Lu Fu

Norovirus P particle: an excellent vaccine platform for antibody production against Alzheimer's disease.

Active vaccination against amyloid β (Aβ42) is considered a potential therapeutic approach for Alzheimers disease (AD). However, immunization with synthetic human Aβ1-42 has resulted in meningoencephalitis in 6% of patients and generated only low-titer anti-Aβ42 antibodies. In order to develop a safe and effective vaccine against Alzheimers disease, the Aβ1-6 peptide was used as the novel immunogen and Norovirus P particles as the vaccine platform in this study. By inserting and presenting Aβ1-6 on the outermost surface of the P particle, we showed that the chimeric P particle-based AD protein vaccine could elicit a strong immune response, inducing highly specific antibody titers against Aβ42 without causing T-cell activation. Furthermore, antibodies induced by the AD protein vaccines were demonstrated to be effective at the cellular level. In addition, we also compared the immunogenicity of the chimeric P particles with different insertional loci in the loop structure domain and demonstrated that insertion of the antigen into all three loops of the P particle at the same time could significantly improve immune responses to the vaccine. In conclusion, the Norovirus P particle is an excellent vaccine platform for stimulating Aβ42 antibody production, and chimeric P particles may be developed as an effective therapy for AD.

Comparison of neurotoxicity of different aggregated forms of Aβ40, Aβ42 and Aβ43 in cell cultures

The abnormal deposition of amyloid‐β (Aβ) peptides in the brain is the main neuropathological hallmark of Alzheimers disease (AD). Amyloid deposits are formed by a heterogeneous mixture of Aβ peptides, among which the most studied are Aβ40 and Aβ42. Aβ40 is abundantly produced in the human brain, but the level of Aβ42 is remarkably increased in the brain of AD patients. Aside from Aβ40 and Aβ42, recent data have raised the possibility that Aβ43 peptides may be instrumental in AD pathogenesis. Besides its length, whether the Aβ aggregated form accounts for the neurotoxicity is also particularly controversial. Aβ fibrils are generally considered as key pathogenic substances in AD pathogenesis. Nevertheless, recent data implicated soluble Aβ oligomers as the main cause of synaptic dysfunction and memory loss in AD. To further address this uncertainty, we analyzed the neurotoxicity of different Aβ species and Aβ forms at the cellular level. The results showed that Aβ42 could form oligomers significantly faster than Aβ40 and Aβ43 and Aβ42 oligomers showed the greatest level of neurotoxicity. Regardless of the length of Aβ peptides, Aβ oligomers induced significantly higher cytotoxicity compared with the other two Aβ forms. Surprisingly, the neurotoxicity of fibrils in PC12 cells was only marginally but not significantly stronger than monomers, contrary to previous reports. Altogether, our findings demonstrate the high pathogenicity of Aβ42 among the three Aβ species and support the idea that Aβ42 oligomers contribute to the pathological events leading to neurodegeneration in AD. Copyright

Elicitation of HIV-1 neutralizing antibodies by presentation of 4E10 and 10E8 epitopes on Norovirus P particles.

Eliciting efficient broadly neutralizing antibodies (BnAbs) is a major goal in vaccine development against human immunodeficiency virus type 1 (HIV-1). Conserved epitopes in the membrane-proximal external region (MPER) of HIV-1 are a significant target. In this study, Norovirus P particles (NoV PPs) were used as carriers to display conformational 4E10 and 10E8 epitopes in different patterns with an appropriate linker. Immune responses to the recombinant NoV PPs were characterized in guinea pigs and Balb/c mice and could induce high levels of MPER-binding antibodies. Modest neutralizing activities could be detected in sera of guinea pigs but not of Balb/c mice. The 4E10 or 10E8 epitopes dispersed on three loops on the outermost surface of NoV PPs (4E10-loop123 PP or 10E8-loop123 PP) elicited higher neutralizing activities than the equivalent number of epitopes presented on loop 2 only (4E10-3loop2 PP or 10E8-3loop2 PP). The epitopes on different loops of the PP were well-exposed and likely formed an appropriate conformation to induce neutralizing antibodies. Although sera of immunized guinea pigs could neutralize several HIV envelope-pseudoviruses, a vaccine candidate for efficiently inducing HIV-1 BnAbs remains to be developed.

Norovirus P particle-based active Aβ immunotherapy elicits sufficient immunogenicity and improves cognitive capacity in a mouse model of Alzheimer’s disease

Disease-modifying immunotherapies focusing on reducing amyloid-beta (Aβ) deposition are the main treatment for Alzheimer’s disease (AD). However, none of the Aβ immunotherapies has produced clinically meaningful results to date. The main reason for this lack of efficacy is that the vaccine induces insufficiently high antibody titers, as it contains small B-cell epitope of Aβ to avoid Aβ42-specific T-cell activation. With the aim of generating a potent AD vaccine, we designed the protein PP-3copy-Aβ1-6-loop123, comprising three copies of Aβ1-6 inserted into three loops of a novel vaccine platform, the norovirus P particle, which could present Aβ at its surface and remarkably enhance the immunogenicity of the vaccine. We demonstrated that PP-3copy-Aβ1-6-loop123 was able to elicit high antibody titers against Aβ42, without causing T-cell activation, in AD mice regardless of their age. Importantly, PP-3copy-Aβ1-6-loop123 treatment successfully reduced amyloid deposition, rescued memory loss, and repaired hippocampus damage in AD mice. The Aβ antibodies induced by this active immunotherapy reacted with and disrupted aggregated Aβ, reducing its cellular toxicity. In addition, our results suggested PP-3copy-Aβ1-6-loop123 immunization could restore Aβ42 homeostasis in both the serum and brain. Thus, the P particle-based Aβ epitope vaccine is a sufficiently immunogenic and safe immunotherapeutic intervention for Alzheimer’s disease.

Analysis of serum β-amyloid peptides, α2-macroglobulin, complement factor H, and clusterin levels in APP/PS1 transgenic mice during progression of Alzheimer's disease.

As a progressive age-related neurodegenerative disorder, Alzheimer’s disease (AD) is a global health concern. Despite the availability of psychological testing, neuroimaging, genetic testing, and biochemical assays of cerebrospinal fluid, convenient and accurate blood biomarkers for the prediction, diagnosis, and preclinical studies of AD are still lacking. The present study aims to longitudinally evaluate the feasibility of &bgr;-amyloid proteins, &agr;2-macroglobulin (&agr;-2M), complement factor H (CFH), and clusterin as blood biomarkers of AD. Using APP/PS1 transgenic and wild-type mice, cognitive impairment and amyloid plaque counts in the brain were evaluated over a range of ages using the Morris water maze test and immunohistochemistry methods, respectively. Serum A&bgr;40, A&bgr;42, &agr;-2M, CFH, and clusterin levels were measured by enzyme-linked immunosorbent assay and correlated with progression of AD. APP/PS1 transgenic mice presented progressive AD characteristics at the ages of 3, 6, 9, and 12 months. Serum A&bgr;42 levels and A&bgr;42/A&bgr;40 ratios increased significantly in transgenic 3- and 6-month-old mice compared with controls. Serum CFH levels decreased significantly in 3- and 6-month-old transgenic mice compared with controls. Meanwhile, serum clusterin levels increased significantly in 12-month-old transgenic mice compared with controls. The &agr;-2M level was not significantly different between transgenic and wild-type mice. The APP/PS1 transgenic mouse is a model of familial AD. The present study indicated that the serum A&bgr;42 level, A&bgr;42/A&bgr;40 ratio, and CFH level are potential biomarkers in preclinical and early stages of AD, whereas serum clusterin level is a potential biomarker in the late stage of AD.

Establishment of a novel method without sequence modification for developing NoV P particle-based chimeric vaccines

The Norovirus (NoV) P particle (PP) is a subviral particle formed by 24 copies of the protruding (P) domain of the capsid protein. Each P domain has three surface loops that can be used for foreign antigen presentation. Hence, PPs have been demonstrated to be an excellent platform for vaccine development against many pathogens. However, current processes for preparing those chimeric PP vaccines vary and would change the original sequence of the PP. A detailed strategy also has not been reported for inserting a foreign antigen into all three loops. In order to develop a novel method for preparing distinct types of PP-based protein vaccines, we created two restriction enzyme sites (EagI and KpnI) in the P domain by site-directed mutagenesis without changing its original sequence. A synthesized gene with three copies of the Alzheimers disease (AD) immunogen Aβ1-6 was then incorporated in loop2 of the P domain. Additionally, a synthesized gene with one copy of Aβ1-6 was inserted into each loop of the P domain. Furthermore, two recombinant proteins PP-3 copy-Aβ1-6-loop2 and PP-1 copy-Aβ1-6-loop123 were successfully purified without affecting PP formation. Particle size analysis and TEM observations demonstrated that the two chimeric P particles were still able to form 24-mer nanoparticles. Moreover, the two chimeric PP-based AD vaccines could both efficiently elicit strong immune responses in the mouse model. In conclusion, we have successfully established a novel method for preparing vaccines based on the NoV PP which would not affect PP sequence and function.

A novel Aβ epitope vaccine based on bacterium-like particle against Alzheimer’s disease

HighlightsAlzheimers disease vaccine using the A&bgr;1–6 epitope is safe and effective.The BLP‐based AD vaccine induced antibodies against A&bgr;42 without T‐cell activation.A suitable epitope copy number improved the immunogenicity of the vaccine.Antibodies effectively blocked toxicity of A&bgr;42 oligomer at the cellular level. &NA; Amyloid‐beta (A&bgr;) plaque accumulation in the brain is one of the hallmarks of Alzheimers disease (AD). Immunotherapy against A&bgr; was considered a potential strategy for reducing the A&bgr; load in the brain. However, none of the A&bgr; immunotherapies have produced clinically meaningful results to date, due to poor safety or lack of efficacy. Thus, we aimed to design a safe and effective vaccine against AD. In this study, we used bacterium‐like particles (BLPs) as carriers and different copy numbers of the A&bgr; 1‐6 peptide as epitopes to design four A&bgr; active immunization vaccines. The epitopes containing different copy numbers of the A&bgr; 1‐6 peptide were specifically loaded on the surface of BLPs via fusion with a peptidoglycan anchoring domain. These four BLP‐based A&bgr; vaccines successfully induced high levels of A&bgr;42‐specific antibodies in mice. However, none of the vaccines induced a T‐cell‐mediated immune response. Importantly, the antibodies induced by these four vaccines were effective in blocking A&bgr;42 oligomer toxicity at the cellular level. Among the four vaccines, 6copy‐A&bgr; 1‐6 ‐PA‐BLP was the most effective in inducing A&bgr;‐specific antibodies, indicating that a suitable epitope copy number is critical for high immunogenicity of the BLP‐based vaccine. Furthermore, high levels of serum A&bgr;‐specific antibodies could still be detected 3 months after the final administration of 6copy‐A&bgr; 1‐6 ‐PA‐BLP. Thus, 6copy‐A&bgr; 1‐6 ‐PA‐BLP may be a potential therapeutic treatment for AD.

Progressive Spatial Memory Impairment, Brain Amyloid Deposition and Changes in Serum Amyloid Levels as a Function of Age in APPswe/PS1dE9 Mice

BACKGROUND Mice co-expressing human amyloid precursor protein with the Swedish mutation (APPswe) and exon-9-deleted presenilin (PS1dE9) has become one of the most widely used mouse models for studying Alzheimers disease (AD) pathogenesis and preclinical studies of AD therapeutic approaches. OBJECTIVE In this study, we systematically investigated cognitive decline, amyloid-β (Aβ) deposition and cerebral or Aβ serum levels as well as the relationships among these measures in APPswe/PS1dE9 transgenic mice. METHOD APPswe/PS1dE9 mice were separated into four equal age cohorts (4, 6, 9, and 12 months). We assessed cognitive capacity, deposited plaques, and the levels of Aβ40/Aβ42 in brain tissue and serum of mice at different ages. RESULTS APPswe/PS1dE9 mice exhibited declined memory beginning at 6 months of age, with cognitive capacity remarkably impaired at 12-months. Coincidently, amyloid deposits began to develop in transgenic mice brain at 6-months and increased with age. In addition, Aβ42 levels in brains of APPswe/ PS1dE9 mice increased with age with no parallel increase in Aβ40. The concentration of serum Aβ42 declined from 4 to 6 months of age, but a similar age-dependent decrease was not observed for Aβ40. CONCLUSION APPswe/PS1dE9 transgenic mice began to develop amyloid plaques at 6 months of age and exhibited a corresponding impairment of spatial learning capacity. Serum Aβ42 level decreased remarkably from 4 to 6 months, at which stage Aβ42 began to accumulate in the brain and deposit as plaques.

PRECLINICAL STUDIES OF NOROVIRUS P PARTICLE-BASED ACTIVE Aβ IMMUNOTHERAPIES FOR ALZHEIMER’S DISEASE

Background: Focused Ultrasound (FUS)-induced blood-brain barrier opening has been shown to eliminate 52% of the amyloid plaque load and reduce the misfolded tau protein from both the entorhinal cortex and the axonal compartment of the hippocampus. Since the effect of ultrasound has been proven beneficial when studying the AD pathologies separately, it is essential to investigate, for the first time, the interaction of FUS-induced BBB opening in the presence of both amyloid-b and tau. Moreover, the unilateral sonication of the transgenic brain provides a unique opportunity to explore potential bilateral effects. Methods: For this study 9 mice of the 3xTg line (11 months old) were randomly assigned to the control and treatment group with the later receiving a double sonication covering the hippocampal region once per week for 4 consecutive weeks. Following the last sonication the mice were sacrificed and counterstained for tau protein (AT8), amyloid-b (Ab42-specific antibody) as well as microglia activation (CD68). The images were acquired by means of confocal microscopy and customized algorithms were constructed to quantify the number of plaques, cells and the axonal distribution of the tau-marker. The same brain slices were utilized to quantify the hippocampal density of the CD68 marker by intensity-based quantification. Results: In Figure 1, the phosphorylated tau is shown to be present in both the somatodendritic and the axonal compartment (red) in the contralateral side. Although the cell bodies affected by

Eliciting 10E8-like antibodies by the membrane proximal external region peptide of HIV-1 in guinea pigs

ObjectiveTo develop an immunotherapy for HIV that can elicit 10E8-like broadly-neutralizing antibodies in guinea pigs, using a multiple antigen peptide (MAP) system as the platform and 10E8 peptide as the epitope.ResultsThe immunogen, 10E8-MAP4, was synthetized using the MAP system. The synthetic 10E8-MAP4 was stable, and the epitopes could be exposed for recognition. In addition, the 10E8 epitope was present in an α-helical structure, which was hypothesized to aid in the generation of neutralizing antibodies. In vivo analysis showed that 10E8-MAP4 could efficiently elicit HIV binding antibodies in guinea pigs, although only weak neutralizing activities were observed.ConclusionsMultiple antigen peptide is an excellent vaccine platform for generating binding antibodies, but may elicit weak neutralizing antibodies for HIV.