinchoninic
acid protein assay kit (Pierce, Rockford, IL, USA). The
purity of the recombinant protein was analyzed by 15%
SDS-PAGE. Full-length tau isoform 2N4R construct
was gifted by Professor Virginia M.-Y. Lee [
Mis-disordered tau (151–391/2N4R) and full-length tau
isoform 2N4R were prepared with E. coli BL21 (DE3) as
described previously, with some modifications [
TEM assay of VLPs
VLPs (10 μl; 0.2 mg/ml) were applied to 200 mesh
copper grids for 5 minutes, blotted with filter paper, and
negatively stained with 2% uranyl acetate for 1 minute,
then blotted and air-dried. VLPs were imaged using a
TEM system (Hitachi, Tokyo, Japan) at 80 kV and
60,000 × magnification. Particle diameter was measured
with Nano Measurer 1.2 (n = 100).
Animals were generated by breeding Tau.P301S(1N4R)
transgenic male mice with wild-type (WT) female mice
under the original C57BL/6×C3H background. All
experimental protocols were approved by the institutional
animal care and use committee of Tsinghua University.
All experiments were performed in accordance with the
China Public Health Service Guide for the Care and Use
of Laboratory Animals. Offspring were genotyped by
polymerase chain reaction of tail DNA. Tau.P301S
transgenic mice (5 months old) were randomly assigned to
treatment with adjuvant (n = 7), HBc (n = 7), or
T294-HBc (n = 7), and their WT littermates (n = 7) were
used as a positive control in the behavior test. Mice were
inoculated subcutaneously four times at 2-week or 3-week
intervals (Fig. 2a). The vaccine and HBc group consisted
of 25 μl of Alum Adjuvant (Thermo Fisher Scientific,
Waltham, MA, USA) and 75 μl of T294-HBc or HBc
(1.33 mg/ml). The adjuvant group was immunized with
25 μl of Alum diluted in 75 μl of PBS. Serum samples
were collected before each inoculation and 10 days after
the final boost. The effects of T294-HBc on the behavioral
and cognitive abilities of Tau.P301S mice were tested
20 days after the last administration.
Indirect enzyme-linked immunosorbent assay
Serum antibody specific for mis-disordered truncated
tau (151–391/2N4R) was detected by enzyme-linked
immunosorbent assay (ELISA). Ninety-six-well plates
(Dynex Technologies, Chantilly, VA, USA) were coated
with 250 ng of recombinant full-length tau isoform
2N4R or pathological truncated tau (151–391/2N4R) per
well at 4 °C overnight, then washed twice with PBS and
blocked with 3% bovine serum albumin (in 0.05%PBS
with Tween-20) for 2 hours at 37 °C. After blocking, the
plates were incubated with serial dilutions of the serum
(100 μl/well in twofold or fivefold dilution steps) for
1 hour at 37 °C. The bound serum antibodies were
detected with horseradish peroxidase (HRP)-conjugated
goat antimouse immunoglobulin G (IgG) (Zhongshan
Golden Bridge Biotechnology Co., Beijing, China) and
chromogenic substrate 3,3′,5,5′-tetramethylbenzidine
(Thermo Fisher Scientific).
To determine the isotypes of the specific antibodies
produced in response to vaccine, mis-disordered
truncated tau (151–391/2N4R) was coated onto ELISA
plates, and sera from immunized mice were diluted at
1:6000 and added to the plate, followed by the addition
of HRP-conjugated IgG1, IgG2a, IgG2b, and IgG3
(Abcam, Cambridge, UK).
Ninety-six-well plates (Dynex Technologies) were coated
with 250 ng/well of mis-disordered tau (151–391/2N4R).
Peptide competitors (> 95% purity; GL Biochem
[Shanghai] Ltd., Shanghai, China) were dissolved in PBS to a final
concentration of 5 mM. The serum was diluted from
1:100 to 1:97,200 in threefold or twofold dilution in PBS,
and 60 μl of the diluted serum solution was mixed with
40 μl of peptide solution (200 μM) in 1.5-ml Eppendorf
tubes (Eppendorf, Hamburg, Germany). Antibody/peptide
mixtures (100 μl) were then transferred onto ELISA
plates and incubated for 1 hour at 37 °C. Bound
serum antibodies were detected with HRP-conjugated
goat antimouse IgG (Zhongshan Golden Bridge
Forced Y-maze test
Forced Y-maze test was conducted using a symmetrical
Y-maze made of gray wood, covered with black paper,
and consisting of three arms with an angle of 120
degrees. Each arm was marked at the end with a different
black-and-white pattern. The three identical arms were
randomly designated as the start arm, in which the
mouse started to explore (always open); the novel arm,
which was blocked during the first trial but open during
the second trial; and the other arm (always open). The
Y-maze test consisted of two trials separated by an
intertrial interval (ITI) to assess spatial recognition
memory. The first trial (training) had a 5-minute
duration and allowed the mouse to explore only two arms
(start arm and other arm) of the maze, with the third
arm (novel arm) being blocked. After a 30-minute ITI,
the mice were placed back in the maze in the starting
arm, with free access to all three arms for 5 minutes for
the second trial. All trials were recorded on a
videocassette recorder using a ceiling-mounted charge-coupled
device (CCD) camera, and the number of entries and
time spent in each arm in the video recordings were
Novel object recognition test
Novel object recognition (NOR) is based on the
spontaneous tendency of mice to exhibit more interactions
with a novel object rather than a familiar object. In the
habituation phase, each mouse was allowed to freely
explore the open-field arena (a white box 40 cm wide,
40 cm deep, 40 cm high) in the absence of objects. Then,
the mouse was removed from the box and placed in its
housing cage. During the familiarization period, each
mouse was placed in the white box containing two
identical objects for 5 minutes and returned quickly to its
housing cage. Recognition memory was tested after
24 hours by exposing the mouse to one familiar object
and one novel object. The time spent exploring and
sniffing each object was recorded.
Morris water maze test
On training days 1–5, four groups of mice were habituated
to a 1.2-m-diameter circular pool with opaque water
maintained at 22 ± 1 °C. The mice were allowed to find a
platform hidden below the water surface by swimming for
60 seconds for two trials per day. If they didn't found the
platform, the mice were guided to the platform. All the
mouse were allowed to stay on the platform for 10 seconds.
The swimming activity of each mouse was automatically
recorded via a video tracking system using a video camera
(Sony Corp., Tokyo, Japan) mounted overhead. At 24 hours
after the last learning trial, the mice were tested for
memory retention in a probe trial without the platform.
Spontaneous Y-maze test
We recorded spontaneous alternation behavior in a
Y-maze test to assess short-term memory
performance. The maze was same as the forced Y-maze,
except that the marker at the end of each arm was
changed to eliminate the effects of the former
forced Y-maze. This test consisted of a single
5-minute trial in which the mouse was allowed to
move freely to all three arms of the Y-maze. The
series of arm entries, including possible returns into
Fig. 2 Humoral immune responses induced by tau294–305 epitope to hepatitis B core immunodominant region (T294-HBc) virus-like particle (VLP)
vaccine. a Schematic diagram of the time points for treating Tau.P301S mice (5 months old) (n = 7). b The antibody titer induced by T294-HBc
VLPs. Sera from immunized mice were serially diluted from 1:100 to 1:819,200 in twofold dilution steps and tested in duplicates by enzyme-linked
immunosorbent assay (ELISA) against mis-disordered tau (151–391/2N4R). c The isotypes of vaccine-induced antibodies. The diluted sera were
added to mis-disordered tau (151-391/2N4R)-coated plate and followed by adding HRP–conjugated secondary antibodies. d Different binding of
serum antibodies to mis-disordered tau (151-391/2N4R) and full-length tau 2N4R. Sera from immunized mice were serially diluted from 1:100 to
1:3906250 in fivefold dilution steps and tested in duplicates by ELISA against mis-disordered tau (151-391/2N4R) and full length tau 2N4R,
respectively. EC50, Half-maximal effective concentration. (Statistics were analyzed by student’s t-test, *P<0.05). e The binding activity of the
antibody elicited by T294-HBc to different tau fragments. The mixture of 200 μM of different tau fragments with diluted sera was added to the
mis-disordered tau-coated plate, and then HRP-conjugated goat anti-mouse immunoglobulin G was added. f The binding activity of the
antibodies elicited by T294-HBc to the brain tissues of Tau.P301S mice. Brain tissue was detected via IHC using serum from HBc (1:200)- or
T294HBc (1:200)-treated mice. Scale bar is 200 μm
the same arm, was recorded with a CCD camera
connected to a computer. An alternation was
defined as entry into all three arms on consecutive
occasions. The number of maximum alternations
was therefore the total number of arm entries
minus 2, and the percentage of alternations was
calculated as (actual alternations/maximum
alternations) × 100% as described previously [
Mice were deeply anesthetized with sodium
pentobarbital and killed after cardiac perfusion with ice-cold PBS
containing heparin (10 U/ml) at the end of the behavior
test. The left half of the brain was fixed in 4%
paraformaldehyde and embedded in paraffin. Sagittal serial
sections of 5-μm thickness were cut on a Leica CM1850
microtome (Leica Biosystems, Buffalo Grove, IL, USA).
The sections were dewaxed with antigen retrieval and
then treated briefly with 80% (vol/vol) methanol
containing 0.3% H2O2 to prevent endogenous peroxidation. The
sections were then blocked with 10% normal goat serum
and 0.3% Triton X-100 in PBS to prevent nonspecific
protein binding and penetrate cell membranes.
Subsequently, the sections incubated with primary
antibodies AT8 (1:500; Thermo Fisher Scientific),
anti-ionized calcium-binding adaptor molecule-1
(anti-Iba-1) (1:100; GeneTex, Irvine, CA, USA),
anti-glial fibrillary acidic protein (anti-GFAP) (1:100;
Cell Signaling Technology, Danvers, MA, USA),
anti-synaptophysin (1:100; Abcam), sera from
vaccineand HBc-treated mice (1:200) at 37 °C for 1 hour,
respectively, followed by incubation with an
HRP-labeled secondary antibody at 37 °C for 1 hour.
The targets were visualized with 3′-diaminobenzidine
substrate. Alexa Fluor 488-labeled secondary antibody
(Thermo Fisher Scientific) was used for the detection
of synaptophysin levels in the brain sections. All
images were collected using a BX60 microscope
(Olympus Corp., Shinjuku, Tokyo) with 4 × and 10 × lens
objectives. The right half was stored at − 80 °C for
Western blot analysis.
Western blot analysis
The right half brain tissues were dounce-homogenized in
radioimmunoprecipitation assay (RIPA) buffer (containing
protease inhibitor mixture, 50 mM Tris, pH 7.2, 150 mM
NaCl, 5 mM EDTA, and 0.1% SDS) and then centrifuged
at 12,000 × g for 1 hour at 4 °C. The supernatant (i.e.,
RIPA-soluble fraction) containing soluble tau was
collected. The RIPA-insoluble pellets were washed with
1 M sucrose in RIPA buffer to remove myelin and
associated lipids by centrifugation at 100,000 × g for 30 minutes
at 4 °C. The RIPA-insoluble pellets were then extracted
with 2% SDS buffer (50 mM Tris, pH 7.6) [
and insoluble fractions were separated by SDS-PAGE and
then transferred onto nitrocellulose membranes. After
blocking for 2 hours at room temperature with 5% nonfat
milk, the membranes were probed with AT8 (1:1000),
tau5 (1:2000), HT7 (1:3000), anti-Iba-1 (1:1000),
anti-GFAP (1:1000), anti-synaptophysin (1:1000),
anti-GAPDH (1:1000; Cell Signaling Technology), and
anti-β-actin (1:1000; MBL, Nagoya, Japan) at room
temperature for 1 hour, respectively, and detected with
HRP-conjugated anti-mouse or anti-human IgG
(1:10,000). The blots were developed using
electrochemiluminescence according to the manufacturer’s instructions
(Thermo Fisher Scientific), and the optical densities of the
bands were determined using IPwin5 Image-Pro Plus
software (Media Cybernetics, Rockville, MD, USA).
The data were expressed as mean ± SEM. The time per
experiment and samples per group depended on the
experiments. We performed ELISAs at least three times,
but seven mice per group were analyzed for behavior
tests, IHC, and Western blot analysis. To compare the
adjuvant group and the vaccine group, one-way analysis
of variance (ANOVA) with LSD, two-way ANOVA,
Mann-Whitney U test, or Student’s t test was applied
using Prism 5.0 (GraphPad Software, La Jolla, CA, USA)
or SPSS 16.0 software (SPSS, Chicago, IL, USA). P < 0.05
was considered significant.
The preparation of T294-HBc
To improve the immunogenicity of tau294–305, we fused it
to the HBc MIR region. The epitope was inserted between
amino acids 78 and 79 with a GSG linker at each side
(Fig. 1a). After three purification steps, SDS-PAGE analysis
showed that the purity of T294-HBc was > 95% (Fig. 1b).
T294-HBc was then subjected to negative staining for TEM
to confirm VLP particle formation. The results showed that
the recombinant T294-HBc automatically assembled into
VLPs with a diameter of 33.55 ± 2.79 nm (Fig. 1c and d).
T294-HBc VLPs effectively elicit high-titer antibodies
against mis-disordered tau (151–391/2N4R)
Tau.P301S transgenic mice were immunized
subcutaneously with 100 μg of T294-HBc VLPs for four times, and
the serum antibody titer was detected by ELISA (Fig. 2a).
The results demonstrated that the T294-HBc vaccine
induced a robust antibody response in mice. In contrast,
the adjuvant or HBc alone did not elicit specific
antibodies against mis-disordered tau 151–391/2N4R
(Fig. 2b). The titers increased with the immunization
times and reached a plateau at the third inoculation
(Fig. 2b). The geometric mean titer (GMT) of antibodies
specific to mis-disordered tau 151–391/2N4R reached
high values at 178,700. On the contrary, the EC50 of
antibodies recognizing full-length tau 2N4R was much
lower than tau 151–391/2N4R, suggesting that the
induced antibodies exhibited significantly high binding
activity to the mis-disordered tau 151–391/2N4R relative
to physiological tau 2N4R (P < 0.05) (Fig. 2d).
The isotypes of the antibodies in response to T294-HBc
vaccine were detected by ELISA. The results indicated
that vaccination of mice with T294-HBc preferentially
induced the generation of IgG1 antibody isotypes (Fig. 2c),
suggesting that predominant Th2 immune response was
involved in T294-HBc immunization.
To detect the ability of serum antibodies to bind to
different fragments of tau protein, a competitive ELISA
was carried out by mixing 200 μM tau263–274 (MTBR1),
tau294–305 (MTBR2), tau325–336 (MTBR3), tau357–368
(MTBR4), and tau294–305 (P301S) with serum antibodies
at different dilutions. As shown in Fig. 2e,
besides tau294–305, the antibodies elicited by T294-HBc
markedly bound to tau357–368 and tau294–305 (P301S).
Moreover, the antibodies also recognized tau263–274 and
tau325–336, but with lower affinity than tau294–305,
suggesting the therapeutic potential of T294-HBc vaccine in
the treatment of AD and FTD.
To further characterize the immune response, we
performed IHC using antibody HT7 and the sera from
vaccine- and HBc-treated mice. The results showed that
HT7 and the sera induced by vaccine bound to the brain
tissues of Tau.P301S mice, whereas no positive signal
was observed with the addition of the sera from
HBc-treated mice, indicating that antibodies induced by
T294-HBc vaccine recognized tau pathology (Fig. 2f ).
T294-HBc VLP immunization improved cognitive capacity in Tau.P301S mice
To evaluate the effect of T294-HBc immunization on
Tau.P301S mice, we carried out a behavioral test battery,
including a forced Y-maze test, NOR, Morris water maze
(MWM), and spontaneous Y-maze test (Fig. 3a) [
forced Y-maze test was performed to examine the effects of
vaccine treatment on short-term memory. Compared with
the adjuvant- and HBc-treated mice, T294-HBc-immunized
mice showed a significant increase (P < 0.05) in both time
spent and number of entries in the new arm, indicating that
T294-HBc improved short-term memory in Tau.P301S
mice (Fig. 3b, c).
NOR was performed to further evaluate the effects of
the vaccine on mouse memory. Compared with the
adjuvant- and HBc-treated mice, the mice treated with
T294-HBc VLPs spent more time on the novel object
than on the familiar object (P < 0.05) (Fig. 3d).
The MWM test was conducted to assess the effects of
T294-HBc immunization on the spatial memory and
learning ability of Tau.P301S mice. During the
acquisition phase of the test, the mice were trained to
search for the hidden platform for 5 days. Compared
with the adjuvant- and HBc-treated Tau.P301S mice,
mice treated with T294-HBc readily found the location
of the hidden platform after 2 days of training (P < 0.05)
(Fig. 3e). After the last training, the platform was
removed, and the mice were given 1 minute to find the
location of the missing platform for the probe trial.
T294-HBc-treated mice exhibited spatially oriented
swimming behavior and shorter escape latencies (Fig. 3f )
and increased number of target crossings (Fig. 3g), and
they spent more time in the target quadrant (Fig. 3h),
indicating that T294-HBc substantially improved the
spatial memory of Tau.P301S transgenic mice. No
significant difference in the swimming speed was observed
within mouse groups in the training period and the
probe trial session.
Spontaneous alternation using a Y-maze is a further
test for habituation and spatial working memory. The
percentage of alternation of T294-HBc-treated mice in
the spontaneous Y-maze test was significantly increased
compared with that of adjuvant- and HBc-treated mice,
indicating that short-term memory was rescued in
Tau.P301S model mice by T294-HBc VLP immunization
(Fig. 3i). Compared with the adjuvant group, the HBc
group did not show a significant effect on antibody
response and animal behavior, and we did not further
detect the pathology in the brains of the HBc group.
T294-HBc VLP immunization reduced tau aggregates in
We detected AT8-positive aggregates in mouse brains by
IHC and Western blotting using anti-phosphorylated tau
antibody AT8 to investigate the therapeutic effect of
vaccine on the Tau.P301S mice. WT mice did not show any
tau pathology in the brain, whereas Tau.P301S mice
showed severe AT8-positive aggregates in the cortex and
in the hippocampal CA1 and dentate gyrus (DG) regions
(Fig. 4). However, T294-HBc VLP vaccine significantly
reduced AT8-positive aggregates in these regions.
Western blotting also showed that T294-HBc VLPs
significantly decreased the levels of AT8-positive aggregates in
the brains of mice (Fig. 4e). These results demonstrate
that T294-HBc VLPs decreased tau pathology in the
T294-HBc VLP immunization reduced RIPA-insoluble tau levels in Tau.P301S mice
To assess the effects of T294-HBc immunization on the
levels of different tau species in mouse brains, we detected
the RIPA-insoluble tau with human tau-specific antibody
HT7 and total tau antibody tau5. Consistent with previous
results, T294-HBc significantly decreased the levels of
highly phosphorylated forms of truncated tau (30 to
36 kDa), tau oligomers (above 36 kDa), and full-length tau
in insoluble tau (Fig. 5a, b). Western blot analysis probed
by tau5 antibody showed that WT mice had a band of
about 70 kDa corresponding to the molecular weight of
mouse tau. Compared with adjuvant-treated mice,
T294-HBc-immunized mice had a significant decrease in
both human and phosphorylated tau.
T294-HBc VLP immunization reduced glial cell activation in Tau.P301S mice
The infiltration of activated astrocytes and microglia is
linked to the pathogenesis of Tau.P301S mice. We
stained astrocytes and microglia with antibodies against
GFAP and Iba-1, respectively, to address whether
vaccine treatment was effective for astrogliosis and
microgliosis in the brains of Tau.P301S transgenic mice.
The results showed that T294-HBc active
immunotherapy reduced the number of activated astrocytes in the
cortex and in the hippocampal CA1 and DG regions
(Fig. 6). Consistently, our Western blotting results also
showed that T294-HBc immunization decreased GFAP
protein levels (Fig. 6e). Moreover, T294-HBc reduced
the activated microglial levels in the cortex and in
the hippocampal CA1 and DG regions (Fig. 7). The
results of Western blotting also showed a decrease in
microgliosis (Fig. 7e). These results indicated that
T294-HBc immunization attenuated
neuroinflammation in Tau.P301S mice by targeting pathogenic
mis-disordered tau [
Active immunotherapy by T294-HBc VLP vaccine rescued synaptic deficits in Tau.P301S mice
With the development of tauopathies in Tau.P301S mice,
synapses are damaged owing to the toxicity of
pathogenic tau [
]. To assess whether the reduction of
pathogenic mis-disordered tau can attenuate synaptic
deficits, in the present study we used antisynaptophysin
antibody to detect the effect of T294-HBc VLP vaccine
on the synapses. Significant increases in synaptophysin
levels were observed in the cortex and in the
hippocampal CA1 and DG regions in T294-HBc-treated mice
compared with adjuvant-treated mice (Fig. 8). Western
blot analysis also showed an increase in synaptophysin
levels in brain homogenates of T294-HBc-treated mice
relative to adjuvant-treated mice (Fig. 8d). These results
indicate that T294-HBc VLP immunization rescued
synaptic deficits in Tau.P301S mice.
Both hyperphosphorylated and truncated tau play a
critical role in AD pathogenesis. Authors of many reports
selectively targeted phosphorylated tau (phospho-tau)
epitopes, including phospho-Ser396/phospho-Ser404
], phospho-Ser422 [
]. However, most phosphoepitopes of tau
such as phospho-Ser404 are present in healthy human
], which raises concerns about the safety of
immunotherapies targeting those phospho-tau species.
Truncated tau is a pathogenic tau present in AD brains
but not in normal human brains; thus, targeting
truncated tau may be a more promising approach [
this study, we chose tau294–305 as our targeting epitope
because it is a structural determinant of the truncated
tau protein for the pathological tau-tau interaction. This
epitope contains a motif, “HXPGGG,” that localizes not
only in tau299–304 (within MTBR2) but also in tau268–273
(within MTBR1), tau330–335 (within MTBR3), and
tau362–367 (within MTBR4) [
]. Therefore, the
antibodies induced by tau294–305 may simultaneously bind to
HBc VLP is a widely used carrier to generate putative
vaccines. HBc-based malaria and influenza vaccines have
entered into clinical trials, and they were well tolerated.
To avoid T-cell autoimmunity likely induced by
full-length Aβ, we developed a tau vaccine (T294-HBc
VLP vaccine) by genetically fusing a B-cell epitope of
tau (tau294–305) to HBc MIR. In our tau vaccine, the
Th epitopes were derived exclusively from the VLP
carrier protein, and the vaccine was formulated with
Alum adjuvant that promoted the Th2 immune
response. As expected, the antibody induced by
T294-HBc VLPs was predominantly the IgG1 isotype,
which indicates that the immune response to the
vaccine mainly involved the Th2 phenotype and that
T294-HBc VLPs may be a safe vaccine type. Moreover,
T294-HBc VLPs formed uniform nanoparticles with a
diameter of approximately 33.55 nm and elicited
robust and specific antibodies against mis-disordered
tau in mice.
FTD, the second most common form of dementia
before the age of 65, is caused by P301S/L mutated tau. A
previous report showed that tau-targeting vaccine
AADvac1 improved cognition and reduced tauopathy in a
transgenic rat model expressing human truncated tau
]. Although this transgenic rat model displayed some
tau pathology and motor and behavioral deficits, it still
could not mimic some incidence and progression of
tauopathy in AD and FTD. Moreover, AADvac1 induced
antibodies against tau294–305, but whether the vaccine
has a beneficial effect on an FTD model with P301S/L
mutation remains unknown. To explore the effects of
T294-HBc VLP vaccine on the animal models of FTD
and AD, we applied the vaccine to the Tau.P301S
transgenic mouse model, which is widely used to mimic
the incidence and progression of FTD and AD and
recapitulates the essential molecular and cellular features of
the human tauopathies, including truncated tau
generation, hyperphosphorylation, tau filament formation, and
]. Woerman et al.’s report
showed that the PS19 mouse model exhibited great
variability in pathology onset at ages over 31 weeks, but it
was relatively uniform before age 30 weeks ; the
mice at 22 weeks of age that we used conformed the
latter age range. Our results showed that T294-HBc
vaccine improved cognition and memory of Tau.P301S
mice and reduced the levels of truncated tau monomer,
oligomer, and hyperphosphorylated tau. Our
observations are in agreement with previous findings that
passive immunization with tau antibodies against
pathological tau forms improved cognition of AD mice and
reduced the levels of hyperphosphorylated tau [
Although the antibodies induced by T294-HBc VLPs
recognized tau294–305 (P301S) with lower affinity, they
could also bind to tau294–305, tau263–274, tau325–336,
and tau357–368, resulting in beneficial effects on
The accumulation of NFTs can cause inflammation
and synapse loss in Tau.P301S mice [
]. However, the
T294-HBc VLP-immunized mice exhibited lower levels
of astrogliosis and microgliosis and higher levels of
synaptophysin than adjuvant-treated mice, leading to
attenuation of cognitive deficits and neuropathology in the
transgenic mice. Several mechanisms could explain the
antibody-mediated clearance of the mis-disordered tau
in vivo [
]. The direct targeting mechanism
proposes that a small amount of serum antibodies can cross
the blood-brain barrier, bind to the mis-disordered tau,
and then induce the phagocytosis of the antigen-antibody
complexes via the Fc portion of the antibody . The
pathologic tau can propagate from cell to cell and induce
the aggregation of pathologic tau in the other neurons
], and the clearance of mis-disordered tau by our
vaccine results in the inhibition of the NFT formation.
In summary, our study indicates that the HBc
VLP-based T294-HBc vaccine exerted favorable effects
on cognition and neuropathology in the Tau.P301S
transgenic mouse model by inducing high titers of
antibodies against truncated tau; decreasing the levels of
truncated tau monomer, oligomer, and
hyperphosphorylated tau; increasing synaptophysin levels; and
suppressing microgliosis and astrogliosis in mouse brains.
Moreover, T294-HBc VLP-induced antibodies could
simultaneously bind to MTBR 1–4 [tau263–274, tau294–305,
tau325–336, tau357–368, and tau294–305(P301S)], indicating
that this vaccine has promising therapeutic potential for
the treatment of FTD and AD.
AD: Alzheimer’s disease; ANOVA: Analysis of variance; Aβ: β-Amyloid peptide;
CCD: Charge-coupled device; DG: Dentate gyrus; EC50: Half-maximal effective
concentration; ECL: Enhanced chemiluminescence;
EDTA: Ethylenediaminetetraacetic acid; ELISA: Enzyme-linked immunosorbent
assay; FTD: Frontotemporal dementia; GFAP: Glial fibrillary acidic protein;
GMT: Geometric mean titer; HBc: Hepatitis B virus core protein;
HRP: Horseradish peroxidase; Iba-1: Ionized calcium-binding adaptor
molecule-1; IgG: Immunoglobulin G; ITI: Intertrial interval; MIR: Major
immunodominant region; MTBR: Microtubule-binding region; MWM: Morris
water maze; NFTs: Neurofibrillary tangles; NOR: Novel object recognition;
RIPA: Radioimmunoprecipitation assay; Th: Helper T cell; VLPs: Virus-like
particles; WT: Wild type
This work was supported by grants from the Strategic Leading Project of
China Academy of Sciences (XDA 2040215) and the National Science and
Technology Major Projects of New Drugs (2015ZX09102015).
This study was supported by the Strategic Leading Project of China
Academy of Sciences (XDA 2040215) and the National Science and
Technology Major Projects of New Drugs (2015ZX09102015).
Availability of data and materials
The data analyzed during the present study are available from the
corresponding author on reasonable request.
RL designed the study and revised the manuscript. MJ carried out the
experiments involving vaccine design and protein purification, performed
the statistical analysis, and wrote the manuscript. XX participated in the
design of the study and in manuscript writing. DL participated in the design
of the study and the statistical analysis. XY participated in experiments
involving mouse breeding and behavior testing. YZ and SW participated in
experiments involving mouse breeding and behavior testing. LZ participated
in the biochemical and histochemical analyses. YH participated in the
biochemical and histochemical analyses. All authors read and approved the
Ethics approval and consent to participate
The animal treatment, husbandry, and experimental protocols of the present
study received the approval of the Tsinghua University Animal Care and Use
Committee (reference number 15-LRT1). All animal tests were carried out in
accordance with the China Public Health Service Guide for the Care and Use
of Laboratory Animals.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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