Vaccination with a non-human random sequence amyloid oligomer mimic results in improved cognitive function and reduced plaque deposition and micro hemorrhage in Tg2576 mice

We synthesized a series of 20 random sequence peptides from a restricted set of 8 amino acids and tested their ability to form prefibrillar oligomers by dot blot analysis with A11. Of these peptides, only 10 were sufficiently soluble to work with. One of these sequences, 3A, formed oligomers that react strongly with A11. We prepared 3A oligomer mimetic antigen by coupling 3A to colloidal gold particles and vaccinated rabbits with this immunogen. Serum from 3A vaccinated rabbits and A11 was used to probe dot blots containing Aβ monomer, Aβ prefibrillar oligomers, Aβ fibrils, calcitonin prefibrillar oligomers, IgG light chain prefibrillar oligomers and synthetic KK(Q40)KK prefibrillar oligomers. Like A11, which is raised against Aβ oligomer mimic antigen, serum from 3A vaccinated rabbits reacted with all prefibrillar oligomer samples, including Aβ, but not Aβ monomer or Aβ fibrils (Figure 1). No immunoreactivity was observed for preimmune serum from the rabbit immunized with 3A oligomers. These results demonstrate that vaccination with a non-human random peptide sequence oligomer mimic give rise to an immune response that recognizes many different types of prefibrillar oligomers, including Aβ and are consistent with our previous observations that serum from rabbits vaccinated with IAPP oligomer mimics is indistinguishable from A11 [13].

To evaluate the therapeutic effectiveness of the 3A antigen, we immunized Tg2576 mice with 3A, Aβ, and IAPP oligomer mimetics and Aβ fibrils. Mice were immunized at monthly intervals beginning at 3 months up to 14 months. We performed ELISA assays to analyze the reactivity of the sera obtained from all groups of immunized mice against monomeric Aβ, Aβ PFOs, IAPP PFOs, 3A PFOs and Aβ fibrils (Figure 2). For all vaccinated groups, the highest reactivity is observed for the antigen they were immunized with. In addition, generic reactivity for prefibrillar oligomers samples is observed in the groups immunized with prefibrillar oligomer mimics. The immune response to the 3A peptide oligomer vaccine was specific for prefibrillar oligomers and did not display significant reactivity to monomeric or fibrillar Aβ (Figure 2). In contrast, the serum from Aβ fibrillar vaccinated mice shows immunoreactivity with Aβ fibril and Aβ PFOs (Figure 2). It shows that immunization with Aβ fibrils produced antibodies that reacted primarily with their own antigen and displayed some cross-reactivity with Aβ PFOs. Cross-reactivity could be due to small quantities of fibril-like structures in the PFO sample or small amounts of antibodies against PFOs in the serum. The titer of each vaccinated-group is expressed as the log10 of the IC50 values to normalize the distribution and statistical analysis on the transformed values has been done accordingly. As a result all the vaccinated groups, including IAPP oligomer, showed a significant immune response not only against their own antigen, but also against oligomers as compared to non-vaccinated mice. Control vaccinated mice displayed very low titers (<50) towards all antigens. None of the mice developed significant quantities of antibodies that react with Aβ40 monomer. These results indicate that the antibodies developed by the immunized mice in response to both Aβ fibrils and oligomers are predominantly conformation-dependent, aggregation specific and recognize generic epitopes that are independent of the precise amino acid sequence.

We evaluated the reference memory of Tg2576 mice at the age of 14 months in a Morris water maze test. At the age of 14 months, mice were trained on the spatial reference version of the Morris water maze task after 11 months of vaccination. 3A peptide, Aβ oligomer, Aβ fibril and IAPP oligomer vaccinated mice all demonstrate a significant improvement (***p < 0.001) in behavioral acquisition and retention in Morris Water Maze (Figure 3A). At 90 minutes and 24 h in probe trials with the platform removed, mice immunized with Aβ oligomer, IAPP oligomer, 3A oligomer mimics and Aβ fibrils show significant improvement (***p < 0.001) in memory retention on the latency to cross the platform location and the number of platform crosses during 90 minute and 24 h tests as compared to controls (Figure 3B). Contextual learning and memory was evaluated using the passive inhibitory avoidance task (Figure 3C). Passive avoidance memory retention (mean ± S.E.M.) as measured by the ability of mice to remember an electrical shock after 24 hrs. Passive-avoidance tests were carried out in Tg2576 mice at 14 months of age. In the first acquisition trial of the learning stage, all mice (14 months old) entered the dark compartment immediately after being placed in the illuminated compartment. In retention trials the step-through latency of the immunized mice with Aβ oligomer, IAPP oligomers and 3A oligomer mimics was significantly increased (*p < 0.05 for Aβ oligomer and ***p < 0.001 for IAPP and 3A peptide, ) as compared to that of the control immunized group (Figure 3C). We evaluated visual recognition memory at the ages of 14 months in a novel object recognition test (Figure 3D). This test is believed to be primarily dependent on cortex. The time that the mice explored the novel object versus the familiar object is called the recognition index (RI). 50% RI means that mice are not specifically interested in either of the objects. At 14 months, Tg2576 mice immunized with 3A oligomer mimics vaccinated mice shows statistically significant improvement in novel object recognition memory at both 1.5 hrs and 24 hrs (3D), to control vaccinated mice (*p < 0.05).

Vaccination against fibrillar Aβ is known to reduce plaque deposition in transgenic animals [10]. Amyloid deposition in 14 months Tg2576 mice was assessed using the human Aβ specific monoclonal antibody, 6E10. We compared the effectiveness of the immunization on amyloid deposition by quantifying the amount of anti- Aβ (6E10) immunoreactive material. Figures 4A show representative photomicrographs from the hippocampus and cortex of control and immunized animals. Image analysis of sections from multiple animals demonstrated that Aβ deposits in Aβ oligomer, IAPP oligomer, 3A oligomers mimics and Aβ fibril immunized mice were decreased significantly as compared to control vaccinated group. (Figure 4B) shows that all the immunized groups displayed a significant reduction in Aβ plaque load in both hippocampus and cortex as compared to the respective controls (*p < 0.05, **p < 0.01).

To evaluate whether vaccination with 3A peptide oligomer not only reduced plaque load but also affected cerebral total Aβ levels, brain homogenates were processed to prepare insoluble amyloid deposits. Subsequently, Aβ levels were quantified using a high-sensitivity sandwich ELISA measuring Aβ40 and Aβ42 separately as described in methods. Consistent with the reduced plaque by 3A oligomer mimics, we observed a significant reduction in soluble and insoluble levels of Aβ40 and Aβ42 (Figure 5). In Aβ fibril vaccinated mice, the insoluble levels of Aβ40 were not significantly decreased as compared to other groups, where as insoluble Aβ42 levels were significantly decreased. Immunization with the 3A peptide oligomer mimic antigen induces an immune response that significantly reduced insoluble levels of cerebral Aβ40 and Aβ42.

Microglia play a major role in regulating homeostasis in the brain and have the ability to activate phagocytose, secrete cytokines, and to present antigens to T cells depending on their stimulatory environment [15]. However, some of these same neuroprotective functions are detrimental if dysregulated [16]. One of the main characteristics accompanying accumulation of Aβ plaques in both human Alzheimer’s brain and transgenic mouse models of AD is an enhanced neuroinflammatory response characterized by activation of microglia. It has been previously reported that immunization with Aβ reduces the microglial activation [12]. We assessed the effect of immunization by 3A oligomer mimics on microglial reactivity as measured by CD45 immunohistochemistry (Figure 6A). All the immunized groups displayed a significant decrease in CD45 immunostaining relative to the respective controls (***p < 0.001) (Figure 6B).

Activation of astrocytes is another characteristic feature of amyloid deposition. It has been observed that activated astrocytes are reduced in the same proportion as the amyloid by immunization with Aβ [12]. To assess the involvement of activated astrocytes following vaccination, immunohistochemical analysis of GFAP, a marker for astroglia was performed. Quantitative analysis showed that there was a significant reduction in GFAP immunoreactivity. 3A peptide oligomer mimic immunized mice display a significant reduction of activated astrocytes in the hippocampus and cortex (Figure 7A-J). The quantification was statistically significant as compared to controls (*p < 0.05). Activated astrocytes were also significantly decreased in hippocampus of mice vaccinated with Aβ oligomer and IAPP oligomer, however in cortex there was no significant decrease. The explanation for this difference may be that in Aβ oligomer and IAPP oligomer vaccinated mice where we observed a decrease in cortical plaques, there is GFAP immunoreactivity that is not associated with plaques.

An increased incidence of micro hemorrhage has been observed in both active and passive Aß immunotherapy [17]. We therefore investigated the incidence of micro hemorrhage in the vaccinated and control animals. The method we used categorizes micro hemorrhages in 4 different stages of increasing size and vessel involvement [18]. The number of micro hemorrhages per section is reported in Table 1 for each stage. As previously reported, most of the micro hemorrhages observed in Aß vaccinated Tg2576 animals were stage 1, consisting of 1 to 5 grains of iron or small vessel involvement and are shown in Figure 8A. We found that the 3A and IAPP vaccinated mice showed a striking reduction in parenchymal iron deposition consistent with a lower incidence of micro hemorrhages compared to the Aβ oligomer and Aβ fibril vaccinated groups (***P < 0.001) (Figure 8B). The 3A and IAPP vaccinated groups were not statistically different from the control vaccinated group. Stage 2 micro hemorrhages, consisting of multiple grains of iron and micro vessel involvement were also significantly elevated in the Aß fibril and Aß oligomer vaccinated groups (Table 1, P < 0.001, P < 0.05 respectively). Stage 3 micro hemorrhages, consisting of several positive micro vessels in 1 area, were significantly elevated only in the Aß fibril vaccinated group. Stage 4 micro hemorrhages with large blood vessel involvement were rarely observed in the Aß vaccinated groups and none were observed in the control, 3A and IAPP vaccinated groups although this difference did not reach statistical significance. In Aß vaccinated fibril and oligomer groups, micro hemorrhages were observed in the somatosensory and auditory cortices, the CA1, CA3 and dente gyrus of the hippocampus, lateral and ventral hypothalamic areas, and the ventral posterior medial thalamic nucleus. No preferential localization of micro hemorrhages in a specific brain area was observed for any groups. These results indicate that the non-Aβ antigens may have a better safety profile with respect to micro hemorrhage than Aβ containing antigens.

We synthesized a series of 8 random sequence 20mers using a restricted set of 8 amino acids, threonine, tyrosine, serine, histidine, isoleucine, valine, phenylalanine and leucine. The sequences of the 20mers were randomly drawn and checked against the human non-redundant protein sequence data base with BLAST to select for sequences that had the lowest homology with human protein sequences. The sequences were synthesized and tested for the ability to form prefibrillar amyloid oligomers using the A11 prefibrillar oligomer specific polyclonal antibody. One of the sequences (peptide 3A: TYLIHVHIITIYHISIYYIV) formed A11 positive oligomers in a time dependent fashion and was selected for further analysis.

Aβ, IAPP and 3A peptide oligomer molecular mimic antigens were prepared by synthesizing carboxyl-terminal thiol derivatives and coupling them to colloidal gold particles as previously described [46].

Aβ40 peptides were lyophilized in 50% acetonitrile/ddH₂O. Soluble oligomers were prepared by dissolving 1.0 mg of peptide in 400ul hexafluoroisopropanol (HFIP) for 10–20 min at room temperature. 100 μl of the resulting seedless solution was added to 900 μl milliQ H₂O in a siliconized Eppendorf tube. The samples were then stirred at 500 RPM using a Teflon coated micro stir bar for 24–48 h at 22°C. The samples were centrifuged for 15 min. at 14,000 × G and the supernatant fraction (pH 2.8–3.5) was transferred to a new siliconized tube and subjected to a gentle stream of N₂ for 5–10 min to evaporate the HFIP. Formation of oligomers were confirmed by atomic force microscopy (AFM), electron microscopy (EM) and size exclusion chromatography (SEC) as described [40]. Fibrils were formed by dissolving Aβ42 in 50% HFIP/ddH₂O and stirred with closed caps for 7 days. Solution was stirred again for 2 days using open caps to evaporate the HFIP. Fibrils were sedimented, washed, and resuspended in PBS at 2 mg/ml.

New Zealand white rabbits were immunized with 3A oligomer mimics as previously described and the serum collected after significant titer against Aβ oligomers was detected [46]. Preimmune serum was collected from the same rabbits prior to immunization. We immunized young Tg2576 mice (3 months old) [5] with one of the following antigens: Aβ oligomer, IAPP oligomer, Aβ fibril, random peptide 3A oligomers or control with colloidal gold in PBS. The mice were equally distributed in each group of eight (4 males and 4 females). The antigens were mixed with incomplete Freund’s adjuvant (1:1,v/v) and immunization was done subcutaneously (100 μg/immunization) every month up to 14 months. For controls a mixture of PBS and colloidal gold was used.

Three common tests for cognitive dysfunctions in AD model mice, the Morris Water Maze (MWM) [47, 48] novel-object recognition [48, 49], and passive inhibitory avoidance tests [10], were performed according to previous reports with minor modifications.

ELISA assays were performed as previously described [40]. Briefly, 200 ng/100 μl of antigen (Aβ oligomer, IAPP oligomer, 3A oligomer mimic, Aβ fibril and Aβ monomer) was plated on ELISA wells and blocked with BSA. Serum samples were serially diluted to an end point of 1:100,000. The secondary antibody used for detection is peroxidase conjugated AffiniPure Goat Anti-mouse IgG (H + L) (Jackson ImmunoResearch). Titer was determined from the midpoint of the dilution curve (IC50).

After mice were anesthetized with pentobarbital (150 mg/kg, IP), blood was collected by cardiac puncture, and mice were perfused transcardially with cold phosphate-buffered saline (PBS). Brain tissues were fixed overnight with 4% paraformaldehyde in PBS, pH 7.4 at 4°C and stored in PBS/0.02% sodium azide (NaN₃) at 4°C until use. Fixed brain tissues were sectioned (40 μm) with a vibratome. Coronal sections were collected in PBS (containing 0.02% sodium azide) and stored at 4°C prior to staining. To stain for Aβ plaques, sections were immersed in 70% formic acid for 5 min. Endogenous peroxidase in tissue was blocked by treating with 3% H₂O₂ in PBS for 10 min at room temperature. Nonspecific background staining was blocked by 1 h incubation in 2% BSA, 0.3% Triton X-100 (TX) at room temperature. Tissues were incubated with primary antibodies (6E10, GFAP and CD45) overnight at 4°C, rinsed 3 times with PBS,0.1% TX, followed by biotinylated secondary antibodies (anti-rabbit, anti-mouse and anti-rat), detection with an ABC peroxidase kit, and visualization with a 3,3'-diaminobenzidine (DAB) substrate kit (Vector, Burlingame, CA). CD45 (Serotec, Raleigh, NC; 1:3000) staining was done as described previously [22]. Control experiments with primary or secondary antibody omitted resulted in negative staining.

Coronal brain sections were double-immunostained for astrocytes and Aβ plaques. Sections were permeabilized in 0.1% Triton X-100 for 15 min at RT, blocked in 5% bovine albumin/PBS for 1 hour, and then probed with anti-mouse glial fibrillary acidic protein, GFAP (1:1000, Sigma), rabbit anti-fibril OC (1:4000) [13] for 24 h at RT. Fluorescent-conjugated secondary antibodies (anti-mouse, anti-rabbit Alexa, 1:400, Invitrogen Molecular Probes) were used for detection. Double immunofluorescent images were captured at a magnification of 10x and anayzed by the z-stack confocal microscopic system using Axio Vision (Zeiss).

Immunostaining was observed under a Zeiss Axiovert-200 inverted microscope (Carl Zeiss, Thornwood, NY) and images were acquired with a Zeiss Axiocam high-resolution digital color camera (1300x1030 pixel) using Axiovision 4.1 or 4.6 software. The same software (Carl Zeiss) was used to analyze the digital images. Percent of immunopositive area (% Field Area) (immunopositive area/total image area × 100) was determined for all the markers studied by averaging images of the cortex, hippocampus and subiculum area from 2–3 sections per animal. Digital images were obtained using the same settings and the segmentation parameters constant within a range per given marker and experiment. The mean value of the% Field Area for each marker in each animal was averaged per genotype group with the number of animals per group indicated in Figure legends.

Soluble and insoluble Aβ fractions were isolated from whole brain homogenates using four step extraction protocol [24]. Frozen hemibrains were sequentially extracted. At each step, sonication in an appropriate buffer was followed by centrifugation at 100,000 x g for 1 hr at 4°C. The supernatant was then removed, and the pellet was sonicated in the next solution used in the sequential extraction process. For four-step extraction, sonication of the frozen brain (150 mg/ml wet weight) began in Tris-buffered saline (TBS) (20 mM Tris and 137 mM NaCl, pH 7.6), which contained protease inhibitors (Protease inhibitor cocktail from Sigma St. Louis USA). The next three sequential extraction steps used 1% Triton X-100 in TBS with protease inhibitors, 2% SDS in water with the same protease inhibitors, and 70% formic acid (FA) in water. Soluble fractions were loaded directly onto ELISA plates, whereas insoluble fractions were diluted 1:20 in a neutralization buffer (1 mol/L Tris base, 0.5 mol/L NaH2PO4) before loading. MaxiSorp immunoplates (Nunc, Rochester, NY) were coated with Mab 20.1 antibody (a kind gift from Dr. David Cribbs University of California Irvine) at a concentration of 25 μg/ml in coating buffer (0.1 M/L Na2CO3, pH 9.6) and blocked with 3% bovine serum albumin. Standard solutions for both Aβ40 and Aβ42 were made in the antigen capture buffer (20 mmol/L NaH2PO4, 2 mmol/EDTA, 0.4 M NaCl, 0.05% 3-[(3- cholamidopropyl) dimethylammonio] propanesulfonate, and 1% bovine serum albumin, pH 7.0) and loaded onto ELISA plates in duplicate. Samples were then loaded (also in duplicate) and incubated overnight at 4°C. Plates were then washed and probed with either horseradish peroxidase conjugated anti-Aβ40(C49) or anti-Aβ42(D32) overnight at 4°C. The chromogen was 3,3_,5,5 tetramethylbenzidine, and the reaction was stopped by 30% phosphoric acid. The plates were read at 450 nm using a plate reader (Molecular Dynamics, Sunnyvale, CA). The readings were then normalized to protein concentrations of the samples.