IDENTIFICATION OF IMMUNOGENIC GLYCOLIPIDS FROM BORRELIA BURGDORFERI

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 莱姆病是由壁虱传播的伯氏疏螺旋体(BB)引起的。在美国，一小部分莱姆关节炎患者在接受2-3个月的口服和/或静脉抗菌素治疗后，会持续几个月甚至几年的关节炎，称为抗生素难治性莱姆关节炎。在这些患者中，尽管在抗生素后时期关节液和滑膜组织中的伯氏杆菌DNA检测结果为阴性，但关节炎仍然存在。这项工作的重点是鉴定一种BB糖脂，它能在莱姆病患者中诱导免疫反应，但在螺旋体被杀死后，这种反应可以预见地消失。为了实现这一目标，我们正在提取和分级BB脂类，并使用来自莱姆病患者的血清或来自未感染的捐赠者的对照血清的ELISA法测试单个组分。正相色谱用于脂肪的初始分离。这将按(头组)类别分离脂类。那些酶联免疫吸附试验呈阳性的组分通过反相色谱进行细分，反相层析法通过脂类的疏水部分来分离脂类。这些亚组分再次用酶联免疫吸附试验进行检测。在血清反应性的指导下，复杂的脂类提取物正在被分级，以便离线质谱学可以用于鉴定脂类结构，包括免疫原性成分的结构。我们从BB中提取了总脂，并用正相色谱分离。我们用MALDI-TOF和ESI qoTOF质谱仪分析了脂组分，以评估它们的复杂性，并为随后的串联MS结构鉴定鉴定完整的脂类。这种方法已经通过完整的质量鉴定了几种已知的BB脂，这将被用于鉴定螺旋体可能的免疫原性脂或脂蛋白。我们还对从BB的免疫原性糖脂脂组分中纯化的酰化胆固醇半乳糖苷(ACG)进行了血清ELISA法检测。初步分析表明，这种脂质具有免疫原性，我们目前正在开发一种可靠的ELISA法来评估对这种抗原的抗体反应。描述脂质分析结果的手稿已被《临床免疫学》接受发表(KL Jones等人，莱姆关节炎患者中对伯氏疏螺旋体糖脂的强免疫球蛋白抗体反应)，莱姆关节炎是这种感染的晚期表现。2009、132、93-102)。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Lyme disease is caused by the tick-transmitted spirochete Borrelia burgdorferi (Bb). In the United States, a small percentage of patients with Lyme arthritis have persistent arthritis for months or even several years after treatment with 2-3 months of oral and/or IV antibotic therapy, termed antibiotic-refractory Lyme arthritis. In these patients, the arthritis persists despite negative results for B. burgdorferi DNA in joint fluid and synovial tissue in the post-antibiotic period. This work is focused on identification of a Bb glycolipid that induces an immune response in patients with Lyme disease, but the response disappears predictably after spirochetal killing. Towards this goal, we are extracting and fractionating Bb lipids, and testing individual fractions using ELISA assays with sera from Lyme disease patients or control sera from uninfected donors. Normal phase chromatography is employed for the initial lipid fractionation. This separates the lipids by (headgroup) class. Those fractions that are ELISA-positive are sub-fractionated by reversed-phase chromatography, which separates lipids by their hydrophobic moieties. These sub-fractions are again be tested by ELISA. Guided by serum reactivity, the complex lipid extract is being fractionated, so that off-line mass spectrometry can be used to identify the lipid structures, including that of the immunogenic component. We have extracted total lipids from Bb and separated them by normal phase chromatography. We have analyzed lipid fractions by MALDI-TOF and ESI qoTOF mass spectrometry to assess their complexity and to identify intact lipids for subsequent tandem MS structural identification. This approach has identified, by intact mass, several known Bb lipids, which will be used to identify possible immunogenic lipids or lipoproteins of the spirochete. We have also performed serum ELISA assays on acylated cholesteryl galactoside (ACG) purified from lipid fractions, an immunogenic glycolipid of Bb. Preliminary analysis shows that this lipid is immunogenic, and we are now working on the development of a reliable ELISA to assess the antibody response to this antigen. A manuscript describing the results from the lipid analyses has been accepted for publication in Clinical Immunology (KL Jones et al, Strong IgG antibody responses to Borrelia burgdorferi glycolipids in patientswith Lyme arthritis, a late manifestation of the infection.Clin Immunol. 2009, 132, 93-102).