Identification and characterization of L. major T cell epitopes based on quantitative proteomics

利什曼病是一种在皮肤中接种原虫寄生虫后观察到的疾病，会引起从局部自限性皮损到危及生命的内脏的各种症状。针对利什曼原虫的保护性免疫是由感染无鞭毛体的树突状细胞启动的抗原特异性CD8+和CD4+T细胞介导的。由于目前还没有疫苗存在，识别保护性T细胞表位是必要的，但到目前为止只有两个CD4表位被鉴定。因此，我们的目标是鉴定和鉴定来自大利什曼原虫的潜在保护性T细胞表位。为此，我们将利用层析和超速离心技术来分离主要前鞭毛和无鞭毛体的蛋白质组。利用无标记定量质谱仪，我们将鉴定构成T细胞表位潜在来源的丰富的和/或阶段特异的蛋白质。然后，我们将选择适用于MHC结合亲和力的电子预测或通过肽库筛选的候选表位。然后，候选人将在体外(与MHC I和II类的结合亲和力，诱导细胞因子释放和T细胞增殖)和体内(免疫)进行免疫反应性分析，然后详细分析免疫优势，抗原摄取机制，寄生虫生命形式特定表位的处理，以及潜在的免疫逃避机制。

Leishmaniasis is a disease observed after inoculation of a protozoan parasite into the skin inducing symptoms ranging from local, self-limiting lesions to life-threatening visceralisation. Protective immunity against Leishmania is mediated by antigen specific CD8+ and CD4+ T cells which are primed by dendritic cells infected with the amastigote life form. As currently no vaccine exists, identification of protective T cell epitopes is essential, but so far only two CD4 epitopes have been characterized. Therefore, we aim to identify and characterize potential protective T cell epitopes derived from Leishmania major. Towards this purpose, we will partition the proteome of L. major promastigote and amastigote life forms by chromatographic and ultracentrifugation techniques. Using label-free quantitative mass spectrometry, we will identify abundant and/or stage-specific proteins constituting potential sources for T cell epitopes. We will then select epitope candidates applying in silico predictions for MHC binding affinity or by peptide library screening. Candidates will then be assayed for immunoreactivity both in vitro (binding affinity to MHC class I and II, induction of cytokine release and proliferation of T cells), and in vivo (immunizations) followed by detailed analyses of immunodominance, mechanisms of antigen uptake, processing of parasite life form-specific epitopes, and potential immune evasion mechanisms.