Reduction of amyloid β-peptide accumulation in Tg2576 transgenic mice by oral vaccination

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Abstract

Alzheimer’s disease (AD) is pathologically characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles. Amyloid β-peptide (Aβ) is the main component of senile plaques, and the pathological load of Aβ in the brain has been shown to be a marker of the severity of AD. Aβ is produced from the amyloid precursor protein by membrane proteases and is known to aggregate. Recently, immune-mediated cerebral clearance of Aβ has been studied extensively as potential therapeutic strategy. In previous studies that used a purified Aβ challenge in a mouse model of AD, symptomatic improvement was reported. However, a clinical Alzheimer’s vaccine trial in the United States was stopped because of severe side effects. Immunization with the strong adjuvant used in these trials might have activated an inflammatory Th1 response.

In this study, to establish a novel, safer, lower-cost therapy for AD, we tested an oral vaccination in a wild-type and a transgenic mouse model of AD administered via green pepper leaves expressing GFP-Aβ. Anti-Aβ antibodies were effectively induced after oral immunization. We examined the immunological effects in detail and identified no inflammatory reactions. Furthermore, we demonstrated a reduction of Aβ in the immunized AD-model mice. These results suggest this edible vehicle for Aβ vaccination has a potential clinical application in the treatment of AD.

Research highlights

► A novel therapy for Alzheimer disease (AD) using oral vaccination. ► Oral Abeta immunization is less likely to induce inflammatory reactions. ► Food vaccination is easy to administer, without a need for refrigeration. ► 70% Reduction of senile plaques was observed in transgenic AD mice.

Introduction

Alzheimer’s disease (AD) is one of the major causes of chronic and progressive age-associated cognitive decline, with characteristic pathological hallmarks including extracellular senile plaques, intracellular hyperphosphorylated tau in neurofibrillary tangles, and brain weight loss [1], [2].

Amyloid β-peptide (Aβ), which is reported to aggregate spontaneously, is the major constituent of the senile plaques in patients with AD. Aβ has been reported to accumulate on the outside of nerve cells or in the cerebral vascular walls. According to the amyloid cascade hypothesis, the gradual cerebral accumulation of soluble and insoluble assemblies of Aβ in the limbic and association cortices triggers a cascade of biochemical and cellular alterations that produce the clinical phenotype of AD. Therefore, soluble or insoluble Aβ is regarded as a therapeutic target of the progression of AD.

Aβ is generated by the sequential proteolytic processing of the amyloid precursor protein (APP) by membrane proteases. In human brains, the longer Aβ peptide, Aβ42, has a greater propensity to aggregate than the shorter Aβ40.

In 1999, a vaccination therapy to Aβ was developed as a novel therapeutic method against AD. These experiments employed APP transgenic mouse model of AD (PDAPP) mice, which overexpress mutant human APP and progressively develop senile plaques or neurofibrillary tangles in an age- and brain region-dependent manner. In the study, transgenic mice were immunized with synthesized Aβ42 either before (at 6 weeks of age) or after (11 months) the onset of neuropathologies. The study reported that the antibody titer against Aβ42 was elevated, and immunization of the young animals essentially prevented the development of senile plaques, neurofibrillary tangles, and neuritic atrophy. Treatment of the older animals also markedly reduced the extent and progression of these AD-like neuropathologies [3]. This reduction in the number of senile plaques was observed not only with active immunization but also with passive immunization [4] and with the mucosal immunization delivered via the nose [5].

Transgenic mouse models of AD, such as PDAPP mice, begin to display a learning disability derived from the cognitive disorder in a water maze test at around 15 months of age, but this learning disorder improves with the Aβ42 immunization [6], [7], [8], [9]. In 2001, the Elan Corporation in the United States examined the clinical application of Aβ immunization. However, during the phase II trial, some patients presented with acute meningitis as a side effect after the second immunization, and this clinical test was suspended [10]. Adjuvants used in the muscular injection of the synthesized Aβ42 have high immunological activity and induce cell-mediated immune responses such as T-lymphocyte activation. In some cases, Aβ or APP-reactive killer T cells may have invaded the brain causing allergic meningitis. Despite the suspension of the clinical trial, some data suggest that the vaccination therapy may be effective for Aβ clearance [11]. Overall, these results indicate that safer and cheaper vaccination methods are needed, in particular those that do not require injection, strong adjuvants, and expensive synthesized peptides.

As for the safety of the vaccine, physiological immunoreactivity against the vaccine is of great importance. In the human body, immunoreactivity against “the self” is usually suppressed, a phenomenon called immunotolerance. “Self” immunotolerance is affected by the balance between Th1 and Th2 helper T cells. When the balance between Th1 and Th2 T cells changes and Th1 cells are activated, cellular reactions to “the self” increase, tending to give rise to autoimmune diseases such as meningitis. When Th2 cells are activated, antibody production is induced and anti-inflammatory cytokines are released to alleviate the inflammation induced by Th1 cells. Safer vaccine therapies that induce the Th2 reaction via mucosal immunization are now being tested in animal models. In mucosal immunization, immune activity tends to be feeble, immunotolerance can be induced, and adjuvants delivered with the target antigen proteins must be taken into consideration. In an experiment in which a DNA vaccine was administered via the nose, Kim et al. delivered Aβ and adenovirus vectors coding granulocyte colony-stimulating factor (G-CSF) as the adjuvant, and effective elevation of anti-antibody titer and continual induction of Th2 reaction was reported [14].

Many studies of vaccination therapy for AD have been conducted, such as subcutaneous or nasal mucosal vaccination using synthetic peptide or DNA [5], [15], [16], but oral vaccination using an edible, virally transduced plant as a vehicle has some benefits [17], [18]. First, because only the target subunits or fragments are expressed, and not all the pathogenic virus or proteins, it is a safer vaccine. Furthermore, purification of the target protein and cold storage are not required, and the cost is low. Plant biotechnology provides methods for introducing animal genes into plants.

In this study, we tested a plant-based vaccination therapy in an animal model of AD. We expressed green fluorescent protein (GFP)-conjugated Aβ in pepper leaves using a plant virus and fed the leaves to wild-type or transgenic mice. We used the cholera toxin B subunit (CTB) as the oral adjuvant. CTB suppresses Th1 cell reactivity and is known to form a pentamer, binding to GM1 gangliosides on the cell surface. Then we examined the immunological reaction in mice and checked the effectiveness and safety of this plant-based vaccination.

We succeeded in stably expressing GFP-conjugated Aβ in green pepper leaves using a plant virus, TocJ. Oral vaccination of wild-type and Tg2576 transgenic mice resulted in the elevation of Aβ antibody titer without significant side effects.

Section snippets

Mice

We used female Tg2576 transgenic mice (670th amino acid of APP mutated from Lys to Asn, and 671st from Met to Leu), a model of Swedish familial AD, and female wild-type B6 mice.

Plants

For the plant-based vaccine, we used leaves of Capiscum annuum var. angulosum virally transduced with GFP-conjugated Aβ42 using a plant virus, TocJ [18], [21]. Viral transduction with GFP alone was used as a negative control.

Quantification of Aβ expression in leaves of Capiscum annuum var. angulosum

The leaves were suspended in phosphate-buffered saline (PBS), homogenized, and centrifuged at

Quantification of Aβ expression in plant leaves

We performed SDS–PAGE and Western blotting for green pepper leaf extracts in PBS. We used the mouse monoclonal antibody 6E10 as the primary antibody to detect Aβ. Then we quantified the expression of Aβ using synthesized mouse IgG as the protein standard. The amount of Aβ was 100–600 μg for 1 g of plant leaves, depending on the preparation.

Production of anti-Aβ antibodies in wild-type B6 mice

To study the course of antibody production after vaccination and the precise immunological response, subcutaneous or oral immunization of wild-type B6 mice was

Discussion

The hypothesis that Aβ vaccination should prevent senile plaque formation was raised in an experiment by Schenk et al. The authors put several kinds of antibodies against Aβ on frozen brain slices with senile plaques either from transgenic mice or from human patients with AD. Then the brain slices were incubated with a reagent containing microglia and phagocytes. If the antibodies were specific to Aβ, the microglia phagocytized Aβ on the brain section. This did not happen with antibodies to

Acknowledgments

We thank Miss Hisaka Sato for valuable discussions. This work was supported in part by the Human Frontier Science Program and a research grant from the Ministry of Health, Labor and Welfare, Japan to S.I.

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    These authors are equally contributed to this work.

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