Neurobiology of Disease 134 (2020) 104636

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Neurobiology of Disease
journal homepage: www.elsevier.com/locate/ynbdi

Review

Active immunization with tau epitope in a mouse model of tauopathy T

induced strong antibody response together with improvement in short
memory and pSer396-tau pathology
A. Joly-Amadoa, ,1, H. Davtyanb,c,1, K. Serraneaua, P. Julesa, A. Zitnyara, E. Pressmana,
⁎

K. Zagorskib, T. Antonyanb, A. Hovakimyanb, H.J. Paeke, M.N. Gordona,2, D.H. Cribbsc,

N. Petrovskyd, M.G. Agadjanyanb, A. Ghochikyanb,3, D. Morgana,2,3
a
USF Health Byrd Alzheimer's Institute, Tampa, FL 33613, USA
b
The Institute for Molecular Medicine, Huntington Beach, CA 92647, USA
c
Institute for Memory Impairments and Neurological Disorders, University of California Irvine, Irvine, CA 92697, USA
d
Flinders Med. Ctr., Bedford Park, Adelaide 5042, Australia
e
Molecular Pharmacology and Physiology, College of Medicine, University of South Florida, Tampa, FL, USA

ABSTRACT

Abnormal tau hyperphosphorylation and its aggregation into neurofibrillary tangles are a hallmark of tauopathies, neurodegenerative disorders that include Alzheimer's
disease (AD). Active and passive Tau-immunotherapy has been proposed as a therapeutic approach to AD with mixed results. One of the limitations of active im-
munotherapy may be associated with the mediocre immunogenicity of vaccines that are not inducing therapeutically potent titers of antibodies. The aim of this study was to
test the efficacy of an anti-tau vaccine, AV-1980R/A composed of N terminal peptide of this molecule fused with an immunogenic MultiTEP platform and formulated in a
strong adjuvant, AdvaxCpG in a Tg4510 mouse model of tauopathy. Experimental mice were immunized with AV-1980R/A and a control group of mice were injected with
adjuvant only. Nontransgenic and tetracycline transactivator (tTA) transgenic littermates were included as baseline controls to contrast with the tau phenotype. Active
immunization with AV-1980R/A induced very strong anti-tau humoral immune responses in both nontransgenic and transgenic mice with evidence of IgG in brains of AV-
1980R/A vaccinated mice. These experimental animals displayed an improvement in short-term memory during a novel object recognition test. However, impairments in
other behavioral tasks were not prevented by AV-1980R/A vaccinations. At the same time, high titers of anti-tau antibodies reduced hyperphosphorylated pSer396 tau but
did not lower the level of other phosphorylated tau species in the brains of AV-1980R/A vaccinated mice. These data indicate that active immunotherapy with an N-
terminal Tau epitope was only partially effective in improving cognition and reducing pathology in the stringent Tg4510 mouse model of tauopathy.

1. Introduction individuals developed a detrimental T cell- mediated inflammatory re-

Alzheimer's disease is the most common form of dementia, in which
progressive accumulation of amyloid plaques and hyperphosphorylated
tau aggregated into neurofibrillary tangles (NFTs) results in progressive
cognitive impairments. Active and passive immunotherapy targeting Aβ
has been successful in multiple AD animal models (reviewed in (Morgan
et al., 2005, Wisniewski and Boutajangout, 2010, Agadjanyan et al.,
2015) and has been tested in an increasing number of clinical trials
without much success (Lobello et al., 2012; Lannfelt et al., 2014; Winblad
et al., 2014; Wisniewski and Goni, 2014; Agadjanyan et al., 2015;
Schilling et al., 2018; Selkoe, 2018; Bachmann et al., 2019; Panza et al.,
2019). In the first active immunization trial with full-length fAβ42 some
sponse (aseptic meningoencephalitis) leading to early termination of the
trial (Orgogozo et al., 2003; Ferrer et al., 2004) indicating that activation
of autoreactive T cells may infiltrate the brain and cause serious adverse
events. In addition, we now understand that even though amyloid de-
position occurs early in the disease, it doesn't account for clinical
symptoms by itself (Jack et al., 2009). Brain atrophy, neuronal and sy-
naptic losses appear to be the key components of cognitive impairments
in AD (DeKosky and Scheff, 1990; Terry et al., 1991; Fox et al., 1999),
and are more likely to be caused by tau pathology.
Indeed, NFTs are observed early in the pathogenesis of AD and in-
crease during aging (Braak and Braak, 1991). NFTs progression is
correlated with cognitive deficits (Duyckaerts et al., 1997), supporting

⁎
Corresponding author.
E-mail address: ajoly@usf.edu (A. Joly-Amado).
1
A.J-A. and H.D. contributed equally to this study
2
Present address: 400 Monroe Ave NW, Michigan State University, Grand Rapids MI 49503
3
These senior authors contributed equally to this work

https://doi.org/10.1016/j.nbd.2019.104636
Received 28 May 2019; Received in revised form 30 August 2019; Accepted 7 October 2019
Available online 17 October 2019
0969-9961/ © 2019 Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
A. Joly-Amado, et al. Neurobiology of Disease 134 (2020) 104636

Fig. 1. Design of the Vaccine Study Conducted in Tg4510 (tetO-MAPT*P301L) Transgenic Mice. Starting at 3 months of age, 3 groups of Tg4510 mice received a total of
7 immunizations with either adjuvant AdvaxCpG (n = 12) or tau vaccine, AV1980R/A (n = 12). Intramuscular injections 1,2,3 were performed every other week at age
3 months, 3.5 months and 4 months. Injection 4,5,6 were performed every other month at 5 months, 6 months and 7 months of age. A final dose was given at 8 months,
one week before euthanization. Due to high immune response, vaccine dose was decreased from 40 micrograms to 20 micrograms after injection 4. Additional groups of
non-transgenic and tTA littermates were used as control for behavior and vaccine titers. Non-transgenic and tTA littermates were not injected with either adjuvant or
antigen. A battery of behavior tests was performed 15 days after injection 6 (end timepoint, at 7 months of age). To assess the efficacy of the vaccine while stopping
transcription of tau, doxycycline was given to the mice in the water during 8 days at a concentration of 200 mg/kg at the end of the last battery of behavior tests. An
additional reversal testing was performed after treatment with doxycycline. Mice were euthanized at 8.5 months of age, 2 weeks after the seventh injection.

a pivotal role for tau pathology and spreading in AD-related memory
impairments (Dujardin et al., 2015). As such, there is a possibility that
targeting tau may represent a more effective method of treating AD
than removing Aβ if a patient is already exhibiting clear signs of cog-
nitive impairments. Therefore, development of safe and efficient im-
munotherapy targeting pathological tau could not only benefit AD pa-
tients, but also may become a useful tool against tauopathies in general.
However, as with Aβ immunotherapy, some studies reported in-
creased neuroinflammation and encephalopathy following active im-
munization with full length tau (Rosenmann et al., 2006) or phospho-
tau epitopes (Rozenstein-Tsalkovich et al., 2013). Furthermore, phos-
phorylation is essential for the regulation of tau's normal physiological
role in microtubule spatial organization. Therefore, one significant
concern that is associated with active tau immunotherapy is that
phospho-tau peptides may induce an immune response against phy-
siological tau species (Kontsekova et al., 2014). Targeting non-phos-
phorylated epitopes or toxic tau conformation may be necessary.
Changes in the conformations of tau are particularly important because
they can directly affect the function of the protein and its toxic role in
disease.
Recently, the phosphatase-activating domain (PAD) motif, a non-
phosphorylated epitope, was identified in the extreme N-terminus of
tau (Kanaan et al., 2011). This domain is normally hidden in a paper-
clip-like conformation of the native protein, but becomes exposed in
aggregated, pathological tau (Jeganathan et al., 2006). Abnormal ex-
posure of this motif has been linked to dysregulation of axonal transport
and neuronal function (LaPointe et al., 2009; Kanaan et al., 2011; Ward
et al., 2012). Immunohistochemical studies of human postmortem tis-
sues in AD patients demonstrated that exposure of the N-terminal re-
gion of tau is an early event in AD that increases with progression of the
disease (Kanaan et al., 2012; Ward et al., 2012). Therefore, this PAD
motif is a reasonable target epitope for active immunotherapy. In fact,
intracranial administration of monoclonal antibody targeting PAD
motif reduced total tau and phosphorylated forms of tau in brains of
Thy-Tau22 tau transgenic mice (Agadjanyan et al., 2017). In addition,
we previously reported that a MultiTEP-based recombinant protein
vaccine, AV-1980R/A targeting PAD was highly immunogenic in
wildtype C57BL6 mice (Davtyan et al., 2016). Here we are reporting for
the first time on immunogenicity of this vaccine in a mouse model of
tauopathy and the efficacy of AV-1980R/A on pathology and cognitive
impairments in Tg4510 mice.
2. Materials and methods
2.1. Animals
All animal testing procedures were approved by the Institutional
Animal Care and Use Committee of the University of South Florida and
were performed in accordance with the eighth edition “Guide for the
Care and Use of Laboratory Animals,” published by the National
Academy of Science, the National Academies Press, Washington, DC
(2011).
Parental mutant tau and tetracycline-controlled transactivator (tTA;
129S6 background) protein transgenic mouse lines were maintained
separately and bred to produce Tg4510 mice, tTA only mice, and
nontransgenic littermates as described (Santacruz et al., 2005; Dickey
et al., 2009). Mice were housed individually and maintained on a
twelve-hour light/dark cycle. Food (Envigo 2018–18% protein- diet)
and water were given ad libitum.
2.2. Epitope vaccines and purification of proteins
Recombinant protein was purified from E. coli BL21 (DE3) cells
transformed with previously constructed pET24a/3Tau2-18-MultiTEP
plasmids as described (Davtyan et al., 2016). The final recombinant
protein was analyzed in 10% Bis-Tris gel electrophoresis (NuPAGE
Novex Gel, Invitrogen, CA). Protein bands were visualized by Coo-
massie dye and specificity of the bands was confirmed by Western Blot
(WB) with anti-tau2-18 1C9 monoclonal antibodies (Davtyan et al.,
2016). The level of endotoxin was measured using E-TOXATE kits, as
recommended by the manufacturer (Sigma, St Louis, MO). For vaccine
preparation the AV-1980R protein was mixed with AdvaxCpG adjuvant.
Mice were immunized with 40 μg or 20 μg protein (per injection) for-
mulated in 1 mg adjuvant (Fig. 1).

2
A. Joly-Amado, et al.
2.3. Experimental design
Tg4510 mice aged 3 months old received a total of 7 immunizations
with either AdvaxCpG adjuvant (n = 12) or the tau vaccine, AV-1980R/
A (n = 12). Mice were injected with 50 μl of indicated antigen in-
tramuscularly (into right hind legs tibialis anterior muscle) using in-
sulin syringes (3/10 ml/cc) with the BD Ultra-Fine needle (30G).
Injection 1, 2, and 3 were performed at biweekly intervals starting at 3
mo. of age (Fig. 1). Injections 4, 5, and 6 were performed at 5 mo., 6
mo. and 7 mo. of age. A final dose was given after 8 mo, one week prior
to tissue collection. Due to the strong antibody response (see below),
the vaccine dose was decreased from 40 micrograms to 20 micrograms
after injection 4. Additional groups of non-transgenic and tTA litter-
mates were used as controls for behavior and vaccine titers. A battery of
behavioral tests was performed 15 days after injection 6 (at 7 mo. of
age). To assess the efficacy of the vaccine while stopping transcription
of tau, doxycycline was given to the Tg4510 mice via drinking water for
8 days at a concentration of 200 mg/kg at the end of the second battery
of behavior tests. An additional reversal testing in the radial arm water
maze was performed after treatment with doxycycline. Mice of both
sexes were included in each treatment condition.
2.4. Behavioral testing
All behavioral testing was performed in groups of mice balanced for
gender by an observer unaware of the treatment/genotype of the mice.
The open field was used as a standard test of general activity. Animals
were monitored for 15 min in a 40 cm square open field with video
tracking software (ANY-Maze, Stoelting, IL), under moderate lighting.
General activity levels were evaluated by measurements of horizontal
and vertical activity.
Each animal was placed in a walled Y-maze for a single 5 min trial.
The sequence of arm entries and total number of arm choices were
recorded. Spontaneous alternation (entering all three arms sequentially
without repetition) was expressed as a percentage, as calculated ac-
cording to the method of (Anisman, 1975).
Short term memory was evaluated by the novel object recognition
test (NOR) which consists of a 40 × 40 cm arena monitored and
quantified by video tracking (ANY-Maze, Stoelting, IL). Two objects
similar in scale to the mouse were placed along the center line of the
arena approximately 3–5 cm from the outside wall. Each animal was
given three acclimation trials of 5 min each with a 5 min inter-trial
interval. After each trial, the arena and object cues were cleaned with
70% ethanol to minimize olfactory cues. After the acclimation trials,
one of the acclimated objects was replaced with a novel object. Animals
were given a 5 min exploratory trial during which object exploration
was monitored by video recording. Working memory was evaluated by
the discrimination ratio, which is defined as the time exploring the
novel object minus the time spent with familiar and divided by com-
bined time spent exploring both novel and familiar objects.
A detailed description of radial arm water maze (RAWM) has been
published previously (Alamed et al., 2006). For fear conditioning, a
0.5 mA mild foot shock was paired with an auditory conditioned sti-
mulus within a novel environment. Freezing on the training day in
response to the foot shock was used as an estimate of learning during
the acquisition trial. Animals were placed in the fear conditioning ap-
paratus for 3 min, and then a 30 s noise at 70 dB was delivered with a
0.5-mA shock applied to the floor grid during the last 2 s of the con-
ditioned stimulus. Training consisted of two mild shocks paired with
two conditioned stimuli with a 2 min interval between each shock. For
contextual memory, the mice were placed in the acquisition chamber
and monitored for freezing to the context 24 h after training (no shocks
or auditory cue given) and tested for 3 min. Immediately after the
contextual test, mice were placed in a novel environment, consisting of
a chamber with different shape, floor and olfactory cues from the
training chamber. Mice were allowed to explore it for 3 min (cued no
3
Neurobiology of Disease 134 (2020) 104636
tone) and exposed to the conditioned stimulus for 3 min (cued tone).
Learning was assessed by measuring freezing behavior (i.e. motionless
position) every second.
2.5. Tissue collection
To assess antibody titers over the course of the experiments, blood
samples were collected from the submandibular vein before (pre-bleed)
and after immunizations 2, 3, 4 and by intracardiac puncture at eu-
thanasia. Blood was collected without anti-coagulant, incubated one
hour at room temperature and then kept overnight at 4 °C. The next day
samples were centrifuged for 10 min at 4000 rpm at room temperature
(RT), in a bench-top centrifuge. The serum (upper phase) was carefully
collected and centrifuged for 10 min at 7000 rpm. The supernatant was
collected and kept at -80 °C for antibody titer measurements.
One week after the last injection (injection 7), mice were euthanized
at 8.5 months of age with a solution containing pentobarbital and
phenytoin, then transcardially perfused with 25 ml of 0.9% normal
saline solution. Before perfusion, spleens were collected and single cell
suspension was prepared as described in (Davtyan et al., 2014a, 2014b)
using RBC (Red Blood Cells) Lysing Buffer (Sigma-Aldrich).
Brains were collected immediately following perfusion. The brain
was trimmed to exclude olfactory bulbs or spinal cord and weighed.
One hemisphere was dissected and frozen for western blot analysis and
the second hemisphere was immersion fixed in freshly-prepared 4%
phosphate-buffered paraformaldehyde for 24 h. The fixed hemispheres
were cryoprotected in successive incubations of 10%, 20% and 30%
sucrose solutions for 24 h each. Subsequently, brains were frozen on a
cold stage and sectioned in the horizontal plane (25 μm thickness) on a
sliding microtome and stored in Dulbecco's phosphate buffered saline
with 10 mM sodium azide solution at 4 °C.
2.6. Detection of splenocytes producing cytokines
Cytokine CBA (Cytometric bead array) assay. Concentrations (pg/
ml) of Th1 [IFN-γ, IL-2 and THF-α] and Th2 [IL-4 and IL-5] cytokines
were detected using Mouse Th1/Th2 Cytokine CBA kit by flow cyto-
metry according to manufacturer's instructions. Splenocytes were re-
stimulated in vitro with a cocktail of 12 Th epitope peptides (2 μg/ml
each) for 24 h and supernatant was collected for assay.
ELISpot assay. Analysis of IFN-γ producing T cells was performed in
splenocyte cultures from immunized mice by ELISpot assay (BD
Biosciences, CA), as previously described (Cribbs et al., 2003,
Petrushina et al., 2007, Davtyan et al., 2013, Davtyan et al., 2014a,
2014b). Cultures of splenocytes were re-stimulated in vitro with a
cocktail of 12 peptides representing the Th epitopes in the MultiTEP
vaccine (Davtyan et al., 2014a, 2014b) (2 μg/ml of each peptide), and
several individual peptides [PADRE, P23, P30, P17, P28] incorporated
in MultiTEP platform, as well as Tau2-18 peptides at concentration of
10 μg/ml for 20 h. The numbers of IFN-γ spot-forming cells (SFC) per
106 splenocytes stimulated by different peptides were then counted.
2.7. Detection of B cells producing tau-specific antibodies
Antibody-secreting B cells specific to tau were detected in spleno-
cytes by ELISpot (Mabtech Inc., Cincinnati, OH) as described in
Davtyan et al. (Davtyan et al., 2016). Briefly, splenocytes from ex-
perimental and control mice were incubated for 24 h in 96-well plates
coated with tau2-18 peptides. After incubation the assay was performed
as recommended by the manufacturer.
2.8. Detection of tau-specific antibodies
The concentrations of anti-tau antibodies in serum was determined
by ELISA, as described previously (Davtyan et al., 2016). To measure
anti-tau antibody concentration plates were coated with 1 μg/per well
A. Joly-Amado, et al.
with tau2-18 peptide (GenScript, NJ). Anti-tau antibody concentration
was calculated using a calibration curve generated with 1C9 mAb
(generated at The Institute for Molecular Medicine, Huntington Beach,
CA). HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch La-
boratories, ME) was used as secondary antibody.
2.9. Histopathology
Stereological principles were employed to select evenly spaced
sections to be stained for each marker. Immunohistochemical proce-
dural methods were as described previously (Gordon et al., 2002).
Sections from all animals were placed in a multi-sample staining tray
and endogenous peroxidase was blocked (10% methanol, 3% H2O2 in
phosphate buffered saline, 10 mM NaPO4, 154 NaCl, pH 7.4,
PBS;30 min). Tissue samples were permeabilized with 0.2% lysine,
1%Triton X-100 in PBS solution and incubated overnight in anti-tau
phosphorylated at Ser396 (rabbit polyclonal, Anaspec), total tau H150
(rabbit polyclonal, SantaCruz Biotechnology), anti-tau phosphorylated
at AT8 (Thermo scientific, Waltham, MA), Iba-1 (Wako), GFAP (Dako),
CD86 (Novus biologicals, Littleton CO). Additionally, we examined the
staining for mouse IgG with biotinylated horse anti-mouse IgG specific
secondary antibody (Vector Laboratories, Burlingame, CA, USA) to
detect whether the systemically injected antibodies may have bound to
brain tissue. Sections were washed in PBS, and then incubated in bio-
tinylated secondary antibody (Vector Laboratories, Burlingame, CA).
The tissue was again washed after 2 h and incubated with Vectastain®
Elite® ABC kit (Vector Laboratories, Burlingame, CA) for enzyme con-
jugation. Finally, sections were stained using 0.05% diaminobenzidine,
0.5% nickel ammonium sulfate and 0.03% H2O2. Tissue sections were
mounted onto slides, dehydrated, cover slipped and scanned for ana-
lysis using a digital scanning microscope Axio Scan Z.1 (Zeiss Inc).
Digitized sections were annotated for regions of interest and positively
stained area was quantified with Nearcyte software. Values for all
sections from the same mouse were averaged to represent a single value
for that region in subsequent statistical analysis.
2.10. Biochemical analyses
Tissues for Western blot analysis were prepared as previously de-
scribed (Brownlow et al., 2014). Dissected hippocampi (HPC) and pos-
terior cortex (PCX) were homogenized then sonicated in RIPA buffer
containing protease inhibitor cocktail (Sigma Aldrich) and phosphatase
inhibitor cocktails I and II (Sigma Aldrich) and centrifuged at 40, 000 ×sg
for 30 min at 4 °C. The supernatant was collected (RIPA soluble fraction)
and protein concentrations were determined by the BCA protein assay kit
(Pierce, Rockford, IL). The remaining pellet was digested with 70% formic
acid according to the wet tissue weight, and then neutralized with NaOH
to analyze RIPA-insoluble proteins (RIPA insoluble fraction). Equal
amounts of proteins according to BCA (5 μg/well for soluble fraction,
1 μg/well for insoluble fraction) were loaded in each well of a 4–12% Bis-
tris gels and transferred to a 0.2 μm pore size nitrocellulose membrane
and immunoblotted with H150 (Santacruz biotechnology, Dallas, TX),
pSer396-tau (Anaspec, Fremont, CA), N-term Tau 12 (biolegend, San
Diego, CA) and actin (Sigma Aldrich, St Louis, MO) at 1:1000-fold dilu-
tion. Fluorescently tagged secondary antibodies (IRDye 800CW, LI-COR
Biosciences) were used at a dilution of 1:10,000. Western Blot results
were quantified by scanning with a LI-COR Odyssey fluorescent scanner.
Band intensities were quantified by densitometric analysis using the
Odyssey imaging system (LI-COR Biosciences) and normalized to the band
intensity of β-actin (for RIPA soluble fraction) or to total protein (RIPA
insoluble fraction). Total protein was detected using REVERT reagent (LI-
COR) according to the manufacturer's instructions.
In addition, concentrations of human total and phosphorylated tau in
soluble brain extracts were determined by Tau (total) Human ELISA kit,
Tau [pS396] Human ELISA Kit, Tau [pS199] Human ELISA Kit, Tau
[pT181] Human ELISA Kit, and Tau [pT231] Human ELISA Kit (all from
4
Neurobiology of Disease 134 (2020) 104636
ThermoFisher Scientific, MA), according to the manufacturer's instruc-
tions.
2.11. Statistical methods
Data were analyzed by ANOVA or repeated measures ANOVA fol-
lowed by Fisher's protected least significant difference post hoc tests
(statview) or ANCOVA. In all tests, a P-value < 0.05 was considered
significant. All data are presented as mean values ± standard error of
the mean (SEM) unless specified otherwise.
3. Results
3.1. Immunogenic efficacy of AD vaccine in Tg4510 mice
Recently we demonstrated that MultiTEP-based recombinant pro-
tein vaccine AV-1980R/A (tau vaccine) is highly immunogenic in
wildtype C57BL6 mice (Davtyan et al., 2016). To test the immunogenic
and therapeutic efficacy of tau vaccination in tau transgenic mice, 3-
month-old Tg4510 mice were immunized with AV-1980R/A, while
control groups injected with adjuvant (AdvaxCpG). Mice received seven
injections at the time points shown in Fig. 1.
Cellular immune responses were measured in splenocytes of vacci-
nated mice by detection of production of Th1/Th2 cytokines (Fig. 2). As
expected, AV-1980R/A vaccine generated a strong cellular immune
response specific to the Th cell epitopes incorporated in the MultiTEP
platform. Next, we tested Th cell immune responses specific to in-
dividual epitopes incorporated into the MultiTEP vaccine platform.
ELISpot data presented in Fig. 3 demonstrated that vaccination with
AV-1980R/A stimulated Th cells specific to epitopes PADRE, P23, P30,
P17 and P28 in this mouse strain possessing an H-2b/q immune haplo-
type. Interestingly, this repertoire was slightly different in C57BL6 (H-2b)
and in THY-Tau22 strain, which was created on H-2b/k background and
was backcrossed with C57BL6 mice (Schindowski et al., 2006). Of note,
we detected significantly higher numbers of Th cells specific to PADRE,
which is a promiscuous universal synthetic epitope, compared with P23,
P30, P17 and P28 peptides in immune splenocytes isolated from tau
vaccinated mice. High numbers of activated Th cells producing both Th1
and Th2 types of cytokines indicated that tau vaccine induced mixed
Th1/Th2 types of immune responses. Importantly, no activation of po-
tentially harmful autoreactive Th cells was detected after re-stimulation
of immune splenocytes with tau self-epitope by ELISpot assay (Fig. 3). As
might be expected, strong cellular immune responses specific to the
MultiTEP vaccine platform should stimulate B cells to produce antibodies
specific to tau2-18 epitope incorporated into AV-1980R/A vaccines. The
ELISpot data confirms this assumption and demonstrates that vaccination
generated a large numbers of anti-tau antibody-secreting B cells (ASC)
after immunization with AV-1980R/A (Fig. 4A). No cross-reactive anti-
body-producing B cells were detected in splenocytes isolated from im-
munized mice (Fig. 4A). More importantly, these tau specific B cells
activated by vaccination generated extremely strong humoral immune
responses after only two immunizations with average concentrations of
antibodies close to 2 mg/ml, > 10% of total serum IgG. This level of
antibodies was maintained throughout the entire study (Fig. 4B).
Finally, to characterize the type of humoral immune responses, we
measured the production of IgG1, IgG2ab, IgG2b, and IgM isotypes in mice
immunized with AV-1980R/A vaccine. Mice from both groups generated
mostly IgG antibodies (IgG1 > IgG2b > IgG2ab), while only negligible
titers of IgM antibodies were detected in the sera of immunized mice (data
not shown). Of note, IgG1/IgG2ab is equal to 4 for AV-1980R/A indicating
that the immune response is biased towards Th2 type.
3.2. Impact of vaccination on cognitive impairments
Spatial memory deficits were assessed by the 2-day radial arm water
maze test. As previously shown, repeated measure ANOVA revealed a
A. Joly-Amado, et al. Neurobiology of Disease 134 (2020) 104636

Fig. 2. AV-1980R/A formulated in AdvaxCpG adjuvant generated both Th1 and Th2 immune responses in Tg4510 mice. Concentrations (pg/ml) of Th1 cytokines (A, B, C) and
Th2 cytokines (D, E) in response to AdvaxCpG adjuvant (open circle) and AV-1980R/A tau vaccine (black square) in Tg4510 mice. [IFN-γ: Interferon-gamma, IL-2: Interleukin 2,
TNF-α: Tumor necrosis factor alpha, IL-4: Interleukin 4, IL-5: Interleukin 5]. Cytokines were detected using Mouse Th1/Th2 Cytokine CBA kit by flow cytometry. Splenocytes
were re-stimulated in vitro with a cocktail of 12 Th epitope peptides (2 μg/ml each) for 24 h and supernatant was collected for assay. Bars represent mean ± SEM (n = 6 mice/
per group). Statistical significance was calculated against AdvaxCpG group using unpaired t-test. (*P < 0.05, ***P < 0.001 and ****P < 0.0001).

genotype effect: non-transgenic and tTA control mice learned the lo-
cation of the platform by the end of day 1 and made few errors on day
2. Tg4510 mice treated with adjuvant were not able to remember the
location of the platform as they made significantly more errors on day 1
and day 2 when compared to non-transgenic or tTA control mice
(Fig. 5A). There was no significant improvement in the number of er-
rors in Tg4510 vaccinated with AV-1980R/A compared to adjuvant
treated Tg4510 mice. All Tg4510 mice performed worse than the non-
transgenic and tTA mice, indicating no effect of the vaccine on spatial
navigation memory.
Conversely, we observed an improvement in short term memory
assessed by novel object recognition (NOR) in mice vaccinated against
tau. As expected, two-way ANOVA revealed a genotype effect as non-
transgenic and tTA control mice spent more time with the novel object
than did the adjuvant Tg4510 mice (Fig. 5B). However, Tg4510 mice
vaccinated with AV-1980R/A displayed a significant increase in time

Fig. 3. AV-1980R/A induced strong cellular response specific to Th epitopes incorporated in the MultiTEP platform without activating potentially harmful auto-
reactive Th cells specific to tau. Splenocytes from individual mice were re-stimulated in vitro with a cocktail of 12 T-helper epitope peptides (2 μg/ml each) and
several individual peptides [PADRE, P23, P30, P17, P28] incorporated in MultiTEP platform, as well as Tau2-18 peptide (10 μg/ml). IFNγ spot-forming colonies (SFC)
were detected by ELISPOT assay and error bars indicate mean ± SEM (n = 11 per group).

5
A. Joly-Amado, et al. Neurobiology of Disease 134 (2020) 104636

Fig. 4. AV-1980R/A induced specific B cells producing very high concentrations of antibodies.
Evaluation of humoral immune responses generated in Tg4510 transgenic mice after immunizations with AV-1980R vaccine formulated with AdvaxCpG adjuvant. (A)
Detection of anti-tau antibody-secreting cells (ASC), visualized as spots, was done in splenocyte cultures obtained from experimental and control mice using ELISpot
assay. (B) Concentrations of anti-tau antibodies in mouse sera were determined by ELISA. Plates were coated with Tau2-18 peptide (Genscript). Anti-tau antibody
concentrations were calculated using a calibration curve generated with 1C9 anti-tau2-18 monoclonal antibody. Bars represent average ± SD (n = 11/per group).

attending to the novel object when compared to Tg4510 adjuvant
treated mice. In addition, AV-1980R/A vaccinated mice achieved the
same discrimination ratio as tTA and non-transgenic control mice. A
battery of other cognitive tests was performed (Table 1). As previously
shown, we observed a genotype effect in total distance travelled during
open field, number of entries in Y maze, contextual fear conditioning
and nesting behavior. Vaccination with AV-1980R/A had not a sig-
nificant influence on these cognitive deficits.
3.3. Vaccination with Tau epitope induced increased levels of mouse IgG in
the brain
As shown in Fig. 4, very high concentrations of antibodies were
produced after vaccination with AV-1980R/A. To investigate whether
the antibodies produced during vaccination were reaching the brain,
we incubated brain sections with an anti-mouse IgG specific secondary
antibody. Quantification of positive area stained with anti-mouse IgG
was performed in anterior cortex (ACX), posterior cortex (PCX), and
hippocampus (HPC) calculated from digitized images is shown in
Fig. 6A. Tg4510 mice vaccinated with tau epitope exhibited sig-
nificantly higher levels of IgG in the ACX, HPC and PCX when compared
to Tg4510 mice treated with adjuvant. When the parenchymal staining
in the posterior cortex was viewed at higher magnification, there was
staining of the neuropil only in Tg4510 mice vaccinated with AV-
1980R/A (Fig. 6B, panels b, d). No positive staining was detected in
Tg4510 mice treated with AdvaxCpG adjuvant (Fig. 6B, panels a, c).
Mouse IgG staining was also absent in non-transgenic and tTA control
mice (data not shown).

Fig. 5. Vaccination with AV-1980R/A did not improve spatial memory during radial arm water maze testing but rescued the short-term memory deficit observed in
Tg4510 during the novel object recognition test.
(A) Tg4510 mice made significantly more errors regardless of treatment with AdvaxCpG adjuvant or AV-1980R/A when attempting to locate a hidden platform during
the 2-day radial arm water maze (RAWM) test when compared to non-transgenic (Ntg) and tTA littermates. Data are presented as mean ± S.E.M., n = 12/group.
***p < 0.001. (B) Tg4510 mice treated with AdvaxCpG adjuvant spent less time with novel object compared to non-transgenic (Ntg) and tTA littermates as they
displayed a lower discrimination ratio (which is defined as the time exploring the novel object minus the time spent with familiar and divided by combined time
spent exploring both novel and familiar objects), indicating short term memory deficit. Tg4510 mice vaccinated with AV-1980R/A displayed a significant increase in
discrimination ratio compared to control Tg4510 mice treated with AdvaxCpG adjuvant indicating an improvement in short memory. Data are presented as
mean ± S.E.M., n = 12/group. *p < 0.05. 2-way ANOVA test was used for RAWM with Tukey's LSD means comparisons for significant overall effects. A t-test was
performed to compare the two Tg4510 groups for NOR.

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A. Joly-Amado, et al. Neurobiology of Disease 134 (2020) 104636

Table 1
Summary of behavior tests performances in non-transgenic (Ntg), tet only (tTA) and Tg4510 mice treated with AdvaxCpG adjuvant or AV-1980R/A. Open field total
distance travelled in meters (TDT), Ymaze entries and alternations, Radial arm water maze (RAWM) total errors, Fear conditioning (FC) percentage time freezing and
nesting behavior.
Ntg Tta AdvaxCpG AV-1980R/A

a,b a,b
Open field (TDT) 36.7 +/− 2.8 46.9 +/− 2.5 150.3 +/− 35.3 122.9 +/− 32.2
Ymaze entries 32.8 +/− 1.9a,b 34.3 +/− 1.3a,b 57.8 +/− 9 59.5 +/− 7.9
Ymaze alternation 60.7 +/− 3.2 59.9 ± 1.6 65.1 +/− 2.3 68.2 +/− 4.8
RAWM reversal total errors 25 +/− 4.7 a,b,c 40.1 +/− 2.7a,b 92.8 +/− 13.3 93.4 +/− 14.6
RAWM post dox reversal total errors 29.5 +/− 5.4a,b 52.9 +/− 8.3 81.9 +/− 14.3 70.5 +/− 7.8
FC context 25.6 +/− 5.4a,b 20.7 +/− 4.7a,b 6.6 +/− 2.3 2.6 +/− 1.2
FC cued (tone) 42.1 +/− 7 26.4 +/− 5.1 27.7 +/− 8 33.6 +/− 6.7
Nesting behavior 3.8 +/− 0.3a,b 3.9 +/− 0.2a,b 1.1 +/− 0.5 0.9 +/− 0.4

p < 0.05 when compared to AdvaxCpG adjuvant.

a

b
p < 0.05 when compared to AV-1980R/A.
p < 0.05 when compared to tTA. As expected, Tg4510 treated with AdvaxCpG adjuvant displayed hyperactivity and cognitive impairments when compared to
c

non-transgenic or tTA littermates. Immunization with AV-1980R/A did not affect these phenotypes.

Fig. 6. Tg4510 mice vaccinated with Tau vaccine AV-1980R/A displayed increased levels of mouse IgG in the brain compared to AdvaxCpG.
(A) Quantification of positive area stained for mouse IgG in anterior cortex (ACX), hippocampus (HPC) and posterior cortex (PCX) in Tg4510 treated with AdvaxCpG
adjuvant or with AV-1980R/A mice. Non-transgenic and tTA littermates displayed similar levels to Tg4510 treated with AdvaxCpG adjuvant (data not shown).
Immunostaining quantification was performed utilizing Mirax software (Zeiss Inc.). (B) Micrographic representation of Mouse IgG immunostaining in the posterior
cortex of Tg4510 mice treated with AdvaxCpG adjuvant (a, c) or AV-1980R/A (b, d). c and d are magnification of square area in a, b, respectively. Tg4510 mice
immunized with AV-1980R/A seemed to display positive staining in cell bodies particularly in the posterior cortex. Data are presented as mean ± S.E.M., n = 12/
group. Statistical significance was calculated against AdvaxCpG group using unpaired t-test *p < 0.05.

3.4. Tau pathology in Tg4510 vaccinated mice
We then evaluated the impact of the different vaccines on tau pa-
thology. Quantification of positive immunostaining for pSer396-tau
(phosphorylated tau) and H150 (total tau) in the entire hemisection
(total), anterior cortex (ACX), hippocampus (HPC) and posterior cortex
(PCX) are shown in Fig. 7. There was a trend towards decrease in total
area as well as ACX and PCX in AV-1980R/A vaccinated mice when
compared to AdvaxCpG adjuvant-treated mice, but the difference did not
reach significance (Fig. 7A). A similar trend was observed in AT8 or
Gallyas silver stained sections (Table 2). No difference was observed in
total tau levels detected by immunohistochemistry as shown in Fig. 7B,
D. As expected, no positive staining was detected in non-transgenic
animals for pSer396-tau or H150 (data not shown). The adjuvant
treated Tg4510 mice had values similar to historical values for un-
treated Tg4510 mice of the same age (Fig. S3).
Western blot analysis revealed a decrease in pSer396-tau levels
(Fig. 8B,E) but not H150 (Fig. 8A,D) or Tau12 (anti Nterm tau aa6-18;
Fig. 8C,F) in the RIPA- soluble fraction of the posterior cortex in AV-
1980R/A vaccinated mice compared to AdvaxCpG adjuvant treated mice
(see Fig. S1 for western immunoblot of all samples). This was the case
whether normalized to actin (Fig. 8) or when the phospho-epitopes
were normalized to total tau (Fig. 8G,H and Fig. S2). There were no
differences in the levels of these markers in PCX RIPA insoluble fraction
(data not shown). In the hippocampus RIPA insoluble fraction, there
was a trend towards decrease in levels of pSer396-tau in AV-1980R/A
vaccinated mice compared to AdvaxCpG adjuvant treated mice that did
not reach significance (Fig. 9).
ELISA assay performed for total tau and several phosphorylated tau
forms in soluble extractions revealed a significant decrease in pSer396-
tau and total tau but not in pSer199-, pT181- and pT231-tau, in AV-
1980R/A vaccinated mice when compared to AdvaxCpG adjuvant treated
mice (Fig. 10).
Inflammation markers and brain weight, which have been pre-
viously shown to be affected by tau pathology in Tg4510 (Wes et al.,
2014; Joly-Amado et al., 2016) were also analyzed (Table 2). Im-
munohistochemistry for Iba-1 and CD86 (microglia) as well as GFAP
(astrocytes) and brain weight showed genotype effects but no treatment
effect.
4. Discussion
The “amyloid cascade hypothesis” considers pathological Aβ as a
triggering toxic event in AD and most clinical immunotherapy ap-
proaches have been aimed at reducing Aβ deposits. However, accu-
mulating evidence show that pathological tau correlates with the onset
of symptoms and the degree of dementia better than Aβ deposition. It is
important to note that the lessons from clinical trials of active and
passive immunization targeting Aβ suggest that anti-Aβ im-
munotherapy may be effective in the early/preclinical stages of AD,

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A. Joly-Amado, et al. Neurobiology of Disease 134 (2020) 104636

Fig. 7. Vaccination with AV-1980R/A did not affect levels of pathology in Tg4510 mice when assessed by immunohistochemistry.
Quantification of positive area stained for pSer396 (A) and H150 (B) in total area (total), anterior cortex (ACX), hippocampus (HPC) and posterior cortex (PCX) in
Tg4510 mice treated with AdvaxCpG adjuvant or with AV-1980R. Immunostaining quantification was performed utilizing Mirax software (Zeiss Inc.). Micrographic
representation of pSer396 (C) and H150 (D) immunostaining in the hippocampus of Tg4510 mice treated with AdvaxCpG adjuvant (C, D left panel), and AV-1980R (C,
D right panel). Data are presented as mean ± S.E.M., n = 12/group. Statistical significance was calculated against AdvaxCpG group using unpaired t-test *p < 0.05.

whereas in the later stages, clearing of Aβ alone may not be sufficient to
stop/delay the disease. Therefore, anti-tau immunotherapy (passive
and active immunizations) was proposed as an additional therapeutic
approach.
The first attempts at tau immunotherapy involved the active im-
munization of wild type mice with full-length recombinant human tau
protein (Rosenmann et al., 2006), followed by experiments using a
variety of immunogens of tau and adjuvants in many different animal
models of tauopathy (reviewed in (Schroeder et al., 2016). In general,
active immunization was efficient at reducing tau pathology in mice as
indicated by decreased phosphorylated tau (Boutajangout et al., 2010;
Troquier et al., 2012; Selenica et al., 2014) and NFTs (Boimel et al.,
2010). Some studies also demonstrated cognitive improvements
(Boutajangout et al., 2010; Benhamron et al., 2018) or improved motor
performance (Asuni et al., 2007). Passive immunotherapy studies in
animal models also showed promising results when administered early in
the disease process. Passive immunization with PHF1 or MC-1 antibody
decreased tau pathology in JNPL3 (Boutajangout et al., 2011; Chai et al.,
2011; d'Abramo et al., 2013) and P301S (PS19) mice (Chai et al., 2011).
Targeting oligomeric tau was efficient in JNPL3 mice (Castillo-Carranza
et al., 2014) but not in Tg4510 (Schroeder et al., 2017).
More recent work has identified some intriguing abilities of tau im-
munotherapy to clear not only tau deposits, but also Aβ deposits in mice.
In 3xTg mice, Dai et al. (Dai et al., 2017) found that an N-terminal anti-
tau antibody was more effective than a mid-domain antibody in reducing
tau, but especially in reducing amyloid deposits also in these mice. Both
antibodies rescued behavioral deficits. In follow up work, Dai et al. (Dai
et al., 2018) also demonstrated the N-terminal antibody could block tau
propagation in 3xtg mice caused by injecting AD-tau seeds. Rajamoha-
medsait H et al. (Rajamohamedsait et al., 2017) found that active im-
munization of 3xTg mice partially reduced tau deposition but con-
siderably blocked amyloid deposition.Benhamron et al. (Benhamron
et al., 2018) found that a phospho-tau vaccine was capable of reducing
Aβ and improving memory in an APP/PS1 mouse model known to have
minimal tau pathology. In a different comparison of N-terminal and mid-
domain antibodies, Albert et al. (Albert et al., 2019) found the mid-
domain antibodies more effective in blocking tau seeding and propaga-
tion. Thus, it seems likely that the properties of the antibody other than
its domain specificity may have more influence on efficacy. However, it
seems quite feasible from the mouse studies that anti-tau immunotherapy
could reduce both tau and amyloid pathologies in cases of AD.
Two active vaccines (Godyn et al., 2016; Novak et al., 2019) are
currently being tested in clinical trials with AD patients. Although these
trials are in the early stages and are intended for safety studies, some
preliminary efficacy data were published from AADVac1 trial, which
reported that a trend towards slower cognitive decline and lower hip-
pocampal atrophy rate was observed in patients with higher antibody
titers (Novak et al., 2018).
There are advantages and disadvantages of both active and passive
immunotherapy. Active immunotherapy has the advantage of requiring
fewer doses and is more cost effective than passive immunotherapy. The
disadvantages are that the antibody response is variable both quantita-
tively and qualitatively (especially in older adults) and adverse effects
are challenging to reverse. Passive immunotherapy has the advantage of
known dosing with an antibody with known characteristics, and if ad-
verse events ensue, the antibody can be discontinued. Disadvantages
include a high frequency of dosing (typically monthly by infusion) and
the considerable costs associated with antibody production and delivery.
In this study, we tested the immunogenicity and therapeutic efficacy
of AV-1980R/A MultiTEP platform based active vaccine targeting PAD
(Tau2-18) region of tau in Tg4510 mice. MultiTEP platform composed
of 12 foreign Th cell epitopes, was specifically designed to enhance
immune responses in elderly patients with immunosenescence and
provide a broad coverage of human MHC class II polymorphism uti-
lizing the wide array of tetanus toxin, hepatitis B and influenza Th
epitopes incorporated into the MultiTEP platform.
In Tg4510 mice, the vaccine induced strong cellular responses
specific to foreign Th epitopes that provided help to B cells to be acti-
vated and produce very high titers of antibodies against tau, achieving
2 mg/ml or approximately 10% of the IgG concentration in mice.
Moreover, no tau specific T cell response was detected indicating that
the likelihood that this vaccine would produce autoreactive

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A. Joly-Amado, et al. Neurobiology of Disease 134 (2020) 104636

(caption on next page)

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A. Joly-Amado, et al. Neurobiology of Disease 134 (2020) 104636

Fig. 8. Tg4510 mice vaccinated with tau vaccine AV-1980R/A display decreased levels of phospho tau in the posterior cortex soluble fraction when compared to
AdvaxCpG adjuvant treated mice.
Phospho tau (pSer396), total tau (H150) and Nterm tau (Tau12) were assessed by western blot analysis (A, B, C representative WB images) in posterior cortex in RIPA
soluble fraction.55 kda, 64 kda and combination of 55-64kda band intensity were quantified for H150 (D), pSer396 (E) and Tau12 (F) in Tg4510 mice treated with
AdvaxCpG adjuvant or with AV-1980R. All groups were normalized to the adjuvant group and to actin. No bands were detected in non-transgenic (Ntg) and tet only
(tTA) mice, as shown in image A, B, C. Ratio of Pser396 and Tau12 to H150 was calculated for bands 55–64 (G, H) and for bands 55 and 64 separately (Fig. S2). Data
are presented as mean ± S.E.M., n = 12/group. Statistical significance was calculated against AdvaxCpG group using unpaired t-test *p < 0.05.

Fig. 9. Tg4510 mice vaccinated with tau vaccine AV-1980R/A display a trend towards decrease of phospho Tau in the hippocampus insoluble fraction when
compared to AdvaxCpG.
Phospho Tau (pSer396) and total Tau (H150) was assessed by western blot analysis (A, representative WB images) in hippocampus in RIPA insoluble fraction. Briefly,
after high centrifugation, pellet was treated with formic acid and neutralized with NaOH. Band intensity was quantified for H150 (B) and pSer396 (C) as well as ratio
of pSer396 over H150 (D) in Tg4510 mice treated with AdvaxCpG adjuvant, or AV-1980R/A. All groups were normalized to the adjuvant group. No bands were
detected in non-transgenic (Ntg) and tet only (tTA) mice, as shown in image A. Data are presented as mean ± S.E.M., n = 12/group. Statistical significance was
calculated against AdvaxCpG group using non parametric Mann Whitney test.

Table 2
Brain weight at euthanasia (in mg).
Ntg tTA AdvaxCpG AV-1980R/A

Brain weight (mg) 503.9 +/− 6.1a,b,c 474.6 +/− 6.5a,b,c 427.9 +/− 13 426 +/− 11.6
Gallyas stain – – 1.0 ± 0.21 0.92 ± 0.12
Immunohistochemistry AT8 – – 1.0 ± 0.16 0.84 ± 0.12
gfap 0.54 ± 0.05a,b 0.56 ± 0.05a,b 1.0 ± 0.10 1.13 ± 0.06
iba-1 0.77 ± 0.04a,b 0.76 ± 0.05a,b 1.0 ± 0.04 0.96 ± 0.07
cd86 total 1.04 ± 0.11 0.81 ± 0.09b 1.0 ± 0.08 1.12 ± 0.09

Positive total area stained with Gallyas, or for AT8 (phospho Tau Ser202/Thr205), Glial fibrillary acidic protein (GFAP), Ionized calcium binding adaptor molecule 1
(Iba-1) and cluster of differentiation 86 (CD86). As expected, Tg4510 treated with AdvaxCpG adjuvant displayed decreased brain weight and increased positive area
ratio for GFAP, Iba-1 and CD86 when compared to non-transgenic or tTA littermates. Immunization with AV-1980R/A did not affect these phenotypes nor did it
affect levels of phospho tau (pSer396), and total tau (H150) assessed by western blot analysis in hippocampus in RIPA soluble fraction, or positive area ratio for AT8.
Statistical significance was calculated against using unpaired t-test or ANOVA.
p < 0.05 when compared to AdvaxCpG adjuvant.
a

b
p < 0.05 when compared to AV-1980R/A.
c
p < 0.05 when compared to tTA.

10
A. Joly-Amado, et al.
Fig. 10. Tg4510 mice vaccinated with AV-1980R/A display decreased levels of phosphorylated tau and total tau in the posterior cortex soluble fraction when
compared to AdvaxCpG adjuvant treated mice when analyzed by ELISA.
Level of total (A) and several phosphorylated tau (pSer396 (B), pSer199 (C), pT181(D), pT231(E), in brain soluble extractions of posterior cortex from Tg4510 mice
treated with AdvaxCpG adjuvant or AV-1980R/A, were analyzed by ELISA. Error bars represent average ± SEM (n = 11–12) Statistical significance was calculated
against AdvaxCpG group using unpaired t-test. **p < 0.01.
inflammatory T cell responses against tau in brain is very low.
The Tg4510 mice developed the expected behavioral phenotype, with
impaired performance in open field, contextual fear conditioning, novel
object recognition, radial arm water maze and nesting behavior. There
was a significant rescue of the novel object recognition behavior back to
levels similar to nontransgenic mice. However, there was no indication of
improvements in any of the other behavioral tests. These results are
consistent with our prior work. In Brownlow et al. (2014), we found that
caloric restriction in Tg4510 mice caused significant improvements in
the novel object task but not in other measures of the behavioral phe-
notype. This occurred with modest trends in reversal of the tau pa-
thology, none of which were statistically significant (Brownlow et al.,
2014). These results would suggest that the relatively simple novel object
recognition task is easier for mice to perform. Hence even small changes
in the tau deposition are capable of rescuing this deficit, but not the
deficits associated with more complex behaviors.
Histological analysis indicated the presence of mouse IgG only in
the tau mice vaccinated against tau. This suggested that these anti-
bodies were most likely retained by the CNS in tau deposits that de-
veloped in these mice.
This could explain the reduction of phopho-tau epitopes at pSer396
observed in vaccinated mice. There was no effect on Gallyas stained tau
deposits or brain atrophy with the tau epitope vaccine. We also observed
reductions in pSer396-tau on western blots from dissected brain regions
and with ELISA measurements. Thus, by several measures, vaccination
against the AD domain of tau caused modest reduction in phospho-tau.
The Tg4510 mouse is a very aggressive model and manipulations
aimed at rescuing the phenotype, short of turning off the transgene
(Santacruz et al., 2005), have failed. In prior work with a tau oligomer
antibody, we found little impact on the tau phenotype in this mouse
(Schroeder et al., 2017). To our knowledge, only one immunotherapy has
resulted in reduction of tau pathology in this mouse model. Sankar-
anarayan et al. (Sankaranarayanan et al., 2015) observed 20% reductions
in tau deposition using a monoclonal antibody. However, in the same paper
they reported much larger reductions in the phenotype of the P301S-tau
model PS19. The Tg4510 mouse has 13-fold over expression of tau, while
the PS19 mouse has 5-fold over expression (Roberson, 2012). Recently it
has been identified that some of the Tg4510 phenotype may be related to
insertional disruption of the Fgf14 gene by the transgene (Gamache et al.,
2019). Thus, this contribution to the phenotype could not be readily re-
versed by preventing tau deposition. We conclude that the modest reduc-
tions in the phenotype seen here may reflect the Tg4510 model and per-
haps not the overall efficacy of the vaccine or its intended target.
5. Conclusions
Altogether our results show that a highly immunogenic tau vaccine
11
Neurobiology of Disease 134 (2020) 104636
using the phosphatase-activating domain (PAD) of tau protein as im-
munogen was efficient at causing high titers of anti-tau antibodies with
little risk of developing auto-reactive T cells. The vaccine was effective
at modestly reducing some aspects of tau pathology and improving
some components of cognitive performance in an aggressive model of
tauopathy, the Tg4510 mouse.
Declaration of Competing Interest
MGA and AG are co-founders of Capo Therapeutics that licensed
MultiTEP vaccine platform technology from the Institute for Molecular
Medicine. The remaining authors declare no financial, commercial and
non-financial conflict of interests.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.nbd.2019.104636.
Acknowledgments
This work was supported by funding from NIH (R01-NS050895, R01-
AG020241, U01-AG060965, R21-NS101504, R01-AG061895, R01-
AG055524, R01-NS076308) and Byrd Alzheimer's Institute. Development
of AdvaxCpG adjuvant was supported by funding from NIAID/NIH
(Contract No. HHS-N272201400053C, HHS-N272200800039C and U01-
AI061142). The content is solely the responsibility of the authors and does
not necessarily represent the official views of the NIH.
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