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

利什曼病是在将原生动物寄生虫接种到皮肤后观察到的一种疾病，可引起从局部自限性病变到危及生命的内脏化等症状。对利什曼原虫的保护性免疫是由抗原特异性CD8+和CD4+ T细胞介导的，这些细胞是由感染了无尾线虫生命形式的树突状细胞引发的。由于目前没有疫苗存在，鉴定保护性T细胞表位是必不可少的，但到目前为止，只有两个CD4表位被表征。因此，我们的目标是鉴定和表征来自利什曼原虫的潜在保护性T细胞表位。为此，我们将利用色谱和超离心技术对L. major promastigote和amastigote生命形式的蛋白质组进行划分。使用无标记定量质谱法，我们将确定丰富的和/或阶段特异性蛋白质构成T细胞表位的潜在来源。然后，我们将应用MHC结合亲和力的计算机预测或通过肽库筛选来选择候选表位。然后将在体外（与MHC I类和II类的结合亲和力，细胞因子释放和T细胞增殖的诱导）和体内（免疫）检测候选药物的免疫反应性，随后详细分析免疫优势，抗原摄取机制，寄生虫生命形式特异性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.