AT8 (recognises pSer202/pThr205) was purchased from Innogenetics (Ghent, Belgium). The phosphorylation-specific monoclonal antibodies (mAbs) DC209 (recognises pThr231), DC217 (recognises pThr217) and DC179 (recognises pThr181) were prepared by AXON Neuroscience SE (Bratislava, Slovak Republic). As a pan-tau antibody, mAb DC25 was used (AXON Neuroscience SE) [23–29]. DC25 recognises a stretch of seven amino acid residues (347 to 353, the longest human tau isoform numbering) in the fourth microtubule-binding repeat of tau, which is common to all neuronal tau protein isoforms (human, mouse, rat and other) and recognises all forms of physiological and pathological tau proteins.
Design and construction of the active vaccine
Tau peptide sequence 294KDNIKHVPGGGS305 was derived from the regulatory domain driving the oligomerisation of tau . An extra N-terminal cysteine residue was added with the aim of obtaining oriented attachment of the peptide on the surface of the keyhole limpet haemocyanin (KLH) protein. The peptide was coupled to the KLH carrier (Calbiochem, San Diego, CA, USA) via the bifunctional cross-linker N-[γ-maleimidobutyryloxy]succinimide ester (GMBS). KLH (20 mg) was dissolved in conjugation buffer (phosphate-buffered saline (PBS) with 0.9 M NaCl) to a concentration of 10 mg/ml. GMBS (2 mg) was dissolved in 50 μl of anhydrous dimethylformamide and mixed with the KLH solution for 1 hour at room temperature (RT). Subsequently, unreacted GMBS was removed on a 5-ml HiTrap desalting column (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) equilibrated in the conjugation buffer. The peptide (20 mg) was dissolved in the maleimide-activated KLH solution and the reaction proceeded for 2 hours at RT. The resulting conjugate was dialysed against PBS. The conjugate was aliquoted and stored at 2°C to 8°C until use.
Transgenic rats used in this study expressed human truncated tau protein in the brain and spinal cord . All transgenic rats used in this study were hemizygous for the transgene construct. Prior to the experiments, all animals were housed under standard laboratory conditions with free access to water and food and were kept under diurnal lighting conditions (12-hour light–dark cycles with light starting at 7:00 AM). All experiments on animals were carried out according to institutional animal care guidelines conforming to international standards and were approved by the State Veterinary and Food Committee of the Slovak Republic (Ro-2426/08-221) and by the Ethics Committee of the Institute of Neuroimmunology (15 October 2008), Slovak Academy of Sciences, Bratislava. Efforts were made to minimise the number of animals utilised.
To prepare the tau peptide vaccine, 100 μg of tau peptide conjugate (dissolved in 150 μl of PBS) was mixed at a 1:1 (vol/vol) ratio with Adju-Phos adjuvant (Brenntag Biosector, Frederikssund, Denmark) in a final dose volume of 300 μl. The suspension was mixed by end-over-end rotation during incubation at 4°C for 6 hours to allow the peptide conjugate to adsorb onto the adjuvant. The transgenic rats received five subcutaneous injections of vaccine (100 μg of peptide-KLH conjugate/dose) starting at 2 months of age, followed by the second injection 3 weeks later and thereafter on a monthly schedule. The control group of transgenic rats received adjuvant mixed 1:1 with PBS in a final dose volume of 300 μl. Sera were collected 2 weeks after the last booster dose.
Determination of antibody response to vaccine
Tau peptide (294 to 305), mis-disordered tau (151-391/4R) and recombinant full-length tau isoform 2N4R were separately coated onto 96-well plates (SARSTEDT, Nümbrecht, Germany) at an amount of 250 ng/well. After blocking, the plates were incubated with the serial dilutions of the sera (50 μl/well 1:100 to 1:51,200 in PBS, in twofold dilution steps) for 1 hour at 37°C. Bound serum antibodies were detected with peroxidase-conjugated secondary antibody (goat anti-rat immunoglobulin (Ig), Dako, Glostrup, Denmark) using chromogenic substrate o-phenylenediamine (Sigma-Aldrich, St Louis, MO, USA).
Determination of isotypic profile of vaccine-induced antibodies
To determine the isotypes of the specific antibodies produced in response to vaccine, sera from immunised rats were serially diluted from 1:100 to 1:12,800 in twofold dilution steps and tested in duplicates by enzyme-linked immunosorbent assay (ELISA) against mis-disordered tau (151-391/4R). To detect rat IgG1, IgG2a, IgG2b, IgG2c and IgM isotypes, anti-rat subclass-specific horseradish peroxidase (HRP)–conjugated secondary antibodies were diluted 1:5,000 in PBS (Pierce Biotechnology, Rockford, IL, USA). Antibody isotype levels were compared based on the half-maximal effective concentration value of the dilution factor.
Isolation of soluble tau and sarkosyl-insoluble tau
Brain tissue was homogenised in a tenfold weight excess of ice-cold extraction buffer (20 mM Tris, pH 7.4, 800 mM NaCl, 1 mM ethylene glycol tetraacetic acid), 1 mM ethylenediaminetetraacetic acid, 0.5% β-mercaptoethanol, 10% sucrose and 1 mM Na3VO4; 20 mM NaF, supplemented with cOmplete Protease Inhibitor Cocktail Tablet (Roche Diagnostics, Indianapolis, IN, USA). After incubation on ice for 5 minutes, the homogenates were cleared by centrifugation at 20,000 g for 20 minutes at 4°C. The supernatants were collected, and the total protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). This supernatant (designated 1S) contained soluble tau fraction. Subsequently, solid sarkosyl (N-lauroylsarcosine, sodium salt; Sigma-Aldrich) was added to the 1S supernatant to achieve a 1% concentration and stirred for 1 hour. Thereafter it was centrifuged at 100,000 g for 1.5 hours at RT. Following centrifugation, pellets were gently rinsed with 1 ml of the extraction buffer and centrifuged at 100,000 g for 20 minutes at RT. The pellets containing sarkosyl-insoluble tau fractions were dissolved in SDS-PAGE loading buffer to a final volume corresponding to the 1/50 of the volume of 1S supernatant used for their preparation. For Western blot analysis, 6 μl of the sarkosyl-insoluble fraction was loaded, which corresponds to 30 mg of tissue.
Samples of sarkosyl-insoluble tau fractions  were dissolved in 1× SDS sample loading buffer in 1/50 volume of the soluble fraction and heated at 95°C for 5 minutes. Each sample in a quantity of 6 μl was then loaded onto 5–20% gradient SDS-polyacrylamide gels and electrophoresed in a Tris-glycine-SDS buffer system for 40 minutes at 25 mA. Proteins were transferred to PVDF membranes (1 hour at 150 mA in 10 mM N-cyclohexyl-3-aminopropanesulfonic acid, pH 12). After the transfer, the membranes were stained with Ponceau S (Additional file 1) for verification of loading of an equal amount of sarkosyl-insoluble proteins and efficiency of electroblotting. Subsequently, the membranes were blocked in 5% nonfat dry milk in PBS for 1 hour at RT and then incubated for 1 hour with primary (tau-specific) monoclonal antibodies, followed by three washes with a large volume of PBS. After washes, HRP-conjugated goat anti-mouse Ig (Dako) diluted 1:4,000 with PBS was used as a secondary antibody. Incubation (1 hour at RT) was followed by washing (three times) with 0.2% Igepal in PBS. The blots were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology), and the signal was detected using the LAS-3000 Imaging System (FUJI Photo Film Co, Tokyo, Japan). The signal intensities were quantified using AIDA Image Analyzer software (Raytest, Straubenhardt, Germany) and then statistically evaluated. Foetal tau (prepared as described in ) in the amount of 0.6 μg/lane was used as an internal standard for quantification.
Immunohistochemistry of rat brain tissue
Transgenic rats were deeply anaesthetised with Zoletil-xylasine and perfused intracardially using a peristaltic pump for 2 minutes with PBS. The brain was frozen in liquid nitrogen and transferred to dry ice. Sagittal brainstem sections (10 μm thick) were cut on a Leica CM1850 cryomicrotome (Leica Biosystems, Buffalo Grove, IL, USA). The sections were postfixed with 4% paraformaldehyde (PFA) in PBS, pH 7.2 (4% PFA), for 10 minutes. Tissue sections were incubated with primary antibodies AT8 (Pierce Endogen), pT212 and pS214 (Invitrogen, Carlsbad, CA, USA), Tau5 (a gift from Dr Lester I Binder) and HT7 (Pierce/Thermo Scientific, Rockford, IL, USA) overnight at 4°C. Sections were immunostained using the standard avidin-biotin-peroxidase method (VECTASTAIN ABC Kit; Vector Laboratories, Burlingame, CA, USA) with VIP as chromogen.
Immunohistochemistry of human brain tissue
Human postmortem AD brain tissue (Braak stage VI) was obtained from the Netherlands Brain Bank in accordance with local ethical approval and written consent from the donor or the donor’s next of kin. Ethical approval was obtained for the analyses carried out using this tissue (Institute of Neuroimmunology, Slovak Academy of Sciences, 5/2011).
Tissue sections from human AD brain were treated with cold (+4°C) 99% formic acid for 1 minute at RT (25°C). Brain sections were incubated for 20 minutes at RT in 0.01 M of PBS, pH 7.4, containing 0.3% Triton X-100 and 1% H2O2, followed by a 30-minute incubation in the blocking solution (0.01 M of PBS, containing 0.3% Triton X-100, 1% horse serum), followed by overnight incubation with sera from transgenic rats immunised with the tau peptide vaccine (diluted 1:1,000) at 4°C. After washing, the sections were immunostained using the standard avidin biotin peroxidase method (ABC Elite Kit; Vector Laboratories). The reaction product was visualised using Vector VIP as a chromogen (Vector Laboratories). Sections were then examined with an Olympus BX51 microscope.
For testing of sensorimotor functions a multi-test battery with a novel sensitive scoring system—NeuroScale —was chosen. Briefly, NeuroScale consists of three variants of beam walking test (square cross-section of 3 cm × 3 cm, rectangular cross-section of 4 cm × 2 cm, round cross-section with diameter of 3.5 cm), a prehensile traction test and a rapid neuromuscular and neurological examination. Animal performance was evaluated and scored according to a predefined rating scale. The maximal number of points possible to achieve in the beam walking and prehensile traction tests was five each. Performance on a simple reflex response was scored with a maximum of one point, except the hindlimb escape extension reflex, for which we used a 3-point rating scale.
Statistical analysis was carried out using the Prism statistical software package (GraphPad Software, La Jolla, CA, USA). To compare two groups, the Mann–Whitney U test or an unpaired t-test was applied. The results are presented as mean ± standard error of the mean unless otherwise specified. Differences were considered significant at the level of P < 0.05.
Preclinical toxicology and safety pharmacology studies
Studies were conducted at good laboratory practice level at Harlan Laboratories SA (Correzzana, Italy). Four separate formulations of AADvac1 vaccine (10, 40 and 160 μg of peptide coupled to KLH formulated with aluminium hydroxide containing 1.5 mg of aluminium per dose and 40 μg of peptide coupled to KLH formulated with aluminium hydroxide containing 0.5 mg of aluminium per dose) were compared to placebo.
Single-dose studies consisted of three separate rat studies with 14 days or 21 days of postadministration daily clinical observation. Animals were weighed twice per week, and food consumption was recorded weekly. The 14-days studies featured complete haematology, urinalysis, coagulation and clinical biochemistry panels. Animals were killed on day 15, organ weights of all internal organs were measured and histopathological analysis of all tissues was performed. The 21-days study featured clinical observation after administration of four times the intended human clinical dose (160 μg).
Chronic toxicity testing was conducted in New Zealand white rabbits. Animals received 9 doses of AADvac1 or placebo in 3-week intervals over the course of 26 weeks. Animals were clinically observed twice daily. Food consumption and body weights were recorded weekly. The study featured complete haematology, urinalysis, coagulation and clinical biochemistry panels. Ophthalmoscopic examinations were performed at baseline and after 13 and 26 weeks. After the animals were killed, organ weights of all internal organs were measured and histopathological analysis of all tissues was performed.
An acute central nervous system (CNS) safety pharmacology study was conducted in ICR (CD-1) outbred mice according to a modified Irwin screen test paradigm. Animals were subjected to detailed neurological examination for 24 hours after drug administration. An acute cardiorespiratory safety pharmacology study was conducted in Beagle dogs. The cardiovascular (arterial pressure, heart rate, cardiac contractility (dP/dtmax), systemic blood flow and electrocardiography) and respiratory parameters (rate, tidal volume and minute volume) were recorded and analysed before and 5, 15, 30, 45 and 60 minutes after placebo or vaccine administration. Histopathological analysis was peer-reviewed by AnaPath GmbH, Oberbuchsiten, Switzerland. Vaccine and placebo for all toxicology and safety pharmacology studies was provided by Bachem Distribution Services GmbH, Weil am Rhein, Germany.