Lyme Disease Diagnostics Using In Vivo Expressed Borrelia Burgdorferi Antigens

描述（由申请人提供）：莱姆病，由于感染硬蜱传播的螺旋体伯氏疏螺旋体，是美国最常见的病媒传播疾病，每年向CDC报告的病例超过20，000例。在皮肤沉积后，螺旋体可在多个部位传播并引起临床上明显的疾病，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，但那些患有播散性感染或治疗延迟的患者可能会出现不确定病因的挥之不去的体征和症状。莱姆病的及时准确诊断对于优化治疗反应和预防疾病的长期后遗症至关重要。检测抗B抗体的血清学试验（ELISA和免疫印迹）。目前，莱姆病的最敏感和广泛可用的实验室测试。这些试验利用培养的B实验室菌株的全细胞裂解物。作为抗原来源的伯氏螺旋体，因此不能检测到B的抗体。Burgdorferi蛋白只在体内表达。推荐的两层方法（通过免疫印迹确认阳性或可疑ELISA）在感染早期仅具有约30%的灵敏度，并且由于B的交叉反应性，特异性降低。伯氏抗原与其他传染性病原体的抗原。此外，目前的莱姆病ELISA和免疫印迹试验不能区分以前接触过B。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）鉴定免疫显性B。体内表达的伯氏螺旋体蛋白，和B）评估它们在B的各个阶段作为诊断抗原的效用。伯氏感染这种方法的关键是使用突变小鼠品系，其中螺旋体可以在保留抗体介导的对生物体的选择压力的条件下在组织中实现高病原体负荷。这些新的突变小鼠将被用作宿主适应性B的组织来源。Burgdorferi及其体内表达的抗原。将使用二维差异荧光凝胶电泳、免疫印迹和MALDI-MS/MS鉴定免疫显性B。在早期和晚期媒介传播的感染中，伯氏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，并检测与代表不同B期的一组血清的反应性。伯氏感染这项I期研究的结果将为开发新的敏感和特异性莱姆病血清学检测奠定基础，这些检测更准确地反映B。在感染宿主中激发免疫应答的伯氏抗原。 莱姆病，由于感染螺旋体伯氏疏螺旋体，可导致多系统疾病，涉及皮肤，心脏，关节和神经系统。及时诊断这种疾病对于优化治疗和预防长期后遗症至关重要。该建议将使用蛋白质组学方法来鉴定新的免疫显性B。主要在哺乳动物宿主内表达的伯氏螺旋体蛋白。这些蛋白质的鉴定和表征将为莱姆病早期和晚期感染的改进血清学检测的发展奠定基础。

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.