A new DNA vaccine fused with the C3d-p28 induces a Th2 immune response against amyloid-beta

To enhance anti-amyloid-beta(Aβ) antibody generation and induce a Th2 immune response,we constructed a new DNA vaccine p(Aβ3-10)10-C3d-p28.3 encoding ten repeats of Aβ3-10 and three copies of C3d-p28 as a molecular adjuvant.In this study,we administered this adjuvant intramuscularly to female C57BL/6J mice at 8-10 weeks of age.Enzyme linked immunosorbent assay was used to detect the titer of serum anti-Aβ antibody,isotypes,and cytokines in splenic T cells.A 3(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to detect the proliferation rate of splenic T cells.Brain sections from a 12-month-old APP/PS1 transgenic mouse were used for detecting the binding capacities of anti-Aβ antibodies to Aβ plaques.The p(Aβ3-10)10-C3d-p28.3 vaccine induced high titers of anti-amyloid-β antibodies,which bound to Aβ plaques in APP/PS1 transgenic mouse brain tissue,demonstrating that the vaccine is effective against plaques in a mouse model of Alzheimer’s disease.Moreover,the vaccine elicited a predominantly IgG1 humoral response and low levels of interferon-γ in ex vivo cultured splenocytes,indicating that the vaccine could shift the cellular immune response towards a Th2 phenotype.This indicated that the vaccine did not elicit a detrimental immune response and had a favorable safety profile.Our results indicate that the p(Aβ3-10)10-C3d-p28.3 vaccine is a promising immunotherapeutic option for Aβ vaccination in Alzheimer’s disease.     

Nasal mucosal inhalation of amyloid-beta peptide 3–10 defective adenovirus attenuates cytotoxicity induced by beta-amyloid(1–42)

Three-month-old Alzheimer's disease model transgenic mice were immunized with Aβ1–42, Plp-Adenovirus [Ad]-X-CMV-(Aβ3–10)10-CpG [AdCpG-(Aβ3–10)10] or AdCpG virus fluid via nasal mucosal inhalation, respectively. ELISA analysis of serum showed Aβ42 antibody titers were significantly increased in mice immunized with Aβ1–42 and AdCpG-(Aβ3–10)10. Concanavalin A and AdCpG-(Aβ3–10)10 stimulation significantly increased the number of proliferating spleen cells cultured from AdCpG(Aβ3–10)10 and Aβ42 groups compared with the control group. In the AdCpG(Aβ3–10)10 group, levels of interleukin(IL)-4 and IL-10 were increased, while those of IL-2 and interferon-γ were decreased. In the Aβ42 group, levels of IL-4, IL-10, IL-2 and interferon-γ were all increased. Experimental findings indicate that AdCpG-(Aβ3–10)10 vaccine can produce strong T helper 2(Th2) humoral immune responses in addition to the production of Aβ42 antibody. The cellular immunologic response was weak and avoided Aβ1–42-mediated cytotoxicity.     

Early active immunization with Aβ_(3–10)-KLH vaccine reduces tau phosphorylation in the hippocampus and protects cognition of mice

Active and passive anti-Aβ immunotherapies have successfully been used for the prevention and treatment of Alzheimer's disease animal models. However, clinical use of these immunotherapies is not effective, because the vaccination is administered too late. At 1 month of age, 100 μL of Aβ3–10-KLH peptide(vaccine, 2 μg/μL) was subcutaneously injected into the neck of an amyloid precursor protein/presenilin-1/tau transgenic(3×Tg-AD) mouse model. Aβ3–10-KLH peptide was re-injected at 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5 months of age. Serum levels of Aβ antibody were detected by enzyme-linked immunosorbent assay, while spatial learning and memory ability were evaluated by Morris water maze. Immunohistochemistry was used to detect total tau with HT7 and phosphorylated tau with AT8(phosphorylation sites Ser202 and Thr205) and AT180(phosphorylation site Thr231) antibodies in the hippocampus. In addition, western blot analysis was used to quantify AT8 and AT180 expression in the hippocampus. The results showed that after vaccine injection, mice produced high levels of Aβ antibody, cognitive function was significantly improved, and total tau and phosphorylated tau levels were significantly reduced. These findings suggest that early active immunization with Aβ3–10-KLH vaccine can greatly reduce tau phosphorylation, thereby mitigating the cognitive decline of 3×Tg-AD mice. This study was approved by the Animal Ethics Committee of China Medical University, China(approval No. 103-316) on April 2, 2016.     

Corrigendum: Early active immunization with Aβ3–10-KLH vaccine reduces tau phosphorylation in the hippocampus and protects cognition of mice

doi:10.4103/1673-5374.280331 In the article titled“Early active immunization with Aβ3–10-KLH vaccine reduces tau phosphorylation in the hippocampus and protects cognition of mice”published on pages 519–527,Issue 3,Volume 15 of Neural Regeneration Research(Wang et al.,2020),the affiliation of the first author Jin-Chun Wang was written incorrectly.The correct author affiliations are as follows.     

Loss of canonical Wnt signaling is involved in the pathogenesis of Alzheimer's disease

Alzheimer's disease(AD) is the most common form of dementia in the older population, however, the precise cause of the disease is unknown. The neuropathology is characterized by the presence of aggregates formed by amyloid-β(Aβ) peptide and phosphorylated tau; which is accompanied by progressive impairment of memory. Diverse signaling pathways are linked to AD, and among these the Wnt signaling pathway is becoming increasingly relevant, since it plays essential roles in the adult brain. Initially, Wnt signaling activation was proposed as a neuroprotective mechanism against Aβ toxicity. Later, it was reported that it participates in tau phosphorylation and processes of learning and memory. Interestingly, in the last years we demonstrated that Wnt signaling is fundamental in amyloid precursor protein(APP) processing and that Wnt dysfunction results in Aβ production and aggregation in vitro. Recent in vivo studies reported that loss of canonical Wnt signaling exacerbates amyloid deposition in a transgenic(Tg) mouse model of AD. Finally, we showed that inhibition of Wnt signaling in a Tg mouse previously at the appearance of AD signs, resulted in memory loss, tau phosphorylation and Aβ formation and aggregation; indicating that Wnt dysfunction accelerated the onset of AD. More importantly, Wnt signaling loss promoted cognitive impairment, tau phosphorylation and Aβ1–42 production in the hippocampus of wild-type(WT) mice, contributing to the development of an Alzheimer's-like neurophatology. Therefore, in this review we highlight the importance of Wnt/β-catenin signaling dysfunction in the onset of AD and propose that the loss of canonical Wnt signaling is a triggering factor of AD.     

Autophagy is involved in oral rAAV/Aβ vaccine-induced Aβ clearance in APP/PS1 transgenic mice

The imbalance between β-amyloid(Aβ) generation and clearance plays a fundamental role in the pathogenesis of Alzheimer 's disease(AD). The sporadic form of AD is characterized by an overall impairment in Aβ clearance. Immunotherapy targeting Aβ clearance is believed to be a promising approach and is under active clinical investigation. Autophagy is a conserved pathway for degrading abnormal protein aggregates and is crucial for Aβ clearance. We previously reported that oral vaccination with a recombinant AAV/Aβ vaccine increased the clearance of Aβ from the brain and improved cognitive ability in AD animal models, while the underlying mechanisms were not well understood. In this study, we first demonstrated that oral vaccination with rAAV/Aβ decreased the p62 level and up-regulated the LC3BII/LC3B-I ratio in APP/PS1 mouse brain, suggesting enhanced autophagy. Further, inhibition of the Akt/m TOR pathway may account for autophagy enhancement. We also found increased anti-Aβ antibodies in the sera of APP/PS1 mice with oralvaccination, accompanied by elevation of complement factors C1 q and C3 levels in the brain. Our results indicate that autophagy is closely involved in oral vaccination-induced Aβ clearance, and modulating the autophagy pathway may be an important strategy for AD prevention and intervention.     

Aquaporin 4 deficiency eliminates the beneficial effects of voluntary exercise in a mouse model of Alzheimer’s disease

Regular exercise has been shown to reduce the risk of Alzheimer’s disease(AD).Our previous study showed that the protein aquaporin 4(AQP4),which is specifically expressed on the paravascular processes of astrocytes,is necessary for glymphatic clearance of extracellular amyloid beta(Aβ)from the brain,which can delay the progression of Alzheimer’s disease.However,it is not known whether AQP4-regulated glymphatic clearance of extracellular Aβis involved in beneficial effects of exercise in AD patients.Our results showed that after 2 months of voluntary wheel exercise,APP/PS1 mice that were 3 months old at the start of the intervention exhibited a decrease in Aβburden,glial activation,perivascular AQP4 mislocalization,impaired glymphatic transport,synapse protein loss,and learning and memory defects compared with mice not subjected to the exercise intervention.In contrast,APP/PS1 mice that were 7 months old at the start of the intervention exhibited impaired AQP4 polarity and reduced glymphatic clearance of extracellular Aβ,and the above-mentioned impairments were not alleviated after the 2-month exercise intervention.Compared with age-matched APP/PS1 mice,AQP4 knockout APP/PS1 mice had more serious defects in glymphatic function,Aβplaque deposition,and cognitive impairment,which could not be alleviated after the exercise intervention.These findings suggest that AQP4-dependent glymphatic transport is the neurobiological basis for the beneficial effects of voluntary exercises that protect against the onset of AD.     

Sex Differences in Neuropathology and Cognitive Behavior in APP/PS1/tau Triple-Transgenic Mouse Model of Alzheimer’s Disease

Alzheimer's disease(AD) is the most common form of dementia among the elderly, characterized by amyloid plaques, neurofibrillary tangles, and neuroinflammation in the brain, as well as impaired cognitive behaviors.A sex difference in the prevalence of AD has been noted,while sex differences in the cerebral pathology and relevant molecular mechanisms are not well clarified. In the present study, we systematically investigated the sex differences in pathological characteristics and cognitive behavior in12-month-old male and female APP/PS1/tau triple-transgenic AD mice(3×Tg-AD mice) and examined the molecular mechanisms. We found that female 3×Tg-AD mice displayed more prominent amyloid plaques, neurofibrillary tangles, neuroinflammation, and spatial cognitive deficits than male 3×Tg-AD mice. Furthermore, the expression levels of hippocampal protein kinase A–cAMP response element-binding protein(PKA-CREB) and p38–mitogen-activated protein kinases(MAPK) also showed sex difference in the AD mice, with a significant increase in the levels of p-PKA/p-CREB and a decrease in the p-p38 in female, but not male, 3×Tg-AD mice. We suggest that an estrogen deficiency-induced PKA-CREB-MAPK signaling disorder in 12-month-old female 3×Tg-AD mice might be involved in the serious pathological and cognitive damage in these mice. Therefore, sex differences should be taken into account in investigating AD biomarkers and related target molecules, and estrogen supplementation or PKA-CREBMAPK stabilization could be beneficial in relieving the pathological damage in AD and improving the cognitive behavior of reproductively-senescent females.     

Comparative study of histopathology changes between the PS1/APP double transgenic mouse model and Aβ_(1-40)-injected rat model of Alzheimer disease

Objective To identify the genetype of the PS1/APP double transgenic mouse model, then to analyse the histopathological changes in the brain and compare the differences between the transgenic mice models and Aβ_(1-40)-injected rats models of Alzheimer disease. Methods The modified congo red staining, Nissl's staining and immunohistology staining was used to observe the Aβ deposits, activation of astrocyte respectively. Results ①The PS1/APP transgenic mouse extensively displayed Aβ deposits in the cortex and hippocampal structures, and GFAP positive cells were aggregated in mass and surrounded the congo red-positive plaque. ②The Aβ_(1-40)-intrahippocampal-injected rat model showed the Aβ plaque deposits in the dentate gyrus of the hippocampus, with the astrocyte surrounded. The neurons loss was significant in the injection point and pin hole of injection with Nissl's staining methods. GFAP-positive cells increased significantly compared with the uninjected lateral of the hippocampus. Conclusion Although Aβ_(1-40)-injected rat models could simulate some characteristic pathological features of human Alzheimer diseases, Aβ deposits and neurons loss in partial hippocampal, it would not simulate the progressive degenenration in the brain of AD. The double transgenie PS1/APP mice could simulate the specific pathogenesis and progressive changes of AD, mainly is Aβ deposits and the spongiocyte response, while no neurons loss were observed in this model.     

Comparative study of histopathology changes between the PS1/APP double transgenic mouse model and Aβ1-40 -injected rat model of Alzheimer disease

Objective To identify the genetype of the PS1/APP double transgenie mouse model, then to analyse the histopathological changes in the brain and compare the differences between the transgenie mice models and Aβ1-40-injeeted rats models of Alzheimer disease. Methods The modified congo red staining, Nissl＇s staining and immunohistology staining was used to observe the Aβ deposits, activation of astrocyte respectively. Results ①The PS1/APP transgenic mouse extensively displayed Aβ deposits in the cortex and hippocampal structures, and GFAP positive cells were aggregated in mass and surrounded the congo red-positive plaque. ②The Aβ1-40-intrahippocmnpal-injeeted rat model showed the Aβ plaque deposits in the dentate gyrus of the hippocampus, with the astrocyte surrounded. The neurons loss was significant in the injection point and pin hole of injection with Nissl＇s staining methods. GFAP-positive cells increased significantly compared with the uninjected lateral of the hippocampus. Conclusion Although Aβ1-40-injected rat models could simulate some characteristic pathological features of human Alzheimer diseases, Aβ deposits and neurons loss in partial hippocampal, it would not simulate the progressive degenenration in the brain of AD. The double transgenie PS1/APP mice could simulate the specific pathogenesis and progressive changes of AD, mainly is Aβ deposits and the spongiocyte response , while no neurons loss were observed in this model.     

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。     

Immunization with a new DNA vaccine for Alzheimer's disease elicited Th2 immune response in BALB/c mice by in vivo electroporation

Immunization with synthetic amyloid β-protein (Aβ) peptide has resulted in preventing and clearing Aβ deposits as well as improving cognitive function in transgenic mouse models of Alzheimer's disease (AD). But similar immunization studies in humans were halted due to the risk of inducing T cell-mediated meningoencephalitis. A safe and effective vaccine for AD requires not only therapeutic levels of anti-Aβ antibodies but also the prevention of an adverse T cell-mediated, proinflammatory autoimmune response. In this study, we developed a DNA vaccine, p(Aβ(3-10))(10)-IL-4, encoding ten tandem repeats of Aβ(3-10) fused with mouse cytokine interleukin-4 (IL-4) as a molecular adjuvant. Wild-type mice were injected intramuscularly with p(Aβ(3-10))(10)-IL-4 followed by in vivo electroporation. The p(Aβ(3-10))(10)-IL-4 vaccine elicited high titer anti-Aβ antibodies which bound to Aβ plaque in brain tissue from a ten-month-old APP/PS1 transgenic mouse. The antibody isotype was mainly IgG(1) and the IgG(1)/IgG(2a) ratio in the p(Aβ(3-10))(10)-IL-4 group was approximately eight times greater than that of the Aβ(42) group. Ex vivo cultured splenocytes isolated from mice immunized with p(Aβ(3-10))(10)-IL-4 exhibited a low IFN-γ response and a high IL-4 response compared with the control group. These results indicate that immunization with the p(Aβ(3-10))(10)-IL-4 vaccine induced effective anti-Aβ antibodies and elicited a Th2-polarized immune response that had a lower potential to cause an inflammatory T cell response. Thus, the DNA vaccine, p(Aβ(3-10))(10)-IL-4, may be a safe and efficient 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.     

Aβ_(3-10)多价腺病毒疫苗鼻粘膜免疫阿尔茨海默病转基因鼠的炎症反应研究

目的探讨基因重组腺病毒疫苗Ad-Aβ_((3-10)10)-CpG鼻粘膜免疫APPswe/PSEN1d E9双转基因鼠诱导的炎症反应。方法 18只雄性10月龄APPswe/PSEN1dE9鼠,随机分为3组,分别以Aβ_(3-10)多价腺病毒疫苗AdAβ_((3-10)10)-CpG、空腺病毒载体鼻粘膜免疫及Aβ_(1-42)肽肌内注射免疫,用MTT法检测脾细胞增殖反应、ELISA法检测脾细胞培养上清和脑组织匀浆中IL-4和INF-γ水平,免疫组化法检测脑内星形胶质细胞及淋巴细胞浸润。结果 Ad-Aβ_((3-10)10)-CpG组和Aβ_(1-42)组,在相对应的免疫原刺激孔产生较高水平的增殖率,高于非相应免疫原刺激孔(P0.05),但低于Con A刺激孔(P0.05)。皮质和海马区GFAP阳性细胞所占面积百分比为:空腺病毒载体组Aβ_(1-42)组Ad-Aβ_((3-10)10)-CpG组。各组小鼠脑组织在血管内和脑实质偶尔发现个别CD3、CD5阳性细胞,3组没有显著差异。Ad-Aβ_((3-10)10)-CpG组和Aβ_(1-42)组中在其相对应的免疫原刺激时检测到较高水平的IL-4、IFN-γ。Aβ_(1-42)组在Aβ_(1-42)肽刺激孔的IFN-γ水平显著高于Ad-Aβ_((3-10)10)-CpG组在Aβ_(3-10)肽刺激孔的IFN-γ水平(P0.05)。AdAβ_((3-10)10)-CpG组脑组织匀浆IL-4水平大于Aβ_(1-42)组,但没有显著性差异(P0.05),Ad-Aβ_((3-10)10)-CpG组脑组织匀浆中IFN-γ水平显著小于Aβ_(1-42)组(P0.05)。结论腺病毒疫苗Ad-Aβ_((3-10)10)-CpG鼻粘膜免疫APPswe/PSEN1dE9双转基因鼠主要产生Th2型免疫应答,可以减少脑内星形胶质细胞活化,未引起脑内炎症反应,即避免了Aβ_(1-42)全肽段所引起的炎症反应。     

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。     

Reduced Th1 and enhanced Th2 immunity after immunization with Alzheimer's beta-amyloid(1-42)

It has been demonstrated that immunization of transgenic mouse models of Alzheimer's disease (AD) with amyloid-beta(1-42) peptide (Abeta(1-42)) results in prevention of Abeta plaque formation and amelioration of established plaques in the brain. As the response of the T lymphocyte helper (Th) arm of the immune response had not yet been investigated after Abeta immunization, we i.p. immunized C57BL/6 mice with Abeta(1-42), Abeta(1-40), or phosphate-buffered saline (PBS), and examined markers of Th1 and Th2 immune responses in spleen and in splenocytes from these mice. Spleens from Abeta(1-42)-immunized mice demonstrated decreased interleukin-12 receptor beta chain expression compared to mice immunized with Abeta(1-40) or PBS. Consistently, following stimulation with concanavalin A or anti-CD3 antibody, primary splenocytes from Abeta(1-42)-immunized mice demonstrated elevated secretion of interleukin-4 and interleukin-10, and decreased levels of interferon-gamma. To validate this Th1-->Th2 shift in a transgenic mouse model of AD, we immunized Tg APP(sw) mice (line 2576) with Abeta(1-42) and found decreased Th1 (interleukin-2 and interferon-gamma) and elevated Th2 (interleukin-4 and interleukin-10) cytokines in their stimulated primary splenocytes. Interferon-gamma was markedly reduced and interleukin-10 was increased in blood plasma from these mice, effects that were associated with dramatically mitigated Abeta deposition after Abeta(1-42) immunization. Taken together, these results show enhanced Th2 and down-regulated Th1 immunity following immune challenge with Abeta(1-42).     

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.     

Vaccine efficacy of transcutaneous immunization with amyloid β using a dissolving microneedle array in a mouse model of Alzheimer's disease

Vaccine therapy for Alzheimer's disease (AD) based on the amyloid cascade hypothesis has recently attracted attention for treating AD. Injectable immunization using amyloid β peptide (Aβ) comprising 1–42 amino-acid residues (Aβ1–42) as antigens showed therapeutic efficacy in mice; however, the clinical trial of this injected Aβ1–42 vaccine was stopped due to the incidence of meningoencephalitis caused by excess activation of Th1 cells infiltrating the brain as a serious adverse reaction. Because recent studies have suggested that transcutaneous immunization (TCI) is likely to elicit Th2-dominant immune responses, TCI is expected to be effective in treating AD without inducing adverse reactions. Previously reported TCI procedures employed complicated and impractical vaccination procedures; therefore, a simple, easy-to-use, and novel TCI approach needs to be established. In this study, we investigated the vaccine efficacy of an Aβ1–42-containing TCI using our novel dissolving microneedle array (MicroHyala; MH) against AD. MH-based TCI induced anti-Aβ1–42 immune responses by simple and low-invasive application of Aβ1–42-containing MH to the skin. Unfortunately, this TCI system resulted in little significant improvement in cognitive function and Th2-dominant immune responses, suggesting the need for further modification.     

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.     

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。