T cell cytokine assay for the diagnosis of disseminated Lyme borreliosis

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，每年报告超过28,000例。感染可局限于皮肤或传播，导致皮肤、心脏、关节和神经系统疾病。螺旋体的OspC基因可能决定了它的侵袭性和传播倾向。虽然抗生素在感染的早期阶段就能达到临床治愈，但播散性感染或延误诊断可能会导致相当大的发病率和医疗费用。及时和准确地诊断莱姆病对于优化治疗和预防该病的长期后遗症至关重要。由于在受感染的组织中几乎没有发现螺旋体，宿主的免疫反应为大多数支持莱姆病诊断的商业实验室测试提供了基础。检测伯氏杆菌反应性抗体的血清学测试(ELISA和免疫印迹)是最广泛使用的测试，但在早期感染时敏感性和特异性较低，在后期可能不确定，特别是在使用抗生素的情况下。此外，目前的莱姆血清学检测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检测可以用来评估对治疗的反应。这项第一阶段的建议旨在通过评估一种新的使用全血的T细胞细胞因子检测用于诊断播散性伯氏杆菌感染和监测治疗反应的可行性，来改进目前可用的莱姆诊断测试。我们方法的关键是使用建立感染(OspC)和持久性(VlsE)所需的两种独特的伯氏杆菌蛋白作为细胞因子诱导的抗原刺激。将产生重组形式的OspC和VlsE，并在小鼠体内进行局部感染、传播和持续一段时间后绘制T细胞细胞因子图谱。将对抗原进行优化，以检测伯氏杆菌感染引起的标志性细胞因子反应，其中5株代表与传播相关的主要OspC基因类型(基因A、B、I、K和N)。使用优化抗原的分析将用于评估标志性细胞因子的下降率是否预测小鼠感染的抗生素消除。最后，将进行试点研究，使用患有莱姆病和不患有莱姆病的受试者的血液样本来评估反应的特异性。这项第一阶段研究的结果将为莱姆病新的快速和特异性细胞免疫分析的开发奠定基础，以补充目前和未来的血清学测试，以加强早期诊断和监测对治疗的反应。 公共卫生相关性：莱姆病是美国最常见的病媒传播疾病，由壁虱传播的伯氏疏螺旋体感染引起。及时诊断感染对于最佳治疗反应和防止长期后遗症非常重要。该项目将评估T细胞细胞因子检测在检测播散性伯氏杆菌感染和监测治疗效果方面的应用。

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 more than 28,000 cases reported annually. The infection can remain localized to the skin or disseminate to cause disease in the skin, heart, joints and nervous system. The ospC genotype of the spirochete may determine its invasiveness and propensity to disseminate. Although antibiotics achieve clinical cure when administered in early stages of infection, disseminated infection or delay in diagnosis can lead to substantial morbidity and health care expenditures. Timely and accurate diagnosis of Lyme disease is essential for optimizing treatment and for preventing long-term sequelae of the disease. Because few spirochetes are found in infected tissues, the host immune response provides the basis for most commercial laboratory tests that support a diagnosis of Lyme disease. Serologic tests (ELISA and immunoblot) that detect B. burgdorferi-reactive antibodies are the most widely used tests, but have lower sensitivity and specificity in early infection and can be indeterminate in later stages, particularly if antibiotics have been administered. In addition, current Lyme serologic 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 1 proposal seeks to improve upon the currently available Lyme diagnostic tests by evaluating the feasibility of a novel T cell cytokine assay using whole blood for the diagnosis of disseminated B. burgdorferi infection and for monitoring response to therapy. The key to our approach is the use of two unique B. burgdorferi proteins required for establishment of infection (OspC) and persistence (VlsE) as antigenic stimuli for cytokine induction. Recombinant forms of OspC and VlsE will be produced and T cell cytokine profiles mapped after tick-borne infection in mice over a period encompassing localized infection, dissemination and persistence. Antigens will be optimized for detection of signature cytokine responses elicited by B. burgdorferi infection with 5 strains representing the main ospC genotypes associated with dissemination (genotypes A, B, I, K, and N). Assays with optimized antigens will be used to assess whether the rate of decline of signature cytokines predicts antibiotic elimination of infection in mice. Finally, pilot studies will be performed to assess the specificity of the response using blood samples from subjects with and without Lyme disease. The results of this Phase 1 study will set the stage for the development of new rapid and specific cellular immune assays for Lyme disease that complement current and future serologic tests to enhance early diagnosis and to monitor response to therapy. PUBLIC HEALTH RELEVANCE: Lyme disease, due to infection with the tick-borne spirochete Borrelia burgdorferi, is the most common vector-borne disease in the United States. Timely diagnosis of infection is important for optimal response to therapy and to prevent long-term sequelae. This project will assess the utility of a T cell cytokine assay for the detection of disseminated B. burgdorferi infection and for monitoring efficacy of treatment.