Aβ3-10基因疫苗对幼年鼠脑内老年斑沉积及记忆障碍预防作用的研究

目的构建表达重复10次的Aβ3-10肽段的DNA疫苗,并探讨其对幼年APP/PS1转基因鼠脑内Aβ沉积的预防及对其延迟记忆障碍的作用。方法将该疫苗用体外电穿孔的方法肌肉免疫3月龄的APP/PS1双转基因鼠,并分别做行为学、Aβ抗体、脾细胞培养上清细胞因子、脑内Aβ沉积及星型胶质细胞测定。结果 p(Aβ3-10)10-MT和Aβ42组免疫一次后即产生抗体,并随着免疫次数增多而增加,类型主要为IgG1,IgG1/IgG2a明显高于Aβ42组。在Morris水迷宫中,p(Aβ3-10)10-MT和Aβ42组潜伏期明显减短,空间探索实验在平台象限所在时间均较pc DNA3.1组长。p(Aβ3-10)10-MT和Aβ42组脾细胞培养上清IL-4和IFN-γ均增高,而在p(Aβ3-10)10-MT组,IL-4较高,IFN-γ较低。免疫组化结果提示皮质和海马区老年斑沉积减少。结论 DNA疫苗p(Aβ3-10)10-MT免疫幼年APP/PS1双转基因鼠后能产生高滴度的抗体,免疫反应为Th2型,预防脑内Aβ的聚集的同时延缓了记忆障碍的发生与发展,避免了副反应的发生,有待成为预防阿尔茨海默病的有效疫苗。     

缺陷腺病毒载体表达重复10次的Aβ3-10基因疫苗鼻黏膜免疫APPswe, PSEN1dE9转基因小鼠可诱导出Th2型免疫反应

Immunotherapy for Alzheimer’s disease (AD) is effective in improving cognitive function in transgenic mouse models of AD. Because the AN1792 [beta-amyloid (Aβ) 1-42] vaccine was halted because of T cell mediated meningoencephalitis, many scientists are searching for a novel vaccine to avoid the T cell mediated immune response caused by the Aβ1-42. Importantly, the time when the immunization is begun can influence the immune effect. In this study, an adenovirus vaccine was constructed containing 10 × Aβ3-10 repeats and gene adjuvant CpG DNA. Transgenic AD mice were immunized intranasally for 3 months. After 10 × Aβ3-10 vaccine immunization, high titers of anti-Aβ42 IgG1 predominant antibodies were induced. In spatial learning ability and probe tests, the 10 × Aβ3-10 immunized mice showed significantly improved memories compared to control mice. The 10 × Aβ3-10 vaccine resulted in a robust Th2 dominant humoral immune response and reduced learning deficits in AD mice. In addition, the 10 × Aβ3-10 vaccine might be more efficient if administered before Aβ aggregation at an early stage in the AD mouse brain. Thus, the adenovirus vector encoding 10 × Aβ3-10 is a promising vaccine for AD.     

Intranasal inoculation with an adenovirus vaccine encoding ten repeats of Aβ3-10 reduces AD-like pathology and cognitive impairment in Tg-APPswe/PSEN1dE9 mice

To develop a safe and efficient vaccine for AD treatment, we constructed an adenovirus vector vaccine encoding ten repeats of Aβ3-10 and CpG motif as a molecular adjuvant. We demonstrated that therapeutic immunization with Ad-10×Aβ3-10-CpG elicits Aβ3-10 specific Th2-polarized immune response with high titers of anti-Aβ antibodies in APPswe/PSEN1dE9 mice, which in turn reduced Aβ deposits in brains and cognitive impairment. In addition, Ad-10×Aβ3-10-CpG reduced astrocytosis without increasing the incidence of microhemorrhage. Our findings of this study raise the possibility that the adenovirus vaccine Ad-10×Aβ3-10-CpG would be a safe and effective alternative for AD immunotherapy.     

An effector-reduced anti-β-amyloid (Aβ) antibody with unique aβ binding properties promotes neuroprotection and glial engulfment of Aβ

Passive immunization against β-amyloid (Aβ) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aβ monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aβ, protected against Aβ1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aβ oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aβ, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aβ antibody that takes advantage of a unique Aβ binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.     

Distinct Patterns of Antiamyloid-β Antibodies in Typical and Atypical Alzheimer Disease

OBJECTIVE To compare serum antiamyloid-β (Aβ) antibodies in typical and atypical Alzheimer disease (AD). DESIGN Preliminary observations. SUBJECTS Thirteen patients with AD, 8 patients with posterior cortical atrophy with evidence of AD (PCA-AD) pathophysiological process by both cerebrospinal fluid (CSF) biomarkers and amyloid imaging, and 12 age-matched control individuals. INTERVENTIONS The class and subclass levels of serum anti-Aβ antibodies were measured using an oligomer-based enzyme-linked immunosorbent assay. This method allowed measuring both free antibodies and, after acidic treatment, the total fraction that includes all antibodies complexed with circulating Aβ40/42 and any cross-reacting antigen. RESULTS Anti-Aβ IgG were restricted to the IgG1 and IgG3 subclasses. Their total levels were strikingly lower and more homogeneous in patients with PCA compared with both typical AD and controls, while biomarkers of amyloid deposition (CSF Aβ42 and positron emission tomography amyloid imaging) were similar in patients with AD and patients with PCA. CONCLUSIONS Serum anti-Aβ IgG1 and IgG3 antibodies differ between distinct forms of AD. Its significance is discussed for possible implications as immune effectors in the specific pathophysiology of AD variants.     

补体C3d-p28增强阿尔茨海默病DNA疫苗Th2型免疫反应的研究

目的 探讨补体C3d-p28作为分子佐剂,在阿尔茨海默病DNA疫苗免疫反应中的作用。方法 分别将重组质粒p(Aβ3-10)10,p(Aβ3-10)10-C3d-p28.3和空载体pc DNA3.1(+)用肌肉注射的方法免疫8~10周龄的雌性BALB/c鼠。质粒注射前24 h,布比卡因肌肉注射诱导轻微的肌肉变性。应用ELISA方法检测血清抗Aβ抗体的滴度、抗体分型、体外脾细胞培养上清液中IL-4和IFN-γ的含量。免疫组织化学染色法检测免疫血清与转基因鼠脑内Aβ斑的结合能力。结果 重组质粒疫苗p(Aβ3-10)10仅诱导出低滴度的抗Aβ抗体,产生了Th1/Th2混合型的免疫反应。而重组质粒疫苗p(Aβ3-10)10-C3d-p28.3诱导出较高滴度的抗Aβ抗体,体外脾细胞培养上清液中IFN-γ低和IL-4高,即引起了Th2型细胞免疫反应,同时产生的抗Aβ抗体能够与双转基因鼠APP/PS1脑中沉积的Aβ斑块结合。结论 补体C3d-p28分子佐剂能够增强抗Aβ抗体的产生并且诱发Th2型的免疫反应。     

Epitope-based DNA vaccine for Alzheimer's disease: Translational study in macaques

BackgroundClinical trials with passive and active Alzheimer's disease (AD) vaccines suggest that early interventions are needed for improvement of cognitive and/or functional performance in patients, providing impetus for the development of safe and immunologically potent active vaccines targeting amyloid β (Aβ). The AN-1792 trial has indicated that Aβ-specific T cells may be unsafe for humans; therefore, other vaccines based on small Aβ epitopes are undergoing preclinical and clinical testing.MethodsHumoral and cellular immune responses elicited in response to a novel DNA epitope-based vaccine (AV-1955) delivered to rhesus macaques using the TriGrid electroporation device were evaluated. Functional activities of anti-Aβ antibodies generated in response to vaccination were assessed in vitro.ResultsAV-1955 generates long-term, potent anti-Aβ antibodies and cellular immune responses specific to foreign T-helper epitopes but not to self-Aβ.ConclusionsThis translational study demonstrates that a DNA-based epitope vaccine for AD could be appropriate for human clinical testing.     

Anti-β-amyloid immunotherapy for Alzheimer's disease: focus on bapineuzumab

Recent advances in our understanding of the neurobiology of Alzheimer's disease (AD) have led to the development of putative disease-modifying treatments. The most revolutionary of these approaches consists in the removal of brain β-amyloid (Aβ) via anti-Aβ antibodies. Brain imaging and neuropathological studies have shown the ability of both active and passive anti-Aβ immunotherapies of clearing Aβ deposits from the brain of the AD patients. An active anti-Aβ vaccine preparation, AN1792, has been used in AD patients with some clues of clinical efficacy but causing meningoencephalitis in about 6% of patients and it has been abandoned. Several second-generation active Aβ vaccines and passive Aβ immunotherapies have been developed and are under clinical investigation with the aim of accelerating Aβ clearance from the brain of the AD patients. The most advanced of these immunological approaches is bapineuzumab, composed of humanized anti-Aβ monoclonal antibodies, that has been tested in two Phase II trials, demonstrating to reduce Aβ burden in the brain of AD patients. However, the preliminary cognitive efficacy of bapineuzumab appears uncertain. The occurrence of vasogenic edema, especially in apolipoprotein E 4 carriers, may limit its clinical use and have led to abandon the highest dose of the drug (2 mg/kg). The results of four ongoing large Phase III trials on bapineuzumab will tell us if passive anti-Aβ immunization is able to alter the course if this devastating disease.     

补体C3d-p28对阿尔茨海默病DNA疫苗的免疫增强作用

目的探讨补体C3d-p28作为分子佐剂,在阿尔茨海默病DNA疫苗基因免疫中的作用。方法在第1、8、22、43、64、85、106、127天,将重组质粒p(Aβ3-10)10、p(Aβ3-10)10-C3d-p28.3和pcDNA3.1(+)肌肉注射于APP/PS1双转基因鼠后腿股四头肌内。疫苗接种前、自第2次注射开始每次接种后7天取鼠眶静脉血共8次,以ELISA法检测抗Aβ抗体的滴度和分型;第8次(最后1次)眶静脉取血后进行6d的Morris水迷宫实验,通过定位航行和空间探索实验评估小鼠空间学习记忆能力。水迷宫实验结束后处死小鼠,以ELISA法检测小鼠脾细胞培养上清液中白细胞介素4(IL-4)和干扰素γ(IFN-γ)水平,免疫组化染色法检测小鼠脑内Aβ斑的表达。结果 p(Aβ3-10)10-C3d-p28.3组抗Aβ抗体水平高于p(Aβ3-10)10组[(55.03±8.93)μg/mLvs.(27.32±7.69)μg/mL,t=-4.455,P0.05],p(Aβ3-10)10-C3d-p28.3组抗体类型主要是IgG1型[(50.64±6.96)μg/mL],明显高于p(Aβ3-10)10组[(14.15±3.16)μg/mL,P0.05]。与p(Aβ3-10)10组比较,p(Aβ3-10)10-C3dp28.3组Morris水迷宫实验平均逃避潜伏期变短、穿越平台次数和穿越平台所在象限停留的时间比例明显增多(均P0.05);脾细胞培养上清液中IL-4水平增高[(110.22±18.12)pg/mL vs.(170.12±22.16)pg/mL,P0.05]、IFN-γ水平减低[(800.12±80.11)pg/mL vs.(640.12±70.53)pg/mL,F=6.152,P0.05];脑内沉积的Aβ斑块明显减少(P0.05)。结论补体C3d-p28分子佐剂使p(Aβ3-10)10-C3d-p28.3抗Aβ抗体的产生增加、Th2型免疫反应增强,转基因鼠空间学习记忆能力提高。     

High-affinity rabbit monoclonal antibodies specific for amyloid peptides amyloid-β40 and amyloid-β42

Antibodies that specifically bind to either amyloid-β peptide (Aβ) isoform Aβ?? or Aβ?? contribute to the study of Alzheimer's disease (AD) pathology and to the development of cerebrospinal fluid-based tests for the probable diagnosis of AD. Polyclonal rabbit anti-Aβ antibodies possess high affinity and specificity, but their generation requires a long immunization period, and the resulting antibodies exhibit variable specificities and affinities. To secure a continuing supply of antibodies with uniform properties, we generated and partially characterized rabbit monoclonal antibodies specific for either Aβ?? or Aβ??. These antibodies possess nanomolar or sub-nanomolar dissociation constants and are at least 3,000-fold more selective for one isoform over the other. These antibodies are suitable for immunoblotting and, in a sandwich ELISA, RabmAb42 (anti-Aβ??) is sensitive enough to measure plasma levels of Aβ??. In addition, these antibodies have been applied to the immunohistology of Down syndrome and AD brain tissues, where they reveal fibrillar and diffuse amyloid deposits and are almost free of non-specific staining. The data indicate that diffuse amyloid deposits contain only minute amounts of Aβ??. Thus these rabbit monoclonal anti-Aβ antibodies can be widely applied in AD and Down syndrome research and diagnosis.     

Aβ_(3-10)多价腺病毒疫苗鼻粘膜免疫AD转基因鼠的治疗效果研究

目的探讨Aβ_(3-10)多价腺病毒疫苗鼻粘膜免疫AD转基因鼠的治疗效果。方法 18只雄性10月龄AD转基因鼠,随机分为3组,分别以Aβ_(3-10)多价腺病毒疫苗Ad-Aβ_((3-10)10)-Cp G、空腺病毒载体及Aβ1-42免疫,ELISA法检测血清抗Aβ抗体滴度及亚型,Morris水迷宫检测转基因鼠的学习记忆能力,免疫组化法检测转基因鼠脑内Aβ沉积;ELISA法检测转基因鼠脑组织匀浆和血清中可溶性Aβ42水平。结果 Ad-Aβ_((3-10)10)-Cp G组和Aβ1-42组抗体水平随着免疫次数逐渐增加,第7次免疫后Ad-Aβ_((3-10)10)-Cp G组和Aβ1-42组血清中抗Aβ抗体水平分别为(67.42±13.68)μg/ml和(94.41±14.01)μg/ml,而空载体组一直在基线水平。Ad-Aβ_((3-10)10)-Cp G组Ig G1/Ig G2a比值明显高于Aβ1-42组(P0.05)。在Morris水迷宫实验中Ad-Aβ_((3-10)10)-Cp G组的逃避潜伏期明显小于空载体组(P0.01);Ad-Aβ_((3-10)10)-Cp G组在靶象限的停留时间明显长于空载体组(P0.01);Ad-Aβ_((3-10)10)-Cp G组穿越平台所在位置的次数明显多于空载体组(P0.05)。Ad-Aβ_((3-10)10)-Cp G组脑组织Aβ沉积所占面积百分比与空载体组比较明显减少(P0.01)。Ad-Aβ_((3-10)10)-Cp G组脑组织匀浆和血清中可溶性Aβ42水平明显高于空载体组(P0.01)。结论 Aβ_(3-10)多价腺病毒疫苗鼻粘膜免疫AD转基因鼠,主要引起Th2型免疫应答,可以改善AD转基因鼠学习和记忆能力,促进转基因鼠脑内Aβ清除,可以减少由细胞免疫应答引起的炎症反应。Aβ_(3-10)多价腺病毒疫苗是AD免疫治疗的安全有效的候选疫苗。     

Neutralization of soluble, synaptotoxic amyloid β species by antibodies is epitope specific

Several anti-amyloid β (Aβ) antibodies are under evaluation for the treatment of Alzheimer's disease (AD). Clinical studies using the N-terminal-directed anti-Aβ antibody bapineuzumab have demonstrated reduced brain PET-Pittsburg-B signals, suggesting the reduction of Aβ plaques, and reduced levels of total and phosphorylated tau protein in the CSF of treated AD patients. Preclinical studies using 3D6 (the murine form of bapineuzumab) have demonstrated resolution of Aβ plaque and vascular burdens, neuritic dystrophy, and preservation of synaptic density in the transgenic APP mouse models. In contrast, few studies have evaluated the direct interaction of this antibody with synaptotoxic soluble Aβ species. In the current report, we demonstrated that 3D6 binds to soluble, synaptotoxic assemblies of Aβ(1-42) and prevents multiple downstream functional consequences in rat hippocampal neurons including changes in glutamate AMPA receptor trafficking, AD-type tau phosphorylation, and loss of dendritic spines. In vivo, we further demonstrated that 3D6 prevents synaptic loss and acutely reverses the behavioral deficit in the contextual fear conditioning task in transgenic mouse models of AD, two endpoints thought to be linked to synaptotoxic soluble Aβ moieties. Importantly C-terminal anti-Aβ antibodies were ineffective on these endpoints. These results, taken with prior studies, suggest that N-terminal anti-Aβ antibodies effectively interact with both soluble and insoluble forms of Aβ and therefore appear particularly well suited for testing the Aβ hypothesis of AD.     

Gantenerumab: a novel human anti-Aβ antibody demonstrates sustained cerebral amyloid-β binding and elicits cell-mediated removal of human amyloid-β

The amyloid-β lowering capacity of anti-Aβ antibodies has been demonstrated in transgenic models of Alzheimer's disease (AD) and in AD patients. While the mechanism of immunotherapeutic amyloid-β removal is controversial, antibody-mediated sequestration of peripheral Aβ versus microglial phagocytic activity and disassembly of cerebral amyloid (or a combination thereof) has been proposed. For successful Aβ immunotherapy, we hypothesized that high affinity antibody binding to amyloid-β plaques and recruitment of brain effector cells is required for most efficient amyloid clearance. Here we report the generation of a novel fully human anti-Aβ antibody, gantenerumab, optimized in vitro for binding with sub-nanomolar affinity to a conformational epitope expressed on amyloid-β fibrils using HuCAL(?) phage display technologies. In peptide maps, both N-terminal and central portions of Aβ were recognized by gantenerumab. Remarkably, a novel orientation of N-terminal Aβ bound to the complementarity determining regions was identified by x-ray analysis of a gantenerumab Fab-Aβ(1-11) complex. In functional assays gantenerumab induced cellular phagocytosis of human amyloid-β deposits in AD brain slices when co-cultured with primary human macrophages and neutralized oligomeric Aβ42-mediated inhibitory effects on long-term potentiation in rat brain. In APP751(swedish)xPS2(N141I) transgenic mice, gantenerumab showed sustained binding to cerebral amyloid-β and, upon chronic treatment, significantly reduced small amyloid-β plaques by recruiting microglia and prevented new plaque formation. Unlike other Aβ antibodies, gantenerumab did not alter plasma Aβ suggesting undisturbed systemic clearance of soluble Aβ. These studies demonstrated that gantenerumab preferentially interacts with aggregated Aβ in the brain and lowers amyloid-β by eliciting effector cell-mediated clearance.     

Combined treatment of Aβ immunization with statin in a mouse model of Alzheimer's disease

We evaluated the therapeutic efficacy of combined treatment of Aβ-immunization with simvastatin in an Alzheimer mouse model at age 22 months. DNA prime-adenovirus boost immunization induced modest anti-Aβ titers and simvastatin increased the seropositive rate. Aβ-KLH was additionally administered to boost the titers. Irrespective of simvastatin, the immunization did not decrease cerebral Aβ deposits but increased soluble Aβ and tended to exacerbate amyloid angiopathy in the hippocampus. The immunization increased cerebral invasion of leukocytes and simvastatin counteracted the increase. Thus, modest anti-Aβ titers can increase soluble Aβ and simvastatin may reduce inflammation associated with vaccination in aged Alzheimer mouse models.     

Short amyloid-β immunogens with spacer-enhanced immunogenicity without junctional epitopes for Alzheimer's disease immunotherapy

Induction of an immune response to amyloid-β (Aβ) protein is effective in treating animal models of Alzheimer's disease. The Aβ1-15 sequence contains the antibody epitope(s), but lacks the T-cell reactive sites of full-length Aβ1-42. We tested two alternative peptide immunogens encompassing either a tandem repeat of GPGPG-linked Aβ1-15 sequences (2Aβ15-linker) or a tandem repeat Aβ1-15 without the spacer sequence (2Aβ15). Titers of the immunized sera were measured by indirect ELISA. We analyzed the production of interferon-γ and interleukin-4 cytokine by lymphocytes and CD4 T-cells using ELISPOT and FACS assays; we then measured CD4 T-cell proliferation using a CFSE-based lymphoproliferation assay. Immunization with 2Aβ15-linker resulted in a high anti-Aβ titer of the noninflammatory T-helper 2 isotype, a lack of lymphocyte proliferation against the spacer part peptide. We observed much lower titers against the Aβ protein after immunization with 2Aβ15. Restimulation of lymphocytes with the corresponding immunogens resulted in proliferative responses, which showed that the sequential arrangement of the epitopes created junctional epitopes. The disruption of junctional epitopes through the introduction of a GPGPG spacer restored the immunogenicity against all the epitopes. Our novel immunogen with spacer may be a safer alternative to a peptide-based vaccine.     

Gene vaccination to bias the immune response to amyloid-beta peptide as therapy for Alzheimer disease

BACKGROUND: The amyloid-beta (Abeta) peptide has a central role in the neurodegeneration of Alzheimer disease (AD). Immunization of AD transgenic mice with Abeta(1-42) (Abeta(42)) peptide reduces both the spatial memory impairments and AD-like neuropathologic changes in these mice. Therapeutic immunization with Abeta in patients with AD was shown to be effective in reducing Abeta deposition, but studies were discontinued owing to the development of an autoimmune, cell-mediated meningoencephalitis. We hypothesized that gene vaccination could be used to generate an immune response to Abeta(42) that produced antibody response but avoided an adverse cell-mediated immune effect. OBJECTIVE: To develop an effective genetic immunization approach for treatment and prevention of AD without causing an autoimmune, cell-mediated meningoencephalitis. METHODS: Mice were vaccinated with a plasmid that encodes Abeta(42), administered by gene gun. The immune response of the mice to Abeta(42) was monitored by measurement of (1) antibody levels by enzyme-linked immunosorbent assay (ELISA) and Western blot and (2) Abeta(42)-specific T-cell response as measured by interferon-gamma enzyme-linked immunospot (ELISPOT) assay. RESULTS: Gene-gun delivery of the mouse Abeta(42) dimer gene induced significant humoral immune responses in BALB/c wild-type mice after 3 vaccinations in 10-day intervals. All 3 mice in the treated group showed significant humoral immune responses. The ELISPOT assay for interferon-gamma release with mouse Abeta(42) peptide and Abeta(9-18) showed no evident cytotoxic T-lymphocyte response. We further tested the responses of wild-type BALB/c mice to the monomer Abeta(42) gene vaccine. Western blot evaluation showed both human and mouse Abeta monomer gene vaccine elicited detectable humoral immune responses. We also introduced the human Abeta(42) monomer gene vaccine into AD double transgenic mice APPswe/PSEN1(A246E). Mice were vaccinated with plasmids that encode Abeta(1-42) and Abeta(1-16), or with plasmid without the Abeta gene. Treated mice showed significant humoral immune responses as demonstrated by ELISA and by Western blot. These mice also showed no significant cellular immune response as tested by ELISPOT. One of the treated mice was killed at 7 months of age for histological observations, and scattered amyloid plaques were noted in all layers of the cerebral cortex and in the hippocampus in both Abeta(42)- and control-vaccinated mice. No definite difference was discerned between the experimental and control animals. CONCLUSIONS: Gene-gun-administered genetic immunization with the Abeta(42) gene in wild-type BALB/c and AD transgenic mice can effectively elicit humoral immune responses without a significant T-cell-mediated immune response to the Abeta peptide. This immunotherapeutic approach could provide an alternative active immunization method for therapy and prevention of AD.     

Biofunctionalized magnetic nanoparticles for specifically detecting biomarkers of Alzheimer's disease in vitro

Magnetic nanoparticles biofunctionalized with antibodies against β-amyloid-40 (Aβ-40) and Aβ-42, which are promising biomarkers related to Alzheimer's disease (AD), were synthesized. We characterized the size distribution, saturated magnetizations, and stability of the magnetic nanoparticles conjugated with anti-Aβ antibody. In combination with immunomagnetic reduction technology, it is demonstrated such biofunctionalized magnetic nanoparticles are able to label Aβs specifically. The ultralow-detection limits of assaying Aβs in vitro using the magnetic nanoparticles via immunomagnetic reduction are determined to a concentration of ～10 ppt (10 pg/mL). Further, immunomagnetic reduction signals of Aβ-40 and Aβ-42 in human plasma from normal samples and AD patients were analyzed, and the results showed a significant difference between these two groups. These results show the feasibility of using magnetic nanoparticles with Aβs as reagents for assaying low-concentration Aβs through immunomagnetic reduction, and also provide a promising new method for early diagnosis of Alzheimer's disease from human blood plasma.     

Effects of epitopes combination and adjuvants on immune responses to anti-Alzheimer disease DNA vaccines in mice

Alzheimer disease (AD) is a neurodegenerative disorder characterized by neuropathological hallmarks including deposits of the beta-amyloid peptide (AssP). Studies have shown that immunization with Abeta42 peptide reduces both the spatial memory impairments and Alzheimer disease-like neuropathologic changes in Alzheimer disease transgenic mice, but can cause side effect of a cell-mediated autoimmune meningoencephalitis. Recently, some studies showed that DNA vaccination could be used to generate an antibody response to Abeta without the adverse cell-mediated immune effect. In the current study, we generate four DNA vaccine plasmids (pV-GE1, pV-GE2, pV-GE3, and pV-GE4) against Alzheimer disease by separately fusing Abeta epitope sequences (coding for EFGH, DAEFGH, EFGH+EFGH, and EFGH+DAEFGH) with IgG heavy chain coding region of mouse. Meanwhile, the full-length gene Abeta encoding plasmid (pV-Abeta), empty vector (pVAX) and synthetic AssP were also included as control. The sera of BALB/c mice immunized via intramuscular with plasmids and peptide were tested by indirect ELISA for auto-AssP immunoreactivity. The results showed that all the DNA vaccine plasmids induced AssP-specific antibodies; moreover pV-GE2 and pV-Abeta constructs elicited higher antibody titers than other constructs (P < 0.05). To further enhance the immune response, GM-CSF encoding plasmid (pGM-CSF) and purified BCG-DNA were used as molecular adjuvants. BCG-DNA could enhance humoral and cellular immune responses simultaneously and did not alter the phenotype of the immune responses, whereas pGM-CSF showed no obvious effect on immune response. These results suggest that this immunization strategy of using Abeta epitope encoding plasmid plus BCG-DNA adjuvant may serve as the basis for developing anti-Alzheimer disease vaccines.     

A mimotope peptide of Aβ42 fibril-specific antibodies with Aβ42 fibrillation inhibitory activity induces anti-Aβ42 conformer antibody response by a displayed form on an M13 phage in mice

In Alzheimer's disease (AD), amyloid-β (Aβ) peptides accumulate in the brain in different forms, including fibrils and oligomers. Recently, we established three distinct conformation-dependent human single-chain Fv (scFv) antibodies, including B6 scFv, which bound to Aβ42 fibril but not to soluble-form Aβ, inhibiting Aβ42 fibril formation. In this study, we determined the mimotopes of these antibodies and found a common mimotope sequence, B6-C15, using the Ph.D.-C7C phage library. The B6-C15 showed weak homology to the C-terminus of Aβ42 containing GXXXG dimerization motifs. We synthesized the peptide of B6-C15 fused with biotinylated TAT at the N-terminus (TAT-B6-C15) and characterized its biochemical features on an Aβ42-fibrillation reaction in vitro. We demonstrated that, first, TAT-B6-C15 inhibited Aβ42 fibril formation; secondly, TAT-B6-C15 bound to prefibril Aβ42 oligomers but not to monomers, trimers, tetramers, fibrils, or ultrasonicated fragments; thirdly, TAT-B6-C15 inhibited Aβ42-induced cytotoxicity against human SH-SY5Y neuroblastoma cells; and, fourthly, when mice were administered B6-C15-phages dissolved in phosphate-buffered saline, the anti-Aβ42 conformer IgG antibody response was induced. These results suggested that the B6-C15 peptide might provide unique opportunities to analyze the Aβ42 fibrillation pathway and develop a vaccine vehicle for Alzheimer's disease.     

Vaccination of Alzheimer's model mice with adenovirus vector containing quadrivalent foldable Abeta(1-15) reduces Abeta burden and behavioral impairment without Abeta-specific T cell response

Active amyloid beta (Abeta) vaccination has been shown to be effective in clearing cerebral Abeta and improving cognitive function in mouse models of Alzheimer's disease (AD). The meningoencephalitis observed in AD vaccination trial was likely related to excessive T cell-mediated immunity caused by the immunogen Abeta(1-42). To avoid this toxicity, previous researchers have been using synthetic truncated Abeta derivatives that promote humoral immunity. In this study, we develop a novel adenovirus vaccine, which can express quadrivalent foldable Abeta(1-15) (4xAbeta(15)) and gene adjuvant GM-CSF in vivo. Importantly, the 4xAbeta(15) sequence includes an Abeta-specific B cell epitope but lacks the reported T cell epitope. The 4xAbeta(15) adenovirus vaccine induces an Abeta-specific IgG1 predominant humoral immune response, and reduces brain Abeta deposition and cognition deficits in Tg2576 mice. Detection of IL-4 and IFN-gamma in restimulated splenocytes shows a significant Th2-polarized immune response. Stimulation of splenocytes with 4xAbeta(15) peptides results in robust proliferative responses, whereas proliferation is absent after stimulation with full-length Abeta, which indicates that the 4xAbeta(15) adenovirus vaccine does not induce Abeta-specific T cellular immune response. Thus, our results raise the possibility that adenovirus vector encoding 4xAbeta(15) would be a promising candidate for future AD vaccination program.