Hayk Davtyan

A transgenic Alzheimer rat with plaques, tau pathology, behavioral impairment, oligomeric aβ, and frank neuronal loss.

Alzheimers disease (AD) is hallmarked by amyloid plaques, neurofibrillary tangles, and widespread cortical neuronal loss (Selkoe, 2001). The “amyloid cascade hypothesis” posits that cerebral amyloid sets neurotoxic events into motion that precipitate Alzheimer dementia (Hardy and Allsop, 1991). Yet, faithful recapitulation of all AD features in widely used transgenic (Tg) mice engineered to overproduce Aβ peptides has been elusive. We have developed a Tg rat model (line TgF344-AD) expressing mutant human amyloid precursor protein (APPsw) and presenilin 1 (PS1ΔE9) genes, each independent causes of early-onset familial AD. TgF344-AD rats manifest age-dependent cerebral amyloidosis that precedes tauopathy, gliosis, apoptotic loss of neurons in the cerebral cortex and hippocampus, and cognitive disturbance. These results demonstrate progressive neurodegeneration of the Alzheimer type in these animals. The TgF344-AD rat fills a critical need for a next-generation animal model to enable basic and translational AD research.

Reducing AD-Like Pathology in 3xTg-AD Mouse Model by DNA Epitope Vaccine — A Novel Immunotherapeutic Strategy

Background The development of a safe and effective AD vaccine requires a delicate balance between providing an adequate anti-Aβ antibody response sufficient to provide therapeutic benefit, while eliminating an adverse T cell-mediated proinflammatory autoimmune response. To achieve this goal we have designed a prototype chemokine-based DNA epitope vaccine expressing a fusion protein that consists of 3 copies of the self-B cell epitope of Aβ42 (Aβ1–11) , a non-self T helper cell epitope (PADRE), and macrophage-derived chemokine (MDC/CCL22) as a molecular adjuvant to promote a strong anti-inflammatory Th2 phenotype. Methods and Findings We generated pMDC-3Aβ1–11-PADRE construct and immunized 3xTg-AD mouse model starting at age of 3–4 months old. We demonstrated that prophylactic immunizations with the DNA epitope vaccine generated a robust Th2 immune response that induced high titers of anti-Aβ antibody, which in turn inhibited accumulation of Aβ pathology in the brains of older mice. Importantly, vaccination reduced glial activation and prevented the development of behavioral deficits in aged animals without increasing the incidence of microhemorrhages. Conclusions Data from this transitional pre-clinical study suggest that our DNA epitope vaccine could be used as a safe and effective strategy for AD therapy. Future safety and immunology studies in large animals with the goal to achieve effective humoral immunity without adverse effects should help to translate this study to human clinical trials.

Alzheimer's Disease Peptide Epitope Vaccine Reduces Insoluble But Not Soluble/Oligomeric Aβ Species in Amyloid Precursor Protein Transgenic Mice

Active vaccination of elderly Alzheimers disease (AD) patients with fibrillar amyloid-β peptide (Aβ42), even in the presence of a potent Th1 adjuvant, induced generally low titers of antibodies in a small fraction (∼20% responders) of those that received the AN-1792 vaccine. To improve the immunogenicity and reduce the likelihood of inducing adverse autoreactive T-cells specific for Aβ42, we previously tested in wild-type mice an alternative approach for active immunization: an epitope vaccine that selectively initiate B cell responses toward an immunogenic self-epitope of Aβ in the absence of anti-Aβ T cell responses. Here, we describe a second generation epitope vaccine composed of two copies of Aβ1–11 fused with the promiscuous nonself T cell epitope, PADRE (pan human leukocyte antigen DR-binding peptide) that completely eliminates the autoreactive T cell responses and induces humoral immune responses in amyloid precursor protein transgenic 2576 mice with pre-existing AD-like pathology. Based on the titers of anti-Aβ1–11 antibody experimental mice were divided into low, moderate and high responders, and for the first time we report a positive correlation between the concentration of anti-Aβ1–11 antibody and a reduction of insoluble, cerebral Aβ plaques. The reduction of insoluble Aβ deposition was not associated with adverse events, such as CNS T cell or macrophage infiltration or microhemorrhages. Surprisingly, vaccination did not alter the levels of soluble Aβ. Alternatively, early protective immunization before substantial neuropathology, neuronal loss and cognitive deficits have become firmly established may be more beneficial and safer for potential patients, especially if they can be identified in a preclinical stage by the development of antecedent biomarkers of AD.

Immunogenicity, Efficacy, Safety, and Mechanism of Action of Epitope Vaccine (Lu AF20513) for Alzheimer’s Disease: Prelude to a Clinical Trial

The Alzheimers disease (AD) process is understood to involve the accumulation of amyloid plaques and tau tangles in the brain. However, attempts at targeting the main culprits, neurotoxic Aβ peptides, have thus far proven unsuccessful for improving cognitive function. Recent clinical trials with passively administrated anti-Aβ antibodies failed to slow cognitive decline in mild to moderate AD patients, but suggest that an immunotherapeutic approach could be effective in patients with mild AD. Using an AD mouse model (Tg2576), we tested the immunogenicity (cellular and humoral immune responses) and efficacy (AD-like pathology) of clinical grade Lu AF20513 vaccine. We found that Lu AF20513 induces robust “non-self” T-cell responses and the production of anti-Aβ antibodies that reduce AD-like pathology in the brains of Tg2576 mice without inducing microglial activation and enhancing astrocytosis or cerebral amyloid angiopathy. A single immunization with Lu AF20513 induced strong humoral immunity in mice with preexisting memory T-helper cells. In addition, Lu AF20513 induced strong humoral responses in guinea pigs and monkeys. These data support the translation of Lu AF20513 to the clinical setting with the aims of: (1) inducing therapeutically potent anti-Aβ antibody responses in patients with mild AD, particularly if they have memory T-helper cells generated after immunizations with conventional tetanus toxoid vaccine, and (2) preventing pathological autoreactive T-cell responses.

The adaptive immune system restrains Alzheimer’s disease pathogenesis by modulating microglial function

Significance Neuroinflammation and activation of innate immunity are pathological hallmarks of Alzheimer’s disease (AD). In contrast, very few studies have examined the impact of the adaptive immune system in AD pathogenesis. Here, we find that genetic ablation of peripheral immune cell populations significantly accelerates amyloid pathogenesis, worsens neuroinflammation, and alters microglial activation state. Critically, it appears that loss of IgG-producing B cells impairs microglial phagocytosis, thereby exacerbating amyloid deposition. Conversely, replacement of IgGs via direct injection or bone marrow transplantation reverses these effects and reduces Aβ pathology. Together, these results highlight the importance of the adaptive immune system and its interactions with microglia in the pathogenesis of AD. The innate immune system is strongly implicated in the pathogenesis of Alzheimer’s disease (AD). In contrast, the role of adaptive immunity in AD remains largely unknown. However, numerous clinical trials are testing vaccination strategies for AD, suggesting that T and B cells play a pivotal role in this disease. To test the hypothesis that adaptive immunity influences AD pathogenesis, we generated an immune-deficient AD mouse model that lacks T, B, and natural killer (NK) cells. The resulting “Rag-5xfAD” mice exhibit a greater than twofold increase in β-amyloid (Aβ) pathology. Gene expression analysis of the brain implicates altered innate and adaptive immune pathways, including changes in cytokine/chemokine signaling and decreased Ig-mediated processes. Neuroinflammation is also greatly exacerbated in Rag-5xfAD mice as indicated by a shift in microglial phenotype, increased cytokine production, and reduced phagocytic capacity. In contrast, immune-intact 5xfAD mice exhibit elevated levels of nonamyloid reactive IgGs in association with microglia, and treatment of Rag-5xfAD mice or microglial cells with preimmune IgG enhances Aβ clearance. Last, we performed bone marrow transplantation studies in Rag-5xfAD mice, revealing that replacement of these missing adaptive immune populations can dramatically reduce AD pathology. Taken together, these data strongly suggest that adaptive immune cell populations play an important role in restraining AD pathology. In contrast, depletion of B cells and their appropriate activation by T cells leads to a loss of adaptive–innate immunity cross talk and accelerated disease progression.

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

Clinical trials with passive and active Alzheimers 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.

Cancer-testis antigen, BORIS based vaccine delivered by dendritic cells is extremely effective against a very aggressive and highly metastatic mouse mammary carcinoma

Here, we analyze for the first time the immunological and therapeutic efficacy of a dendritic cell (DC) vaccine based on a cancer-testis antigen, Brother of regulator of imprinted sites (BORIS), an epigenetically acting tumor-promoting transcription factor. Vaccination of mice with DC loaded with truncated form of BORIS (DC/mBORIS) after 4T1 mammary tumor implantation induced strong anti-cancer immunity, inhibited tumor growth (18.75% of mice remained tumor-free), and dramatically lowered the number of spontaneous clonogenic metastases (50% of mice remained metastases-free). Higher numbers of immune effector CD4 and CD8 T cells infiltrated the tumors of vaccinated mice vs. control animals. Vaccination significantly decreased the number of myeloid-derived suppressor cells (MDSCs) infiltrating the tumor sites, but not MDSCs in the spleens of vaccinated animals. These data suggest that DC-based mBORIS vaccination strategies have significant anti-tumor activity in a therapeutic setting and will be more effective when combined with agents to attenuate tumor-associated immune suppression.

Refinement of a DNA based Alzheimer disease epitope vaccine in rabbits

We previously demonstrated that our second-generation DNA-based Alzheimer disease (AD) epitope vaccine comprising three copies of a short amyloid-β (Aβ) B cell epitope, Aβ11 fused with the foreign promiscuous Th epitope, PADRE (p3Aβ11-PADRE) was immunogenic in mice. However, since DNA vaccines exhibit poor immunogenicity in large animals and humans, in this study, we sought to improve the immunogenicity of p3Aβ11-PADRE by modifying this vaccine to express protein 3Aβ11-PADRE with a free N-terminal aspartic acid fused with eight additional promiscuous Th epitopes. Generated pN-3Aβ11-PADRE-Thep vaccine has been designated as AV-1955. We also delivered this vaccine using the TriGrid electroporation system to improve the efficiency of DNA transfection. This third-generation DNA epitope vaccine was evaluated for immunogenicity in rabbits in comparison to the parent construct p3Aβ11-PADRE. AV-1955 vaccination induced significantly stronger humoral immune responses in rabbits compared with p3Aβ11-PADRE vaccine. Anti-Aβ11 antibodies recognized all forms of human β-amyloid peptide (monomers, oligomers and fibrils), bound to amyloid plaques in brain sections from an AD case and reduced oligomer- and fibril-mediated cytotoxicity ex vivo. These findings suggest that AV-1955 could represent an effective DNA epitope vaccine for AD therapy, pending safety and efficacy studies that are currently being conducted in Rhesus monkeys.

Delivery of a DNA vaccine for Alzheimer's disease by electroporation versus gene gun generates potent and similar immune responses.

Background: Induction of a humoral response against amyloid-β peptide may be beneficial for Alzheimer’s disease (AD) patients and may alleviate the onset and progression of AD. DNA-based vaccination provides a unique alternative method of immunization for treatment and prevention of AD. Currently, the two major delivery methods used for enhancing DNA uptake and immune responses to DNA vaccines in humans are electroporation (EP) and gene gun (GG). Objective: The goal of this translational study was to evaluate the efficacy of an AD DNA epitope vaccine (DepVac) delivered intramuscularly by EP or intradermally by GG. Methods: Humoral and cellular immune responses to immunization with DepVac were evaluated by ELISA and ELISPOT, respectively. Functional activity of the antibodies was also assessed. Results: EP- and GG-mediated immunizations with DepVac induced similar anti-amyloid-β (Aβ) antibody and T cell responses. Anti-Aβ antibodies bound to amyloid plaques in AD brain tissue and to toxic forms of Aβ42 peptide. Conclusion: Both delivery methods are effective at promoting potent antibodies specific for Aβ.

Epitope analysis following active immunization with tau proteins reveals immunogens implicated in tau pathogenesis

BackgroundAbnormal tau hyperphosphorylation and its accumulation into intra-neuronal neurofibrillary tangles are linked to neurodegeneration in Alzheimer’s disease and similar tauopathies. One strategy to reduce accumulation is through immunization, but the most immunogenic tau epitopes have so far remained unknown. To fill this gap, we immunized mice with recombinant tau to build a map of the most immunogenic tau epitopes.MethodsNon-transgenic and rTg4510 tau transgenic mice aged 5 months were immunized with either human wild-type tau (Wt, 4R0N) or P301L tau (4R0N). Each protein was formulated in Quil A adjuvant. Sera and splenocytes of vaccinated mice were collected to assess the humoral and cellular immune responses to tau. We employed a peptide array assay to identify the most effective epitopes. Brain histology was utilized to measure the effects of vaccination on tau pathology and inflammation.ResultsHumoral immune responses following immunization demonstrated robust antibody titers (up to 1:80,000 endpoint titers) to each tau species in both mice models. The number of IFN-γ producing T cells and their proliferation were also increased in splenocytes from immunized mice, indicating an increased cellular immune response, and tau levels and neuroinflammation were both reduced. We identified five immunogenic motifs within either the N-terminal (9-15 and 21-27 amino acids), proline rich (168-174 and 220-228 amino acids), or the C-terminal regions (427-438 amino acids) of the wild-type and P301L tau protein sequence.ConclusionsOur study identifies five previously unknown immunogenic motifs of wild-type and mutated (P301L) tau protein. Immunization with both proteins resulted in reduced tau pathology and neuroinflammation in a tau transgenic model, supporting the efficacy of tau immunotherapy in tauopathy.