Brucella epitope recognition by CD8+ T cells

描述(申请人提供)：布鲁氏菌是一种人类细胞内的病原体，主要由CD8+T细胞控制，但含有保护CD8+T细胞识别的多肽的布鲁氏菌蛋白尚不清楚。我们的目标是识别特定的布鲁氏菌CD8+T细胞表位，并测试它们保护小鼠免受毒力布鲁氏菌感染的能力。要做到这一点，我们将首先分离和鉴定自然加工和呈递的布鲁氏菌特异性T细胞表位，结合受感染巨噬细胞上的主要组织相容性复合体I类(MHC I)分子。将使用质谱仪(MS)和生物信息学来确定T细胞表位，以将表位与相应的布鲁氏菌蛋白相关联。其次，我们将使用侵入性大肠杆菌载体免疫小鼠，评估针对布鲁氏菌抗原的CTL活性。第三，我们将评估抗原性布鲁氏菌蛋白和T细胞表位对清除小鼠体内毒力布鲁氏菌的影响。根据表明我们在每个具体目标方面的能力的初步数据，我们有信心实现这些目标。此外，我们还对重组大肠杆菌载体免疫的小鼠的抗原产生了显著的CTL反应。我们相信，我们的目标是开发和测试这一新的方法来阐明CD8+CTL表位及其对体内清除布鲁氏菌的贡献，这将填补在鉴定布鲁氏菌蛋白方面的空白，这些蛋白对于细胞毒性CD8+T细胞清除这种细胞内细菌至关重要。我们的具体目标是：1.鉴定天然加工和MHC I呈递的布鲁氏菌多肽。小鼠巨噬细胞RAW264.7细胞将感染羊布鲁氏菌。MHC I-布鲁氏菌多肽复合物将从细胞膜上免疫共沉淀，并纯化多肽。随后将对多肽进行LC-MS，并将分析布鲁氏菌T细胞表位的数据。布鲁氏菌多肽与MHC I分子结合的亲和力将被用来确认高结合肽的鉴定。2.评估CTL对含有特异性T细胞表位的布鲁氏菌蛋白的杀伤活性。侵袭性大肠杆菌载体将被改造成表达含有T细胞表位的布鲁氏菌蛋白。这些重组大肠杆菌载体将用于免疫小鼠。表达布鲁氏菌多肽的H-2单倍型匹配细胞系将作为刺激/靶细胞，用于从免疫小鼠分离的脾T细胞的CTL检测。CTL数据分析将确定包含CD8+T细胞表位的布鲁氏菌蛋白，这对清除细胞内的布鲁氏菌很重要。3.评价侵袭性大肠杆菌载体携带的布鲁氏菌蛋白诱导CTL活性和宿主保护作用。在用表达布鲁氏菌蛋白的侵袭性大肠杆菌载体免疫的小鼠中，将确定表达LuxCDABE毒力的羊细小杆菌的清除情况，清除情况将与CTL活性相关。将布鲁氏菌特异性CD8+T细胞过继转移到初治受体小鼠，将证实不同的布鲁氏菌特异性CTL对清除布氏杆菌强毒力感染的有效性。因此，这一目的将把CTL活性与清除布鲁氏菌感染的生物学相关性联系起来。鉴定含有CD8+T细胞表位的布鲁氏菌蛋白对于理解布鲁氏菌蛋白质组在免疫识别和宿主保护中的作用至关重要。

DESCRIPTION (provided by applicant): Brucella melitensis, an intracellular human pathogen, is largely controlled by CD8+ T cells, but the Brucella proteins containing peptides recognized by protective CD8+ T cells remain unknown. Our goal is to identify specific Brucella CD8+ T cell epitopes and test their ability to protect mice against infection with virulent Brucella. To do this we will first isolate and identify naturally processed and presented Brucella-specific T cell epitopes bound to Major Histocompatibility Complex class I (MHC I) molecules on infected macrophages. T cell epitopes will be identified using Mass Spectrometry (MS) and bioinformatics to correlate an epitope with the respective Brucella protein. Second, we will assess CTL activity against the Brucella antigens using an invasive E. coli vector to immunize mice. Third, we will assess the impact of antigenic Brucella proteins and T cell epitopes on clearance of virulent Brucella from mice. We are confident of achieving these goals based on preliminary data that demonstrate our ability in each specific aim. Also, we have shown significant CTL response to antigen from mice immunized with the recombinant E.coli vectors. We believe our goal of developing and testing this novel approach to elucidate CD8+ CTL epitopes and their contributions to clearance of Brucella in vivo will fill the void in identifying Brucella proteins important for clearance of this intracellular bacterium by cytotoxic CD8+ T cells. Our specific aims are: 1. Identify naturally processed and MHC I presented Brucella peptides. Mouse macrophage RAW264.7 cells will be infected with Brucella melitensis. MHC I-Brucella peptide complex will be co- immunoprecipitated from the cell membrane and the peptide purified. Peptides will then be subject to LC-MS, and data will be analyzed for Brucella T cell epitopes. Affinity of Brucella peptide binding to MHC I molecules will be used to confirm the identify of high binding peptides. 2. Assess CTL activity against Brucella proteins containing specific T cell epitopes. Invasive E. coli vectors will be engineered to express Brucella proteins containing T cell epitopes. These recombinant E. coli vectors will be used to immunize mice. H-2 haplotype matched cell lines expressing the Brucella peptides will be used as stimulator/target cells for CTL assays of splenic T-cells isolated from the immunized mice. CTL data analysis will identify Brucella proteins containing CD8+ T cell epitopes important for clearance of intracellular Brucella. 3. Evaluate Brucella proteins delivered by invasive E. coli vectors to induce CTL activity and host protection. Clearance of luxCDABE-expressing virulent B. melitensis will be determined in mice immunized with invasive E. coli vectors expressing Brucella proteins and clearance will be correlated to CTL activity. Adoptive transfer of Brucella specific CD8+ T cells to naive recipient mice will confirm the effectiveness of different Brucella-specific CTLs to clear virulent B. melitensis infection. Thus, this aim will link CTL activity to biologic relevance in clearing Brucella infection. Identifying Brucella proteins that contain CD8+ T cell epitopes would be crucial to understanding the contribution of the Brucella proteome to immune recognition and host protection.