Mice

Thy-Tau22-5xFAD (T5x) double transgenic AD mice were generated as described in [11]. Briefly, Thy-Tau22 mice express human 4 repeat tau with two frontotemporal dementia-associated point mutations (G272V and P301S) under control of the neuronal driven promoter Thy1.2 and are maintained on a C57Bl6/J background [13]. The 5xFAD mice used in this study are also maintained on a congenic C57Bl6/J and co-expresses human amyloid precursor protein (APP695) carrying the Swedish, Florida, and London mutations and a human presenilin-1 (PS1) transgene carrying the M146L and L286V mutations under the Thy-1 promoter. Both APP and PS1 transgenes are co-integrated and thus co-inherited. Heterozygous Thy-Tau22 and 5xFAD mice were crossed to create Thy-Tau22-5xFAD (T5x) mice that were genotyped via PCR amplification of human tau, PS1, and APP transgenes. Both female and male animals were used in this study and sex-dependent effects examined. All animals were housed in a temperature and light cycle-controlled facility, and their care was under the guidelines of the National Institutes of Health and an approved IACUC protocol at University of California, Irvine.

Epitope vaccines and purification of proteins

To prepare two recombinant proteins, minigenes encoding 3Aβ 1–11 -MultiTEP or 3Tau 2–18 -MultiTEP were cloned into the modified Escherichia coli expression vector pET11 (for AV-1959R; Novagen, MA) or pET24a (for AV-1980R; Novagen, MA) in frame with 6xHis-Tag at the C-terminus. Gene encoding 2N4R tau protein was amplified from human whole brain Marathon®-Ready cDNA library (Clontech) using primers 5′-catatggctgagccccgccaggagttcgaagtgatg (forward) and 5′-ctcgagtcacaaaccctgcttggccagggaggcagac (reverse) and cloned into the pET24a + E. coli expression vector in frame with 6xHis-tag at the C-terminus using restriction sites NdeI and XhoI. DNA sequencing was performed to confirm that the generated plasmids contained the correct sequences. Recombinant proteins were purified from E. coli BL21 (DE3) cells transformed with pET11/3Aβ 1–11 -MultiTEP, pET24a/3Tau 2–18 -MultiTEP, or pET24a/Tau plasmids as described [10, 14, 15] for epitope vaccines and in [16] for Tau protein. The final recombinant protein was analyzed in 10% Bis-Tris gel electrophoresis (NuPAGE Novex Gel, Invitrogen, CA). Protein bands were visualized by Coomassie dye, and specificity of the bands was confirmed by Western blot (WB) using 6E10 and anti-tau 2–18 1C9 monoclonal antibodies [10]. The level of endotoxin was measured using E-TOXATE kits, as recommended by the manufacturer (Sigma, St Louis, MO).

Preparation of oligomeric recombinant tau

Oligomeric forms of tau protein were prepared as described by Combs et al. [17]. Briefly, arachidonic acid in ethanol was added to recombinant tau protein in polymerization buffer (5 mM DTT, 100 mM NaCl, 100 μM EDTA, and 10 mM HEPES at pH 7.64) to a final concentration of 75 μM in order to induce tau polymerization. The reaction was allowed to proceed overnight, and the extent of aggregation was confirmed by western blotting. The aggregated tau sample was aliquoted and was stored at − 80 °C until used in SPR assay.

Experimental protocols

Three groups of T5x mice were immunized with AV-1959R (20 μg/per mouse/per injection), AV-1980R (20 μg/per mouse/per injection), or a mixture of AV-1959R and AV-1980R proteins (20 μg protein/mouse/injection), all formulated with AdvaxCpG adjuvant (Vaxine Pty Ltd., Adelaide Australia) at 1 mg/mouse/injection. Control groups of T5x mice were injected with AdvaxCpG adjuvant only or PBS. All mice were injected four times intramuscularly. Sera were collected 14 days after third immunizations, and anti-Aβ and anti-tau antibody responses were analyzed. At age of 8 months old, mice were terminated and brains were collected for biochemical and immunohistological analysis. More detailed experimental protocols are provided in Fig. 1.

Fig. 1 Design of immunization study of T5x mice. Experimental protocol in T5x mice vaccinated with AV-1959R and AV-1980R proteins separately or mixed together formulated in AdvaxCpG adjuvant. Control mice were injected either with PBS or AdvaxCpG adjuvant only Full size image

Detection of Aβ- and tau-specific antibodies

The concentrations of anti-Aβ and anti-tau antibodies in serum were determined by ELISA, as described previously [15, 18, 19]. To measure anti-Aβ and anti-tau antibody concentration, plates were coated with 1 μg/per well Aβ 42 peptide (American Peptide, CA), tau 2–18 peptide (GenScript, NJ), or full-length recombinant tau protein (purified at The Institute for Molecular Medicine, Huntington Beach, CA), respectively. Anti-Aβ and anti-tau antibody concentrations were calculated using a calibration curve generated with affinity-purified polyclonal antibodies from sera of mice vaccinated with AV-1959R/A and AV-1980R/A, respectively. HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, ME) was used as secondary antibody.

Epitope mapping of tau-specific antibodies

Epitope mapping of anti-tau antibodies was performed by “alanine scanning” using competitive ELISA. Briefly, 17 peptides spanning tau 2–18 sequence, but possessing one alanine substitution in each position were synthesized. Ninety-six-well plates (Immulux HB; Dynex Technologies, Inc., VA) were coated with 1 μg/well (in 100 μl; Carbonate-Bicarbonate buffer, pH 9.6, o/n at 4 °C) tau 2–18 peptide (GenScript, NJ). Next day coated plates were blocked with blocking buffer (3% dry, non-fat milk in TBST, 300 μl/well). Serial dilutions of reference wild type (tau 2–18 ) or mutated test peptides (corresponding to 0.02 μM, 0.1 μM, 0.5 μM, 2.5 μM, 5 μM, 12.5 μM, and 25 μM final concentrations) were incubated with immune sera diluted 1: 300,000 (corresponding to the linear region of the curve for binding to Tau 2–18 peptide) for 1.5 h at 37 °C. After incubation, 100 μl of antibody/peptide mixture was added into the wells. HRP-conjugated goat anti-mouse IgG (1:2500; Jackson ImmunoResearch Laboratories, PA) were used as secondary antibodies. The reaction was developed by adding 3,3′,5,5′tetramethylbenzidine (TMB) (Pierce, IL) substrate solution and stopped with 2 M H 2 SO 4 . The optical density (OD) was read at 450 nm (Biotek, Synergy HT, VT). The percent of binding of sera blocked with wild type or mutated peptides to tau 2–18 was calculated relative to the binding of sera without competing peptides to tau 2–18 as 100%. The half maximal inhibitory concentration (IC 50 ) for each peptide was calculated.

Surface plasmon resonance (SPR)

Binding studies were performed at 25 °C using a Biacore 2000 optical biosensor equipped with a Protein A-coated sensor chip and equilibrated with running buffer (10 mM HEPES, 150 mM NaCl, 0.01% Tween-20, 0.1 mg/mL BSA, pH 7.4). The surfaces were regenerated with two 12-s injections of 150 mM phosphoric acid after each binding cycle.

Antibody captures for kinetic analysis of antigen binding. For each binding cycle, the antibodies were diluted into running buffer and injected across individual Protein A surfaces. These injections produced capture levels of ~ 55–75 RU (resonance units). Using a short-and-long injection approach, tau monomer and oligomer were tested in triplicate in a threefold dilution series starting at 40 nM (this concentration was established using an estimated average molecular weight of 138 kDa for the oligomer). For each tau/antibody interaction, the responses from the three runs were globally fitted to a 1:1 interaction model (shown as the overlaid smooth red lines in the figures) to obtain the binding parameters listed in the table in Fig. 4.

Detection of Aβ plaques and tau tangles in human brain tissues by IHC and confocal microscopy

Sera from mice immunized with AV-1959R/A, AV-1980R/A, and mixture of AV-1959R/A and AV-1980R/A, as well as control mice injected with AdvaxCpG only, were screened for the ability to bind to human Aβ plaques or/and tau tangles using 40-μm brain sections of formalin-fixed cortical tissues from a severe AD case (received from Brain Bank and Tissue Repository, MIND, UC Irvine) using immunohistochemistry, as described previously [20,21,22]. In addition, brain sections were stained with anti-Aβ (beta-amyloid (1–42), 1:250, Invitrogen, CA) and humanized anti-tau (Armanezumab, 1:1000; Institute for Molecular Medicine, CA) antibody as positive controls. Sections were imaged using an Olympus FX1200 confocal microscope, with identical laser and detection settings across a given immunolabel.

Mouse brain tissue preparation, immunohistochemistry, and confocal microscopy

Following perfusion, one hemisphere from each mouse was postfixed in 4% paraformaldehyde for 48 h then stored in PBS + 0.05% sodium azide. Fixed half-brains were placed in 30% sucrose for at least 48 h before being cut in the coronal plane (40-μm sections) using a freezing sliding microtome. Brain sections were rinsed in PBS before blocking in PBS + 0.05% Triton-X with 10% goat serum for 1 h. First, samples were stained with Amylo-Glo™ RTD Amyloid Plaque Stain Reagent (Biosensis, Australia) for 15 min, washed three times, and then incubated in pS199 (Abcam, UK, 1:1000) and PHF-1 (gift from Dr. Peter Davis, 1:1000) phospho-tau primary antibodies at 4 °C overnight. The next day, sections were washed three times with PBS and placed in appropriate Alexa Fluor-conjugated secondary antibody solutions at room temperature for 1 h. Sections were rinsed three additional times, mounted onto slides, and coverslipped using Fluoromount-G. For confocal microscopy, immunofluorescent staining was performed on equivalent brain sections and imaged on the Olympus FX1200 confocal microscope. Tau tangles and β-amyloid plaques were visualized using Z-stack maximum-projection images taken through the entire depth of the section at 1-μm intervals.

Biochemical analyses

Right hemispheres, previously frozen on dry ice and stored at − 80 °C, were crushed on dry ice using mortar and pestle, then homogenized in solution of T-PER (Thermo Scientific, Waltham, MA) and phosphatase and protease inhibitor mixtures (Thermo Scientific, MA and Roche, CA) and processed as previously described [22,23,24]. Quantitative biochemical analysis of human Aβ was conducted using commercially available electrochemiluminescent multiplex assay system [Meso Scale Discovery (MSD)]. Human Aβ triplex (6E10 capture antibody) was used for simultaneous measurement of Aβ38, Aβ40, and Aβ42 in both soluble and insoluble protein fractions [24].

Concentrations of human total and phosphorylated tau in samples (soluble and insoluble 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 ThermoFisher Scientific, MA), according to the manufacturer’s instructions.

Soluble SDS-PAGE WB and quantifaction was performed following standard protocols as previously described [22,23,24]. Primary antibodies used for WB analysis included the following: Armanezumab (1:2000; Institute for Molecular Medicine, Huntington Beach, CA), anti-GFAP (1:500; Millipore-Sigma, MO), anti-P2RY12 (1:500; Millipore-Sigma, MO), and anti-CD45 (1:500; Bio-Rad, CA). All blot membranes were also labeled with anti-β-actin antibodies (1:1000; Millipore-Sigma, MO) as a loading control.

Statistical analysis

All statistical parameters [mean, standard deviation (SD), significant difference, etc.] used in experiments were calculated using Prism 6 software (GraphPad Software, Inc.). Statistically significant differences were examined using unpaired t test or one-way ANOVA with Tukey’s multiple comparisons test (p value < 0.05 was considered as statistically different).