Lyme Disease Diagnostics Using In Vivo Expressed Borrelia Burgdorferi Antigens

描述(申请人提供)：莱姆病，由于感染硬蜱传播的伯氏疏螺旋体螺旋体，是美国最常见的媒介传播疾病，每年向疾控中心报告超过20,000例。螺旋体在皮肤中沉积后，可在几个部位传播和煽动临床上明显的疾病，特别是皮肤、心脏、关节和神经系统。虽然抗生素在大多数早期莱姆病患者中成功地实现了临床治愈，但那些播散性感染或治疗延迟的患者可能会出现病因不明的挥之不去的迹象和症状。莱姆病的及时和准确诊断对于优化治疗反应和预防该病的长期后遗症至关重要。检测伯氏杆菌抗体的血清学试验(酶联免疫吸附试验和免疫印迹试验)是目前莱姆病最敏感和最广泛使用的实验室试验。这些检测利用培养的伯氏杆菌实验室菌株的全细胞裂解物作为抗原源，因此不能检测到针对体内表达的伯氏杆菌蛋白的抗体。推荐的两级法，即用免疫印迹法确认阳性或可疑的ELISA，在感染的早期阶段只有~30%的敏感性，而且由于伯氏杆菌抗原与其他感染源的交叉反应而降低了特异性。此外，目前的莱姆酶联免疫吸附试验和免疫印迹试验不能区分以前接触伯氏杆菌和活动性感染，而且到目前为止还没有血清学试验可以用来评估对治疗的反应。这一第一阶段的提案旨在通过蛋白质组学方法改进目前可用的莱姆病血清学测试，该方法将a)鉴定在体内表达的免疫优势伯氏杆菌蛋白，以及b)评估它们作为伯氏杆菌感染不同阶段的诊断抗原的有效性。这种方法的关键是使用一种突变的小鼠品系，在这种品系中，螺旋体可以在保持抗体介导的对有机体的选择压力的条件下，在组织中实现高病原体负担。这些新的突变小鼠将被用作宿主适应的伯氏杆菌及其体内表达抗原的组织来源。2D-差示荧光凝胶电泳法、免疫印迹法和MALDI-MS/MS法将被用来鉴定在媒介传播感染的早期和晚期，免疫优势伯氏杆菌抗原仅在哺乳动物组织中表达。候选诊断抗原将以重组蛋白的形式生产，并与代表不同阶段伯氏杆菌感染的一组血清进行反应性测试。这项第一阶段研究的结果将为莱姆病新的敏感和特异的血清学测试的开发奠定基础，这些测试将更准确地反映在受感染宿主中激发免疫反应的伯氏杆菌抗原。 莱姆病是由于感染伯氏疏螺旋体引起的，可导致皮肤、心脏、关节和神经系统等多系统疾病。及时诊断该病对于优化治疗和预防长期后遗症至关重要。这项建议将使用蛋白质组学方法来鉴定新的免疫优势伯氏杆菌蛋白，这些蛋白主要在哺乳动物宿主中表达。对这些蛋白质的鉴定和鉴定将为改进莱姆病早期和晚期感染的血清学测试奠定基础。

DESCRIPTION (provided by applicant): Lyme disease, due to infection with the Ixodes tick-transmitted spirochete Borrelia burgdorferi, is the most common vector-borne disease in the United States, with over 20,000 cases reported to the CDC annually. After deposition in the skin, spirochetes can disseminate and incite clinically apparent disease in several sites, especially the skin, heart, joints and nervous system. While antibiotics are successful in achieving clinical cure in the majority of patients with early Lyme disease, those with disseminated infection or in whom treatment is delayed can experience lingering signs and symptoms of uncertain etiology. The timely and accurate diagnosis of Lyme disease is essential for optimizing the response to therapy and for preventing long-term sequellae of the disease. Serologic tests (ELISA and immunoblot) that detect antibodies against B. burgdorferi are currently the most sensitive and widely available laboratory tests for Lyme disease. These tests utilize whole-cell lysates of cultured laboratory strains of B. burgdorferi as a source of antigens, and therefore do not detect antibodies to B. burgdorferi proteins expressed exclusively in vivo. The recommended two-tiered approach in which a positive or equivocal ELISA is confirmed by immunoblot has only ~30% sensitivity in early stages of infection, and specificity is reduced due to cross-reactivity of B. burgdorferi antigens with those of other infectious agents. Moreover, the current Lyme ELISA and immunoblot tests do not distinguish previous exposure to B. burgdorferi from active infection, and no serologic test to date can be used to assess response to therapy. This Phase I proposal seeks to improve upon the currently available serologic tests for Lyme disease through a proteomic approach that will a) identify immunodominant B. burgdorferi proteins expressed in vivo, and b) evaluate their utility as diagnostic antigens at various stages of B. burgdorferi infection. The key to this approach is the use of a mutant mouse strain in which spirochetes can achieve high pathogen burden in tissues under conditions in which antibody-mediated selection pressure on the organism is preserved. These novel mutant mice will be used as a tissue source of host-adapted B. burgdorferi and its in vivo expressed antigens. 2D-differential fluorescence gel electrophoresis, immunoblot, and MALDI-MS/MS will be used to identify immunodominant B. burgdorferi antigens expressed exclusively in mammalian tissues in early and late stage vector-borne infection. Candidate diagnostic antigens will be produced as recombinant proteins and tested for reactivity with a panel of sera representative of various stages B. burgdorferi infection. The results of this Phase I study will set the stage for the development of new sensitive and specific serologic tests for Lyme disease that more accurately reflect the B. burgdorferi antigens inciting immune responses in the infected host. Lyme disease, due to infection with the spirochete Borrelia burgdorferi, can cause a multi system illness involving the skin, heart, joints and nervous system. Timely diagnosis of the disease is critical for optimizing treatment and for the prevention of long-term sequellae. This proposal will use a proteomics approach to identify novel immunodominant B. burgdorferi proteins that are predominantly expressed within the mammalian host. Identification and characterization of these proteins will set the stage for the development of improved serologic tests for Lyme disease in both early and late stage infection.