Multiple Approaches to Abeta Vaccination in Animal Models

描述（由申请人提供）：在社区居住的老年人中，混合脑病理占大多数痴呆症病例。细致的神经病理学研究表明，¿-syn、A -¿和tau的聚集出现在相同的神经元结构中，为PD/DLB与AD重叠的临床观察提供了病理基础。对转基因（Tg）小鼠的研究表明，人类A¿和¿-的神经元表达与双Tg小鼠类似阿尔茨海默病的路易体变异（39）。这些小鼠在学习和记忆方面有严重缺陷，在-syn - Tg小鼠之前出现运动缺陷，并表现出明显的年龄依赖性神经变性。最近，突变体（A53T）¿-syn Tg小鼠与3xTg-AD Tg小鼠（DLB-AD小鼠）杂交。DLB-AD小鼠表现出加速的认知衰退，并伴有a¿、tau和¿-syn病理的显著增强（40）。因此，促进¿-syn清除的方法可能为PD和DLB以及AD提供治疗益处。目前已有两项研究表明，主动和被动抗-syn免疫治疗可以降低神经元细胞体和突触中聚集的-syn，并改善行为（1,2）。在这一竞争更新的目标是设计多表位抗-syn DNA疫苗，并测试其在Tg模型中的治疗潜力，这些模型会产生类似人类的神经元-syn病理和混合病理。为了充分实现这些目标，我们提出了以下4个目标：目标1：在DNA免疫应答中精细定位¿-syn的b细胞和t细胞表位。我们将分析野生型小鼠对抗-syn DNA免疫的B细胞和T细胞免疫反应。小鼠将接种编码全长人类¿-syn蛋白的DNA。抗-syn抗体滴度和b细胞和t细胞表位将被鉴定。目的2：在野生型小鼠中检测候选多表位DNA疫苗。多表位设计，其中¿-syn b细胞表位（12,15,52,54）将与PADRE融合并作为外源T辅助细胞表位。抗体滴度和亲和力都将被测量。目标3。γ -syn DNA表位疫苗对Tg小鼠的预防和治疗效果检验。将使用人类野生型¿-syn （hSYN线D）小鼠(3)。我们将评估最佳多表位疫苗在2月龄hSYN Tg小鼠病理前诱导“保护性”免疫反应的能力，以及在6月龄hSYN Tg小鼠中诱导预先存在的a-syn病理的“治疗性”反应的能力。目标4。在混合病理Tg小鼠中检测¿-syn DNA表位疫苗的疗效。我们将测试最佳的多表位-syn DNA疫苗在病理前2月龄混合病理DLB-AD - Tg小鼠中诱导“保护性”免疫反应的能力。DLB-AD小鼠表现出加速的认知衰退，与a¿，tau和¿-syn病理的显著增强有关。将免疫DLB-AD - Tg小鼠与未免疫小鼠进行认知能力下降和A¿，tau和¿-syn病理的比较。最后，我们将在混合病理DLB-AD - Tg小鼠中比较最佳多表位-syn DNA疫苗和最佳多表位A - DNA疫苗（40）。

DESCRIPTION (provided by applicant): Mixed brain pathologies account for most dementia cases in community-dwelling older persons. Careful neuropathological studies have shown that aggregations of ¿-syn, A¿ and tau appear in the same neuronal structures, providing a pathological basis for the clinical observations of the overlap between PD/DLB and AD. Studies with transgenic (Tg) mice with neuronal expression of human A¿ and ¿-syn the doubly Tg mice resembled the Lewy-body variant of Alzheimer's disease (39). These mice had severe deficits in learning and memory, developed motor deficits before ¿-syn Tg mice, and showed prominent age- dependent neurodegeneration. More recently, mutant (A53T) ¿-syn Tg mice were crossed onto 3xTg-AD Tg mice (DLB-AD mice). The DLB-AD mice exhibit accelerated cognitive decline associated with a dramatic enhancement of A¿, tau, and ¿-syn pathologies (40). Thus approaches that promote the clearance of ¿-syn may provide therapeutic benefit for PD and DLB, as well as AD. Two studies have now shown that active and passive anti-¿-syn immunotherapy can decreased aggregated ¿-syn in neuronal cell bodies and synapses and improve behavior (1, 2). In this competitive renewal the goals are to design multi-epitope anti-¿-syn DNA vaccines and test their therapeutic potential in Tg models that develop human-like neuronal ¿-syn pathology and mixed pathology. To fully achieve these goals we propose the following 4 Aims: Aim 1: Fine mapping of the B-cell and T-cell epitope(s) of ¿-syn in response to DNA Immunization. We will analyze the B- and T- cell immune responses to anti-¿-syn DNA Immunization in wild-type mice. Mice will be vaccinated with DNA encoding full-length human ¿-syn protein. Anti- ¿-syn titers and the B-cell and T-cell epitopes will be identified. Aim 2: Testig of candidate multiple-epitope DNA vaccines in wild-type mice. A multi-epitope design where ¿-syn B-cell epitopes (12, 15, 52, 54) will be fused with PADRE and as the foreign T helper cell epitopes. Both antibody titers and affinity will be measured. Aim 3. Testing prophylactic and therapeutic efficacy of ¿-syn DNA epitope vaccines Tg mice. Human wild-type ¿-syn (hSYN line D) mice (3) will be used. We will assess the ability of the best multi-epitope vaccines to induce "protective" immune response in pre-pathology 2 months old hSYN Tg mice, and "therapeutic" response to pre-existing a-syn pathology in 6 months old hSYN Tg mice. Aim 4. Testing the efficacy of ¿-syn DNA epitope vaccine(s) in mixed pathology Tg mice. We will test the ability of the best multi-epitope ¿-syn DNA vaccine to induce "protective" immune response in pre-pathology 2 months old mixed pathology DLB-AD Tg mice. The DLB-AD mice exhibit accelerated cognitive decline associated with a dramatic enhancement of A¿, tau, and ¿-syn pathologies. Immunized DLB-AD Tg mice will be compared against non-immunized mice for cognitive decline and A¿, tau, and ¿-syn pathologies. Finally, we will compare the best multi-epitope ¿-syn DNA vaccine against our best multi-epitope A¿ DNA vaccine in the mixed pathology DLB-AD Tg mice (40). PUBLIC HEALTH RELEVANCE: Mixed brain pathologies account for most dementia cases in community-dwelling older persons, and careful neuropathological studies have shown that aggregations of alpha-synuclein, amyloid-beta and tau proteins appear in the same neuronal structures, providing a pathological basis for the clinical observations of the overlap between dementia with Lewy bodies disease (DLB) and Parkinson's disease (PD) and Alzheimer's disease (AD). Taken together, these studies indicate that the pathological process of PD and DLB may accelerate that of AD, while that of AD may also drive PD and DLB. In this competitive renewal we propose to utilize advanced immunotherapeutic strategies to target alpha-synuclein neuropathology. Thus the goal is to design multi-epitope anti-alpha-synuclein DNA vaccines, and test their effectiveness in transgenic mouse models that develop human-like neuronal alpha-synuclein pathology, as well as in mixed pathology transgenic mice with alpha-synuclein pathology and amyloid-beta and tau pathology. In summary, our advanced multiple component DNA vaccine designs are engineered to promote a strong anti-alpha-synuclein antibody response without the risk of producing an adverse autoimmune response.