Vaccination with MHC-II restricted ApoB100 peptides to prevent atherosclerosis

描述（由申请人提供）：接种MHC-II限制性ApoB100肽预防动脉粥样硬化。动脉粥样硬化是一种由低密度脂蛋白（LDL）水平升高引发的大中型动脉慢性炎症性疾病。最近的证据表明，先天和适应性免疫反应都有助于这种疾病。通过体外筛选实验，我们从小鼠载脂蛋白（Apo）B100中发现了34条高亲和力结合主要组织相容性复合体(MHC)- ii （Apoe-/-小鼠中I-Ab）的肽段，并对其中5条进行了体内实验。我们发现，在Apoe-/-小鼠动脉粥样硬化模型中，接种与i - ab结合的ApoB100肽具有很强的动脉粥样硬化保护作用。动脉粥样硬化病变大小的减小与接种小鼠主动脉中IL-10表达的增加有关。由于我们在接种疫苗的小鼠的主动脉中发现IL-10升高，我们假设用ApoB100肽免疫诱导产生IL-10的Tregs和/或产生IL-10的巨噬细胞。具体目的1是测试哪种细胞类型负责接种小鼠主动脉中IL-10的产生。我们将Apoe-/-小鼠与IL-10报告小鼠杂交以鉴定产生IL-10的细胞。细胞类型特异性IL-10敲除小鼠（Il10fl/fl x LysM-Cre或x FoxP3- Cre）将确定这一拟议机制的相关性。根据初步数据，我们假设Tregs对动脉粥样硬化具有保护作用。特异性目的2是测试调节性T细胞（Tregs）在ApoB100肽保护动脉粥样硬化中的作用。我们建议通过表面表型和细胞因子分泌来定义抗原特异性CD4 T细胞，测试它们在动脉粥样硬化小鼠主动脉中的归巢，并通过活细胞多光子成像研究它们与动脉粥样硬化斑块中树突状细胞（DCs）的相互作用。基于初步数据鉴定了30个与人类MHC-II的许多等位基因结合的人类ApoB100肽，我们提出基于ApoB100的动脉粥样硬化保护性疫苗策略可翻译为人类。具体目的3是将预防动脉粥样硬化疫苗应用于人类。我们发现了一个人-鼠ApoB100序列相同的肽，并与许多人类MHC-II等位基因结合，包括DRB1*0101。我们将在现有的DRB1*0101转基因小鼠中测试这种序列相同的ApoB100肽对动脉粥样硬化的影响，优化疫苗接种方案（佐剂、频率、剂量），并评估四聚体和肽特异性抗体作为成功疫苗接种的生物标志物的潜力。当这项工作完成后，我们将知道(1)IL-10在疫苗诱导的动脉粥样硬化保护中的作用(2)Tregs在动脉粥样硬化保护中的作用及其在主动脉中的定位和维持机制，以及(3)这种方法是否确实实用并可用于人类。我们已经有了一个体外筛选试验，并建议开发生物标志物来测试成功的疫苗接种。最终，一种安全有效的动脉粥样硬化疫苗可能会对国家和世界范围的公共卫生产生巨大影响。

DESCRIPTION (provided by applicant): Vaccination with MHC-II restricted ApoB100 peptides to prevent atherosclerosis. Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries triggered by elevated levels of low density lipoprotein (LDL). Recent evidence shows that both innate and adaptive immune responses contribute to the disease. Using an in vitro screening assay, we found 34 peptides from mouse apolipoprotein (Apo)B100 that bind major histocompatibility complex (MHC)-II (I-Ab in Apoe-/- mice) with high affinity and tested 5 of them in vivo. We discovered that vaccination with I-Ab-binding ApoB100 peptides is strongly atheroprotective in the Apoe-/- mouse model of atherosclerosis. The reductions of atherosclerotic lesion size are associated with increased IL-10 expression in the aortas of vaccinated mice. Since we find elevated IL-10 in aortas of vaccinated mice, we hypothesize that immunization with ApoB100 peptides induces IL-10-producing Tregs and/or IL-10-producing macrophages. Specific aim 1 is to test which cell type is responsible for IL-10 production in aortas of vaccinated mice. We have crossed Apoe-/- mice to IL-10 reporter mice to identify the IL-10-producing cells. Cell type-specific IL-10 knockout mice (Il10fl/fl x LysM-Cre or x FoxP3- Cre) will identify the relevance of this proposed mechanism. Based on preliminary data, we hypothesize that Tregs afford protection from atherosclerosis. Specific aim 2 is to test the role o regulatory T cells (Tregs) in atheroprotection by ApoB100 peptides. We propose to define the antigen-specific CD4 T cells by surface phenotype and cytokine secretion, test their homing to atherosclerotic mouse aortas and study their interaction with dendritic cells (DCs) in atherosclerotic plaque by live cell multiphoton imaging. Based on preliminary data identifying 30 human ApoB100 peptides that bind many alleles of human MHC-II, we propose that ApoB100-based atheroprotective vaccination strategies are translatable to humans. Specific aim 3 is to translate atheroprotective vaccination to humans. We discovered one peptide that is human-to-mouse ApoB100 sequence-identical and binds to many human MHC-II alleles including DRB1*0101. We will test the effect of this sequence-identical ApoB100 peptide on atherosclerosis in existing mice transgenic for DRB1*0101, optimize vaccination regimens (adjuvant, frequency, dose) and evaluate the potential of tetramers and peptide- specific antibodies as biomarkers reporting successful vaccination. When the proposed work is completed, we will know (1) the role of IL-10 in vaccination-induced atheroprotection (2) the role of Tregs in atheroprotection and the mechanism of their homing and maintenance in the aorta and (3) whether this approach is indeed practical and translatable to humans. We already have an in vitro screening assay and propose to develop biomarkers to test for successful vaccination. Ultimately, a safe and effective atherosclerosis vaccine could have a very large national and world-wide public health impact.