A multi-targeting immunotherapy ameliorates multiple facets of Alzheimer’s disease in 3xTg mice

Mice

Eight-week-old female C57BL/6 wide-type mice were used for the immunogenicity analysis and 3xTg mice model were utilized for the evaluation of therapeutic effects of the vaccines. The 3xTg mice harboring PS1 M146V, APPswe, and Tau P301L transgenes were purchased from Laboratory animal origin and production license number Changzhou Cavens Biotechnology. All animals were reared in individually ventilated cages (IVC) in the Animal Experimental Platform, Core Facilities for Life Science, Jilin University. In a 12 h light/dark cycle with ad libitum access to food and water, Mice used in this study were not used for breeding and were reared by sex at a density of four to five mice per cage.

Epitope vaccine preparation—two recombinant proteins

The Aβ vaccine, named PP-3copy- Aβ1–6, is comprised of three copies of human Aβ1–6 inserted into three loops of the norovirus P particle. The exact amino acid sequence of the Aβ epitope is: GGGDAEFRHGGGDAEFRHGGGDAEFRHGGG. For the generation of the Aβ vaccine recombinant plasmid, we used the engineered pET28a+ expression vector, which contains 6xhistidine on the C-terminal region of its polyclonal site to carry the coding sequence of Aβ vaccine protein. Subsequent purification of his-tagged Aβ vaccine protein involved the application of cell lysate supernatant to a HiSTrap FF chromatography column (GE Healthcare), followed by elution of the target protein using a linear imidazole gradient ranging from 50 mM to 300 mM. The Aβ vaccine protein was successfully eluted with 300 mM imidazole fraction. We have confirmed the existence of Aβ vaccine protein in that eluant by running SDS-PAGE gel and western blot analysis using anti-His antibody (Supplementary Fig. 1a, b). The particle size and TEM analysis also showed that Aβ vaccine protein could form the nanoparticles around 22 nm (Supplementary Fig. 1c, d).

For the generation of the pTau vaccine, we first used site-directed mutagenesis to replace the protruding amino acid of three loops of P protein with lysine (K) and named it as PP-3K carrier protein. Then the PP-3K particles were produced in Escherichia coli by Ni-NTA purification (Supplementary Fig. 1e–g). The target antigen, a phosphorylated Tau (pTau30) peptide, encompassing four phosphorylation sites (pS202/T205/S396/S404) of human Tau protein, was synthesized (GL Biochem) and modified to incorporate a C-terminal cysteine. The exact amino acid sequence and the four phosphorylation sites for pTau epitope is: sspgSpgTpgsrivykSpvvsgdtSprhlc (Capital letter means phosphorylated Tau site, represents pS202, pT205, pS396, and pS404, respectively). This pTau30 peptide was conjugated to the surface-exposed lysines of PP-3K (as indicated in the additional image) via the bifunctional crosslinker SMCC (Thermo Fisher Scientific, cat. no. 22360), with the conjugation efficiency confirmed through SDS-PAGE analysis. The size of the PP-3K carrier protein and pTau30 peptide is about 34 KD and 3 KD, respectively. After linking PP-3K and pTau30 by SMCC, the size of the main pTau vaccine protein product is 37 KD, which is equivalent to that one pTau30 peptide is attached to one PP-3K molecule. We also observed a small number of PP-pTau linkage products with 2 or 3 pTau peptides attached to the PP-3K carrier (Supplementary Fig. 1h, right panel, lane 2 and lane 3). In addition, since PP-3K protein itself contains lysine and cysteine, it is inevitable that pTau vaccine will self-assemble into aggregates through SMCC linking system. Therefore, there are many pTau vaccine polymers ranging from 75 to 250 KD after connecting PP-3K with pTau30. Thus, if we combine the whole linkage products containing more than one pTau30 peptide, the linking efficiency of the pTau vaccine is about 50%.

Experimental protocols/vaccine immunization

Premorbid group: “In the premorbid phase, 3xTg mice, both male and female, were categorized into four cohorts. The groups received either Aβ vaccine (25 μg protein/dose, n = 12), pTau vaccine (25 μg protein/dose, n = 10), a combination of Aβ and pTau vaccines (25 μg + 25 μg protein/dose, n = 12), or the vector (PP particles) group (25 μg protein/dose, n = 12), each vaccine was formulated with CpG and MF59 adjuvant. Immunization commenced at 3–4 months of age with a bi-weekly intramuscular regimen for four doses, followed by booster doses at weeks 4 and 18 post-initial immunization. Serum samples were obtained prior to each immunization for anti-Aβ and anti-tau antibody assessment. At 11 months, after behavioral assessments were conducted, heart, liver, spleen, lung, kidney, and brain tissues were harvested following humane euthanasia.

Onset group, immunization was initiated at 6.5 months. This cohort included PBS (n = 14, 100 μL/dose), Vector (n = 15, 100 μL/dose, with CpG+MF59 adjuvant), Aβ vaccine (n = 15, 25 μg/dose, with CpG+MF59 adjuvant), pTau vaccine (n = 14, 25 μg/dose, with CpG+MF59 adjuvant), and dual vaccine groups (n = 15, 25 μg + 25 μg/dose, with CpG+MF59 adjuvant), maintaining a gender balance in each. Considering the temporal disparity in Tau and Aβ pathologies and the immunization frequency, a modified regimen was adopted. Concurrent immunization with Aβ and pTau vaccines was performed at 6, 6.5, and 7 months. However, the fourth and subsequent booster doses were administered separately: the Aβ vaccine at 7.5 and 9 months and the pTau vaccine at 8 and 10.5 months. Serum for antibody detection and inflammatory protein analysis was collected bi-weekly post-first immunization and continued until 13 months. Behavioral evaluations were conducted between 13.5 and 14.5 months, followed by humane euthanasia at 15 months for brain and organ harvest for homogenization and histopathological analysis.

All the experiments were performed in accordance with legal and institutional guidelines and were carried out under ethics, consent, and permissions of the Ethical Committee of Care and Use of Laboratory Animals at Jilin University (S2021065). Two weeks after the MWM test, the mice were deeply anesthetized with pentobarbital (100 mg/kg) and perfused with phosphate-buffered saline (PBS), followed by cervical dislocation. Animal experiments were performed in full compliance with the ARRIVE reporting guidelines43.

Characterization of serum antibody response

The quantification of serum antibodies specific to Aβ and pTau30 was performed utilizing enzyme-linked immunosorbent assay (ELISA). ELISA plates, pre-coated with Aβ 1–42 peptide or pTau202/205-BSA, pTau396-BSA, and pTau404-BSA peptides (100 ng/well), facilitated this analysis. For calibration of Aβ antibody levels, mouse monoclonal antibody 6E10 (1:200 dilution, 803001, Biolegend, San Diego, CA, USA) was employed. Concurrently, antibodies targeting pTau202/205,396,404 were calibrated using AT8 (1:200 dilution, MN1020, Thermo Fisher Scientific, Waltham, MA, USA), PHF-13 (1:200 dilution, 829001, BioLegend, San Diego, CA, USA), and Phospho-Tau (Ser404) Polyclonal Antibody (1:200 dilution, 44–758 G, Thermo Fisher Scientific, Waltham, MA, USA), respectively.

Determination of the isotypic profile of vaccine-induced antibodies

The mouse monoclonal antibody isotyping reagent (Sigma-Aldrich, USA) was used to analyze the isotypes of antibodies generated against the vaccine.

Hindlimb clamping

Muscle atrophy in mice was assessed through the hindlimb clamping test. This involved suspending mice by their tails for 10 s while observing the contraction of their hind limbs. The contraction intensity was evaluated by three independent experimenters using pre-established scoring criteria, detailed in ref. 44.

Nest

Nest building, crucial for insulation, reproduction, and shelter in mice, was analyzed to understand complex physiological behaviors linked to various brain regions, including the hippocampus. Individual 3xTg mice were provided with 1.8 g of cotton (two 5 cm × 5 cm pieces) in a single cage, after changing the bedding material. Nest construction was evaluated by three trained experimenters 24 h later, using a scoring system ranging from 1 (no nest) to 4 (nearly perfect or perfect nest).

Novel object recognition task (NOR)

The NOR task, designed to assess learning and memory, comprises three phases: adaptation, training, and testing. During the adaptation phase, mice explored an object-free environment for 10 min, akin to the open field test. In the training phase, mice were exposed to two identical, odorless, and immovable objects. The testing phase, conducted an hour after the training, involved replacing one of the objects with a new one. The discrimination score for novel object exploration was calculated using the formula: (time exploring novel object/(time exploring novel object + time exploring familiar object)) × 100%.

Morris water maze test

The MWM test, a predominant behavioral experimental method in Alzheimer’s disease (AD) research, evaluates spatial learning and memory in mice. The protocol involves an acquisition phase over five consecutive days, followed by a probe trial on the sixth day. The circular pool, sectioned into four quadrants and adorned with three distinct patterns on the walls, conceals a platform submerged 2 cm underwater in one quadrant. During the acquisition phase, mice, starting from different quadrants, navigate to find the hidden platform within 60–90 s, with the average latency across four daily trials recorded. On the probe trial day, with the platform removed, the subjects’ spatial orientation is assessed by measuring the duration spent in the platform’s quadrant and the frequency of crossing the former platform location.

Western blotting for detection of pathological proteins in brain homogenatesPathologic examination of the brain and other organs

The mouse brains were separated into two hemispheres after removal. One hemisphere was deep-frozen in liquid nitrogen and homogenized, and further divided into reassembly buffer (RAB). Then 1 mL brain homogenate suspension was transferred to a 1.5 mL microfuge tube and centrifuged at 50,000×g for 20 min at 4 °C (Optima Max-XP, Beckman Coulter, Inc., CA). The pellet was resuspended with 1 mL RIPA buffer with protease and phosphatase inhibitor MIX and centrifuged at 50,000×g for 20 min at 4 °C to collect the supernatant as RIPA-soluble fraction (soluble fraction). The pellets were further disposed by 0.5 mL urea buffer (8 M urea and 5% (w/v) SDS, pH 7.2) at 2–8 °C overnight and centrifuged to separate the supernatant as urea-soluble fraction (insoluble fraction. The procedure details (the RAB and the RIPA solution formulations) are also described before45,46. The brain homogenate fraction samples of each mouse from the same group were mixed in a ratio of 1:1 before western blot analysis, which represents the average total Tau protein of pTau protein levels of the corresponding group. The other hemisphere was immersed in 4% paraformaldehyde for immunohistochemistry (IHC) to detect the levels of Aβ plaque, NFT, microglia, and astrocytes. The brain homogenates were used for western blot analysis, flow cytometry, ElisaKit, and the detection of inflammatory factors and chemokines by Elisakit.

For pathological examination of brain tissues, samples were homogenized in ice-cold RAB and RIPA buffers. Solution preparation methods are mentioned in the supplement. Western blot analysis involved resolving proteins on a 12% SDS-PAGE, followed by their transfer onto membranes in transfer buffer, and overnight immunoblotting with phospho-tau (Ser202/Thr205) mAb (AT8, 1:1000 dilution, MN1020, Thermo Fisher Scientific, Waltham, MA, USA), Phospho-Tau (Ser396) mAb (PHF-13, 1:1000 dilution, 829001, BioLegend, San Diego, CA, USA), Phospho-Tau (Ser404) Polyclonal Antibody (1:1000 dilution, 44–758 G, Thermo Fisher Scientific, Waltham, MA, USA), GFAP mAb (1:1000 dilution, 2E1.E9, 644702, Biolegend, San Diego, CA, USA), Iba-1 mAb (1:1000 dilution, EPR16588, ab178846, Abcam, Cambridge, UK) or GAPDH mAb (1:1000 dilution, 60004-1-lg, Proteintech, Chicago, IL, USA). Subsequently, the membranes were incubated with Horseradish Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (1:10000 dilution, 111-035-144, Jackson ImmunoResearch, PA, USA) or Horseradish Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (1:10,000 dilution, 111-035-003, Jackson ImmunoResearch, PA, USA) for 1 h at room temperature, and then treated with an enhanced chemiluminescence (ECL) solution for detection.

IHC (Immunohistochemistry)

For immunohistochemical analysis, brain sections underwent a gradient dewaxing process followed by antigen retrieval using citrate buffer. Post retrieval, sections were washed with TBST four times and sequentially treated with 3% hydrogen peroxide and 10% goat serum to block non-specific binding. The sections were then incubated with primary antibody overnight at 4 °C, including anti-β-amyloid 1–16 (1:400 dilution, 6E10, 803001, Biolegend, San Diego, CA, USA), Phospho-Tau (Ser202/Thr205) mAb (AT8, 1:100 dilution, MN1020, Thermo Fisher Scientific, Waltham, MA, USA), Phospho-Tau (Ser396) mAb (PHF-13, 1:500 dilution, 829001, BioLegend, San Diego, CA, USA), Phospho-Tau (Ser404) Polyclonal Antibody (1:300 dilution, 44-758G, Thermo Fisher Scientific, Waltham, MA, USA), GFAP mAb (1:500 dilution, 2E1.E9, 644702, Biolegend, San Diego, CA, USA), Iba-1 mAb (1:400 dilution, EPR16588, ab178846, Abcam, Cambridge, UK) and Anti-FOX3 (NeuN) mAb (1:500 dilution, 1B7, 834501, Biolegend, San Diego, CA, USA). Following primary antibody incubation, sections were washed and incubated with HRP-conjugated anti-mouse and rabbit secondary antibody polymers (abs996, absin) for 30 min at room temperature in the dark. Visualization was achieved using DAB chromogen, with incubation time adjusted based on color development. After drying, the slides were placed in a panoramic scanning image system for whole-slide scanning.

For standard image analysis of IHC images, including the determination of % area for antibodies such as 6E10, anti-pTau202/205, or anti-GFAP), TIFF image files were opened using ImageJ and converted to 8-bit greyscale files. Images displaying the minimum and maximum intensity of staining, along with representative samples from intermediate staining intensities, were utilized to establish an optimal threshold value. This threshold was determined by the investigator to accurately reflect the intended staining pattern across all experimental conditions within the cohort. That threshold was then held constant across all images in the cohort, and black and white images of selected regions of interest were generated and quantified as % area stained using the Analyze Particles function. At least 6 views per mouse per region were analyzed and averaged46.

“Skeleton analysis” was used to measure the morphology of the microglia47,48. Isolating individual microglia in each image, then the images were skeletonized using the ImageJ skeletonize plugin. The particle analysis function was then used to remove background before using the Analyze Skeleton function in ImageJ to analyze the number of branches/endpoints per microglia cell.

Immunofluorescence

One series of the 30 μm sections was permeabilized, subjected to antigen retrieval, blocked, and then incubated with the following primary antibodies mouse anti-β-amyloid 1–16 (1:400 dilution, 6E10, 803001, Biolegend, San Diego, CA, USA) and rabbit anti-IBA1 (1:300 dilution, ab178846, Abcam, Cambridge, UK) at 4 °C for 24 h and followed by incubation with the corresponding fluorescent secondary antibodies at 37 °C for 2 h, including Alexa Fluor@555 Donkey pAb to Rabit IgG(1:400 dilution, ab150074, Abcam, Cambridge, UK) and DyLight-FITC goat anti-mouse IgG(1:400 dilution, IF-0091, Beijing, CHINA). Sections were stained with DAPI and sealed with an anti-fluorescence quencher.

Prussian blue

Paraffin sections were deparaffinized in a gradient for Prussian blue staining. A solution consisting of equal parts potassium ferrocyanide and hydrochloric acid was prepared to form the Prussian blue staining solution. Sections were immersed in this staining solution for one hour, followed by rinsing with distilled water. Subsequently, nuclei were stained with a nuclear fast red solution for 1 to 5 min, then washed under running water. Finally, the sections were dehydrated and mounted.

Determination of Aβ in brain and serum

The levels of Aβ42 in serum and brain homogenates (both soluble and insoluble fractions) of 3xTg mice were determined using Human Aβ42 ELISA kits (Uscn Life Science Inc., China), following the manufacturer’s protocol.”

Flow cytometry for detection of human Aβ40/42 and human tau

RAB and RIPA fraction of brain homogenates were applied for pathologic protein detection (Aβ40/42) with BioLegend LEGENDplex™ Human Neurodegenerative Biomarker Panel 1 (741197, BioLegend, USA). Briefly, the soluble and insoluble brain homogenate are mixed with beads and detection antibodies, and then analyzed by flow cytometer. We use the multi-factor online analysis software of biolegend to calculate the content of factors (Aβ42/Aβ40/Human Tau).

ElisaKit for detection of cytokine and chemokine

Plasma and soluble brain homogenate from mice were diluted twofold. All samples were tested using the following ELISA kits according to the manufacturer’s instructions: TNF-α (Abcam, #ab252354), according to the manufacturer’s instructions, IL-1β (Cloud-clone Corp, #SEA563Si96T) and IFN-α (Chenglin, #AD0081Mk).

Purification of serum antibodies from immunized mice

Serum antibodies from immunized mice were isolated using the saturated ammonium sulfate precipitation method, aiming for downstream in vitro validation. The procedure details are provided in the ref. 29.

Aβ oligomer and NFT preparation

Aβ oligomers were induced in vitro by dissolving 1 mg Aβ42 peptide in 110 μL of pre-cooled hexafluoroisopropanol (HFIP). Evaporate HFIP by placing it on ice with the lid open for 1−2 h. The resultant peptide film was dissolved in DMSO and then diluted with DMEM, followed by incubation at 4 °C for 24 h.

For the preparation of Aβ deposition, an initial Aβ reserve solution of 2 mM concentration was prepared. The Aβ 42 peptide was dissolved in 100 mM NaOH solution and subjected to sonication in a water bath for 30 seconds, thus forming the monomer reserve solution. To this solution, 10 mM Hepes, 100 mM NaCl, and 0.02% sodium azide were added to achieve a final concentration of 25 μM. The mixture was then incubated at room temperature for 21 days to facilitate Aβ deposition or tau fibrils.

To prepare tau fibrilization, the pTau30 peptide was prepared by dilution in a solution containing 10 mM 1,4-Dithiothreitol (DTT), 100 mM NaCl, and 10 mM HEPES, followed by a 1-h incubation at room temperature. Subsequently, heparin was added to achieve a final concentration of 8 mM. The mixture was then incubated with agitation at 37 °C for a period of 7 days. After incubation, the tau solution underwent centrifugation at 100,000×g for 1 h at 4 °C, resulting in a pellet comprised of aggregated tau46. This pellet was subjected to sonication at 65% power/amplitude for 30 s using a water bath sonicator, after which it was diluted for further use.

Aβ uptake assay

As previously described49, FITC-labeled Aβ42 peptide was initially resuspended in 1% NH4OH (10 μL), and then PBS was added to 1 mg/ml. The mixture was incubated at 37 °C for 12 h to promote Aβ42 oligomer. Following incubation, the mixture was diluted in DMEM/F12 medium to 5 µg/ml before it was applied to BV2 cells. After stimulation with 40 ng/ml IL-4 and 100 ng/ml dexamethasone for 24 h, cells were rinsed and incubated with FITC-labeled Aβ42 oligomer (5 μM) and serum antibodies (1 μM) in DMEM for 24 h. The cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min. Following this incubation, cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min. After fixation, cells underwent a final wash and were then treated with an anti-fluorescence quenching sealing solution containing DAPI at room temperature for 5 min. The uptake of Aβ42 oligomer was visualized using a CKX41-32PH automatic microscope (Olympus, Japan), and representative images were captured. The average signal intensity of Aβ42 oligomer in individual cells was quantified and expressed as a fold change relative to control cells, with at least 30 cells analyzed per experimental group.

Dot blot assay

A nitrocellulose membrane was pre-soaked in electroconversion buffer for 5 min before being placed in a dot blot apparatus. Protein samples, diluted to 10 μl, were applied onto the upper wells of the dot blot fixture. The membrane was then incubated in 5% skim milk powder for 30 min at room temperature with agitation. Antibody incubation and ECL detection were performed as described in the Western blot section.

CCK8

SH-SY5Y cells were cultured in 96-well plates, each well containing ~7500 cells in 100 μL of medium. The plates were incubated at 37 °C for 24 h to facilitate cell adhesion. Subsequently, 1 μM of either Aβ oligomers or tau fibrils, with or without antibodies purified from serum, were added to the cultures, followed by further incubation for 72 h at 37 °C. Cell viability was assessed by adding 10 μL cck8 to each well, incubating for 0.5 to 4 h at 37 °C, and measuring the absorbance at 450 nm using an enzyme-linked immunosorbent assay.

Th-T

Additional experiments were conducted to evaluate Aβ fibril formation in vitro using thioflavin T (Th-T) and to ascertain the inhibitory effect of antibodies on fibril formation. The procedure details are provided in ref. 28.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, Inc.) with a probability level set at 95%. Results are described as mean ± SEM. The relations among the measured data of behavioral or pathological monitoring at consecutive time points were analyzed using Mauchly’s test of sphericity. Multivariate ANOVA was employed when p ≤ 0.05. Bonferroni-corrected t-test was otherwise applied. Graphs were carried out using Adobe Illustrator 2020 and BioRender.com.

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