Lyme Disease Diagnostics Using In Vivo Expressed Borrelia Burgdorferi Antigens

描述（由申请人提供）：莱姆病是由蜱虫传播的螺旋体伯氏疏螺旋体感染引起的，是美国最常见的媒介传播疾病，每年向疾病预防控制中心报告的病例超过20,000例。螺旋体沉积于皮肤后，可传播并诱发若干部位的临床明显疾病，尤其是皮肤、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持续体征和症状。及时准确地诊断莱姆病对于优化治疗反应和预防疾病的长期后遗症至关重要。检测伯氏疏螺旋体抗体的血清学检测（ELISA和免疫印迹）是目前最敏感和最广泛使用的莱姆病实验室检测方法。这些测试利用培养的伯氏疏螺旋体实验室菌株的全细胞裂解物作为抗原来源，因此不能检测出体内仅表达的伯氏疏螺旋体蛋白的抗体。推荐的两层方法，即免疫印迹证实阳性或模棱两可的ELISA，在感染的早期阶段只有~30%的敏感性，并且由于伯氏疏螺旋体抗原与其他感染因子的交叉反应性，特异性降低。此外，目前的莱姆病酶联免疫吸附试验和免疫印迹试验不能区分既往接触伯氏疏螺旋体和活动性感染，而且迄今为止没有血清学试验可用于评估对治疗的反应。该I期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检测，该方法将a)鉴定体内表达的免疫优势伯氏疏螺旋体蛋白，b)评估其作为伯氏疏螺旋体感染不同阶段诊断抗原的效用。这种方法的关键是使用一种突变小鼠菌株，在这种菌株中，螺旋体可以在保留抗体介导的有机体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伯氏疏螺旋体及其体内表达抗原的组织来源。2d -差分荧光凝胶电泳、免疫印迹和MALDI-MS/MS将用于鉴定在早期和晚期媒介传播感染中仅在哺乳动物组织中表达的免疫优势伯氏疏螺旋体抗原。候选诊断抗原将作为重组蛋白生产，并与代表不同阶段伯氏疏螺旋体感染的一组血清检测反应性。这项I期研究的结果将为开发新的莱姆病敏感和特异性血清学测试奠定基础，这些测试将更准确地反映伯氏疏螺旋体抗原在受感染宿主中引发的免疫反应。

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.