PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC 2013 September 13.
Published in final edited form as:
PMCID: PMC3634356
NIHMSID: NIHMS455445

Immunogenicity, Efficacy, Safety, and Mechanism of Action of Epitope Vaccine (Lu AF20513) for Alzheimer’s Disease: Prelude to a Clinical Trial

Abstract

Alzheimer’s disease (AD) process is understood to involve the accumulation of amyloid plaques and tau tangles in the brain. However, attempts at targeting the main culprits, neurotoxic Aβ peptides, have thus far proven unsuccessful for improving cognitive function. Recent clinical trials with passively administrated anti-Aβ antibodies failed to slow cognitive decline in mild-moderate AD patients, but suggest that an immunotherapeutic approach could be effective in patients with mild AD. In an AD mouse model (Tg2576) we tested the immunogenicity (cellular and humoral immune responses) and efficacy (AD-like pathology) of clinical grade Lu AF20513 vaccine. Lu AF20513 induces robust “non-self” T cell responses and production of anti-Aβ antibodies that reduce AD-like pathology in the brains of Tg2576 mice without inducing microglial activation and enhancing astrocytosis or CAA. Importantly, a single immunization with Lu AF20513 induces strong humoral immunity in mice with pre-existing memory Th cells. In addition, Lu AF20513 induces strong humoral responses in guinea pigs and monkeys. Collectively, these data suggest translation of Lu AF20513 to clinical setting with aims to (i) induce therapeutically potent anti-Aβ antibody responses in patients with mild AD, particularly if they have memory Th cells generated after immunizations with conventional Tetanus Toxoid vaccine; (ii) exclude likely pathological autoreactive T cell responses.

Introduction

Neuropathological features of AD include deposition of the amyloid-β (Aβ) fragment of amyloid precursor protein (APP) in senile plaques, accumulation of neurofibrillary tangles (NFT) composed of tau protein, and death of neurons (Hardy and Allsop, 1991; Pike et al., 1991; Price and Sisodia, 1994; Hardy and Selkoe, 2002; Nikolaev et al., 2009). For over two decades, Aβ peptides have been thought to play a central role in the onset and progression of AD (Selkoe, 1991, 1994), and it was this proposal from which the ‘amyloid cascade hypothesis’ emerged (Hardy and Higgins, 1992; Golde et al., 2006; Hardy, 2006). According to this hypothesis, in AD, the normally soluble Aβ molecule undergoes conformational changes and is deposited as insoluble fibrils, and soluble oligomers and protofibrils. Importantly, the amyloid cascade hypothesis has evolved to focus mainly on soluble oligomers and protofibrils of Aβ, which are now considered to be the most toxic forms of Aβ, responsible for causing synaptic destruction (Harper et al., 1997; Walsh et al., 1997; Lambert et al., 1998; Yong et al., 2002; Klein et al., 2004; Cleary et al., 2005; Haass and Selkoe, 2007). Accordingly, therapeutic interventions for AD have been directed toward decreasing Aβ production using β- and γ-secretase inhibitors/modulators, or by immunotherapeutic strategies to enhance Aβ clearance and to block tau aggregation (Sigurdsson et al., 2004; Rafii and Aisen, 2009; Holtzman et al., 2011). The first AN1792 vaccine clinical trial in AD patients was unsuccessful due to a small but statistically significant incidence of meningoencephalitis (Orgogozo et al., 2003). However, vaccine strategies for AD treatment will remain highly promising if new-generation vaccines can avoid anti-Aβ T cell responses that may underlie the incidences of meningoencephalitis (Orgogozo et al., 2003), as well as the issue of T cell tolerance, which might have accounted for the low antibody titers in many patients in the AN1792 trial (Nicoll et al., 2003; Ferrer et al., 2004; Gilman et al., 2005; Boche et al., 2008; Holmes et al., 2008). In this translational study we have devised and validated a novel AD epitope vaccine, Lu AF20513, in which the Th cell epitopes of Aβ42 were replaced by two foreign Th epitopes from Tetanus Toxoid (TT), P2 and P30, and the immunodominant B cell epitope of amyloid, Aβ1–12. Our data reveal that the Lu AF20513 (i) overcomes T cell tolerance induced by self-antigen; (ii) greatly reduces the possibility of inducing harmful autoreactive T cell responses that may explain the failure of the AN1792 vaccine; and (iii) may improve the ability of the elderly to mount an effective immune response by stimulation of pre-existing memory Th cells. In this report on the immunogenicity, efficacy, and safety of GMP grade Lu AF20513 in the APP/Tg mouse model of AD, in guinea pigs and in cynomolgus monkeys, our data support the translation to Phase I/IIa clinical trials.

Materials and Methods

Animals, epitope vaccine, and experimental protocols

Mice

Female and male, 4–6 mo old Tg2576 mice (H-2bxs haplotype) were bred at the animal facility of the University of California at Irvine. Female, 6–8 weeks old B6SJL mice (H-2bxs haplotype) were obtained from the Jackson Laboratory. 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.

Guinea pigs

Female and male guinea pigs (albino, Dunkin Hartley) were obtained from Charles River, Germany and were approximately 8–10 weeks of age at commencement of treatment. All animals were housed in a temperature and light-cycle controlled facility at TNO Triskelion B.V., the Netherlands, and their care was under the guidelines of the European Communities (Directive 86/609/EEC) and Dutch legislation (The experiments on Animals Act, 1997).

Cynomolgus monkeys

Female and male, purpose-bred cynomolgus monkeys (Macaca fascicularis), approximately 3 years of age, were obtained from Biodia, Mauritius. All animals were housed in a temperature and light-cycle controlled facility at Ricerca Biosciences LCC, France, and their care was in compliance with the following guidelines: Guide for the care and use of laboratory animals, NRC, 1996, Decree no 2001-464 regarding the experiments with laboratory animals, described in the Journal Officiel de la République Francaise on 29 May 2001, and Decree no 2001-486 relating to the protection of animals used in scientific experiments, described in the Journal Officiel de la République Francaise on 06 June 2001.

Epitope vaccine

GMP grade recombinant protein vaccine (Lu AF20513) was used in this study, which composed of two foreign T helper cell epitopes from TT, P30 and P2, and 3 copies of the B cell epitopes of Aβ42, Aβ1–12 (Fig. 1A) avoiding known Th cell epitopes in Aβ. Lu AF20513 is expressed in E. coli and primarily found in the inclusion bodies. The inclusion bodies were solubilized and the protein was purified through a series of chromatographic methods and the buffer exchanges to the final formulation.

Figure 1
Lu AF20513 vaccine and design of immunization studies of Tg2576 mice. A. Schematic representation of multivalent clinical grade AD epitope vaccine (Lu AF20513). Three copies of B cell epitope of Aβ42 (Aβ1–12) are attached to P30 ...

Experimental protocols

All Tg2576 mice were from the same breeding cohort and were allocated randomly to experimental and control groups. 4–6 mo old mice were immunized with Lu AF20513 as previously described (Cribbs et al., 2003; Petrushina et al., 2003; Agadjanyan et al., 2005). Briefly, Lu AF20513 protein was formulated in CFA/IFA or Quil-A adjuvants, as described previously (Cribbs et al., 2003), and mice were injected subcutaneously (SC) with indicated concentrations of antigen (Fig. 1B–D). Control mice were injected with an adjuvant only. All mice were boosted at monthly intervals. More detailed experimental protocols are described in Fig. 1BD. The guinea pigs and cynomolgus monkeys were immunized monthly (SC) with 20μg and 50μg of Lu AF20513 formulated in Alhydrogel® per animal, respectively. Control groups of guinea pigs and cynomolgus monkeys were injected with adjuvant only. Blood was collected before injections (pre-bleed) or 10–11 days after the second injection with Lu AF20513 formulated in Alhydrogel®.

T cell proliferation and production of cytokines by immune splenocytes

Analysis of T cell proliferation was performed in splenocyte cultures from individual animals using a [3H]-thymidine incorporation assay, as previously described (Cribbs et al., 2003; Agadjanyan et al., 2005; Davtyan et al., 2010). The same splenocytes used to assess T cell proliferation were utilized for detection of T cells producing IFNγ (Th1) or IL-4 (Th2) cytokines by ELISPOT (BD Pharmingen), as previously described (Cribbs et al., 2003; Agadjanyan et al., 2005; Petrushina et al., 2007). The cultures of splenocytes from experimental and control mice were re-stimulated in vitro with P30, P2, Lu AF20513, Aβ40 or an irrelevant peptide (25 μg/ml of each peptide) for 18 hours.

Detection of anti-Aβ antibodies by ELISA

Total anti-Aβ antibodies were detected using ELISA as described previously(Ghochikyan et al., 2006; Petrushina et al., 2007; Davtyan et al., 2010). Anti-Aβ antibody concentrations in mice were calculated using a calibration curve generated with 6E10 monoclonal antibody (Signet). HRP-conjugated anti-IgG1, IgG2ab, IgG2b and IgM specific antibodies (Bethyl Laboratories, Inc.) were used to characterize the isotype profiles of antibodies in pooled sera at dilution 1:200. Anti-Aβ antibodies in guinea pigs and monkeys were detected using 96 wells ELISA plates coated with Aβ1–28 (American Peptide). In case of guinea pigs we used sheep anti-guinea pig IgG-biotin (Nordic Immunology, The Netherlands) coupled to streptavidin-europium (Perkin Elmer) as a secondary antibody, while in case of monkeys we used goat anti-monkey IgG antibodies conjugated with HRP (Nordic Immunology, The Netherlands). The individual samples were diluted 1:100, 1:200, 1:400, 1:800, 1:2400, 1:7200, 1:21600 and the titer was determined by back-calculating the OD of the assay cut point on a 4-parameter logistic curve using the Prism software.

Purification of anti-Aβ1–12 antibody

Anti-Aβ1–12 antibodies were purified from sera of mice immunized with Lu AF20513 by an affinity column (SulfoLink, Pierce) as described previously(Mamikonyan et al., 2007). Column was immobilized with Aβ18-C peptide (GenScript). Purified antibodies were analyzed via 10% Bis-Tris gel (Invitrogen), and the concentration was determined using BCA protein assay kit (Pierce).

Surface Plasmon Resonance (SPR) Analysis

Binding studies were performed on the BIAcore 3000 SPR platform (Biacore) as described previously (Mamikonyan et al., 2007). Monomeric, oligomeric and fibrillar forms of Aβ42 peptides were prepared as described previously and confirmed by Electron Microscopy (EM) and binding of these peptides to 6E10, A11, and OC antibodies by dot blot (Kayed et al., 2007; Mamikonyan et al., 2007; Kayed et al., 2010). These peptides were immobilized to the surface of biosensor chip CM5 (GE Healthcare) via an amine coupling of primary amino groups of the appropriate peptide to carboxyl groups in the dextran matrix of the chip. Serial dilutions of anti-Aβ1–12 antibody, irrelevant mouse IgG (SouthernBiotech), or 6E10 antibody (666.6, 222.2, 74.1, 24.7, 8.23, 2.74 nM) in the running buffer containing 10 mM HEPES, 150 mM NaCl, 0.05% surfactant P20, pH 7.4, were injected at 20 μl/min over each immobilized form of peptide, and the kinetics of binding/dissociation was measured as change of the SPR signal [in resonance units (RU)]. Each injection was followed by a regeneration step of a 25-s pulse of 1 M NaCl, 50 mM NaOH. Fitting of experimental data was done with BIAevaluation 4.1.1 software using 1:1 interaction model to determine apparent binding constants.

Detection of Aβ plaques in human brain tissues

Anti-Aβ1–12 antibodies (1.25μg/ml) were screened for the ability to bind to human Aβ plaques using 50μm brain sections of formalin-fixed cortical tissue from a severe AD case (received from Brain Bank and Tissue Repository, MIND, UC Irvine) using immunohistochemistry as described previously (Ghochikyan et al., 2003; Agadjanyan et al., 2005; Davtyan et al., 2011). A digital camera (Olympus) was used to capture images of the plaques at a 10x magnification.

Neurotoxicity Assay

Cell culture MTT assay was performed as described previously with minor modifications (Davtyan et al., 2011). Aβ42 oligomers and fibrils were incubated alone or with purified anti-Aβ1–12 antibodies or irrelevant antibodies (BD, Biosciences) for 2h at room temperature with occasional mixing to ensure maximal interaction. After incubation, the peptide/immune sera mixtures were diluted into culture media so that the final concentration of peptide and antibodies was 2μM and 0.2μM, respectively. This media was then added (100μl) to SH-SY5Y cells (ATCC). The treatment time was 48h. Untreated controls were run in parallel. Following incubation, neurotoxicity was assayed using the MTT assay according to the manufacturer’s instructions (Promega Corp.). Cell viability was calculated by dividing the absorbance of wells containing samples by the absorbance of wells containing medium alone.

Brain collection, immunohistochemistry, histostaining, quantitative image analysis, biochemical analysis

Brain collection

At 15–17 mo of age, vaccinated and control mice were sacrificed for further neuropathological analysis as described previously (Movsesyan et al., 2008b). The left hemisphere was snap frozen and reserved for measurement both soluble and insoluble Aβ levels by ELISA. The right hemisphere was fixed in 4% paraformaldehyde in PBS at +4°C for 24 hrs for further sectioning.

Immunohistochemistry

50 μm-thick coronal sections of fixed hemi-brains were cut using vibratome. To assess the extent of neuropathology, several sets of free-floating equally spaced sections have been selected for each mouse brain, and immunostained using the antibodies described below. Aβ cored and defused deposits were detected with 6E10 (1:1000, Signet), as described in (Petrushina et al., 2007; Movsesyan et al., 2008b). Activated microglia were stained with the anti-I-A/I-E (marker of MHC II alloantigens, 1:100, BD Pharmingen), and astrocytes were labeled with anti-GFAP (glial fibrillary acidic protein, 1:500, Dako) antibody, both as we described (Petrushina et al., 2007; Movsesyan et al., 2008b)..

Histostaining

Fibrillar Aβ deposits were visualized using Thioflavin S (ThS; Sigma-Aldrich), and hemosiderin deposits were detected via Prussian blue staining. Both assays were performed as described in (Petrushina et al., 2007; Movsesyan et al., 2008b).

Quantitative image analysis

The number of 6E10- or ThS-positive plaques was analyzed through the whole hemisphere, as well as in cortical and hippocampal regions. Measurement of number of blood vessels showing amyloid depositions characteristic of cerebral amyloid angiopathy (CAA) was done in 6E10-stained brain sections; activated microglial and hemosiderin-positive profiles were counted through the whole hemisphere as well. The mean semi-quantitative scores per hemisphere or per neuroanatomical region in those assays were determined based on visual microscopic inspection of sets of 5 coronal sections equally spaced between −0.80 to −2.92 mm with respect to Bregma, performed by three independent observers blinded to the treatment conditions.

NIH Image J 1.45s software was used to analyze the number of GFAP-positive astrocytes. For every animal, total of 19 images (802 x 650 μm each) of cortex, striatum and hippocampus were selected at approximately the same plane (3 sections between −0.82 to −2.75 mm with respect to Bregma). The images were captured using a MD700 video camera (AmScope) and 10x objective.

Biochemical analysis

To determine the levels of both soluble Aβ40 and Aβ42 and insoluble total Aβ, a 10% (w/v) of brain homogenate was prepared from each mouse brain at Amorfix (Canada) using in PBS/2% NP-40 containing proprietary protease inhibitors. Each 10% brain homogenate was centrifuged at 100,000xg for 1 hr at 4°C. The supernatant was collected as the source of soluble aggregates for analysis of soluble Aβ40 and Aβ42 using human β-amyloid ELISA kits (Invitrogen), according to manufacturer’s recommendations. The final values of soluble Aβ were expressed as nanograms (ng) per gram wet weight of hemi-brain. The remaining pellet was solubilized in 70% formic acid by sonication and centrifuged at 100,000xg for 1 hr at 4°C. Avoiding upper lipid layer, the lower aqueous layer was collected and stored at −70°C. Quantification of insoluble Aβ level was done using human β-amyloid ELISA kit (Invitrogen), according to manufacturer’s recommendations. The final values of insoluble Aβ were expressed as micrograms (μg) per gram wet weight of hemi-brain.

Generation and testing of memory T cell

6–8 weeks old B6SJL mice were immunized 3 times with P30 peptides formulated in Quil-A (50μg/mouse and n=30). Control mice were injected with adjuvant only (n=25). After resting period for 6 months, 10 mice from P30 primed and control groups were terminated and memory T cell response was analyzed before and after a single booster injection using ELISPOT assay. Recall responses of CD4+ T cells were detected after isolation of this subpopulation of cells from splenocytes using specific isolation kit and as suggested by the manufacturer (Militenyi Biotec). Isolated CD4+ T cells from splenocytes of primed (n=4) and non-immune mice (n=4) were used for enrichment of naive splenocytes (5x104 CD4+ cells were added into 15x104 splenocytes) in indicated experiments. The purity of isolated CD4+ cells and the efficacy of depletion were analyzed by flow cytometry (Militenyi Biotec). For booster injection we used 100μg/mouse Lu AF20513 and both cellular and humoral immune responses were analyzed in experimental and control mice as indicated in Figure S6.

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 an analysis of variance (ANOVA) and post hoc comparisons were done using Tukey’s test (P value less than 0.05 was considered as statistically different).

Results

Lu AF20513 induces strong Th cell responses specific to foreign Th epitopes from TT

One challenge associated with the clinical use of the Aβ “self” T cell epitopes as part of a vaccine to treat AD patients are the development of undesirable anti-Aβ or anti-APP specific T cell responses (Cribbs et al., 2003; Orgogozo et al., 2003; Ferrer et al., 2004; Agadjanyan et al., 2005; Lemere, 2009). With this in mind, we synthesized Lu AF20513 vaccine, which was designed to activate non-self CD4+ T lymphocytes (Th) specific to TT, pre-existent in most individuals due to prior tetanus immunization, in order to enhance antibody production by Aβ-specific B cells (Fig. 1A).

We first tested the cellular responses to Lu AF20513 vaccination in Tg2576 mice (Fig. 2A,B), an APP over-expressing model of AD (Hsiao et al., 1996). Control groups of mice were injected with adjuvant only (Fig. 1B). Vaccinations of Tg2576 mice with Lu AF20513 led to production of IFNγ and IL-4 after re-stimulation with P30, but not Aβ40 (Fig. 2A). In proliferation assays, Th cells were activated with Lu AF20513 and P30, but not Aβ40 peptide (Fig. 2B). T cell responses were not detected in mice injected with adjuvant only (Fig. 2C,D). Of note, here we tested Th cell responses to only one TT epitope, P30, based on our previous experiments performed in wild-type B6SJL mice, which demonstrated that two immunizations with Lu AF20513 induced equally effective Th cell responses specific to both P2 and P30 epitopes of TT and Lu AF20513 (Fig. 2E,F). It should also be mentioned that re-stimulation of immune splenocytes with Lu AF20513 induced significantly stronger Th cell proliferation (P≤0.001) than when stimulated by P2 and P30 peptides individually (Fig. 2E,F). Thus, splenocytes from Lu AF20513-immunized wild-type and Tg2576 micedid not induce autoreactive anti-Aβspecific Th cell responses (Fig. 2).

Figure 2
Efficacy of Lu AF20513 vaccine: induction of cellular immune responses in Tg2576 and wild-type mice. A, C. Number of IFNγ and IL-4 producing cells is detected by ELISPOT in splenocyte cultures obtained from Lu AF20513 immunized or non-immunized ...

Lu AF20513 immunization induced therapeutically potent anti-Aβ antibody production in Tg2576 mice with very early AD-like pathology

To demonstrate that activation of P30 and P2 specific Th cells culminates in activation of anti-Aβ specific B cells, we measured the anti-Aβ42 antibody concentrations in Tg2576 mice vaccinated with Lu AF20513 formulated in two different adjuvants, CFA/IFA or Quil-A (Fig. 1C). Of note these animals have been in very early stage of AD-like pathology at the start of immunization (Lesne et al., 2006). Control mice injected with CFA/IFA and Quil-A adjuvants did not generate anti-Aβ42 antibodies, while Lu AF20513 formulated in either adjuvant induced robust antibody production after 2–3 immunizations. Of note, no differences were seen in the level and kinetics of antibody production in mice immunized with Lu AF20513 formulated either in CFA/IFA or in Quil-A (Fig. 3A). To characterize the type of humoral immune response, we measured the production of IgG1, IgG2ab, IgG2b, and IgM isotypes of anti-Aβ antibodies in Tg2576 mice immunized with Lu AF20513 formulated in CFA/IFA or Quil-A. We did not observe any differences in the magnitude and the type of antibody responses: mice from both vaccinated groups that received either CFA/IFA or Quil-A generated mostly IgG antibodies (IgG2b>IgG2ab>IgG1) (Fig. 3B). Since CFA/IFA adjuvant is used only for animal studies, while the equivalent of Quil-A for human use, QS21, is already used in many clinical trials, we combined mice from groups immunized with Lu AF20513/Quil-A and Lu AF20513/CFA/IFA as indicated in Fig. 1D and performed subsequent immunizations with Lu AF20513 formulated in Quil-A only. The data demonstrated that the anti-Aβ antibody concentration in vaccinated mice reached the maximum titers after 3 immunizations (Fig. 3A), declined slightly after that and remained at steady state during subsequent immunizations until the end of the experiment (Fig. 3C). The levels of IgG1, IgG2ab, IgG2b immune responses were robust and stable, while the level of IgM was low during the whole vaccination process (Fig. 3B,D). In sum, these results demonstrate that Th cells specific to P30 and P2 epitopes from TT are providing the support necessary for a therapeutically potent anti-Aβ antibody response in Tg2576 mice vaccinated with the Lu AF20513.

Figure 3
Efficacy of Lu AF20513 vaccine: induction of humoral immune responses specific to amyloid. A. Both CFA/IFA and Quil-A adjuvants were equally effective in stimulation of anti-Aβ antibody responses to immunizations with Lu AF20513. B. Lu AF20513 ...

Ex vivo and in vitro characterization of anti-Aβ antibodies generated by Lu AF20513 epitope vaccine

In order to functionally characterize the anti-Aβ1–12 antibodies generated in response to the Lu AF20513, we measured the binding of these antibodies to various forms of β-amyloid including plaques in the brains from a clinical AD case as well as their neuroprotective ability (Fig. 4).

Figure 4
In vitro assays suggest that Lu AF20513 induced production of functionally potent anti-Aβ antibody. A. Purified anti-Aβ antibody binds to monomeric, oligomeric, and fibrillar forms of Aβ42 with high affinity. The black lines represent ...

One of the important features of anti-Aβ antibodies is their capacity to bind the toxic forms of β-amyloid (Glabe, 2008). Our results from an SPR assay showed that polyclonal anti-Aβ antibodies bound with high average affinity to immobilized monomers (KD=2.83x10−8M), oligomers (KD=9.9x10−9M) and fibrils (KD=2.8x10−8M) (Fig. 4A,B), while irrelevant mouse IgG antibody purified from normal mouse serum bound to all these forms of Aβ42 on the base-line level. The binding affinity of purified antibodies to oligomers was higher than binding affinity to fibrils and monomers. Notably, the affinity of binding for control monoclonal antibody, 6E10, is higher for monomeric form of Aβ42 (KD=1.21x10−10M) compared with that of oligomeric (KD=9.37x10−9M) and fibrillar (KD=2.97x10−9M) forms (Fig. 4B). Nevertheless, the affinity of binding to oligomeric forms is similar for anti-Aβ1–12 and 6E10 antibodies (KD=9.9x10−9M vs KD=9.37x10−9M). Additionally, the anti-Aβ antibody generated in mice immunized with Lu AF20513 also bound to Aβ plaques in the brain tissues from an AD case, whereas irrelevant mouse IgG did not bind to AD plaques (Fig. 4C).

Next, we tested the protective effect of anti-Aβ antibodies against neurotoxicity induced by Aβ42 oligomers and fibrils in an in vitro model. Culture of SH-SY5Y neuroblastoma cells with Aβ42 oligomers and fibrils had cytotoxic effects, reducing cell viability to 50% and 63.1%, respectively, whereas pre-incubation with anti-Aβ antibodies rescued the cell viability to 79.5% and 91.6% (Fig. 4D). Binding to amyloid plaques in the brain sections along with the neuroprotective effect of anti-Aβ1–12 antibodies suggest that the Lu AF20513 vaccination strategy is functionally active.

Vaccination with Lu AF20513 significantly reduces AD-like pathology in aged Tg2576 mice

As a pre-clinical test of the therapeutic efficacy of Lu AF20513 vaccination, we studied neurological changes in the brains of aged Tg2576 mice (15–17 mo old) vaccinated with Lu AF20513 (Fig. 1D). Fig. 5A shows a significant decrease in hemi-brain plaque burden in mice immunized with Lu AF20513 as compared to the control adjuvant-only injected group. We detected significantly less 6E10-positive diffuse and cored plaques in both cortical and hippocampal regions of brains of Lu AF20513 vaccinated mice as compared to the same brain regions of control animals (Fig. 5B). Additionally, the results showed a significant reduction of cored Thioflavin S (ThS)-positive Aβ plaques in the hemi-brains as well as in cortical and hippocampal regions of these brains obtained from vaccinated mice versus brains isolated from control Tg2576 mice (Fig. 5C,D). These data were confirmed by detection of insoluble Aβ in the brains of vaccinated and control mice. As demonstrated in Fig. 5E, vaccination with Lu AF20513 induced a significant reduction (P<0.001) of insoluble total Aβ in the brains of immunized mice vs control animals.

Figure 5
Anti-Aβ antibody inhibited AD-like neuropathology in Tg2576 mice vaccinated with Lu AF20513. A–D. Significant inhibition of numbers of 6E10-immunoreactive cored and diffused (A, B) and ThS-positive cored amyloid-β plaques (C, D ...

Increasing evidence suggests that soluble (or diffusible) Aβ oligomers mediate different toxic pathways in AD such as tau hyperphosphorylation (De Felice et al., 2008), impairment of memory, and neuronal death (Lesne et al., 2006; Haass and Selkoe, 2007; Shankar et al., 2008). A potential problem of immunotherapy is that a reduction of insoluble Aβ may lead to increased levels of soluble forms of this peptide (Patton et al., 2006). Importantly, we observed that vaccination significantly reduced (P<0.001) the levels of potentially toxic forms of amyloid, soluble Aβ42 and Aβ40 peptides in the brains of Lu AF20513 immunized Tg2576 mice compared to control (adjuvant injected) animals (Fig. 5F). Hence, Lu AF20513 vaccination reduces the levels of pathologic forms of Aβ in the brains of immunized mice.

Vaccination with Lu AF20513 reduces glial activation without increasing cerebral amyloid angiopathy and microhemorrhages

To detect inflammation-related pathology in the brains of animals immunized with the Lu AF20513 vaccine, microglial (MHC class II) and astrocyte (glial fibrillary acidic protein, GFAP) activation were examined. Semi-quantitative image analysis of MHC class II-positive cells (I-Ab/I-Eb) demonstrated significantly fewer activated (MHC class II+) microglial cells in Tg2576 mice vaccinated with the Lu AF20513 (Fig. 6A, including representative MHC class II staining). Astrocyte-specific marker in the brains of Lu AF20513 immunized mice revealed a significant reduction in GFAP+ cells via quantitative image analysis indicative of the presence of fewer astrocytes in comparison to the control group (Fig. 6B, including representative GFAP staining).

Figure 6
The less activation of microglia and astrocytes in Lu AF20513 vaccinated Tg2576 mice. A, B. Anti-Aβ antibody reduced glial activation (A) and astrocytosis (B) without changing CAA (C) in the brains of 15–17 mo old Tg2576 mice vaccinated ...

One possible side effect of the disruption of amyloid plaques could be the increase of cerebral amyloid angiopathy (CAA), as was observed in pre-clinical trials (Wilcock et al., 2007) and the AN1792 trial (Nicoll et al., 2003; Ferrer et al., 2004; Masliah et al., 2005). To assess the effect of vaccination with Lu AF20513 on vascular deposition of Aβ, we calculated 6E10-positive vessels in the brain sections of immunized and control Tg2576 mice. No differences in the number of amyloid-containing blood vessels were observed in brains of vaccinated and control mice (Fig. 6C).

However, the ratio of amyloid-containing blood vessels to parenchymal plaques was increased in vaccinated mice vs. control animals (5.07±5.6 vs. 0.24±0.15, respectively) suggesting that amyloid or amyloid-antibody immune complexes may be redistributed to the vessels for clearance from the brain.

Next, we analyzed microhemorrhages in brains of Lu AF20513 immunized and control mice. Previously, it was reported that active immunizations with Aβ42 formulated in strong adjuvants, CFA/IFA (Wilcock et al., 2007) may induce cerebral microhemorrhages in ~20 months old APP/Tg mice. However, microhemorrhages have not been reported in 15–17 months old mice. Characteristic blue hemosiderin-positive profiles were not increased in Lu AF20513 immunized mice compared with control animals (Fig. 6D).

Thus, immunization with Lu AF20513 did not exacerbate cerebral amyloid angiopathy or increase microhemorrhages, and had the positive impact of lowering microglial activation and astrocytosis.

Lu AF20513 induces high titers of anti-Aβ antibodies in natural immune tolerant animal models: guinea pigs and cynomolgus monkeys

Encouraged by the results generated in APP/Tg mice, we tested the Lu AF20513 vaccine in guinea pigs and cynomolgus monkeys after formulation in the widely used mild adjuvant Alhydrogel® to strengthen the link to a clinical situation. Fifteen out of sixteen guinea pigs (94%) responded to two immunizations with Lu AF20513 by producing anti-Aβ antibodies with the average titer equal to 1:7954 ± 7676 (Table 1). Of note, data from the first clinical trial with the AN1792 vaccine demonstrated that anti-Aβ antibody titers ≥1:2200 showed some slowing of cognitive decline and localized reduction of plaques (Hock et al., 2003; Gilman et al., 2005; Masliah et al., 2005; Nicoll et al., 2006). Lu AF20513 vaccine generated therapeutically relevant titers of anti-Aβ antibody (>1:2200) in 80% of responding guinea pigs (Table 1). More importantly, all 14 monkeys immunized twice with Lu AF20513 formulated in Alhydrogel® responded to the vaccine (average titer equal to 1:6491 ± 5338) and 71.5% of these animals generated therapeutically relevant titers of anti-Aβ IgG antibody (Table 2). Of note, in pre-bleed sera (Table 1 and and2)2) we detected only a background level of anti-Aβ antibodies.

Table 1
Humoral immune responses in guinea pigs immunized with Lu AF20513 (20μg/guinea pig) formulated in Alhydrogel®.
Table 2
Humoral immune responses in cynomolgus monkeys immunized with Lu AF20513 (50μg/monkey) formulated in Alhydrogel®.

Analysis of immune mechanism associated with Lu AF20513 vaccinations

One important aspect of the design of the Lu AF20513 is that immunization with this vaccine should quickly induce a robust anti-Aβ antibody production in elderly individuals with pre-existing TT-reactive memory Th cells. To test this hypothesis, we immunized two groups of B6SJL mice with either P30 peptide formulated in Quil-A, or Quil-A, only, and first analyzed the Th memory cells specific to P30 after 6 mo of resting period (Fig. 7A). After in vitro re-stimulation of immune, but not control splenocytes with P30, we detected high numbers of cells producing IFNγ (Fig. 7B). Importantly, depletion of almost 95% of CD4+ cells from primed splenocytes completely abrogated detection of IFNγ-producing cells (Fig. 7C). Moreover, after enrichment of splenocytes isolated from naïve (non-immunized) mice with CD4+ T cells isolated from P30 primed splenocytes (~75% non-immune splenocytes mixed with ~25% of P30 primed CD4+ T cells), we restored immune responses to P30 (Fig. 7D). Hence, these data reveal that after a 6 month resting period, mice have functional memory CD4+ Th cells specific to the P30 epitope of TT vaccine.

Figure 7
Testing the effect of pre-existing memory T cells on generation of anti-Aβ antibody response: simulation of vaccination in humans. A. Two groups of B6SJL mice (8 wks old) were injected three times biweekly with P30 peptide formulated in Quil-A ...

Next we tested whether a single booster injection of Lu AF20513 formulated in Quil-A adjuvant could activate pre-existing anti-P30 specific memory Th cells and lead to a quicker and stronger anti-TT cellular responses and anti-Aβ antibody responses. Thus, after a 6 month resting period, we boosted the P30-primed mice with Lu AF20513 formulated in Quil-A and analyzed both cellular and humoral immune responses (Fig. 8). Boosting of experimental mice with epitope vaccine induced strong Th cell responses specific to P30: very large number of cells producing IFNγ was detected in this group of mice with pre-existing memory Th cells vs control mice (Fig. 8A). Most importantly, the single injection with Lu AF20513 formulated in the strong Th1 adjuvant Quil-A led to induction of robust anti-Aβ antibody responses only in mice with pre-existing memory Th cells: concentrations of anti-Aβ antibodies were significantly higher (P≤0.001) than that in control mice (Fig. 8B). Thus, a single immunization with Lu AF20513 strongly activated pre-existing memory CD4+ T cells specific to the Th epitopes of this vaccine and rapidly led to the robust production of antibodies specific to the B cell epitope (Aβ1–12) of the same vaccine.

Figure 8
The generation of strong cellular and humoral immune responses to Lu AF20513 in mice with pre-existing memory T-cells. A. Single boost of experimental, but not control animals with Lu AF20513 leads to robust activation of these pre-existing memory CD4 ...

Discussion

According to the Amyloid Cascade Hypothesis (Hardy and Higgins, 1992) the accumulation of Aβ peptide is a primary pathological event in the development of AD, that precedes tau accumulation and leads to neurodegeneration and dementia. The identification of the Aβ peptide as a target for therapeutic interventions for AD has led to many preclinical and clinical studies. Despite the fact that the recent Pfizer’s and Eli-Lilly’s clinical data with passively administrated anti-Aβ antibodies have been disappointing, there is a consensus in the field that the removal or lowering of Aβ in patients with very early AD pathology or even in pre-symptomatic subjects could be an effective measure. Obviously, for such preventive treatments passive vaccination is not practical while a safe active vaccine might be beneficial. Although the first clinical trial with AN1792 was unsuccessful due to meningoencephalitis (possibly caused by the activation of autoreactive T cells) (Nicoll et al., 2003; Ferrer et al., 2004; Masliah et al., 2005), currently various epitope-based vaccines, similar to one that we suggested earlier (Agadjanyan et al., 2005; Petrushina et al., 2007; Movsesyan et al., 2008b), are in clinical trials (for examples UBITh®, V950, CAD106, ACC-001) (Wang et al., 2007; Lemere and Masliah, 2010; Wiessner et al., 2011; Winblad et al., 2012; http://clinicaltrials.gov). Designs of these vaccines aim to circumvent the problem of T cell autoreactivity and overcome tolerance induction to self-antigen (Nicoll et al., 2003; Ferrer et al., 2004; Masliah et al., 2005). Currently, the published data are available only on clinical trial with CAD106 composed of bacteriophage Qβ expressing Aβ1–6 B cell epitope. Although, the authors reported that anti-Aβ antibodies were detected in 62% of low dose and 82% of high dose subjects, the exact titers of antibodies are not presented, so actual magnitude of the humoral immune responses is not clear (Winblad et al., 2012). Importantly, no data have been presented in this report on antigen-specific cellular immune responses generated after vaccinations of AD patients. Likewise, humoral and cellular immune responses to CAD106 have not been properly reported in pre-clinical mouse and monkey studies (Wiessner et al., 2011).

In this study, we tested a novel clinical grade epitope vaccine, Lu AF20513 composed of the same immunodominant B cell epitope of Aβ42, (Aβ1–12) fused with two Th epitopes from a conventional TT vaccine that are recognized by different human MHC-class II molecules (James et al., 2007). Importantly, an active immunization approach has been tested in Tg2576 mice with very early-stage AD-like pathology. In our previous studies we tested several protein, peptide, and DNA-based AD epitope vaccines containing a synthetic Th cell epitope PADRE and showed that they are immunogenic in mice and do not induce T cell responses against self-Aβ molecules (Mamikonyan et al., 2007; Petrushina et al., 2007; Movsesyan et al., 2008b; Movsesyan et al., 2008a; Ghochikyan, 2009; Davtyan et al., 2010; Davtyan et al., 2012). In the design of the Lu AF20513 vaccine, we replaced PADRE with P2 and P30 epitopes (Fig. 1A) with the goal of activating non-self pre-existing memory Th cells in the general human population, which is immune to TT due to the public health vaccination program. Data from this study demonstrated that Lu AF20513 induced therapeutically potent Aβ-specific humoral immune responses in mice (Fig. 3), and strong anti-P2 and anti-P30 Th cell responses without the activation of autoreactive Th cells (Fig. 2). These anti-Aβ antibodies bound to oligomeric and fibrillar forms of Aβ (Fig. 4A,B) and pathological plaques in the brain sections from an AD patient (Fig. 4C), while also protecting neuronal cells from Aβ42 oligomer- and fibril-mediated toxicity (Fig. 4D). The most striking is our observation that Lu AF20513 vaccination offers protection from the development of cored and diffuse plaques in the brains of Tg2576 mice (Fig. 5A–D).

One potential problem with immunotherapy is the fact that a reduction of insoluble Aβ plaques may lead to increased levels of soluble forms of Aβ, which are toxic to neurons. Two major mechanisms for antibody-mediated clearance of Aβ have been suggested: sequestration of Aβ from the CNS into the periphery (‘peripheral sink’) (DeMattos et al., 2001) and entry of anti-Aβ antibodies into the CNS and clearance of antigen-antibody complexes by microglial cells (Bard et al., 2000). Regardless of the mechanism of action, we observed that anti-Aβ antibodies generated by Lu AF20513 vaccinations inhibited the accumulation of not only insoluble, but also of soluble forms of Aβ42 and Aβ40 peptides in the brains of experimental vs control Tg2576 mice (Fig. 5E,F). Importantly, despite the significant reduction in plaque burden and soluble Aβ42, no differences in CAA was observed in brains of vaccinated mice vs control animals (Fig. 6C). Increased ratio of amyloid-containing blood vessels to the numbers of parenchymal plaques in vaccinated mice suggests that anti-Aβ antibodies were not as effective at inhibiting cerebral vascular deposition of Aβ as they were at blocking amyloid plaque formation. In addition, we demonstrated that the inhibition of Aβ42 and Aβ40 depositions in the brains of Lu AF20513 vaccinated Tg2576 mice is associated with a reduction in the frequencies of activated microglia and astrocytes (Fig. 6A,B).

Because our data on glial activation was collected at the end of 11 months of active immunotherapy we cannot exclude the possibility that glial cells were activated acutely following active immunization protocol due to Fc-mediated microglial activation in response to antibody-Aβ immune complex formation in the CNS as it was observed in old (19–20 mo old) APP Tg mice, with substantial pre-existing parenchymal as well as CAA, in response to immunotherapy (Wilcock et al., 2001). However, we should mention that because immunization was initiated prior to the onset of amyloid deposition (Tg2576 mice were 4–6 mo old) we believe that any acute inflammatory response would be rather mild under these experimental conditions. Moreover, we believe that early immunotherapy intervention may be critical to reduce the incidence of adverse cerebrovascular events, such as microhemorrhages and vasogenic edema.

Collectively, data generated in Tg2576 mice vaccinated with Lu AF20513 suggest that this epitope vaccine may be safe for translation to human clinical studies. Of note, in this protective study the behavioral changes in the cohort of vaccinated and control Tg2576 mice were not analyzed since there are many reports demonstrating that immunization with Aβ42 generates antibodies specific to N-terminus of Aβ (Janus et al., 2000; Lemere et al., 2000; Morgan et al., 2000) that improve cognitive functions in various APP mice including Tg2576, especially if vaccination is started prior to onset of amyloid-like pathology. In addition, results from previous active and passive vaccination clinical trials suggest that early intervention with immunotherapeutic approaches are likely to be most clinically effective (Lemere and Masliah, 2010; Delrieu et al., 2012; Eli Lilly and Company Announcement, 2012; Johnson & Johnson Announcement, 2012).

We have also included analysis of anti-Aβ antibody titers in guinea pigs and monkeys, which demonstrated the immunogenic potential of the Lu AF20513 vaccine in animals that are naturally immune tolerant to Aβ42 and exhibit diverse genetic backgrounds for MHC class II molecules as compared to mice. Two immunizations of guinea pigs where sufficient for the generation of anti-Aβ IgG antibody responses in 15 out of 16 immunized animals, and 80% of responders generated anti-Aβ antibody titers over 1:2,200 (Table 1). These represent therapeutically relevant titers, as was previously reported in the AN1792 trials (Gilman et al., 2005). Perhaps most importantly, Lu AF20513 vaccinations induced robust humoral immune responses in 100% of cynomolgus monkeys and in 71.5% of monkeys the anti-Aβ antibody titers were therapeutically relevant (Table 2). These findings may translate into adequate clinical responses to vaccination in the elderly, where the generation of robust immunity is more difficult due to immunological senescence.

It is important to note that while several different epitope vaccines are currently in clinical trials (Wang et al., 2007; Lemere and Masliah, 2010; Wiessner et al., 2011; Ghochikyan and Agadjanyan, 2012) and (http://clinicaltrials.gov), the immune responses generated by these vaccines have not been fully characterized: to our knowledge, there are no published data on the exact specificity of Th cells that could be activated by these vaccines either in animal models of AD or in humans. Since the antigen-specific T cell responses were never defined, our challenge was to design an active vaccine against AD in humans, with polymorphic MHC genes, that overcomes the safety and efficacy barriers of previous approaches. Here, we revealed the immunological mechanism of action of Lu AF20513 by demonstrating that this vaccine does indeed activate Th cells specific to TT, leading to enhanced anti-Aβ antibody production. The most remarkable finding in this study is that a single injection with Lu AF20513 activates pre-existing memory CD4+ T cells specific to foreign Th epitopes and quickly induces strong anti-Aβ antibody responses (Fig. 8). Therefore, this Lu AF20513 may represent an effective and safe form of active immunotherapy that may overcome the limited ability of the elderly to respond to vaccinations by activating pre-existing anti-P30/P2 memory Th cells (Fig 9) in the general human population vaccinated with a conventional TT vaccine.

Figure 9
Proposed model of early therapeutic or preventive Lu AF20513 vaccination. Model of vaccination of patients with early stage of AD (early therapeutic) or asymptomatic subjects at risk of AD (preventive) possessing pre-existing memory Th cells specific ...

Acknowledgments

This work was supported by funding from NIH (NS-50895, NS-065518, AG-20241 and NS 057395) and Alzheimer’s Association (IIRG-0728314). H.D. and N.M. were supported by NIA T32 training grant (AG000096). Additional support for AD case tissues was provided by University of California, Irvine Alzheimer’s Disease Research Center Grant P50 AG16573. We thank Jasja Wolthoorn (TNO Triskelion), Robert Pels Rijcken (TNO Triskelion), and Hervé Giorgi (Ricerca Biosciences SAS) for their services in conducting the guinea pig and monkey studies.

Footnotes

Conflict of Interest: A.K. Larsen, P.J. Madsen, D.K. Ditlevsen are employees of H. Lundbeck A/S; K.M. Wegener and L.O. Pedersen are employees and shareholders of H. Lundbeck A/S.

Author Contributions

H.D., A.G., A.K.L., D.H.C., L.O.P. and M.G.A. designed research; H.D., A.G., I.P., A.H., A.D., A.P., N.M., A.K., S.R., P.J.M., K.M.W. and D.K.D. performed research; H.D., A.G., I.P., A.M.M, A.K., A.K.L., K.M.W., D.K.D, D.H.C., L.O.P. and M.G.A analyzed data; H.D., A.G., I.P., A.M.M., L.O.P and M.G.A. wrote the paper.

References

  • Agadjanyan MG, Ghochikyan A, Petrushina I, Vasilevko V, Movsesyan N, Mkrtichyan M, Saing T, Cribbs DH. Prototype Alzheimer’s disease vaccine using the immunodominant B cell epitope from beta-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide. J Immunol. 2005;174:1580–1586. [PubMed]
  • Bard F, Cannon C, Barbour R, Burke RL, Games D, Grajeda H, Guido T, Hu K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M, Lieberburg I, Motter R, Nguyen M, Soriano F, Vasquez N, Weiss K, Welch B, Seubert P, Schenk D, Yednock T. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med. 2000;6:916–919. [PubMed]
  • Boche D, Zotova E, Weller RO, Love S, Neal JW, Pickering RM, Wilkinson D, Holmes C, Nicoll JA. Consequence of Abeta immunization on the vasculature of human Alzheimer’s disease brain. Brain. 2008;131:3299–3310. [PubMed]
  • Cleary JP, Walsh DM, Hofmeister JJ, Shankar GM, Kuskowski MA, Selkoe DJ, Ashe KH. Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function. Nat Neurosci. 2005;8:79–84. [PubMed]
  • Cribbs DH, Ghochikyan A, Tran M, Vasilevko V, Petrushina I, Sadzikava N, Kesslak P, Kieber-Emmons T, Cotman CW, Agadjanyan MG. Adjuvant-dependent modulation of Th1 and Th2 responses to immunization with beta-amyloid. Int Immunol. 2003;15:505–514. [PMC free article] [PubMed]
  • Davtyan H, Mkrtichyan M, Movsesyan N, Petrushina I, Mamikonyan G, Cribbs DH, Agadjanyan MG, Ghochikyan A. DNA prime-protein boost increased the titer, avidity and persistence of anti-Abeta antibodies in wild-type mice. Gene Ther. 2010;17:261–271. [PMC free article] [PubMed]
  • Davtyan H, Ghochikyan A, Movsesyan N, Ellefsen B, Petrushina I, Cribbs DH, Hannaman D, Evans CF, Agadjanyan MG. Delivery of a DNA Vaccine for Alzheimer’s Disease by Electroporation versus Gene Gun Generates Potent and Similar Immune Responses. Neurodegener Dis. 2012;10:261–264. [PMC free article] [PubMed]
  • Davtyan H, Ghochikyan A, Cadagan R, Zamarin D, Petrushina I, Movsesyan N, Martinez-Sobrido L, Albrecht RA, Garcia-Sastre A, Agadjanyan MG. The immunological potency and therapeutic potential of a prototype dual vaccine against influenza and Alzheimer’s disease. J Transl Med. 2011;9:127. [PMC free article] [PubMed]
  • De Felice FG, Wu D, Lambert MP, Fernandez SJ, Velasco PT, Lacor PN, Bigio EH, Jerecic J, Acton PJ, Shughrue PJ, Chen-Dodson E, Kinney GG, Klein WL. Alzheimer’s disease-type neuronal tau hyperphosphorylation induced by A beta oligomers. Neurobiol Aging. 2008;29:1334–1347. [PMC free article] [PubMed]
  • Delrieu J, Ousset PJ, Caillaud C, Vellas B. ‘Clinical trials in Alzheimer’s disease’: immunotherapy approaches. J Neurochem. 2012;120(Suppl 1):186–193. [PubMed]
  • DeMattos RB, Bales KR, Cummins DJ, Dodart JC, Paul SM, Holtzman DM. Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases brain A beta burden in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci U S A. 2001;98:8850–8855. [PubMed]
  • Eli Lilly and Company. Announces Top-Line Results on Solanezumab Phase 3 Clinical Trials in Patients with Alzheimer’s Disease. 2012 Available at http://newsroom.lilly.com/releasedetail.cfm?releaseid=702211.
  • Ferrer I, Rovira MB, Guerra MLS, Rey MJ, Costa-Jussa F. Neuropathology and pathogenesis of encephalitis following amyloid-beta immunization in Alzheimer’s disease. Brain Pathol. 2004;14:11–20. [PubMed]
  • Ghochikyan A. Rationale for peptide and DNA based epitope vaccines for Alzheimer’s disease immunotherapy. CNS Neurol Disord Drug Targets. 2009;8:128–143. [PMC free article] [PubMed]
  • Ghochikyan A, Agadjanyan MG. CAD-106, a beta-amyloid-based immunotherapeutic for Alzheimer’s disease. Thompson Reuter; 2012. https://partnering.thomson-pharma.com.
  • Ghochikyan A, Mkrtichyan M, Petrushina I, Movsesyan N, Karapetyan A, Cribbs DH, Agadjanyan MG. Prototype Alzheimer’s disease epitope vaccine induced strong Th2-type anti-Abeta antibody response with Alum to Quil A adjuvant switch. Vaccine. 2006;24:2275–2282. [PMC free article] [PubMed]
  • Ghochikyan A, Vasilevko V, Petrushina I, Tran M, Sadzikava N, Babikyan D, Movsesyan N, Tian W, Ross TM, Cribbs DH, Agadjanyan MG. Generation and chracterization of the humoral immune response to DNA immunization with a chimeric β-amyloid-interleukin-4 minigene. Eur J Immunol. 2003;33:3232–3241. [PMC free article] [PubMed]
  • Gilman S, Koller M, Black RS, Jenkins L, Griffith SG, Fox NC, Eisner L, Kirby L, Rovira MB, Forette F, Orgogozo JM. Clinical effects of Abeta immunization (AN1792) in patients with AD in an interrupted trial. Neurology. 2005;64:1553–1562. [PubMed]
  • Glabe CG. Structural classification of toxic amyloid oligomers. J Biol Chem. 2008;283:29639–29643. [PubMed]
  • Golde TE, Dickson D, Hutton M. Filling the gaps in the abeta cascade hypothesis of Alzheimer’s disease. Curr Alzheimer Res. 2006;3:421–430. [PubMed]
  • Haass C, Selkoe DJ. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid beta-peptide. Nat Rev Mol Cell Biol. 2007;8:101–112. [PubMed]
  • Hardy J. Has the amyloid cascade hypothesis for Alzheimer’s disease been proved? Curr Alzheimer Res. 2006;3:71–73. [PubMed]
  • Hardy J, Allsop D. Amyloid deposition as the central event in the aetiology of Alzheimer’s disease. Trends Pharmacol Sci. 1991;12:383–388. [PubMed]
  • Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science. 2002;297:353–356. [PubMed]
  • Hardy JA, Higgins GA. Alzheimer’s disease: the amyloid cascade hypothesis. Science. 1992;256:184–185. [PubMed]
  • Harper JD, Wong SS, Lieber CM, Lansbury PT. Observation of metastable Abeta amyloid protofibrils by atomic force microscopy. Chem Biol. 1997;4:119–125. [PubMed]
  • Hock C, Konietzko U, Streffer JR, Tracy J, Signorell A, Muller-Tillmanns B, Lemke U, Henke K, Moritz E, Garcia E, Wollmer MA, Umbricht D, de Quervain DJ, Hofmann M, Maddalena A, Papassotiropoulos A, Nitsch RM. Antibodies against beta-Amyloid Slow Cognitive Decline in Alzheimer’s Disease. Neuron. 2003;38:547–554. [PubMed]
  • Holmes C, Boche D, Wilkinson D, Yadegarfar G, Hopkins V, Bayer A, Jones RW, Bullock R, Love S, Neal JW, Zotova E, Nicoll JA. Long-term effects of Abeta42 immunization in Alzheimer’s disease: follow-up of a randomised, placebo-controlled phase I trial. Lancet. 2008;372:216–223. [PubMed]
  • Holtzman DM, Morris JC, Goate AM. Alzheimer’s disease: the challenge of the second century. Sci Transl Med. 2011;3:77sr71. [PMC free article] [PubMed]
  • Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G. Correlative memory deficits, Aβ elevation, and amyloid plaques in transgenic mice. Science. 1996;274:99–102. [PubMed]
  • James EA, Bui J, Berger D, Huston L, Roti M, Kwok WW. Tetramer-guided epitope mapping reveals broad, individualized repertoires of tetanus toxin-specific CD4+ T cells and suggests HLA-based differences in epitope recognition. Int Immunol. 2007;19:1291–1301. [PubMed]
  • Janus C, Pearson J, McLaurin J, Mathews PM, Jiang Y, Schmidt SD, Chishti MA, Horne P, Heslin D, French J, Mount HT, Nixon RA, Mercken M, Bergeron C, Fraser PE, St George-Hyslop P, Westaway D. A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer’s disease. Nature. 2000;408:979–982. [PubMed]
  • Johnson & Johnson. Announces Discontinuation Of Phase 3 Development of Bapineuzumab Intravenous (IV) In Mild-To-Moderate Alzheimer’s Disease. 2012 Found at http://www.jnj.com/connect/news/all/johnson-and-johnson-announces-discontinuation-of-phase-3-development-of-bapineuzumab-intravenous-iv-in-mild-to-moderate-alzheimers-disease.
  • Kayed R, Canto I, Breydo L, Rasool S, Lukacsovich T, Wu J, Albay R, 3rd, Pensalfini A, Yeung S, Head E, Marsh JL, Glabe C. Conformation dependent monoclonal antibodies distinguish different replicating strains or conformers of prefibrillar Abeta oligomers. Mol Neurodegener. 2010;5:57. [PMC free article] [PubMed]
  • Kayed R, Head E, Sarsoza F, Saing T, Cotman CW, Necula M, Margol L, Wu J, Breydo L, Thompson JL, Rasool S, Gurlo T, Butler P, Glabe CG. Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers. Mol Neurodegener. 2007;2:18. [PMC free article] [PubMed]
  • Klein WL, Stine WB, Jr, Teplow DB. Small assemblies of unmodified amyloid beta-protein are the proximate neurotoxin in Alzheimer’s disease. Neurobiol Aging. 2004;25:569–580. [PubMed]
  • Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Liosatos M, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C, Finch CE, Krafft GA, Klein WL. Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A. 1998;95:6448–6453. [PubMed]
  • Lemere CA. Developing novel immunogens for a safe and effective Alzheimer’s disease vaccine. Prog Brain Res. 2009;175:83–93. [PMC free article] [PubMed]
  • Lemere CA, Masliah E. Can Alzheimer disease be prevented by amyloid-beta immunotherapy? Nat Rev Neurol. 2010;6:108–119. [PMC free article] [PubMed]
  • Lemere CA, Maron R, Spooner ET, Grenfell TJ, Mori C, Desai R, Hancock WW, Weiner HL, Selkoe DJ. Nasal Aβ Treatment Induces Anti-Aβ Antibody Production and Decreases Cerebral Amyloid Burden in PD-APP Mice. Annals of the New York Academy of Sciences. 2000;920:328–331. [PubMed]
  • Lesne S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A, Gallagher M, Ashe KH. A specific amyloid-beta protein assembly in the brain impairs memory. Nature. 2006;440:352–357. [PubMed]
  • Mamikonyan G, Necula M, Mkrtichyan M, Ghochikyan A, Petrushina I, Movsesyan N, Mina E, Kiyatkin A, Glabe C, Cribbs DH, Agadjanyan MG. Anti-Abeta 1-11 antibody binds to different beta-amyloid species, inhibits fibril formation, and disaggregates preformed fibrils, but not the most toxic oligomers. J Biol Chem. 2007;282:22376–22386. [PMC free article] [PubMed]
  • Masliah E, Hansen L, Adame A, Crews L, Bard F, Lee C, Seubert P, Games D, Kirby L, Schenk D. Abeta vaccination effects on plaque pathology in the absence of encephalitis in Alzheimer disease. Neurology. 2005;64:129–131. [PubMed]
  • Morgan D, Diamond DM, Gottschall PE, Ugen KE, Dickey C, Hardy J, Duff K, Jantzen P, DiCarlo G, Wilcock D, Connor K, Hatcher J, Hope C, Gordon M, Arendash GW. A beta peptide vaccination prevents memory loss in an animal model of Alzheimer’s disease. Nature. 2000;408:982–985. [PubMed]
  • Movsesyan N, Mkrtichyan M, Petrushina I, Ross TM, Cribbs DH, Agadjanyan MG, Ghochikyan A. DNA epitope vaccine containing complement component C3d enhances anti-amyloid-beta antibody production and polarizes the immune response towards a Th2 phenotype. J Neuroimmunol. 2008a;205:57–63. [PMC free article] [PubMed]
  • Movsesyan N, Ghochikyan A, Mkrtichyan M, Petrushina I, Davtyan H, Olkhanud PB, Head E, Biragyn A, Cribbs DH, Agadjanyan MG. Reducing AD-like pathology in 3xTg-AD mouse model by DNA epitope vaccine- a novel immunotherapeutic strategy. PLos ONE. 2008b;3:e21–24. [PMC free article] [PubMed]
  • Nicoll JA, Wilkinson D, Holmes C, Steart P, Markham H, Weller RO. Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nat Med. 2003;9:448–452. [PubMed]
  • Nicoll JA, Barton E, Boche D, Neal JW, Ferrer I, Thompson P, Vlachouli C, Wilkinson D, Bayer A, Games D, Seubert P, Schenk D, Holmes C. Abeta species removal after abeta42 immunization. J Neuropathol Exp Neurol. 2006;65:1040–1048. [PubMed]
  • Nikolaev A, McLaughlin T, O’Leary DD, Tessier-Lavigne M. APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature. 2009;457:981–989. [PMC free article] [PubMed]
  • Orgogozo JM, Gilman S, Dartigues JM, Laurent B, Puel M, Kirby LC, Jouanny P, Dubois B, Eisner L, Flitman S, Michel BF, Boada M, Frank A, Hock C. Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization. Neurology. 2003;61 (1):46–54. [PubMed]
  • Patton RL, Kalback WM, Esh CL, Kokjohn TA, Van Vickle GD, Luehrs DC, Kuo YM, Lopez J, Brune D, Ferrer I, Masliah E, Newel AJ, Beach TG, Castano EM, Roher AE. Amyloid-beta peptide remnants in AN-1792-immunized Alzheimer’s disease patients: a biochemical analysis. Am J Pathol. 2006;169:1048–1063. [PubMed]
  • Petrushina I, Tran M, Sadzikava N, Ghochikyan A, Vasilevko V, Agadjanyan MG, Cribbs DH. Importance of IgG2c isotype in the immune response to b-amyloid in APP/Tg mice. Neurosci Letters. 2003;338:5–8. [PubMed]
  • Petrushina I, Ghochikyan A, Mktrichyan M, Mamikonyan G, Movsesyan N, Davtyan H, Patel A, Head E, Cribbs DH, Agadjanyan MG. Alzheimer’s Disease Peptide Epitope Vaccine Reduces Insoluble But Not Soluble/Oligomeric A{beta} Species in Amyloid Precursor Protein Transgenic Mice. J Neurosci. 2007;27:12721–12731. [PMC free article] [PubMed]
  • Pike CJ, Walencewicz AJ, Glabe CG, Cotman CW. In vitro aging of beta-amyloid protein causes peptide aggregation and neurotoxicity. Brain Res. 1991;563:311–314. [PubMed]
  • Price DL, Sisodia SS. Cellular and molecular biology of Alzheimer’s disease and animal models. Annu Rev Med. 1994;45:435–446. [PubMed]
  • Rafii MS, Aisen PS. Recent developments in Alzheimer’s disease therapeutics. BMC Med. 2009;7:7. [PMC free article] [PubMed]
  • Selkoe DJ. The molecular pathology of Alzheimer’s disease. Neuron. 1991;6:487–498. [PubMed]
  • Selkoe DJ. Alzheimer’s disease: a central role for amyloid. J Neuropath and Exp Neurology. 1994;53:438–447. [PubMed]
  • Shankar GM, Li S, Mehta TH, Garcia-Munoz A, Shepardson NE, Smith I, Brett FM, Farrell MA, Rowan MJ, Lemere CA, Regan CM, Walsh DM, Sabatini BL, Selkoe DJ. Amyloid-beta protein dimers isolated directly from Alzheimer’s brains impair synaptic plasticity and memory. Nat Med. 2008;14:837–842. [PMC free article] [PubMed]
  • Sigurdsson EM, Knudsen E, Asuni A, Fitzer-Attas C, Sage D, Quartermain D, Goni F, Frangione B, Wisniewski T. An attenuated immune response is sufficient to enhance cognition in an Alzheimer’s disease mouse model immunized with amyloid-beta derivatives. J Neurosci. 2004;24:6277–6282. [PubMed]
  • Walsh DM, Lomakin A, Benedek GB, Condron MM, Teplow DB. Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate. J Biol Chem. 1997;272:22364–22372. [PubMed]
  • Wang CY, Finstad CL, Walfield AM, Sia C, Sokoll KK, Chang TY, Fang XD, Hung CH, Hutter-Paier B, Windisch M. Site-specific UBITh amyloid-beta vaccine for immunotherapy of Alzheimer’s disease. Vaccine. 2007;25:3041–3052. [PubMed]
  • Wiessner C, Wiederhold KH, Tissot AC, Frey P, Danner S, Jacobson LH, Jennings GT, Luond R, Ortmann R, Reichwald J, Zurini M, Mir A, Bachmann MF, Staufenbiel M. The second-generation active Abeta immunotherapy CAD106 reduces amyloid accumulation in APP transgenic mice while minimizing potential side effects. J Neurosci. 2011;31:9323–9331. [PubMed]
  • Wilcock DM, Jantzen PT, Li Q, Morgan D, Gordon MN. Amyloid-b vaccination, but not nitro-NSAID treatment, increases vascular amyloid and microhemorrhage while both reduce parenchymal amyloid. Neuroscience. 2007;144:950–960. [PMC free article] [PubMed]
  • Wilcock DW, Gordon MN, Ugen KE, Gottschall PE, Dicarlo G, Dickey C, Boyett KW, Jantzen PT, Connor KC, Malachrino J, Hardy J, Morgan D. Number of Ab inoculations in APP+PS1 transgenic mice influences antibody titers, microglia activation, and congophilic plaque levels. DNA and Cell Biology. 2001;20:731–736. [PubMed]
  • Winblad B, Andreasen N, Minthon L, Floesser A, Imbert G, Dumortier T, Maguire RP, Blennow K, Lundmark J, Staufenbiel M, Orgogozo JM, Graf A. Safety, tolerability, and antibody response of active Abeta immunotherapy with CAD106 in patients with Alzheimer’s disease: randomised, double-blind, placebo-controlled, first-in-human study. Lancet Neurol. 2012;11:597–604. [PubMed]
  • Yong W, Lomakin A, Kirkitadze MD, Teplow DB, Chen SH, Benedek GB. Structure determination of micelle-like intermediates in amyloid beta -protein fibril assembly by using small angle neutron scattering. Proc Natl Acad Sci U S A. 2002;99:150–154. [PubMed]