Biomarkers for neurodegenerative diseases.

Biomarkers for neurodegenerative diseases are needed to improve the diagnostic workup in the clinic but also to facilitate the development and monitoring of effective disease-modifying therapies. Positron emission tomography methods detecting amyloid-ß and tau pathology in Alzheimer's disease have been increasingly used to improve the design of clinical trials and observational studies. In recent years, easily accessible and cost-effective blood-based biomarkers detecting the same Alzheimer's disease pathologies have been developed, which might revolutionize the diagnostic workup of Alzheimer's disease globally. Relevant biomarkers for α-synuclein pathology in Parkinson's disease are also emerging, as well as blood-based markers of general neurodegeneration and glial activation. This review presents an overview of the latest advances in the field of biomarkers for neurodegenerative diseases. Future directions are discussed regarding implementation of novel biomarkers in clinical practice and trials.

Mild magnetic nanoparticle hyperthermia promotes the disaggregation and microglia-mediated clearance of beta-amyloid plaques.

The formation of beta-amyloid (Aß) plaques is a classical hallmark of Alzheimer's disease (AD) that is associated with the promotion of neuroinflammation and subsequent neurotoxicity. Given the limited therapeutic options for targeting and clearing Aß plaques in AD, there is an urgent need to develop effective approaches to reduce plaque accumulation. The objective of this study was to validate mild magnetic nanoparticle (MNP) hyperthermia technology as a strategy to clear Aß deposits and determine the impact on microglia functionality. Our results demonstrated that the heating of MNPs localized to Aß aggregates upon exposure to high frequency alternating magnetic field (AMF) was sufficient to disrupt Aß plaques, resulting in its fragmentation. Importantly, this could facilitate the phagocytic clearance of Aß as well as attenuate pro-inflammatory responses by human microglial cells. Our results support the feasibility of mild MNP/AMF hyperthermia as a new strategy for reducing beta-amyloid burdens in Alzheimer's disease.

Clinical Relevance of [18F]Florbetaben and [18F]FDG PET/CT Imaging on the Management of Patients with Dementia.

PET of ß-Amyloid plaques (Aß) using [18F]florbetaben ([18F]FBB) and [18F]fluorodeoxyglucose ([18F]FDG) increasingly aid clinicians in early diagnosis of dementia, including Alzheimer's disease (AD), frontotemporal disease, dementia with Lewy bodies, and vascular dementia. The aim of this retrospective analysis was to evaluate clinical relevance of [18F]FBB, [18F]FDG PET and complimentary CSF measurements in patients with suspected dementia. In this study, 40 patients with clinically suspected or history of dementia underwent (1) measurement of Aß peptides, total tau, and p-tau protein levels in the cerebrospinal fluid (CSF) compared with healthy controls (HC); (2) clinical and neuropsychological assessment, which included Consortium to Establish a Registry for Alzheimer's Disease neuropsychological assessment battery (CERAD-NAB); (3) [18F]FBB and [18F]FDG PET imaging within an average of 3 weeks. The subjects were within 15 days stratified using PET, CSF measurements as HC, mild cognitive impaired (MCI) and dementia including Alzheimer´s disease. The predictive dementia-related cognitive decline values were supporting the measurements. PET images were evaluated visually and quantitatively using standard uptake value ratios (SUVR). Twenty-one (52.5%) subjects were amyloid-positive (Aß+), with a median neocortical SUVR of 1.80 for AD versus 1.20 relative to the respective 19 (47.5 %) amyloid-negative (Aß-) subjects. Moreover, the [18F]FDG and [18F]FBB confirmed within a sub-group of 10 patients a good complimentary role by correlation between amyloid pathology and brain glucose metabolism in 8 out of 10 subjects. The results suggest the clinical relevance for [18F]FBB combined with [18F]FDG PET retention and CFS measurements serving the management of our patients with dementia. Therefore, [18F]FBB combined with [18F]FDG PET is a helpful tool for differential diagnosis, and supports the patients' management as well as treatment.

Retinal hyperspectral imaging in the 5xFAD mouse model of Alzheimer's disease.

Hyperspectral imaging of the retina has recently been posited as a potentially useful form of spectroscopy of amyloid-beta (Aß) protein in the eyes of those with Alzheimer's disease (AD). The concept of using the retina as a biomarker for AD is an attractive one, as current screening tools for AD are either expensive or inaccessible. Recent studies have investigated hyperspectral imaging in Aß models however these studies have been in younger mice. Here we characterised hyperspectral reflectance profile in 6 to 17 months old 5xFAD mice and compare this to Aß in isolated preparations. Hyperspectral imaging was conducted across two preparations of Aß using a custom built bench ophthalmoscope. In the in vitro condition, 1 mg of purified human Aß42 was solubilised and left to aggregate for 72 h. This soluble/insoluble Aß mixture was then imaged by suspending the solution at a pipette tip and compared against phosphate buffered saline (PBS) control (n = 10 ROIs / group). In the in vivo condition, a 5xFAD transgenic mouse model was used and retinae were imaged at the age of 6 (n = 9), 12 (n = 9) and 17 months (n = 8) with age matched wildtype littermates as control (n = 12, n = 13, n = 15 respectively). In the vitro condition, hyperspectral imaging of the solution showed greater reflectance compared with vehicle (p < 0.01), with the greatest differences occurring in the short visible spectrum (< 500 nm). In the in vivo preparation, 5xFAD showed greater hyperspectral reflectance at all ages (6, 12, 17 months, p < 0.01). These differences were noted most in the short wavelengths at younger ages, with an additional peak appearing at longer wavelengths (~ 550 nm) with advancing age. This study shows that the presence of Aß (soluble/insoluble mixture) can increase the hyperspectral reflectance profile in vitro as well as in vivo. Differences were evident in the short wavelength spectrum (< 500 nm) in vitro and were preserved when imaged through the ocular media in the in vivo conditions. With advancing age a second hump around ~ 550 nm became more apparent. Hyperspectral imaging of the retina does not require the use of contrast agents and is a potentially useful and non-invasive biomarker for AD.

Expression and Purification of Intrinsically Disordered Aß Peptide and Setup of Reproducible Aggregation Kinetics Experiment.

High purity and sequence homogeneity of intrinsically disordered proteins are prerequisites for reproducible studies of the kinetics and equilibrium of their self-assembly reactions. Starting from the pure state enables quantitative studies of intrinsic and extrinsic factors in the process to understand its molecular determinants. Here we outline detailed protocols for recombinant expression and purification of ultra-pure amyloid ß peptide (Aß) in sequence homogeneous form, which allows for the setup of reproducible kinetic self-assembly experiments.

Amyloid-beta peptide detection via aptamer-functionalized nanowire sensors exploiting single-trap phenomena.

New highly sensitive direct methods for the early detection of peptides involved in Alzheimer's disease (AD) are required in order to prolong effective and healthy memory and thinking capabilities and also to stop the factors resulting in AD. In this contribution, we report the successful demonstration of a label-free approach for the detection of amyloid-beta (Aß) peptides by highly selective aptamers immobilized onto the SiO2 surface of the fabricated sensors. A modified single-stranded deoxyribonucleic acid (ssDNA) aptamer was specially designed and synthesized to detect the target amyloid beta-40 sequence (Aß-40). Electrolyte-insulator-semiconductor (EIS) structures as well as silicon (Si) nanowire (NW) field-effect transistors (FETs) covered with a thin SiO2 dielectric layer have been successfully functionalized with Aß-40-specific aptamers and used to detect ultra-low concentrations of the target peptide. The binding of amyloid-beta peptides of different concentrations to the surface of the sensors varied in the range from 0.1 pg/ml to 10 µg/ml resulting in a change of the surface potential was registered by the fabricated devices. Moreover, we show that the single-trap phenomena observed in the novel Si two-layer (TL) NW FET structures with advanced characteristic parameters can be effectively used to increase the sensitivity of nanoscale sensors. The obtained experimental data demonstrate a highly sensitive and reliable detection of ultra-low concentrations of the Aß-40 peptides. This opens up prospects for the development of real-time electrical biosensors for studying and understanding different stages of AD by utilizing Si TL NW FET structures fabricated on the basis of cost-efficient CMOS-compatible technology.

Improving separation optimization in capillary electrophoresis by using a general quality criterion.

In this paper we extend the use of the quality criterion t' to optimize separations in capillary electrophoresis (CE). The theoretical parameter t' takes into account not only the relative separation between a given pair of compounds but also their separation from the neutral species migrating with the electroosmotic flow (EOF). Furthermore, it can be composed for complex mixtures as a global multicriterium optimization function T', for a rapid, simple and reliable selection of optimized separation conditions by mathematical maximization. Here, we demonstrate the applicability of T' using as a variable the electrophoretic mobility (me) for the optimization of pH in the separation of a mixture of amyloid beta (Aß) peptide fragments. In addition, it is shown the versatility of T' using other variables related to me, which do not require experimental measurements. This is the case with ionizable compounds as the Aß peptide fragments, whose charge-to-mass ratios can be calculated if accurate pKa values are available in the literature. The excellent performance of T' for Aß peptide fragments is further validated optimizing the pH for the separation of mixtures of harmala alkaloids (HAlks) and quinolone antibiotics.

A simple aptasensor for Aß40 oligomers based on tunable mismatched base pairs of dsDNA and graphene oxide.

ß-amyloid 1-40 oligomers (Aß40O) is considered to be one of the important biomarkers for the diagnosis and treatment of Alzheimer's disease (AD). To explore a method with excellent performance is favorable for measuring the low concentration of Aß40O in AD patients. Here, we developed a simple and fast method with a double stranded DNA (dsDNA)/graphene oxide (GO) based sensor, which was a fluorescent probe for a highly sensitive detection of Aß40O down to 0.1 nM with a linear detectable range from 0.1 nM to 40 nM. The proposed sensor effectively reduced non-specific adsorption and improved the specificity of detection because of the covalent conjugation of a binding DNA (bDNA) containing Aß40O-targeting aptamer (AptAß) onto GO surface, as well as the optimization of the number of mismatch base pairs of dsDNA. Moreover, AD patients and healthy persons were distinguished by this present method. All advantages of this method are exactly what the clinical detection of AD biomarkers need. This novel aptasensor might pave a way towards the early diagnosis of AD.

Anti-Alzheimer's Materials Isolated from Marine Bio-resources: A Review.

The most common type of dementia found in the elderly population is Alzheimer's disease. The disease not only impacts the patients and their families but also the society therefore, the main focus of researchers is to search new bioactive materials for treating AD. The marine environment is a rich source of functional ingredients and to date, we can find sufficient research relating to anti- Alzheimer's compounds isolated from marine environment. Therefore, this review focuses on the anti- Alzheimer's material from marine bio-resources and then expounds on the anti-Alzheimer's compounds from marine seaweed, marine animal and marine microorganisms. Moreover, because of the complexity of the disease, different hypothesizes have been elaborated and active compounds have been isolated to inhibit different stages of pathophysiological mechanisms. Sulfated polysaccharides, glycoprotein, and enzymatic hydrolysates from marine seaweeds, peptides, dietary omega-3 polyunsaturated fatty acids and skeletal polysaccharide from marine animals and secondary metabolites from marine microorganism are summarized in this review under the anti-Alzheimer's compounds from the marine.

Platelet-driven formation of interface peptide nano-network biosensor enabling a non-invasive means for early detection of Alzheimer's disease.

Soft material fabricated with DNA origami or peptide cross-linking technique may be promising theranostic platforms in the future; however, their naturally occurring counterparts, such as the peptide aggregates in the neurodegenerative diseases, constitute an increasingly burdensome issue of public health. Thus, a design of artificial peptide nano-network biosensor is conceived, in an attempt to combat the natural pathological peptides, by mimicking their pathogenesis process. Specifically, periphery platelet can secrete A-beta and induce its cross-linking & aggregation to form a surface peptide nano-network, resulting in large numbers of poly-tyrosine strands being covalently trapped in the network to serve as an efficient signal amplifier, through the electrochemical oxidation of tyrosine. This method is sensitive and quantitative in the range of normal and pathological periphery platelet distribution and can effectively discriminate Alzheimer's disease (AD) patients based on the detected potential neurodegenerative activity of platelet. These results may point to some future perspective of this method in the early screening of AD.

An organic electrochemical transistor integrated with a molecularly selective isoporous membrane for amyloid-ß detection.

Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with severe memory loss and impaired cognitive skills. A common pathological change found in AD-affected brains is the accumulation of a peptide named amyloid-ß (Aß) that can form plaques. Aß aggregates are visible to structural scanning tools; however, these bulky and expensive instruments are accessible to trained personnel in clinical settings only, thus hampering timely diagnosis of the disease, particularly in low-resource settings. In this work, we design an organic electrochemical transistor (OECT) for in vitro detection of Aß aggregates in human serum. The OECT channel is integrated with a nanostructured isoporous membrane which has a strong affinity for Aß aggregates. The detection mechanism relies on the membrane capturing Aß aggregates larger than the size of its pores and thus blocking the penetration of electrolyte ions into the channel underneath. Combining the high transconductance of the OECT with the precise porosity and selectivity of the membrane, the device detects the presence of Aß aggregates in human serum samples with excellent sensitivity. This is the first-time demonstration of a biofunctionalized, nanostructured, and isoporous membrane integrated with a high-performance transistor for biosensing. This robust, low-power, non-invasive, and miniaturized sensor aids in the development of point-of-care tools for early diagnosis of AD.

Dielectrophoresis of Amyloid-Beta Proteins as a Microfluidic Template for Alzheimer's Research.

We employed dielectrophoresis to a yeast cell suspension containing amyloid-beta proteins (Aß) in a microfluidic environment. The Aß was separated from the cells and characterized using the gradual dissolution of Aß as a function of the applied dielectrophoretic parameters. We established the gradual dissolution of Aß under specific dielectrophoretic parameters. Further, Aß in the fibril form at the tip of the electrode dissolved at high frequency. This was perhaps due to the conductivity of the suspending medium changing according to the frequency, which resulted in a higher temperature at the tips of the electrodes, and consequently in the breakdown of the hydrogen bonds. However, those shaped as spheroidal monomers experienced a delay in the Aß fibril transformation process. Yeast cells exposed to relatively low temperatures at the base of the electrode did not experience a positive or negative change in viability. The DEP microfluidic platform incorporating the integrated microtip electrode array was able to selectively manipulate the yeast cells and dissolve the Aß to a controlled extent. We demonstrate suitable dielectrophoretic parameters to induce such manipulation, which is highly relevant for Aß-related colloidal microfluidic research and could be applied to Alzheimer's research in the future.

Influx of Tau and Amyloid-ß Proteins into the Blood During Hemodialysis as a Therapeutic Extracorporeal Blood Amyloid-ß Removal System for Alzheimer's Disease.

The accumulation of amyloid-ß protein (Aß) and tau in the brain is a major pathological change related to Alzheimer's disease. We have continued to develop Extracorporeal Blood Aß Removal Systems (E-BARS) as a method for enhancing Aß clearance from the brain. Our previous report revealed that dialyzers effectively remove blood Aß and evoke large Aß influxes into the blood, resulting in a decrease in brain Aß accumulation after initiating hemodialysis, and that patients who underwent hemodialysis had lower brain Aß accumulation than those who did not. Here, plasma total tau concentrations from 30 patients undergoing hemodialysis were measured using an ultrasensitive immunoassay and compared to those from 11 age-matched controls. Plasma total tau concentrations were higher in patients with renal failure regardless of whether they underwent hemodialysis, suggesting the involvement of the kidneys in tau degradation and excretion. Hemodialyzers effectively removed blood Aß but not extracorporeal blood tau. The influx of tau into the blood was observed at around the 1 h period during hemodialysis sessions. However, the influx amount of tau was far smaller than that of Aß. Furthermore, histopathological analysis revealed similar, not significantly less, cerebral cortex phosphorylated tau accumulation between the 17 patients who underwent hemodialysis and the 16 age-matched subjects who did not, although both groups showed sparse accumulation. These findings suggest that hemodialysis may induce both tau and Aß migration into the blood. However, as a therapeutic strategy for Alzheimer's disease, it may only be effective for removing Aß from the brain.

Highly sensitive aptasensor based on interferometric reflectance spectroscopy for the determination of amyloid ß as an Alzheimer's disease biomarkers using nanoporous anodic alumina.

It is well known that Alzheimer's disease is one of the global challenges for the 21st century. Therefore, it is urgent to develop a reliable biosensor for the detection of this disease. Here in, we have developed for the first time, an aptasensor based on interferometric reflectance spectroscopy (IRS) for the determination of amyloid ß (Aß) oligomers that is an Alzheimer's disease biomarker. For this purpose, the nanoporous anodic alumina (NAA) was first fabricated. After that, the pore walls of the NAA were modified with (3-aminopropyl) trimethoxysilane (NAA-NH2). The amino-terminal aptamers probe were then attached to the pore walls of the NAA-NH2 by using glutaraldehyde (GA) as the cross-linking agent. Subsequently, methylene blue (MB) was immobilized into the aptamer as the photo-probe, generating the MB/G-quadruplex complex. Since MB has a high absorption coefficient, the intensity of the reflected white light to the charge-coupled device (CCD) detector decreased. In the presence of the Aß oligomers that have high affinity to the immobilized aptamer, the MB/quadruplex complex broke and MB washed away from the aptasensor. Therefore, the intensity of the reflected white light to the CCD detector increased. The increased signal intensity of the aptasensor has a logarithmic relationship with the concentration of Aß oligomers. The proposed aptasensor exhibited a good response to the concentration of Aß oligomers in the range of 0.5-50.0 µg × mL-1. The experimental detection limit was of 0.02 µg × mL-1 (at 3σ/S). The proposed optical aptasensor exhibited good selectivity, linear range, and stability.

Multi-site dynamic recording for Aß oligomers-induced Alzheimer's disease in vitro based on neuronal network chip.

Alzheimer's disease (AD) is a chronic central neurodegenerative disease. The pathological features of AD are the extracellular deposition of senile plaques formed by amyloid-ß oligomers (AßOs) and the intracellular accumulation of neurofibrillary tangles. However, due to the lack of effective method and experimental models to study the cognitive decline, communication at cell resolution and the implementation of interventions, the diagnosis and treatment on AD still progress slowly. In this paper, we established a pathological model of AD in vitro based on AßOs-induced hippocampal neuronal network chip for multi-site dynamic analysis of the neuronal electrical activity and network connection. The multiple characteristic parameters, including positive and negative spike intervals, firing rate and peak-to-peak values, were extracted through the analysis of spike signals, and two firing patterns from the interneurons and pyramidal neurons were recorded. The spatial firing patterns mapping and cross-correlation between channels were performed to validate the degeneration of neuronal network connectivity. Moreover, an electrical stimulation with frequency at 40 Hz was exerted to preliminarily explore the therapeutic effect on the pathological model of AD. This neuronal network chip enables the implementation of AD models in vitro for studying basic mechanisms of neurodegeneration within networks and for the parallel testing of various potential therapies. It can be a novel technique in the research of AD pathological model in vitro.

Quench-type electrochemiluminescence immunosensor for detection of amyloid ß-protein based on resonance energy transfer from luminol@SnS2-Pd to Cu doped WO3 nanoparticles.

A highly efficient quench-type electrochemiluminescence (ECL) immunosensor was proposed for the trace detection of amyloid ß-protein (Aß1-42). In this work, tin disulfide nanoflowers (SnS2 NFs) with large specific surface area, favorable catalytic property and chemical stability were prepared and used as substrate material. Taking advantage of the excellent catalytic ability and biocompatibility, palladium nanoparticles (Pd NPs) were in situ reduced on SnS2 NFs to obtain SnS2-Pd. Moreover it could combine with large amounts of luminol and achieve a strong ECL signal output. In addition, copper doped mesoporous tungsten trioxide (Cu:WO3) nanoparticles were selected to quench ECL emission of luminol@SnS2-Pd via resonance energy transfer, where luminol@SnS2-Pd was the donor and Cu:WO3 was the acceptor. On this basis, a quench-type ECL immunosensor was constructed for detection of Aß1-42. Under optimum conditions, the fabricated ECL immunosensor showed sensitive response to Aß1-42 concentration from 0.1 pg/mL to 50 ng/mL with a low detection limit of 5.4 fg/mL (S/N = 3). It is expected to be a promising analytical tool for the sensitive detection of Aß1-42 and other biomarkers with high specificity, good reproducibility and long-term stability.

In-capillary immuno-preconcentration with circulating bio-functionalized magnetic beads for capillary electrophoresis.

This study reports on the conception of magneto-Capillary Electrophoresis (magneto-CE), an approach integrating immuno-capture on circulating bio-functionalized magnetic beads into a unique capillary for preconcentration and electrokinetic separation. This hybrid mode is an evolution of in-capillary magnetic bead-based operation from static cluster format to dynamic configuration where beads are allowed to controllably circulate inside a CE capillary for interaction improvement. To implement the magneto-CE operation, a purpose-made instrument was constructed, allowing visual observation of the movement of the magnetic beads. We applied a new methodological strategy for determination of the amyloid ß peptide (Aß 1-42), which is as an established biomarker for molecular diagnosis of Alzheimer's disease (AD). The methodology is based on magneto-immuno-capture of fluorescently labeled Aß 1-42 followed by a chemical elution with a basic solution prior to CE separation with laser induced fluorescent (LIF) detection. The superiority of this dynamic configuration of magneto-CE was demonstrated for this target analyte, with sample pretreatment and separation being performed in-capillary without any delay in between and without any waste of pretreated sample, which otherwise would not be the case with offline/batch-wise operation.

A versatile method for producing labeled or unlabeled Aß55, Aß40, and other ß-amyloid family peptides.

We present a straightforward, versatile method for expressing and purifying ß-amyloid (Aß40) and transmembrane peptides derived from ß-amyloid precursor protein (Aß55). In principle, these methods should be applicable to other types of strongly aggregating peptides. We start with a DNA plasmid encoding a HexaHis tag with a flexible, hydrophilic linker sequence, followed by a cleavage site, and then Aß peptides. The HexaHis tag rather than a protein fusion partner (e.g., GST) obviates the need for a folded protein in affinity purification. Second, we present two cleavage methods, using either Factor Xa or BNPS-Skatole. Although the latter procedure requires subsequent reduction of the product, we describe methods for minimizing side reactions. Because the use of BNPS-Skatole obviates the need for a folded protein in the cleavage reaction, it is compatible with harsh conditions (e.g., inclusion of detergents and denaturants) needed to solubilize the fusion proteins; such conditions tend to inactivate Factor Xa. Finally, we also describe purification strategies for Aß40 and Aß55 using FPLC and/or reverse phase HPLC. Yields of peptide after these BNPS-Skatole cleavage and peptide reduction, though subquantitative, greatly exceed those obtained using Factor Xa cleavage, as the reaction of BNPS-Skatole is insensitive to the presence of detergents and denaturants, and therefore can be used to produce highly aggregative and low solubility peptides such as Aß55. Trp is a low abundance amino acid in proteins generally, and for peptides like Aß55, and other transmembane peptides lacking Trp in relevant positions, this cleavage method remains a useful option.

Ultrasensitive amyloid-ß proteins detection based on curcumin conjugated ZnO nanoparticles quenching electrochemiluminescence behavior of luminol immobilized on Au@MoS2/Bi2S3 nanorods.

This work reported curcumin quenched electrochemiluminescence (ECL) behavior of luminol with H2O2 as coreactant. To achieve excellent ECL signal, luminol was anchored on Au functionalized MoS2/Bi2S3 nanorods (Luminol-Au@MoS2/Bi2S3), which not only exhibited super catalysis for H2O2 decomposition to enhance the ECL intensity of luminol/H2O2 system, but also offered a satisfying substrate material for grafting more Au nanoparticles via Au-S binding which was used to adsorb luminol and primary antibodies. As an antioxidant, the hydrophobicity of curcumin limited its bioavailability. Herein, curcumin was conjugated with ZnO nanomaterials (ZnO-CR) to increase its dispersity in water which can make it more bioavailable. Moreover, the possible quenching mechanism between luminol/H2O2 system and ZnO-CR was ascribed to the consumption of superoxide radical (O2•-) and energy transfer. The consumption amount of O2•- was calculated to be 1.2092 µM/mL through superoxide anion free radical detection kit. Besides, polydopamine was self-polymerized on the surface of ZnO-CR (PDA@ZnO-CR) to anchor secondary antibodies. In optimum parameter, the ECL immunosensor exhibited excellent performance for amyloid-ß(Aß) proteins detection with a wide detection range from 0.05 pg mL-1 to 10 ng mL-1 and a low detection limit of 21 fg mL-1 (S/N = 3).

Active immunization with norovirus P particle-based amyloid-ß chimeric protein vaccine induces high titers of anti-Aß antibodies in mice.

BACKGROUND: Active immunotherapy targeting amyloid-ß (Aß) is a promising treatment for Alzheimer's disease (AD). Numerous preclinical studies and clinical trials demonstrated that a safe and effective AD vaccine should induce high titers of anti-Aß antibodies while avoiding the activation of T cells specific to Aß. RESULTS: An untagged Aß1-6 chimeric protein vaccine against AD based on norovirus (NoV) P particle was expressed in Escherichia coli and obtained by sequential chromatography. Analysis of protein characteristics showed that the untagged Aß1-6 chimeric protein expressed in soluble form exhibited the highest particle homogeneity, with highest purity and minimal host cell protein (HCP) and residual DNA content. Importantly, the untagged Aß1-6 chimeric soluble protein could induce the strongest Aß-specific humoral immune responses without activation of harmful Aß-specific T cells in mice. CONCLUSIONS: The untagged Aß1-6 chimeric protein vaccine is safe and highly immunogenic. Further research will determine the efficacy in cognitive improvement and disease progression delay.